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Structural and functional studies on the role of the outer membrane of Pseudomonas aeruginosa in resistance… Angus, Barbara Lee 1986

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STRUCTURAL AND FUNCTIONAL STUDIES ON THE ROLE OF THE OUTER MEMBRANE OF PSEUDOMONAS AERUGINOSA IN RESISTANCE AND PERMEABILITY TO ANTIBIOTICS by Barbara Lee Angus B.Sc. U n i v e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of M i c r o b i o l o g y ) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1986 (ci) Barbara Lee Angus, 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Hi CjZ-&l£ J 0L-O /ZA/ The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date OCT- /?& /an ABSTRACT The i n t r i n s i c a n t i b i o t i c r e s i s t a n c e of aeruginosa was examined using an a n t i b i o t i c h y p e r s u s c e p t i b l e mutant s t r a i n . This m u l t i p l e mutant (M1) was c h a r a c t e r i s e d with r e s p e c t to a n t i b i o t i c s u s c e p t i b i l i t y , outer membrane p e r m e a b i l i t y and outer membrane s t r u c t u r e . I t was found to be h y p e r s u s c e p t i b l e to a l l a n t i b i o t i c s t e s t e d . Two spontaneous r e v e r t a n t s of the mutant s t r a i n were i s o l a t e d (P1-4 and P1-6), using c a r b e n i c i l l i n or gentamicin as the s e l e c t i v e agent, and c h a r a c t e r i s e d , r e v e a l i n g that these had recovered p a r t i a l r e s i s t a n c e to a l l a n t i b i o t i c s . Two other s t r a i n s , f o r which two g e n e t i c l o c i (apparently r e s p o n s i b l e f o r a n t i b i o t i c h y p e r s u s c e p t i b i l i t y i n the mutant M1) had been s e p a r a t e l y conjugated i n t o a s t r a i n with a w i l d type g e n e t i c background, were a l s o c h a r a c t e r i s e d . One of these transconjugants (P2-6) was found to be p a r t i a l l y h y p e r s u s c e p t i b l e to a wide range of a n t i b i o t i c s , whereas the other (P2-8) was p a r t i a l l y h y p e r s u s c e p t i b l e to B-lactam and aminoglycoside a n t i b i o t i c s but not to the hydrophobic agent, trimethoprim. Outer membrane p e r m e a b i l i t y of the above s t r a i n s was c h a r a c t e r i s e d by three d i f f e r e n t methods. Using an assay i n v o l v i n g p e r i p l a s m i c beta-lactamase, the mutant s t r a i n M1 was found to be f i v e - f o l d more permeable to the chromogenic c e p h a l o s p o r i n n i t r o c e f i n than i t s wild type parent WT1. In a d d i t i o n , mutant M1 was much more permeable to the hydrophobic f l u o r e s c e n t compound 1-N-phenyl-N-naphthylamine than was w i l d type s t r a i n WT1. In c o n t r a s t , i i mutant s t r a i n M1 was l e s s s u s c e p t i b l e to p e r m e a b i l i s a t i o n of the outer membrane to lysozyme by aminoglycoside a n t i b i o t i c s or e t h y l e n e d i a m i n e t e t r a a c e t a t e . P a r t i a l l y r e s i s t a n t mutants were used to confirm and c l a r i f y these r e s u l t s . I t was concluded from these data that the mutations r e s p o n s i b l e f o r a n t i b i o t i c s u s c e p t i b i l i t y i n the mutant were a c t i n g upon one or more of the outer membrane c o n s t i t u e n t s , p r o t e i n and l i p o p o l y s a c c h a r i d e . C h a r a c t e r i s a t i o n of outer membrane p r o t e i n s by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s showed no observable d i f f e r e n c e s between outer membrane p r o t e i n s of the wi l d type and mutant s t r a i n s . Further examination showed that the l i p o p o l y s a c c h a r i d e i n the mutant s t r a i n M1 was a l t e r e d with respect to i t s composition, m o b i l i t y on SDS-polyacrylamide gels and i n i t s M g + + - b i n d i n g p r o p e r t i e s . From these data, i t appeared that at l e a s t three separate mutations e x i s t e d i n the mutant which c o n t r i b u t e d to a n t i b i o t i c s u s c e p t i b i l i t y . Two of these mutations were r e s p o n s i b l e f o r separate a l t e r a t i o n s to l i p o p o l y s a c c h a r i d e , while the t h i r d mutation could not be c h a r a c t e r i s e d . I t was proposed t h a t the c h i e f a l t e r a t i o n l e a d i n g to a n t i b i o t i c s u s c e p t i b i l i t y i n s t r a i n M1 i n v o l v e d p a r t i a l l o s s of an outer membrane-stabilising, M g + + - b i n d i n g s i t e on l i p o p o l y s a c c h a r i d e . In another p o r t i o n of the study, n a t i v e oligomers of three P_j_ aeruginosa outer membrane p r o t e i n s and one E_;_ c o l i p o r i n were demonstrated using a chemical c r o s s l i n k i n g technique. P_^  aeruginosa p r o t e i n F, the major c o n s t i t u t i v e i i i outer membrane p o r i n , was c r o s s l i n k e d to dimers i n outer membranes and whole c e l l c r o s s l i n k i n g experiments. P u r i f i e d p r e p a r a t i o n s of P_^_ aeruginosa p r o t e i n s F, P (phosphate s t a r v a t i o n - i n d u c e d ) and c o l i p r o t e i n PhoE (Ic) were a l s o c r o s s l i n k e d to r e v e a l dimers and t r i m e r s upon two-dimensional SDS-polyacrylamide g e l e l e c t r o p h o r e t i c a n a l y s i s . C r o s s l i n k i n g of p r o t e i n F was a b o l i s h e d by pretreatment of the p r o t e i n with SDS, i n d i c a t i n g that the c r o s s l i n k e d products were due to na t i v e a s s o c i a t i o n s i n the outer membrane. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v L i s t of Tables x L i s t of F i g u r e s x i i L i s t of A b b r e v i a t i o n s x i v ACKNOWLEDGEMENTS xv INTRODUCTION 1 1 . A n t i b i o t i c r e s i s t a n c e and medical importance 1 of Pseudomonas aeruginosa 2. C l a s s e s of a n t i b i o t i c s used a g a i n s t Pseudomonas 4 aeruginosa 3. Mechanisms of a n t i b i o t i c r e s i s t a n c e 8 4. S t r u c t u r e of the gram negative outer membrane 10 a. P r o t e i n s 12 b. L i p o p o l y s a c c h a r i d e 14 5. P e r m e a b i l i t y of the outer membrane 18 6. Role of the outer membrane of Pseudomonas 20 aeruginosa i n permeation of a n t i b i o t i c s 7. A n t i b i o t i c h y p e r s u s c e p t i b l e mutants 21 8. Aim of the study 23 METHODS 24 1. Medium and c u l t u r e c o n d i t i o n s 24 2. B a c t e r i a l s t r a i n s 24 3. A n t i b i o t i c s u s c e p t i b i l i t y t e s t i n g 25 v Page 4. A n t i b i o t i c s and chemicals 26 5. I s o l a t i o n of spontaneous r e v e r t a n t s of M1 27 6. Bacteriophage s e n s i t i v i t y t e s t i n g 28 7. Beta-lactamase assay and c a l c u l a t i o n s 28 8. NPN uptake assay 30 9. P e r m e a b i l i s a t i o n of whole c e l l s to lysozyme 31 10. Dansyl polymyxin b i n d i n g assay 32 11 . Phase p a r t i t i o n i n g of c e l l s 3 12. P r e p a r a t i o n of outer membranes 34 13. P r e p a r a t i o n of c e l l envelopes 34 14. I s o l a t i o n of l i p o p o l y s a c c h a r i d e 34 15. P r e p a r a t i o n of L i p i d A 36 16. Chemical assays 36 a. P r o t e i n assay b. 3-deoxy-D-mannooctulosonic a c i d (KDO) assay c. Phosphate assay 17. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s 37 18. E l e c t r o p h o r e t i c b l o t t i n g of p r o t e i n s and 38 l i p o p o l y s a c c h a r i d e 19. Immunoenzymatic s t a i n i n g of p r o t e i n s or 38 l i p o p o l y s a c c h a r i d e on b l o t s 20. Gel s t a i n i n g procedures 39 a. P r o t e i n b. L i p o p o l y s a c c h a r i d e 21 . Protease d i g e s t i o n of outer membranes 40 22 . Assay f o r LPS carbohydrate 41 23. Gas l i q u i d chromatography of whole c e l l 41 v i f a t t y a c i d s Page. 24. C r o s s l i n k i n g of outer membranes and p u r i f i e d 42 p r o t e i n s CHAPTER ONE C h a r a c t e r i s a t i o n of an A n t i b i o t i c 44 H y p e r s u s c e p t i b l e Mutant of Pseudomonas aeruginosa INTRODUCTION 44 RESULTS 47 PART I. A n t i b i o t i c s u s c e p t i b i l i t y of wi l d type and 47 mutant s t r a i n s of Pseudomonas aeruginosa 1. D e r i v a t i o n of b a c t e r i a l s t r a i n s used i n 47 c h a r a c t e r i s a t i o n of a n t i b i o t i c s u s c e p t i b i l i t y and outer membrane a l t e r a t i o n s 2. S u s c e p t i b i l i t y of w i l d type and mutant s t r a i n s 51 to a v a r i e t y of a n t i b i o t i c s with d i f f e r e n t mechanisms of a c t i o n 3. A n t i b i o t i c s u s c e p t i b i l i t y of transconjugant 56 and t r a n s d u c t a n t s t r a i n s d e r i v e d from mutant s t r a i n M1 4. Bacteriophage s e n s i t i v i t y of wi l d type and 59 mutant s t r a i n s 5. Other p r o p e r t i e s of the a n t i b i o t i c hyper- 62 s u s c e p t i b l e mutant s t r a i n a. Frequency of r e v e r s i o n 62 b. Growth rate of the mutant 63 c. Colony morphology 63 6. Summary 64 PART I I . C h a r a c t e r i s a t i o n of a l t e r a t i o n s i n outer 66 membrane p e r m e a b i l i t y of mutant s t r a i n s 1. H y d r o p h i l i c (porin-mediated) p e r m e a b i l i t y of 67 whole c e l l s to a chromogenic cephalosporin a n t i b i o t i c 2. I n d u c t i o n p r o p e r t i e s of chromosomal beta- 74 v i i Page lactamase from w i l d type and mutant s t r a i n s 3. Outer membrane p e r m e a b i l i t y to hydrophobic 77 compounds 4. Surface charge p r o p e r t i e s of wi l d type and 81 mutant c e l l s 5. Self-promoted outer membrane p e r m e a b i l i t y : 83 Aminoglycoside enhancement of outer membrane p e r m e a b i l i t y to a hydrophobic compound 6. Self-promoted outer membrane p e r m e a b i l i t y : 86 EDTA, aminoglycoside and polymyxin B enhance-ment of lysozyme-mediated c e l l l y s i s 7. Summary 90 PART I I I . C h a r a c t e r i s a t i o n of a l t e r a t i o n s i n outer 92 membrane components i n v o l v e d i n a n t i b i o t i c s u s c e p t i b i l i t y 1. Outer membrane p r o t e i n p a t t e r n s 92 2. Densitometry scans of e l e c t r o p h o r e s e d outer 95 membrane samples s t a i n e d f o r p r o t e i n or carbohydrate 3. Protease d i g e s t i o n patterns and monoclonal 98 antibody r e a c t i v i t y of outer membrane p r o t e i n s 4. Self-promoted outer membrane p e r m e a b i l i t y : 101 i n t e r a c t i o n of polymyxin B with w i l d type and mutant c e l l s u r f a c e s 5. I n t e r a c t i o n of polymyxin B with l i p o p o l y - 102 s a c c h a r i d e : b i n d i n g of dansyl polymyxin 6. I n t e r a c t i o n of polymyxin B with l i p o p o l y - 104 s a c c h a r i d e : b i n d i n g of dansyl polymyxin to the i s o l a t e d L i p i d A f r a c t i o n of LPS 7. Competition by Mg + + of dansyl polymyxin 105 b i n d i n g to i s o l a t e d l i p o p o l y s a c c h a r i d e and L i p i d A 8. Whole c e l l f a t t y a c i d composition 109 9. L i p o p o l y s a c c h a r i d e rough core n e u t r a l sugar 111 composition v i i i Page 10. SDS-PAGE banding p a t t e r n of rough core 115 l i p o p o l y s a c c h a r i d e i n wild type and mutant s t r a i n s 11. SDS-PAGE banding p a t t e r n of major l i p o p o l y - 117 sac c h a r i d e O - a n t i g e n - c o n t a i n i n g species 12. Immunoreactivity of a l t e r e d outer membrane 119 components with monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t l i p o p o l y s a c c h a r i d e 13. LPS phosphate content 121 14. Summary 124 DISCUSSION 126 CHAPTER TWO Chemical C r o s s l i n k i n g of Pseudomonas aeruginosa 147 and E s c h e r i c h i a c o l i Outer Membrane Por i n P r o t e i n s to Reveal Native Oligomers INTRODUCTION 147 RESULTS 151 1. C r o s s l i n k i n g of E_j_ c o l i outer membrane 151 p r o t e i n Pho E 2. C r o s s l i n k i n g of P_j_ aeruginosa p r o t e i n P 155 3. C r o s s l i n k i n g of P_j_ aeruginosa p r o t e i n F 159 4. C r o s s l i n k i n g of p r o t e i n s i n P_j_ aeruginosa 161 outer membranes with v a r i o u s c o n c e n t r a t i o n s of c r o s s l i n k e r DISCUSSION 170 LITERATURE CITED 177 ix LIST OF TABLES Page Table I P_;_ aeruginosa s t r a i n s 50 I I Minimal i n h i b i t o r y c o n c e n t r a t i o n s (MIC) of a 52 v a r i e t y of a n t i b i o t i c s f o r parent and a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s . I l l Minimal i n h i b i t o r y c o n c e n t r a t i o n s (MIC) of three 55 a n t i b i o t i c s i n spontaneous r e v e r t a n t s , t r a n s c o n j u g a n t s and t r a n s d u c t a n t s of Ml. IV S u s c e p t i b i l i t y to v a r i o u s a n t i b i o t i c s of a l l P_^  57 aeruginosa s t r a i n s by d i s c i n h i b i t i o n diameter assay V Bacteriophage s e n s i t i v i t y of wi l d type and 61 a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s . VI Chromosomal beta-lactamase a c t i v i t y and n i t r o - 70 c e f i n permeation through the outer membrane of wild type and a n t i b i o t i c h y p e r s u s c e p t i b l e P.  aeruginosa s t r a i n s . VII Outer membrane p e r m e a b i l i t y of whole c e l l s to 72 n i t r o c e f i n u s ing TEM-2 beta-lactamase VIII I n d u c t i o n of chromosomal beta-lactamase i n P_^  75 aeruginosa w i l d type and a n t i b i o t i c hypersuscept-i b l e s t r a i n s . IX Fluorescence i n c r e a s e due to uptake of NPN i n 78 whole c e l l s . X Phase p a r t i t i o n i n g of wild type and mutant c e l l s 82 of aeruginosa. XI Rate of f l u o r e s c e n c e i n c r e a s e due to uptake of 84 NPN by whole c e l l s a f t e r a d d i t i o n of 4 ug/ml of gentamicin. XII P e r m e a b i l i s a t i o n of whole c e l l s to lysozyme by 88 EDTA, gentamicin or polymyxin B. XIII Binding of dansyl polymyxin B to whole c e l l s , L P S 103 and L i p i d A, and competition of binding by Mg++ XIV F a t t y a c i d composition of whole c e l l s of ?^ 110 aeruginosa s t r a i n s WT1 , P1 — 1 , P1-4 and M1. x Page XV A n a l y s i s of l i p o p o l y s a c c h a r i d e n e u t r a l sugars. 114 XVI Phosphate content of l i p o p o l y s a c c h a r i d e and 122 L i p i d A from w i l d type and mutant s t r a i n s XVII Summary of the c r o s s l i n k e d multimers of p o r i n 171 p r o t e i n s observed i n t h i s study. x i LIST OF FIGURES Page F i g u r e 1. S t r u c t u r e of beta-lactam a n t i b i o t i c s 5 2. Gentamicin enhancement of NPN uptake by whole 79 c e l l s . 3. P e r m e a b i l i s a t i o n of whole c e l l s to lysozyme by 87 EDTA, gentamicin or polymyxin B. 4. SDS-PAGE of outer membranes and c e l l envelopes 93 from wild type and a n t i b i o t i c h y p e r s u s c e p t i b l e mutant s t r a i n s of P_;_ aeruginosa. 5. Densitometry scans of SDS-PAGE of P_^  96 aeruginosa outer membranes s t a i n e d f o r carbohyd-r a t e and/or p r o t e i n . 6. SDS-PAGE of protease d i g e s t i o n and immuno- 99 r e a c t i v i t y of outer membrane samples from s t r a i n s WT1 and M1. 7. Binding of dansyl polymyxin to L i p i d A and 106 competition of b i n d i n g by Mg + +. 8. I n h i b i t i o n by gentamicin of dansyl polymyxin 108 b i n d i n g to L i p i d A. 9. Spectrophotometric scans of carbohydrate assay 112 on p u r i f i e d LPS from P_;_ aeruginosa s t r a i n s WT1 , WT3 and M1. 10. SDS-PAGE of LPS from w i l d type and a n t i b i o t i c 116 s u s c e p t i b l e s t r a i n s . 11. Immunoreactivity of e l e c t r o p h o r e t i c a l l y b l o t t e d 120 LPS from w i l d type and mutant s t r a i n s . 12. Model suggesting how LPS a l t e r a t i o n s i n an 146 a n t i b i o t i c h y p e r s u s c e p t i b l e mutant of Pseudomonas  aeruginosa a f f e c t outer membrane p e r m e a b i l i t y 13- One- and two-dimensional c r o s s l i n k i n g a n a l y s i s 152 of p u r i f i e d E_;_ c o l i p r o t e i n PhoE. 14. One- and two-dimensional c r o s s l i n k i n g a n a l y s i s 156 of p u r i f i e d aeruginosa p r o t e i n P. 15. Two-dimensional c r o s s l i n k i n g a n a l y s i s of p u r i f i e d 160 P. aeruginosa p r o t e i n F. x i i 16. O n e - d i m e n s i o n a l SDS-PAGE o f o u t e r membrane samples t r e a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f DSP f o r two m i n u t e s . 162 17. T w o - d i m e n s i o n a l c r o s s l i n k i n g o f o u t e r membranes 164 u s i n g 40 ug DSP/mg p r o t e i n . 18. T w o - d i m e n s i o n a l c r o s s l i n k i n g o f o u t e r membranes 165 u s i n g 200 ug DSP/mg p r o t e i n . 19. T w o - d i m e n s i o n a l SDS-PAGE o f c r o s s l i n k e d o u t e r 167 membrane samples s t a i n e d f o r c a r b o h y d r a t e . 2 0 . T w o - d i m e n s i o n a l SDS-PAGE o f u n c r o s s l i n k e d c o n t r o l 168 o u t e r membrane s a m p l e s . x i i i L I S T OF ABBREVIATIONS p r o t e o s e p e p t o n e no.2 g r o w t h medium s o d i u m d o d e c y l s u l p h a t e e t h y l e n e d i a m i n e t e t r a a c e t a t e d a n s y l p o l y m y x i n B N - p h e n y l - 1 - n a p h t h y 1 a m i n e l i p o p o l y s a c c h a r i d e m i n i m a l i n h i b i t o r y c o n c e n t r a t i o n A b s o r b a n c e a t 600 nm d i t h i o - b i s - ( s u c c i n i m i d y l p r o p i o n a t e ) c r o s s l i n k e r p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p h o s p h a t e b u f f e r e d s a l i n e n a n o S i e m e n ( u n i t o f e l e c t r i c a l c o n d u c t a n c e , ohm" x i v ACKNOWLEDGEMENTS I would l i k e to acknowledge the f i n a n c i a l support of both the Canadian C y s t i c F i b r o s i s Foundation and Dr. R.E.W. Hancock during my term of Ph. D. candidacy. I would a l s o l i k e to thank the f o l l o w i n g members of the l a b o r a t o r y , who have made l i f e more enj o y a b l e : T h a l i a Nicas, A l i c e Carey, Lucy Mutharia, K e i t h Poole, Kazuhiro Nakajima, Val R a f f l e , Lynn Dent, Lydia Chan, Andrew Wieczorek, Rich Darveau, P i x i e Mouat, Joe Lam, Gord C r o c k f o r d , Rich Moore, Tom Parr , Janet B a t t e r s h i l l , Lynn Vasington Moore, Bernadette Loh, C h r i s Grant, P a t r i c k Wong, Richard S i e h n e l , Niamh K e l l y , Gus B e l l , Betty Worobec, Nancy M a r t i n , Janet Sawyer, Wendy Woodruff and e s p e c i a l l y Bob Hancock. "When you have e l i m i n a t e d the i m p o s s i b l e , whatever remains, however improbable, must be the t r u t h . " (A. C. Doyle) xv INTRODUCTION 1. A n t i b i o t i c r e s i s t a n c e and medical importance of Pseudomonas  aeruginosa. The emergence of r e s i s t a n c e to a n t i b i o t i c s i n pathogenic organisms became a medical concern s h o r t l y a f t e r widespread c l i n i c a l use of a n t i b i o t i c s began. A n t i b i o t i c r e s i s t a n c e f i r s t emerged during therapy due to the a b i l i t y of microorganisms to mutate at a r e l a t i v e l y low frequency. A more recent source of concern i s the advent of s o - c a l l e d " i n f e c t i o u s " or plasmid-mediated r e s i s t a n c e , whereby r e s i s t a n c e to a n t i m i c r o b i a l agents may be t r a n s f e r r e d at much higher f r e q u e n c i e s and with a consequently non-random occurrence. This type of a n t i b i o t i c r e s i s t a n c e has l e d to r a p i d spread of b a c t e r i a l i n f e c t i o n s w i t h i n h o s p i t a l s . For example, i n the 1960's, s t r a i n s of Staphylococcus aureus r e s i s t a n t to p e n i c i l l i n G were able to cause s e r i o u s i n f e c t i o n s i n p a t i e n t s with wounds. Resistance to p e n i c i l l i n s (and other a n t i b i o t i c s ) i n these organisms was due to possession of p l a s m i d - c a r r i e d , b e t a - l a c t a m - i n a c t i v a t i n g enzymes, or m u l t i p l y a l t e r e d p e n i c i l l i n b i n d i n g p r o t e i n s , the t a r g e t s i t e s of p e n i c i l l i n s (Brunton, 1984). Semisynthetic beta-lactams and p e n i c i l l i n a s e i n h i b i t o r s such as c l a v u l a n i c a c i d were l a r g e l y s u c c e s s f u l i n d e a l i n g with these two types of r e s i s t a n c e . Pseudomonas aeruginosa more ^recently j o i n e d S_;_ aureus as one of the major causes of nosocomial ( h o s p i t a l - a c q u i r e d ) 1 i n f e c t i o n s (Lowbury, 1975). P_;_ aeruginosa i s an o p p o r t u n i s t i c pathogen and, although i t poses no danger to healthy i n d i v i d u a l s , i t i s able to cause s e r i o u s i n f e c t i o n s i n p a t i e n t s who are immunosuppressed, e i t h e r due to the nature of t h e i r i n j u r i e s or to drug therapy such as i s used i n treatment of cancer. aeruginosa can cause v a r i o u s types of l o c a l i n f e c t i o n s , f o r example, i n burn wounds, u r i n a r y , i n t e s t i n a l or r e s p i r a t o r y t r a c t s ( c y s t i c f i b r o s i s ) , the c o n j u n c t i v a of the eye, or i n the ear. The organism tends to i n f e c t t i s s u e areas surrounding small blood v e s s e l s i n each case (Lowbury, 1975 ) . P_;_ aeruginosa may a l s o cause g e n e r a l i s e d ( s e p t i c e m i c or toxemic) i n f e c t i o n s i n r a r e cases, and death i s almost c e r t a i n because of i t s high r e s i s t a n c e to a n t i b i o t i c s and the e x t e n s i v e t i s s u e damage caused by the organism p r i o r to onset of s e p t i c e m i a . P. aeruginosa i s a l s o m e d i c a l l y s i g n i f i c a n t with r e s p e c t to v i r u l e n c e f a c t o r s and immunity. The organism produces a l a r g e v a r i e t y of excreted v i r u l e n c e f a c t o r s , whereas other gram negative pathogens do not. Included amongst these f a c t o r s are v a r i o u s proteases ( i n c l u d i n g e l a s t a s e and c o l l a g e n a s e ) , phospholipases A and C ( l e c i t h i n a s e and hemolysin), h y a l u r o n i d a s e , and exotoxins A and S (Lowbury , 1 975 ) . Human immunity to P_;_ aeruginosa i s not yet w e l l understood. Although s i g n i f i c a n t l e v e l s of antibody appear i n the serum of p a t i e n t s i n f e c t e d with P_;_ aeruginosa (Fox and Lowbury, 1953), these do not always appear to be of p r o t e c t i v e value i n combatting i n f e c t i o n . The p r e v a i l i n g 2 t h e o r i e s are e i t h e r that the s i t e of i n f e c t i o n i s somehow i n a c c e s s i b l e to a n t i b o d i e s , that the a n t i b o d i e s are degraded by P_;_ aeruginosa proteases, that the types of anti-Pseudomonal a n t i b o d i e s i n the lung are non-opsonic f o r a l v e o l a r macrophages, or that the organism i s p r o t e c t e d from phagocytic a t t a c k . In a d d i t i o n , at the stage where se p t i c e m i a occurs, when serum antibody could be p r o t e c t i v e , p r i o r t i s s u e damage i s u s u a l l y so ex t e n s i v e that immunity i s no longer of any consequence. The aforementioned approaches to a n t i m i c r o b i a l therapy, which were s u c c e s s f u l i n d e a l i n g with aureus, have had only l i m i t e d success with aeruginosa s i n c e r e s i s t a n c e was, f o r the most p a r t , not dependent on enzymic d e a c t i v a t i o n or t a r g e t a l t e r a t i o n , but on imper m e a b i l i t y to a n t i m i c r o b i a l agents (Angus e_t a_l. , 1982; Yoshimura and Nikaido, 1982) and probably a l s o on i n a c c e s s i b i l i t y to drug therapy i n v i v o (Costerton e_t a l . , 1981). Impermeability (one type of s o - c a l l e d i n t r i n s i c r e s i s t a n c e ) i s mediated by the outer membrane i n gram negative b a c t e r i a , which forms a b a r r i e r to a n t i b i o t i c s and other molecules above a de f i n e d e x c l u s i o n l i m i t ( L e i v e , 1974; Zimmermann, 1980). A n t i b i o t i c s are thus prevented from reaching t h e i r s i t e s of a c t i o n i n the periplasm or w i t h i n the c e l l . This accounts f o r the ge n e r a l r e s i s t a n c e of P.  aeruginosa to a wide v a r i e t y of a n t i b i o t i c s , d e s p i t e t h e i r v a r y i n g s i t e s of a c t i o n w i t h i n the c e l l . P_j_ aeruginosa i s much l e s s s u s c e p t i b l e to most a n t i b i o t i c s than are the e n t e r i c group of b a c t e r i a , such as E s c h e r i c h i a c o l i and S_j_ typhimurium 3 (Brown, 1975; Zimmermann and R o s s e l e t , 1977). Notable exceptions to t h i s r u l e are aminoglycosides and polymyxin B, which w i l l be d i s c u s s e d l a t e r . Newer beta-lactam a n t i b i o t i c s have a l s o been developed s p e c i f i c a l l y f o r use a g a i n s t P.  aeruginosa, and to which the bacterium i s p r o p o r t i o n a l l y more s u s c e p t i b l e than are e n t e r i c b a c t e r i a . Despite the advent of improved a n t i b i o t i c s , the d i f f i c u l t y of a c h i e v i n g t h e r a p e u t i c a l l y u s e f u l l e v e l s of these i n the body, without causing t o x i c i t y , means that P_^  aeruginosa i n f e c t i o n s remain d i f f i c u l t to t r e a t . 2. Cla s s e s of a n t i b i o t i c s used a g a i n s t P. aeruginosa. S e v e r a l d i f f e r e n t types of a n t i b i o t i c s have been used throughout t h i s study f o r the purposes of i l l u s t r a t i n g s u s c e p t i b i l i t y / r e s i s t a n c e and f o r demonstrating s p e c i f i c types of uptake across the outer membrane of P_;_ aeruginosa. The p e r t i n e n t p h y s i c a l p r o p e r t i e s of these a n t i b i o t i c s which lend themselves to each purpose are t h e r e f o r e d e s c r i b e d below. a) Beta-lactams This group of a n t i b i o t i c s a cts by i n h i b i t i n g the c e l l d i v i s i o n process. The s i t e of a c t i o n i s on p e n i c i l l i n b i n d i n g p r o t e i n s at the outer s u r f a c e of the cytoplasmic membrane (Blumberg and Strominger, 1974), and each of these p r o t e i n s has c h a r a c t e r i s t i c a f f i n i t i e s f o r d i f f e r e n t beta-lactam a n t i b i o t i c s . The s t r u c t u r e s of some of the major c l a s s e s of beta-lactams are d e s c r i b e d by F i g u r e 1. S t r u c t u r a l m anipulation of beta-lactam a n t i b i o t i c s to y i e l d many 4 v a r i a t i o n s of these d i f f e r e n t c l a s s e s was designed to i n f l u e n c e a b s o r p t i o n i n the body, p e n e t r a t i o n i n t o b a c t e r i a l c e l l s , s t a b i l i t y to beta-lactamase, and a f f i n i t y f o r p e n i c i l l i n b i n d i n g p r o t e i n s (Abraham, 1982). P_;_ aeruginosa i s more s u s c e p t i b l e to the more recent p e n i c i l l i n a n t i b i o t i c s (eg. c a r b e n i c i l l i n , p i p e r a c i l l i n and t i c a r c i l l i n ) and " t h i r d g e n e r a t i o n " c e p h a l o s p o r i n s . The a c t i v e p o r t i o n of the beta-lactam molecule i s the beta-lactam r i n g , which i s numbered 1 i n f i g u r e 1(a), and which does not vary. The adjacent r i n g (2 i n f i g u r e 1(a)) does vary i n s t r u c t u r e and t h e r e f o r e p l a y s an important r o l e i n the above mentioned p r o p e r t i e s of the a n t i b i o t i c . In a d d i t i o n , there are many types of R-groups which can be s u b s t i t u t e d at the designated p o s i t i o n s to f u r t h e r i n f l u e n c e chemical p r o p e r t i e s . In p e n i c i l l i n G, f o r example, the second r i n g i s five-membered, with sulphur i n the 1 p o s i t i o n , and i s r e f e r r e d to as a "penam" r i n g (Rao and Vasudevan, 1983). Cephalosporins ( F i g . 1(b)) are s i m i l a r , but have a s i x -membered "cepham" r i n g . Newer d e r i v a t i v e s s u b s t i t u t e oxygen or carbon f o r the sulphur atom or have a double bond i n the second r i n g ( F i g . 1, (c-e)) Some of these compounds are u s e f u l as beta-lactamase i n h i b i t o r s , such as c l a v u l a n i c a c i d . In a d d i t i o n , a new c l a s s of these compounds a l s o e x i s t s which only possesses one r i n g s t r u c t u r e , the beta-lactam r i n g , and these compounds are r e f e r r e d to as "monobactams" (Figure 1 ( f ) - Sykes et a l . , 1981). 5 Figure 1. Structure of (3-lactam antibiotics a) i -N-0 ^COOH Penicillin 0 0 N-R COOH Oxypenicillin e) R- R 'COOH b) 0 •N R d) R> COOH Cephalosporin COOH Oxycephalosporin f) R« -»si R Penem Monobactam b) Aminoglycosides and polymyxin The gen e r a l s t r u c t u r e of aminoglycosides i s three carbohydrate r i n g s with three to s i x f r e e amino groups, making these l a r g e , p o l y c a t i o n i c molecules. Aminoglycosides bind to the 30S ribosomal subunit to i n h i b i t p r o t e i n s y n t h e s i s i n prokaryotes, although the a c t u a l b a c t e r i c i d a l event i s thought to i n v o l v e the act of energised uptake i n t o the c e l l (Bryan and van den E l z e n , 1977) > and has been d i s c u s s e d at some length by Hancock (1981). Aminoglycosides have a l s o been rep o r t e d to exert a d e s t a b i l i s i n g e f f e c t on the outer membrane (Nicas and Hancock,1980). Some t h e r a p e u t i c a l l y important aminoglycosides are streptomycin, neomycin and kanamycin, which have only l i m i t e d a c t i v i t y a g a i n s t P^ aeruginosa, as w e l l as gentamicin, tobramycin, sisomycin and n e t i l m i c i n , which are c u r r e n t l y used with some success a g a i n s t aeruginosa. u s u a l l y i n combination with one of the newer c e p h a l o s p o r i n or o x y p e n i c i l l i n a n t i b i o t i c s (Maxwell, 1983). Polymyxin B i s a l s o a p o l y c a t i o n i c a n t i b i o t i c but i t s s t r u c t u r e i s much d i f f e r e n t than that of aminoglycosides. Polymyxin B i s composed of a hydrophobic methyloctanoyl or methylheptanoyl t a i l attached v i a a t r i p e p t i d e to a c y c l i c peptide composed mostly of d i a m i n o b u t y r i c a c i d as w e l l as L-thr e o n i n e , L - l e u c i n e and D-phenylalanine (Weinstein, 1975). Polymyxin B a l s o i n h i b i t s p r o t e i n s y n t h e s i s and d i s r u p t s outer membrane i n t e g r i t y , although the main e f f e c t may be on membrane e n e r g i s a t i o n . c) Others T e t r a c y c l i n e s are l a r g e molecules composed of 7 four six-membered r i n g s . These act to i n h i b i t p r o t e i n s y n t h e s i s but are b a c t e r i o s t a t i c , r a t h e r than b a c t e r i o c i d a l . U n l i k e aminoglycosides, they are not p o l y c a t i o n s and do not d e s t a b i l i s e the outer membrane i n the same f a s h i o n . Chloramphenicol i s a b a c t e r i o s t a t i c agent which binds to the 50S ribosomal subunit of prokaryotes to i n h i b i t p r o t e i n s y n t h e s i s . I t i s a n i t r o b e n z y l d e r i v a t i v e of d i c h l o r o a c e t i c a c i d and i s consequently q u i t e hydrophobic i n nature. Trimethoprim i s a di a m i n o p y r i d i n e composed of two resonant r i n g s t r u c t u r e s , one of which i s a benzyl d e r i v a t i v e and one which c o n t a i n s two n i t r o g e n atoms i n the r i n g and two a s s o c i a t e d f r e e amino groups. This a n t i b i o t i c a cts w i t h i n the b a c t e r i a l c e l l to i n h i b i t d i h y d r o f o l a t e reductase (Hamilton-M i l l e r , 1984), which i s important i n the s y n t h e s i s of n u c l e i c a c i d s and c e r t a i n amino a c i d s . I t i s a l s o very hydrophobic, and aeruginosa i s consequently f a i r l y r e s i s t a n t to i t . 3• Mechanisms of a n t i b i o t i c r e s i s t a n c e . Three types of a n t i b i o t i c r e s i s t a n c e mechanisms are g e n e r a l l y r e c o g n i s e d : a) s p e c i f i c i n a c t i v a t i n g enzymes, i n c l u d i n g beta-lactamases, f o r p e n i c i l l i n s and c e p h a l o s p o r i n s , and ph o s p h o r y l a t i n g or A D P - r i b o s y l a t i n g enzymes, f o r aminoglycosides (Bryan, 1984) and chloramphenicol. A c q u i s i t i o n by a bacterium of a plasmid s p e c i f y i n g one of these enzymes can d r a s t i c a l l y i n c r e a s e the minimal i n h i b i t o r y c o n c e n t r a t i o n values f o r these a n t i b i o t i c s . D erepression of a 8 chromosomally s p e c i f i e d gene f o r beta-lactamase, such as ampC of E_j_ c o l i , or aminoglycoside 3'-phosphotransferase I I of P.  aeruginosa ( O k i i e_t ajl. , 1983) may a l s o i n c r e a s e r e s i s t a n c e . b) t a r g e t a l t e r a t i o n s , such as i n p e n i c i l l i n b i n d i n g p r o t e i n s , ribosomes or s p e c i f i c b i o s y n t h e t i c enzymes, which do not a f f e c t f u n c t i o n of the p r o t e i n s but lower t h e i r a f f i n i t y f o r b i n d i n g of a n t i b i o t i c s . c) p e r m e a b i l i t y , s i n c e the t a r g e t s f o r the a n t i b i o t i c s l i e i n t e r i o r to the outer membrane and many are w i t h i n the c e l l . A decrease i n p e r m e a b i l i t y to a n t i b i o t i c s i s the only mechanism which can e x p l a i n r e s i s t a n c e to a range of a n t i b i o t i c s with v a r y i n g p h y s i c a l p r o p e r t i e s and s i t e s of a c t i o n . I t should be mentioned that s y n e r g i s t i c a c t i o n between two of these parameters, f o r example beta-lactamase and outer membrane p e r m e a b i l i t y , may be very important f o r the observed r e s i s t a n c e l e v e l (Vu and Nikaido, 1985). The outer membrane b a r r i e r may act to slow contact of beta-lactams with t h e i r t a r g e t s i n the perip l a s m s u f f i c i e n t l y such that low l e v e l s of beta-lactamase can dispose of them (Richmond and C u r t i s , 1974). The balance between the amount of beta-lactam necessary to allow i n d u c t i o n of beta-lactamase, the amount of beta-lactamase which can be produced by the c e l l , and the amount of beta-lactam which gains access to the periplasm, might be c r i t i c a l i n determining c e l l s u r v i v a l . For example, a sm a l l change (up or down) i n any of these could upset the 9 balance such that l a r g e changes i n s u s c e p t i b i l i t y are manifested. In P_^_ aeruginosa, the t h i r d mechanism (outer membrane p e r m e a b i l i t y ) appears to be of g r e a t e s t importance i n e x p l a i n i n g high i n t r i n s i c r e s i s t a n c e to a n t i b i o t i c s . 4. S t r u c t u r e of the gram negative outer membrane. Gram negative c e l l s possess an a d d i t i o n a l b i l a y e r envelope o u t s i d e of t h e i r p e p t i d o g l y c a n w a l l and cyto p l a s m i c membrane. The outer membrane i s composed of p h o s p h o l i p i d , p r o t e i n and l i p o p o l y s a c c h a r i d e , i n an ordered conformation, and f u n c t i o n s to allow p a s s i v e d i f f u s i o n of molecules below a c e r t a i n s i z e - e x c l u s i o n l i m i t . The b i l a y e r appears to be organised such that the inn e r monolayer c o n t a i n s most of the ph o p h o l i p i d ( p r i m a r i l y p h o s p h a t i d y l ethanolamine) and the outer monolayer c o n t a i n s l i p o p o l y s a c c h a r i d e occupying an estimated 45$ of the c e l l s u r f a c e (Smit et a l . , 1975). The remaining c e l l s u r f a c e components are p r o t e i n s which have a f u n c t i o n a l s i g n i f i c a n c e i n both outer membrane s t a b i l i t y and pass i v e d i f f u s i o n . The c e l l s u r f a c e of E_^  c o l i appears, from X-ray d i f f r a c t i o n s t u d i e s (Yamada and Mizushima, 1980; Rosenbusch, 1974), to be r i g i d l y organised i n t o a hexagonal a r r a y , based on the p o s i t i o n i n g of p o r i n p r o t e i n molecules i n a t r i m e r i c f a s h i o n with a s s o c i a t e d l i p o p o l y s a c c h a r i d e . The purpose of the gram negative outer membrane seems to be as a s e l e c t i v e p e r m e a b i l i t y b a r r i e r . Since gram negative b a c t e r i a have a much l e s s complex p e p t i d o g l y c a n w a l l , t h i n n e r 10 and w i t h o u t t e i c h o i c a c i d s ( M i r e l m a n , 1979), the o u t e r membrane may be an a l t e r n a t i v e to the t h i c k , h i g h l y c r o s s l i n k e d p e p t i d o g l y c a n l a y e r o f gram p o s i t i v e c e l l s (Nakae and N i k a i d o , 1975). I t performs some f u n c t i o n s , such as r u d i m e n t a r y s i z e e x c l u s i o n and p r o t e c t i o n from d e s t r u c t i v e a g e n t s , s i m i l a r l y to p e p t i d o g l y c a n o f gram p o s i t i v e c e l l s a n d , i n a d d i t i o n , makes p o s s i b l e some a s p e c t s unique to gram n e g a t i v e c e l l s . F o r i n s t a n c e , the p r e s e n c e o f an o u t e r membrane c r e a t e s a p e r i p l a s m i c s p a c e , which a l l o w s the c e l l to c o n c e n t r a t e n u t r i e n t s and s e c r e t e d c a t a b o l i c enzymes o u t s i d e the c e l l i n a p r o t e c t e d e n v i r o n m e n t . The p r o t e i n s which e x i s t i n the p e r i p l a s m i c space are used to break down n u t r i e n t m a c r o m o l e c u l e s i n t o components which may be t r a n s f e r r e d a c r o s s the c y t o p l a s m i c membrane, as w e l l as to s p e c i f i c a l l y b i n d amino a c i d s and s u g a r s , which removes them from s o l u t i o n , a l l o w i n g t h e i r c o n c e n t r a t i o n i n the p e r i p l a s m , and thus f a c i l i t a t e a c t i v e t r a n s p o r t . The o u t e r membrane p r o v i d e s p r o t e c t i o n a g a i n s t s e v e r a l types o f a n t a g o n i s t i c m o l e c u l e s . For example, the p r o t e i n lysozyme (which d e s t r o y s p e p t i d o g l y c a n ) i s e f f e c t i v e l y e x c l u d e d from i t s t a r g e t by the membrane (Sanderson e_t §_1. , 1974), and n o n - i o n i c d e t e r g e n t s and h y d r o p h o b i c a n t i b i o t i c s are e x c l u d e d due to c e r t a i n p r o p e r t i e s o f the unique o u t e r membrane m o l e c u l e , l i p o p o l y s a c c h a r i d e ( N i k a i d o , 1979). The o u t e r membrane components which make these e s s e n t i a l f u n c t i o n s p o s s i b l e are d i s c u s s e d i n d e t a i l below. 1 1 a) P r o t e i n s The outer membrane of a l l s p e c i e s examined so f a r con t a i n s a smal l v a r i e t y of p r o t e i n s which tend to be present i n l a r g e copy number. The most w e l l c h a r a c t e r i s e d of these are the s o - c a l l e d p o r i n p r o t e i n s , which are membrane-spanning, i n t e g r a l p r o t e i n s r e s p o n s i b l e f o r s i z e - l i m i t e d , p a s s i v e d i f f u s i o n through the outer membrane (Lugtenberg, 1983). Other types of p r o t e i n s found i n c o l i (and which o f t e n have analogues i n other s p e c i e s ) are s t r u c t u r a l p r o t e i n s such as OmpA (outer membrane j j r o t e i n ) and l i p o p r o t e i n s . P o r i n p r o t e i n s seem to be common to a l l gram negative s p e c i e s and share some b a s i c c h a r a c t e r i s t i c s . For example, a l l of the p o r i n p r o t e i n s so f a r examined appear to e x i s t i n t h e i r n a t i v e forms as oligomers which vary i n t h e i r s e n s i t i v i t y to s o l u b i l i s a t i o n by detergents (DiRienzo e_t a_l. , 1978). This causes anomalous behaviour on SDS-polyacrylamide g e l s , i n c l u d i n g h e a t - m o d i f i a b i l i t y at v a r y i n g temperatures of s o l u b i l i s a t i o n (a d i s t i n g u i s h i n g c h a r a c t e r i s t i c of outer membrane p r o t e i n s ) . P o r i n s have monomeric molecular weights i n the range of 30,000 - 50,000 d a l t o n s on SDS-PAGE, and are reported to c o n t a i n u n u s u a l l y high amounts of b e t a - s t r u c t u r e f o r i n t e g r a l membrane p r o t e i n s , which u s u a l l y have l a r g e areas of a l p h a - h e l i c a l s t r u c t u r e (Rosenbusch, 1974). Although a l l p o r i n p r o t e i n s operate by pas s i v e d i f f u s i o n , r a t h e r than a c t i v e t r a n s p o r t as i n the cytoplasmic membrane (DiRienzo e_t al^. , 1978), they may be e i t h e r n o n - s p e c i f i c with r e s p e c t to s u b s t r a t e or s e l e c t i v e f o r c e r t a i n s u b s t r a t e s on 12 the b a s i s of s t r u c t u r e or charge. The major c o l i p o r i n p r o t e i n , OmpF, i s an example of a ge n e r a l p o r i n , s i n c e i t i s only weakly c a t i o n - s e l e c t i v e and has not been found to s t r o n g l y p r e f e r c e r t a i n s u b s t r a t e s by any c r i t e r i o n other than s i z e (and h y d r o p h i l i c n a t u r e ) . I t s s i z e e x c l u s i o n l i m i t appears to be approximately 600 d a l t o n s (Decad and Nikaido, 1 976 ; Benz ejt a_l. , 1979), above which d i f f u s i o n i s d r a s t i c a l l y lowered. This measurement was obtained from p o r i n -s u b s t i t u t e d , l i p i d v e s i c l e e x c l u s i o n assays (Nakae, 1976) and by measuring d i f f e r e n t i a l permeation of r a d i o l a b e l l e d o l i g o s a c c h a r i d e s i n t o whole c e l l s (Decad and Nikaido, 1976). E. c o l i a l s o has another g e n e r a l i s e d p o r i n , OmpC, and the r e l a t i v e amounts of these two p o r i n s are c o n t r o l l e d by osmotic f o r c e s and temperature through the r e g u l a t o r y l o c i , ompB (Tol IV - Davies and Reeves, 1975) and envY (Lundrigan and Ea r h a r t , 1984), r e s p e c t i v e l y . Loss by mutation of both g e n e r a l i s e d p o r i n s r e s u l t s i n stro n g s e l e c t i v e pressure f o r mutants s y n t h e s i s i n g new membrane p r o t e i n s (Foulds and Chai, 1978), which t h e r e f o r e i n d i c a t e s the importance of the f u n c t i o n of OmpF and OmpC p o r i n s . Well s t u d i e d p r o t e i n s with s i m i l a r f u n c t i o n s i n other b a c t e r i a i n c l u d e aeruginosa p r o t e i n F (with an appa r e n t l y l a r g e r e x c l u s i o n l i m i t of approximately 3000 d a l t o n s - Benz and Hancock, 1981), as w e l l as p o r i n p r o t e i n s from typhimurium, N. gonorrhoeae , Chlamydiae , H. i n f l u e n z a e , Y. p e s t i s , and R_^  c a p s u l a t a . Porins have been i d e n t i f i e d which d i s p l a y s e l e c t i v i t y toward d i f f e r e n t s u b s t r a t e s . The best known from amongst t h i s 13 c l a s s i s the lambda phage r e c e p t o r p r o t e i n (LamB) of E_^  c o l i , which i s r e l a t i v e l y s p e c i f i c f o r maltose and higher dextrose oligomers and whose s y n t h e s i s i s repressed by g l u c o s e . Another e x t e n s i v e l y s t u d i e d s e l e c t i v e p o r i n i s p r o t e i n P from P • aeruginosa, which i s unusual both f o r i t s small channel s i z e (0.25nS) and f o r i t s high anion and phosphate s e l e c t i v i t y (Hancock e_t a_l. , 1982). Some p r o t e i n s which have not been demonstrated to be p o r i n s are a l s o of note. OmpA of c o l i d i s p l a y s heat m o d i f i c a t i o n and i s thought to be important to the r e c i p i e n t c e l l i n F p i l u s - m e d i a t e d c o n j u g a t i o n (Skurray et a l . , 1974). P r o t e i n H1 of 7^ aeruginosa i s i n d u c i b l e by growth i n low Mg++ and i s p o s t u l a t e d to r e p l a c e the s t a b i l i s i n g e f f e c t of Mg + + i n the outer membrane (Nicas and Hancock, 1980). Murein l i p o p r o t e i n of E_j_ c o l i (Braun and Rehn, 1969 ) i s , i n p a r t , c o v a l e n t l y l i n k e d to the p e p t i d o g l y c a n at i t s c a r b o x y - t e r m i n a l l y s i n e r e s i d u e and to three f a t t y a c i d s at i t s amino terminus, which i s i n s e r t e d i n the outer membrane. The e q u i v a l e n t l i p o p r o t e i n of P^ aeruginosa, p r o t e i n I, was not shown to be c o v a l e n t l y attached to p e p t i d o g l y c a n (Hancock e_t a_l. , 1981a). Numerous minor outer membrane p r o t e i n s have a l s o been i d e n t i f i e d which have some involvement i n i r o n or v i t a m i n B12 t r a n s p o r t (Hancock e_t a_l. , 1977). b) L i p o p o l y s a c c h a r i d e The other major component of the gram negative outer membrane i s a molecule unique to the c e l l envelope, l i p o p o l y s a c c h a r i d e (LPS). LPS, as the name suggests, contains 14 a l i p i d p o r t i o n ( L i p i d A), which i s i n s e r t e d i n t o the outer l a y e r of the outer membrane b i l a y e r , and a p o l y s a c c h a r i d e component (core sugars plus O-antigen) which extends toward the environment from the L i p i d A. The l i p o p o l y s a c c h a r i d e t h e r e f o r e i n t e r a c t s with the c e l l ' s environment and may f u n c t i o n analogously to the capsule seen i n some Gram p o s i t i v e b a c t e r i a . LPS i s r e s p o n s i b l e f o r r e s i s t a n c e to many agents, e s p e c i a l l y hydrophobic ones such as detergents and b i l e s a l t s (Nikaido, 1976). LPS a l s o i n f l u e n c e s i n t e r a c t i o n with other c e l l s , i n c l u d i n g t i s s u e adhesion s i t e s and phagocytes f o r animal pathogens and p l a n t c e l l s f o r p l a n t pathogens. In a d d i t i o n , LPS i s i t s e l f a t o x i c compound, known as endotoxin, capable of e l i c i t i n g a naphylaxis and the r e s u l t i n g t i s s u e damage i f s u f f i c i e n t amounts are presented to the body. The b a s i c s t r u c t u r e of L i p i d A, the t o x i c p o r t i o n of LPS, appears remarkably s i m i l a r amongst a l l gram negative b a c t e r i a (Qureshi e_t a_l. , 1983). I t i s composed of two b e t a - 1 , 6 -l i n k e d glucosamine u n i t s which are s u b s t i t u t e d with between four and s i x long chain f a t t y a c i d s . The number and nature of the f a t t y a c i d s v a r i e s between s p e c i e s however, and r e l a t i v e p r o p o r t i o n s of each f a t t y a c i d may a l s o vary between s t r a i n s . From s t u d i e s of both E_j_ c o l i and Salmonella typhimurium L i p i d A (Rosner e_t a_l. , 1 979 ; Takeyama ejb §JL_. , 1 983 ), the major L i p i d A f a t t y a c i d s p e c i e s seem to be 3-hydroxytetradecanoic (C14) a c i d (both e s t e r i f i e d to glucosamine oxygen and amide l i n k e d ) with s m a l l e r amounts of t e t r a d e c a n o i c , dodecanoic ( C 1 2 ) , and hexadecanoic (C16) a c i d s . The e n t e r i c group of 15 b a c t e r i a seem to have 3 - 0 H - t e t r a d e c a n o i c a c i d i n common, whereas P_;_ aeruginosa, f o r example, s u b s t i t u t e s 2 - 0 H -dodecanoic, 3-OH-dodecanoic and 3-OH-decanoic f o r 3 - 0 H -t e t r a d e c a n o i c a c i d (Hancock and Meadow, 1969 ; Chester e_t a l . , 1973). L i p i d A c o n t a i n s phosphate groups at the 1 (reducing) and 4 ' p o s i t i o n s of the diglucosamine u n i t . These groups may be monophosphoryl or pyrophosphoryl, and may be f u r t h e r modified by l i n k a g e to ethanolamine and 4-aminoarabinose ( i n Salmonella) or other compounds (Osborn, 1979). L i p i d A i s attached to 3-deoxy-D-mannooctulosonic a c i d (ketodeoxyoctonate or KDO) at the 3 ' p o s i t i o n of diglucosamine. There are two to three KDO r e s i d u e s per molecule of LPS depending on the b a c t e r i a l s p e c i e s , and these a l s o seem to be s u b s t i t u t e d by phosphate or phosphorylethanolamine groups (Drewry e_t al., 1 975 ; Nurminen et a l . , 1984). Next to KDO i n the LPS chain i s heptose, a l s o common to most gram negative b a c t e r i a . There may be two to three heptose r e s i d u e s per LPS molecule, s i m i l a r l y to the s i t u a t i o n with KDO. Beyond heptose, a s h o r t chain of v a r i o u s carbohydrates i s at t a c h e d . These sugars u s u a l l y have no net charge, may be branched and vary s u b s t a n t i a l l y more between s p e c i e s than the two p r e v i o u s l y mentioned components (KDO and heptose). T y p i c a l carbohydrates found i n t h i s r e g i o n i n c l u d e g l u c o s e , g a l a c t o s e , rhamnose, N-acetylglucosamine and N-ace t y l f u c o s a m i n e . The above components, i n c l u d i n g L i p i d A, KDO, heptose and n e u t r a l sugars, are together r e f e r r e d to as rough core LPS, due to the rough appearance of c o l o n i e s from 16 Salmonella and E s c h e r i c h i a mutants l a c k i n g O-antigen s u b s t i t u t i o n of t h e i r "core" LPS backbone. 0- (or somatic) antigen was a term o r i g i n a l l y used to d e s c r i b e the major n o n - f l a g e l l a r , h e a t - s t a b l e , c e l l s u r f a c e a n t i g e n of Salmonella s p e c i e s . I t comprises the outer region of the LPS and i s c h a r a c t e r i s e d by r e p e a t i n g u n i t s of three to f i v e carbohydrate m o i e t i e s . In c o n t r a s t to core sugars, these c a r r y more p o t e n t i a l l y charged r e s i d u e s , although,they may be s u b s t i t u t e d to n e u t r a l i s e charge. Some examples of sugars found i n the 0-antigen of "smooth" LPS are N-acetylfucosamine, N-acetylimidomannuronic a c i d , a c e t i m i d o g u l u r o n i c a c i d and 2-amino-2-deoxy-galacturonic a c i d (Wilkinson and Welbourn, 1975; K r o p i n s k i et a l . , 1982; K n i r e l et, al. , 1983). I t i s the high v a r i a b i l i t y of 0-antigen components which makes immunological p r o t e c t i o n a g a i n s t many gram negative b a c t e r i a d i f f i c u l t and causes the multitude of serotypes seen i n b a c t e r i a with r e l a t i v e l y long chain (smooth) LPS (Fensom and Meadow, 1970; Koval and Meadow, 1975). As p r e v i o u s l y mentioned, LPS can play an important r o l e i n immunogenicity and p a t h o g e n i c i t y of an organism. I t a l s o c o n t r i b u t e s to c e l l s u r f a c e r e c o g n i t i o n by being a major r e c e p t o r t a r g e t f o r phages and c o l i c i n s or pyocins (Hancock e_t a l . , 1976). LPS a l s o acts to p r o t e c t c e l l s from serum complement, s i n c e rough mutants of many b a c t e r i a l s p e c i e s are more serum s e n s i t i v e than smooth s t r a i n s ( T a y l o r , 1983; Goldman, e_t a_l. , 1 984). LPS a l s o provides a strong b a r r i e r f u n c t i o n f o r the outer membrane. Whereas a p h o s p h o l i p i d 17 b i l a y e r i s an e f f e c t i v e b a r r i e r to many h y d r o p h i l i c molecules, i t permits the uptake of hydrophobic or a m p h i p h i l i c molecules. L i p o p o l y s a c c h a r i d e , being q u i t e p o l a r i n nature and s t a b i l i s e d by d i v a l e n t c a t i o n s and b i n d i n g to p r o t e i n s , prevents uptake of hydrophobic substances and i s t h e r e f o r e a more e f f i c i e n t b a r r i e r membrane ( L e i v e , 1974; S t a n - L o t t e r e_t a l . , 1979; Hancock, 1984). 5. P e r m e a b i l i t y of the outer membrane. As d i s c u s s e d above, the outer membrane i s an e f f e c t i v e b a r r i e r surrounding the gram negative c e l l , however permeation of n u t r i e n t s can be achieved l a r g e l y by pas s i v e d i f f u s i o n though p o r i n s . Because of the l a r g e d i v e r s i t y of a n t i b i o t i c s t r u c t u r e s , more than one pathway of entry i n t o the c e l l might be expected. To cross the outer membrane, one of two p o s s i b l e routes must be taken: i ) passage through w a t e r - f i l l e d channels made by p o r i n molecules or i i ) uptake across the LPS-ph o s p h o l i p i d b i l a y e r . i ) To cross the outer membrane v i a p o r i n s , a molecule must be r e l a t i v e l y water s o l u b l e and a l s o s m a l l e r than the s i z e e x c l u s i o n l i m i t ( s ) set by the channel s i z e of the p o r i n p r o t e i n s . S t u d i e s by Nikaido and Rosenberg (1981) and Nikaido et a l . , (1983) showed that beta-lactams penetrated the outer membrane of i n t a c t c o l i c e l l s v i a p o r i n s , but that h y d r o p h o b i c i t y (to some extent - Yoshimura and Nikaido, 1982) or negative charge of the beta-lactams a d v e r s e l y a f f e c t e d 18 t h e i r permeation. Other s t u d i e s of c e p h a l o s p o r i n permeation through E_^  c o l i p o r i n s r e c o n s t i t u t e d i n t o liposomes (Kobayashi et a l . , 1982), and of p o r i n d e f i c i e n t mutants (Nikaido e_t a l . , 1977; J a f f e et a l . , 1982; Nikaido et a l . , 1983) confirmed and extended these r e s u l t s . i i ) Because c e r t a i n hydrophobic a n t i b i o t i c s are not very e f f e c t i v e a g a i n s t gram negative b a c t e r i a which possess smooth LPS, they are t h e r e f o r e suspected of c r o s s i n g the outer membrane through the LPS/phospholipid b i l a y e r . Evidence to suggest t h i s comes from deep rough ( 0-antigen and core-d e f i c i e n t ) LPS mutants which are more s u s c e p t i b l e than t h e i r w i l d type c o u n t e r p a r t s with smooth LPS to hydrophobic agents such as Gentian v i o l e t (Gustafsson e_t al_. , 1973 ) and s e v e r a l hydrophobic a n t i b i o t i c s (Nikaido, 1976). Experiments of Leive et a l . , (1984) showed that a) w i l d type E_j_ c o l i c e l l s became l e s s s u s c e p t i b l e to t e t r a c y c l i n e d e r i v a t i v e s of i n c r e a s i n g h y d r o p h o b i c i t y , whereas b) a deep rough mutant was e q u a l l y s u s c e p t i b l e to t e t r a c y c l i n e s r e g a r d l e s s of h y d r o p h o b i c i t y ( 1 0 0-fold lower MIC than w i l d type f o r the most hydrophobic d e r i v a t i v e ) . i i i ) Another pathway has been p o s t u l a t e d f o r uptake of aminoglycoside a n t i b i o t i c s , and which has so f a r only been demonstrated f o r P^ aeruginosa ( Z i m e l i s and Jackson, 1973; Nicas and Hancock, 1980), although polymyxin B a l s o appears to u t i l i s e t h i s pathway to cross the outer membranes of other gram negative b a c t e r i a ( S c h i n d l e r and Teuber, 1975; S c h i n d l e r and Osborn, 1979; Vaara and V i l j a n e n , 1983; Hancock, 1984). 19 This s o - c a l l e d self-promoted uptake i n v o l v e s the replacement of outer membrane s t a b i l i s i n g Mg + + by aminoglycosides at s p e c i f i c b i n d i n g s i t e s on LPS, causing d e s t r u c t i o n of outer membrane i n t e g r i t y . T h i s r e s u l t s i n entry of e i t h e r more aminoglycoside molecules or of any other, impermeable molecules (Hancock e_t a_l. , 1 981 b ). EDTA was a l s o shown to in c r e a s e outer membrane p e r m e a b i l i t y i n E_^  c o l i c e l l s ( L e i v e , 1968), presumably by removing Mg + + from t h i s LPS b i n d i n g s i t e . 6. Role of the P. aeruginosa outer membrane i n permeation of  a n t i b i o t i c s . The outer membrane of P_^  aeruginosa appears to be a p a r t i c u l a r l y e f f e c t i v e b a r r i e r to a n t i b i o t i c s , based on i t s high i n t r i n s i c a n t i b i o t i c r e s i s t a n c e p r o f i l e . I t s r e s i s t a n c e to h y d r o p h i l i c beta-lactam a n t i b i o t i c s o r i g i n a l l y l e d s e v e r a l workers to p o s t u l a t e that c e l l envelope p e r m e a b i l i t y must be lower than that observed i n E_j_ c o l i and S_^  typhimurium (Brown, 1975; Sykes and Matthew, 1976; Suginaka e_t ajl, 1979). I t was subsequently d i s c o v e r e d that the P_^  aeruginosa major p o r i n p r o t e i n F a c t u a l l y has a l a r g e r s i z e e x c l u s i o n l i m i t than do e n t e r i c b a c t e r i a (Hancock and Nikaido, 1978; Hancock, Decad and Nikaido, 1979) but that the a c t i v i t y of these p o r i n s was estimated to be approximately 1 0 0-fold lower than E_j_ c o l i OmpF p o r i n , based on black l i p i d b i l a y e r experiments (Benz and Hancock, 1981), i n v i v o outer membrane p e r m e a b i l i t y experiments ( t h i s t h e s i s ; Nicas and Hancock, 1983), and liposome s w e l l i n g assays (Nikaido and Rosenberg, 1983). 20 P e r m e a b i l i t y through the LPS-phospholipid b i l a y e r of P.  aeruginosa appears to be as low or lower than that of E_;_ o o l i and S_^  typhimurium, based on a n t i b i o t i c MIC data. I t i s i n t e r e s t i n g that the only type of outer membrane permeation which i s r e l a t i v e l y high i n P_^  aeruginosa i s a f f o r d e d by the self-promoted uptake pathway. The a n t i b i o t i c s which use t h i s route (aminoglycosides and polymyxin B) i n c l u d e a number which have been used t h e r a p e u t i c a l l y a g a i n s t P_;_ aeruginosa i n f e c t i o n s with some degree of success. The property of P.  aeruginosa LPS which makes the outer membrane so s t a b l e to many types of agents may a l s o act to d e s t a b i l i s e i t when challe n g e d with the a p p r o p r i a t e molecule. In e f f e c t , the self-promoted uptake pathway proposed by Nicas and Hancock (1980) may prove to be the p r o v e r b i a l "chink i n the armour" i n a n t i b i o t i c treatment of t h i s organism. 7. A n t i b i o t i c h y p e r s u s c e p t i b l e mutants A number of i n t r i n s i c a l l y a n t i b i o t i c h y p e r s u s c e p t i b l e mutants have been d e s c r i b e d i n the l i t e r a t u r e , i n s e v e r a l s p e c i e s . Although not a l l have been c h a r a c t e r i s e d to an equal degree, these n e v e r t h e l e s s show a few gen e r a l t r e n d s . F i r s t l y , of w e l l c h a r a c t e r i s e d mutations to a n t i b i o t i c s u s c e p t i b i l i t y , the m a j o r i t y were mutant i n the LPS of the outer membrane, r a t h e r than i n p o r i n p r o t e i n s (Hancock, 1984). Mutation to p o r i n d e f i c i e n c y was a s s o c i a t e d with c o n v e r s i o n to h y d r o p h i l i c a n t i b i o t i c r e s i s t a n c e i n c o l i and S^ _ 21 typhimurium ( B a v o i l ejt a_l. , 1977; J a f f e e_t a_l. , 1982). A p l a u s i b l e hypothesis to e x p l a i n t h i s o b s e r v a t i o n i s t h a t , i n e n t e r i c b a c t e r i a , p o r i n molecules are r e l a t i v e l y a c t i v e , t h e r e f o r e p o r i n d e f i c i e n c y causes a s i g n i f i c a n t decrease i n outer membrane p e r m e a b i l i t y to a n t i b i o t i c s . Since the outer membrane of P_^  aeruginosa i s estimated to be 10 - 100-fold l e s s permeable to v a r i o u s compounds than that of E_;_ c o l i , P.  aeruginosa may a l r e a d y be considered as a p o r i n a c t i v i t y -d e f i c i e n t mutant and t h e r e f o r e p o r i n p r o t e i n d e f i c i e n c y would not lead to a measurable decrease i n a n t i b i o t i c r e s i s t a n c e ( T . I . N icas, p e r s o n a l communication). Most h y p e r s u s c e p t i b l e mutants so f a r c h a r a c t e r i s e d were determined to be a l t e r e d i n some LPS property and are noteworthy. The best c h a r a c t e r i s e d LPS mutants are the Rfa ( R - f a c t o r ) s e r i e s i n S_^  typhimurium. These mutants have i n c r e a s i n g s e v e r i t y of LPS d e f e c t s , from RfaA, where only the r e p e a t i n g 0-antigen p o r t i o n s of LPS are m i s s i n g , to RfaE, which i s a deep rough ( h e p t o s e l e s s ) LPS mutant. Less severe LPS mutations di d not appear to a l t e r a n t i b i o t i c s u s c e p t i b i l i t y a p p r e c i a b l y , whereas the RfaE mutant had enhanced s u s c e p t i b i l i t y to hydrophobic a n t i b i o t i c s , d e t e r g e n t s , EDTA and phospholipases, and tended to leak p e r i p l a s m i c enzymes (Sanderson e_t ja l . , 1974; Singh and Reithmeier, 1975). S i m i l a r p r o p e r t i e s were found i n c e r t a i n P_;_ aeruginosa s t r a i n s (Meadow and Wells, 1985) which had been s e l e c t e d f o r c a r b e n i c i l l i n s u s c e p t i b i l i t y and s e n s i t i v i t y to rough LPS-22 s p e c i f i c b acteriophages. Of a s e r i e s of mutants with i n c r e a s i n g d e f e c t s i n LPS, only those with 0-antigen plus both rhamnose and glucose missing from the core had i n c r e a s e d s u s c e p t i b i l i t y to a n t i b i o t i c s . In c o n t r a s t , the envA mutant of S_^  typhimurium shows higher p e r m e a b i l i t y to hydrophobic substances such as Gentian v i o l e t , but has no obvious d e f e c t s i n LPS s t r u c t u r e (Grundstrom e_t a l . , 1980). Other mutants have been d e s c r i b e d which have been c h a r a c t e r i s e d with r e s p e c t to e i t h e r t h e i r s u s c e p t i b i l i t y p a t t e r n or LPS chemotype, but a c a u s a l r e l a t i o n s h i p between the two phenotypes was not d e r i v e d . Consequently, a gre a t deal of c o n f u s i o n s t i l l e x i s t s as to the r o l e of each p o r t i o n of the LPS molecule and the i n t e r r e l a t i o n between LPS molecules and between LPS and p r o t e i n molecules i n s t a b i l i t y of the outer membrane. 8. Aim of the study. This study d e s c r i b e s the r o l e of the outer membrane i n a n t i b i o t i c r e s i s t a n c e of P_^  aeruginosa. In the f i r s t p o r t i o n of the study, an a n t i b i o t i c h y p e r s u s c e p t i b l e mutant was c h a r a c t e r i s e d to i d e n t i f y outer membrane components which might be r e s p o n s i b l e f o r a n t i b i o t i c r e s i s t a n c e / s u s c e p t i b i l i t y i n P_^_ aeruginosa. The second p o r t i o n of t h i s study examines the n a t i v e c o n f i g u r a t i o n of two p o r i n p r o t e i n s from P.  aeruginosa and one from E . c o l i u s ing a chemical c r o s s l i n k i n g technique to determine p o s s i b l e t r i m e r i c o r g a n i s a t i o n . 23 METHODS 1. Medium and c u l t u r e c o n d i t i o n s Proteose peptone #2 (1$ [w/v]) ( D i f c o 0121-01) (PP2) was used as a r i c h medium, and BM2 minimal medium ( G i l l e l a n d e_t a l . , 1974), supplemented with 0.5 mM MgSO^f 10 uM FeSO^, and 20 mM of carbon source (glucose, s u c c i n a t e , pyruvate, g l y c e r o l or c i t r a t e ) was used as a d e f i n e d medium. L i q u i d c u l t u r e s were grown i n volumes of from 10 ml to 10 1 i n f l a s k s shaken at 37°C under c o n d i t i o n s of good a e r a t i o n . P l a t e c u l t u r e s were grown on the same media with the a d d i t i o n of 2% agar. 2. B a c t e r i a l S t r a i n s P. aeruginosa s t r a i n s WT1 and M1 were a g i f t from Dr. W. Zimmermann of Ciba Geigy, B a s e l , S w i t z e r l a n d . S t r a i n WT1 had been mutagenised i n four steps with e t h y l methanesulphonate, then once with N-methyl-N'-nitro-N-n i t r o s o g u a n i d i n e to produce mutant s t r a i n M1. S i n g l e c o l o n i e s were s e l e c t e d by r e p l i c a p l a t i n g on r i c h agar c o n t a i n i n g c e p h a l o s p o r i n C. I s o l a t i o n of spontaneous r e v e r t a n t s t r a i n s P1-4 and P1-6 i s de s c r i b e d i n a separate s e c t i o n . S t r a i n s WT3 and AK 43 were a g i f t from Dr. A. K r o p i n s k i , Queen's U n i v e r s i t y , Kingston, O n t a r i o . S t r a i n s WT2, P2-6, P2-6t, WT2-6, P2-8, P2-8t and P2-6,8 were a kind g i f t from Dr. J . Fyfe of Univ. of Edinburgh, U.K. S t r a i n WT2 was a m u l t i p l y auxotrophic PAO d e r i v a t i v e (PA0222). 24 S t r a i n s P2-6 and P2-8 were c o n s t r u c t e d by c o n j u g a t i o n u s i n g , as donor s t r a i n , M1 l e u " (R68.45) and WT2 as r e c i p i e n t . Recombinants were s e l e c t e d by c o n v e r s i o n to prototrophy and subsequently t e s t e d f o r a n t i b i o t i c s u s c e p t i b i l i t y . S t r a i n s P2-6t and P2-8t were c o n s t r u c t e d by phage F116C t r a n s d u c t i o n from s t r a i n s P2-6 and P2-8, r e s p e c t i v e l y , using the same r e c i p i e n t s t r a i n and p r o t o t r o p h i c markers as used f o r c o n j u g a t i o n experiments. Recombinant P2-6,8 was c o n s t r u c t e d by F116C t r a n s d u c t i o n of pro+ prototrophy from s t r a i n P2-6 i n t o s t r a i n P2-8. D e t a i l e d genotypes of these s t r a i n s are given on p. 50. Plasmid RP1-containing s t r a i n s were c o n s t r u c t e d by performing c o n j u g a t i o n experiments between an RP1-containing donor s t r a i n of E_^ c o l i K-12 (JC3272) and each P_^  aeruginosa w i l d type or a n t i b i o t i c h y p e r s u s c e p t i b l e r e c i p i e n t s t r a i n . C e l l s were grown as d e s c r i b e d above i n n u t r i e n t broth to mid-lo g phase, then 0.5 ml donor plus 0.1 ml r e c i p i e n t were placed i n n u t r i e n t broth and shaken f o r three hours. C e l l s were then p l a t e d onto s e l e c t i v e medium. R e c i p i e n t s were s e l e c t e d using a very high c o n c e n t r a t i o n of c a r b e n i c i l l i n (0.5 mg/ml) to which both RP1-carrying c o l i and non-plasmid-carrying P. aeruginosa were s u s c e p t i b l e . 3. A n t i b i o t i c S u s c e p t i b i l i t y T e s t i n g Minimal i n h i b i t o r y c o n c e n t r a t i o n (MIC) values were obtained by p l a t e d i l u t i o n method. A n t i b i o t i c s were i n c o r p o r a t e d at v a r i o u s c o n c e n t r a t i o n s i n t o PP2-agar p l a t e s . 25 B a c t e r i a were grown o v e r n i g h t , d i l u t e d 1 0 0-fold and then 0.01 ml of b a c t e r i a l suspension was spotted onto the p l a t e s . Depending on the number of s t r a i n s to be t e s t e d , the s p o t t i n g was done e i t h e r manually or by using a m u l t i s y r i n g e a p p l i c a t o r ( Z i e r d t e_t a_l. , 1960). P l a t e s were grown overnight at 37°C. MIC values were d e f i n e d as the lowest c o n c e n t r a t i o n of a n t i b i o t i c which produced i n h i b i t i o n of growth. S i n g l e c e l l r e s i s t a n c e l e v e l s were obtained by d i l u t i n g an o v e r n i g h t c u l t u r e 1 0 - 6 and then s p r e a d - p l a t i n g 0.1 ml onto PP2-agar p l a t e s c o n t a i n i n g a n t i b i o t i c . C o n c e n t r a t i o n s used were somewhat lower than those used f o r MIC d e t e r m i n a t i o n s . For d i s c i n h i b i t i o n assays, b a c t e r i a grown ove r n i g h t were d i l u t e d 1/100 then spread evenly onto PP2-agar p l a t e s using a s t e r i l e swab. Resu l t s were obtained by s p o t t i n g 0.01 ml a l i q u o t s of a n t i b i o t i c s o l u t i o n s onto s t e r i l e f i l t e r d i s c s which were immediately placed onto the lawn of each s t r a i n to be t e s t e d . The i n h i b i t i o n zone was expressed as the t o t a l diameter of i n h i b i t i o n around the d i s c , i n c l u d i n g the diameter of the d i s c i t s e l f , which measured 6mm. 4. A n t i b i o t i c s and Chemicals B e n z y l p e n i c i l l i n , a m p i c i l l i n , polymyxin B sulp h a t e , gentamicin s u l p h a t e , streptomycin s u l p h a t e , t e t r a c y c l i n e h y d r o c h l o r i d e , chloramphenicol and trimethoprim were purchased from Sigma Chemical Co. ( S t . L o u i s , USA). N o r f l o x a c i n and N-formyl thienamycin (Imipenem) were obtained from Merck, Sharp 26 and Dohme re s e a r c h lab (New Jersey, USA). Tobramycin and moxalactam were a g i f t from E l i L i l l y and Co. ( I n d i a n a p o l i s , USA). C e f s u l o d i n was a g i f t from Ciba Geigy AG ( B a s e l , S w i t z e r l a n d ) . Cefotaxime was a g i f t of Roussel Canada Inc. ( M o n t r e a l ) . Azthreonam was a g i f t of E.R. Squibb and Sons, Inc. C a r b e n i c i l l i n was purchased from Ayerst L a b o r a t o r i e s (Montreal, Canada). T i c a r c i l l i n was a g i f t from Beecham L a b o r a t o r i e s (Pt. C l a i r e , Canada). C e f t a z i d i m e and n i t r o c e f i n were a g i f t of Glaxo Group Research L t d . , Greenford, England. The c r o s s l i n k e r , d i t h i o - b _ i s - ( s u c c i n i m i d y l propionate) (DSP) was purchased from the P i e r c e Chemical Co., (Rockford USA). D i s u l p h i d e reducing agents d i t h i o e r y t h r i t o l and 2 -mercaptoethanol were obtained from Bio-Rad L a b o r a t o r i e s (Richmond, USA). Acrylamide and b i s - a c r y l a m i d e were purchased from M i l e s L a b o r a t o r i e s (Indiana, USA), Eastman Kodak Co. (Rochester, USA) and BDH Chemicals (Toronto, Canada). Sodium dodecyl sulphate was s p e c i a l l y p u r i f i e d q u a l i t y and was obtained from BDH Chemicals. Ampholines were purchased from LKB (Stockholm, Sweden). N i t r o c e l l u l o s e membrane sheets were purchased from S c h l e i c h e r and S c h u e l l Inc. (Keene, N.H., USA). The f l u o r e s c e n t probe, 1-N-phenylnapthylamine (NPN),and lysozyme were purchased from Sigma Chemical Co. Dansyl polymyxin B was prepared as d e s c r i b e d i n Methods. A l l other chemicals used were of the h i g h e s t standard commercially a v a i l a b l e . 5 . I s o l a t i o n of Spontaneous Revertants of M1 27 C e l l s with w i l d type or p a r t i a l r e s i s t a n c e to a n t i b i o t i c s were i s o l a t e d as spontaneous r e v e r t a n t s of the s u p e r s u s c e p t i b l e mutant M1. S t r a i n M1 was grown overnight i n 1$ PP2 medium and then s u b c u l t u r e d i n t o 10 ml of the same medium and grown to an o p t i c a l d e n s i t y at 600 nm of 1.0. The c e l l s were then c e n t r i f u g e d , resuspended to a 100-fold c o n c e n t r a t i o n i n 0.1 ml of PP2 and p l a t e d on medium c o n t a i n i n g c a r b e n i c i l l i n or gentamicin at c o n c e n t r a t i o n s between the s i n g l e c e l l r e s i s t a n c e l e v e l s f o r s t r a i n s M1 and WT1. 6. Bacteriophage s e n s i t i v i t y t e s t i n g A l l b a c t e r i a l s t r a i n s were t e s t e d f o r s e n s i t i v i t y to a l a r g e v a r i e t y of P_^  aeruginosa phages (Nicas and Hancock, 1980). An a d d i t i o n a l deep r o u g h - s p e c i f i c phage, D8 (0PLS27 -K. J a r r e l l , Ph.D. T h e s i s , Queen's Univ., O n t a r i o , 1980), was a l s o t e s t e d . The assays were done as d e s c r i b e d by Hancock and Reeves (1975), using a m u l t i s y r i n g e phage a p p l i c a t o r . B r i e f l y , phage stoc k s were d i l u t e d to g i v e approximately 10^ PFU/ml, then spotted onto a b a c t e r i a l lawn on PP2-agar. 7. Beta-lactamase assay and c a l c u l a t i o n s Beta-lactamase was assayed with the chromogenic c e p h a l o s p o r i n , n i t r o c e f i n (O'Callaghan £t a^., 1972). C u l t u r e s were grown to an o p t i c a l d e n s i t y at 600 nm of 0.5-0.8 i n PP2 broth and then suspended to one quarter volume i n 50 mM sodium phosphate or 5 mM Na-HEPES b u f f e r (both pH 7.0). One 28 hundred m i e r o l i t r e s of c e l l s were added to 0.65 ml of n i t r o c e f i n s o l u t i o n (0.1 - 0.25 mg/ml i n the same b u f f e r ) i n a semimicro c u v e t t e , and the k i n e t i c s of n i t r o c e f i n h y d r o l y s i s were monitored at A M g 5 u s i n g a P e r k i n Elmer dual beam spectrophotometer coupled to a Sargent-Welch c h a r t r e c o r d e r . Values f o r h y d r o l y s i s of n i t r o c e f i n by sim u l t a n e o u s l y prepared c e l l - f r e e c u l t u r e supernatants were s u b t r a c t e d from whole c e l l h y d r o l y s i s values to c o n t r o l f o r p o s s i b l e leakage of beta-lactamase from the pe r i p l a s m . Assays were performed on whole and broken c e l l s to g i v e a measure of r e l a t i v e c r y p t i c i t y i n the s t r a i n s . C e l l s i n b u f f e r were broken once by French pressure at 20,000 l b / i n 2 , which proved to be more e f f i c i e n t f o r r e l e a s i n g p e r i p l a s m i c beta-lactamase than were MgCl^ o r EDTA treatments. C r y p t i c i t y was c a l c u l a t e d as the r a t i o of the beta-lactamase a c t i v i t y of broken c e l l s to that of whole c e l l s . The r a t i o of the apparent p e r i p l a s m i c c o n c e n t r a t i o n of n i t r o c e f i n ( S g ) over the o u t s i d e medium c o n c e n t r a t i o n ( S Q ) was c a l c u l a t e d u s i n g the Michaelis-Menten equation, s u b s t i t u t i n g the r a t e of h y d r o l y s i s i n i n t a c t c e l l s (v) f o r V and the r a t e of h y d r o l y s i s i n broken c e l l s at high n i t r o c e f i n c o n c e n t r a t i o n s as V max • V " Vmax x s e ^ Km + s e m^ w a s determined from assays performed on broken c e l l s . The p e r m e a b i l i t y parameter "C" was c a l c u l a t e d from the 29 h y d r o l y s i s r a t e "v" of n i t r o c e f i n i n whole c e l l s over the c o n c e n t r a t i o n " S o n Q f n i t r o c e f i n i n the surrounding medium, using F i c k ' s f i r s t law of d i f f u s i o n , as expressed by Zimmermann and Ross e l e t (1977): v = C (S s ) o e' wherein C i s the p e r m e a b i l i t y c o e f f i c i e n t ( p r o p o r t i o n a l to the t o t a l s u r f a c e area across which n i t r o c e f i n d i f f u s e s d i v i d e d by the d i s t a n c e t r a v e l l e d across the membrane) and S w a s c a l c u l a t e d from the above Michaelis-Menten equation. For measurement of induced beta-lactamase, c e l l s were grown f o r 2 h i n PP2 broth c o n t a i n i n g 0.2 to 200 ug of benzyl p e n i c i l l i n per ml as an in d u c e r , broken and assayed as above. Phase p a r t i t i o n i n g of n i t r o c e f i n was performed as de s c r i b e d by T s u j i e_t a_l. , (1977), using equal volumes of 1-o c t a n o l and 5mM Na-HEPES b u f f e r , pH 7.0. N i t r o c e f i n was q u a n t i f i e d by assaying the peak absorbance value f o r unhydrolysed n i t r o c e f i n (390 nm) using the a p p r o p r i a t e blank r e f e r e n c e f o r each phase. The r e s u l t s were presented as the r a t i o of A^g 0 i n the top / bottom phases. 8. NPN uptake assay C e l l s were suspended to an o p t i c a l d e n s i t y of 0.5 at 600 nm i n 5 mM Na-HEPES b u f f e r pH 7.0 - 5mM Na-azide. 1-N-phenylnaphthylamine (NPN) was d i s s o l v e d i n acetone at a c o n c e n t r a t i o n of 0.5 mM and used, as d e s c r i b e d by Loh et a l . 30 (1984), at a f i n a l c o n c e n t r a t i o n ( i n c e l l suspension) of 0.01 mM. Fluorescence i n t e n s i t y was measured with a Perkin-Elmer 650-10S f l u o r e s c e n c e spectrophotometer equipped with a Haake c i r c u l a t i n g water bath to maintain the cuvette h o l d i n g chamber at a steady temperature. E x c i t a t i o n and emission wavelengths f o r NPN were 350 and 420 nm, r e s p e c t i v e l y , with s l i t widths of 5 nm. T o t a l f l u o r e s c e n c e of c e l l suspensions was measured. Gentamicin was added to a f i n a l c o n c e n t r a t i o n of 4 ug/ml i n c e l l suspension and then r a t e s of gentamicin-induced f l u o r e s c e n c e i n c r e a s e were monitored. NPN, gentamicin and MgCl^ alone each had n e g l i g i b l e f l u o r e s c e n c e . Fluorescence caused by c e l l s alone was minimal and was s u b t r a c t e d from t o t a l f l u o r e s c e n c e . 9. P e r m e a b i l i s a t i o n of whole c e l l s to lysozyme C e l l s were grown to midlog phase i n proteose peptone No.2 medium then c e n t r i f u g e d and resuspended i n 5mM Na-azide (to i n h i b i t r e s p i r a t i o n ) , 5 mM Na-Hepes pH 7-0 to an o p t i c a l d e n s i t y at 600nm of approximately 0.5. Lysozyme (Sigma Chemical Co., St. L o u i s ) was added to a c o n c e n t r a t i o n of 50 ug/ml, then the p e r m e a b i l i s i n g compounds EDTA, gentamicin or polymyxin B were added at the given c o n c e n t r a t i o n s ( F i g . 3 ) . L y s i s of c e l l s was f o l l o w e d i n a Perkin-Elmer OV/VIS spectrophotometer as the decrease i n ^ftoo* M i c r o s c o p i c o b s e r v a t i o n was used to check that decrease i n ^^QQ was r e l a t e d to l y s i s , and c o n t r o l s were performed to ensure that the presence of lysozyme or p e r m e a b i l i s e r alone d i d not cause 31 l y s i s . 10. Dansyl polymyxin b i n d i n g assay Dansylated polymyxin (DPX) was prepared by Dr. R. Moore as d e s c r i b e d by S c h i n d l e r and Teuber (1975). Fluorescence of the dansyl group was measured at e x c i t a t i o n and emission wavelengths of 340 and 485 nm, r e s p e c t i v e l y , i n a Perkin-Elmer 650-10S f l u o r e s c e n c e spectrophotometer (Norwalk, Conn, U.S.A.). The assay mixture c o n s i s t e d of a suspension of c e l l s ^ A600=0.5 p r i o r to a d d i t i o n ) [or LPS (3-0 ug/ml) or L i p i d A (4.0 ug/ml)] i n 5mM sodium Hepes b u f f e r pH 7.0 -5mM sodium azide (to i n h i b i t r e s p i r a t i o n ) , to which 500 pmole a l i q u o t s of DPX were added up to the p o i n t of s a t u r a t i o n . Fluorescence i n c r e a s e a f t e r DPX b i n d i n g was recorded i n a r b i t r a r y u n i t s u sing a voltmeter (Beckman I n d u s t r i a l ) . To c a l c u l a t e the amount of dansyl polymyxin which could be bound to a given amount of LPS, a l i q u o t s of DPX were bound to a 100-fold excess of LPS or L i p i d A, to g i v e a f l u o r e s c e n c e value when a l l DPX added was bound. These values were compared to the f l u o r e s c e n c e value at s a t u r a t i o n when DPX was i n excess, to determine what f r a c t i o n of the added DPX was a c t u a l l y bound. I n h i b i t i o n of DPX b i n d i n g by Mg + + or gentamicin was examined by adding a l i q u o t s of i n h i b i t o r to assay mixtures c o n t a i n i n g c e l l s , LPS or L i p i d A, as w e l l as DPX at near 32 s a t u r a t i n g c o n d i t i o n s . Decrease i n observed f l u o r e s c e n c e was measured as d e s c r i b e d and expressed as percent i n h i b i t i o n of f l u o r e s c e n c e . 11. Phase p a r t i t i o n i n g of c e l l s C e l l s were grown to mid log phase, c e n t r i f u g e d and concentrated t e n - f o l d p r i o r to the assay. The aqueous two-phase polymer system d e s c r i b e d by Magnusson e_t a_l. (1977) was prepared by adding volumes of 20$(w/w) p o l y e t h y l e n e g l y c o l (PEG) 6000 (Sigma Chemical Co.) and 20$(w/w) Dextran T500 (Pharmacia, Sweden) to give f i n a l c o n c e n t r a t i o n s of 4.4$ and 6.2%, r e s p e c t i v e l y , i n 30mM T r i s - H C l b u f f e r , pH 7.0. This was e q u i l i b r a t e d at 4°C f o r s e v e r a l hours to allow s e p a r a t i o n i n t o two phases. The assay was performed i n g l a s s tubes by adding 1.0 ml of each of the top and bottom phases as w e l l as 0.1 ml T r i t o n X-100 and 0.1 ml of 10-fold concentrated c e l l s . The tubes were then i n v e r t e d 20 times and l e f t to e q u i l i b r a t e f o r 45 minutes at room temperature. A l i q u o t s were removed from both the top and bottom phases and A^QQ ( j u e to the presence of c e l l s was recorded f o r each i n a Perkin-Elmer UV/VIS Lambda 3 spectrophotometer, using a p p r o p r i a t e b l a n k s . The r e s u l t s were expressed as the r a t i o of A g 0 0 i n t h e top/bottom phases. 12. P r e p a r a t i o n of outer membranes C e l l s were harvested by c e n t r i f u g a t i o n and, to plasmolyse 33 the c e l l s , were resuspended to 100X c o n c e n t r a t i o n i n 20% sucrose-1OmM T r i s - H C l pH 8.0, f o l l o w i n g the procedure of Hancock and Nikaido (1978). C e l l s were t y p i c a l l y f r o z e n to ai d breakage and then t r e a t e d with 0.5 mg/ml p a n c r e a t i c deoxyribonuclease and broken twice i n a French Pressure c e l l at 15,000 l b / s q . i n c h . C e l l d e b r i s was removed by c e n t r i f u g a t i o n at 3000 rpm i n an SS34 r o t o r f o r 10 min and the supernatant was decanted and l a y e r e d onto a two-step g r a d i e n t with T0% sucrose i n the bottom l a y e r and 60% sucrose i n the upper l a y e r . This was c e n t r i f u g e d at 183,000 X g f o r at l e a s t 12 h i n a Beckman SW41 or SW27Ti r o t o r . The lower outer membrane band was c o l l e c t e d from the g r a d i e n t by the bottom d r i p method, concentrated by c e n t r i f u g a t i o n at 160,000 X g i n a Beckman Type 50 or 70 r o t o r and kept f r o z e n at 20°C . 13. P r e p a r a t i o n of c e l l envelopes To prepare c e l l envelopes, m i d - l o g a r i t h m i c phase c e l l s were c e n t r i f u g e d and broken as d e s c r i b e d p r e v i o u s l y , then d i l u t e d i n 10 mM phosphate b u f f e r pH 7.4 and c e n t r i f u g e d at 160,000 X g f o r 1 hour. The envelope p e l l e t was resuspended i n d e i o n i z e d water. 14. I s o l a t i o n of l i p o p o l y s a c c h a r i d e The method used f o r LPS i s o l a t i o n was that of Darveau and Hancock (1983). Mid-log phase c e l l s were harvested as de s c r i b e d above and resuspended to approximately 7 0 - f o l d 34 c o n c e n t r a t i o n i n 10 mM T r i s - H C l b u f f e r pH 8.0 - 2mM M g C l ^ Q < 1 mg/ml deoxyribonuclease and 0.025 mg/ml p a n c r e a t i c r i b o n u c l e a s e . The c e l l suspension was then passed twice through a French pressure c e l l at 15,000 l b / s q . i n . The r e s u l t i n g c e l l l y s a t e was s o n i c a t e d f o r two 30 sec b u r s t s . DNase and RNase were re-added to f i n a l c o n c e n t r a t i o n s of 0.2 and 0.05 mg/ml, r e s p e c t i v e l y , and the suspension was incubated at 37°C f o r 2 h. Tetrasodium - EDTA and SDS were added to f i n a l c o n c e n t r a t i o n s of 0.1 M and 2% (w/v), r e s p e c t i v e l y , ( f i n a l pH was 9 . 5 ) . The sample was vortexed to ensure s o l u b i l i t y and c e n t r i f u g e d at 50 ,000 X g f o r 30 min at 20<>c to remove p e p t i d o g l y c a n . The supernatant was decanted and Pronase was added to a f i n a l c o n c e n t r a t i o n of 0.2 mg/ml and the sample was incubated o v e r n i g h t at 37°C with constant shaking. Two volumes of 0 .375 M MgCl 2 i n g 5 e g e t h a n o l were added, mixed and cooled to 0°C i n an i c e bath . The sample was then c e n t r i f u g e d at 12,000 X g f o r 15 min at 0 - k ° C . The p e l l e t was resuspended i n the same volume of 0.1 M EDTA 2% (w/v) SDS i n 10mM T r i s - H C l pH 8 and was incubated at 85°C f o r 20 min. to ensure d e n a t u r a t i o n of any S D S - r e s i s t a n t p r o t e i n s . Pronase was re-added to a f i n a l c o n c e n t r a t i o n of 0.025 mg/ml and the sample was incubated overnight at 37°C. LPS was p r e c i p i t a t e d with M g + + - e t h a n o l as p r e v i o u s l y d e s c r i b e d , resuspended i n the same volume of 10 mM T r i s - H C l and c e n t r i f u g e d to c o l l e c t the LPS p e l l e t at 200,000 X g f o r 2 h at 15°C i n the presence of 25 mM MgCl 9. 35 15. P r e p a r a t i o n of L i p i d A L i p i d A was prepared using a m o d i f i c a t i o n of the method of R i e t s c h e l e_t al.. (1977). Equimolar amounts (s t a n d a r d i s e d f o r KDO content) of l y o p h i l i s e d LPS prepared from each s t r a i n were resuspended to 15 ml i n 50 mM sodium acetate b u f f e r pH 3.0. The pH was b u f f e r e d at 3.0 i n an attempt to l e s s e n the chance of removing l a b i l e phosphate groups from L i p i d A. The resuspended LPS was heated at 100°C f o r 1.5 h, then f r o z e n and thawed four times to promote aggregation of L i p i d A. Samples were c e n t r i f u g e d i n t h i c k - w a l l e d g l a s s tubes at 9500 rpm f o r 5 minutes and washed once with sodium a c e t a t e b u f f e r , then washed twice more with d e i o n i s e d water to remove sodium a c e t a t e p r i o r to l y o p h i l i s a t i o n . Samples were resuspended i n and assayed f o r l o s s of KDO content to ensure complete removal of LPS carbohydrate from L i p i d A. 1 6 . Chemical assays P r o t e i n assay was performed as d e s c r i b e d by Sandermann and Strominger (1972) using 1? (w/v) SDS to s o l u b i l i s e p r o t e i n s . 3-Deoxy-D-mannooctulosonic a c i d (KDO) was determine by the c o l o u r i m e t r i c t h i o b a r b i t u r i c a c i d assay of Karkhanis e_t a l . (1978) on samples of LPS hydrolysed i n 0.2 N H^ so^  f o r 30 min at 1 0 0 ° ^ The ammonium s a l t of KDO (Sigma) was used as the standard. Phosphate was assayed using the ascorbate/molybdate 36 method of Ames and Dubin (1960). 17. SDS-Polyaorylamide g e l e l e c t r o p h o r e s i s The Laemmli g e l method, as modified by Lugtenberg e_t a l . (1975), was used f o r a l l r o u t i n e g e l e l e c t r o p h o r e s i s of outer membrane p r o t e i n s . Various percentages of acrylamide were used at a constant r a t i o of acrylamide : b i s - a c r y l a m i d e of 44:0.8. Gels contained 0.4? (w/v) SDS i n 0.375 M T r i s - H C l pH 8.8. A l l s t a c k i n g g e l s were 3% (w/v) acrylamide and contained 0.4$ (w/v) SDS i n 0.125 M T r i s - H C l pH 6.8. Samples were r o u t i n e l y s o l u b i l i s e d by heating to 88°C i n the presence of 4$ SDS and 10$ (v/v) 2-mercaptoethanol. Other agents which were o c c a s i o n a l l y added to the s o l u b i l i s a t i o n mix were 0.1 M M g C l 2 > to remove LPS from the g e l by promoting LPS a g g r e g a t i o n , or 40 mM EDTA, to improve LPS banding. In c e r t a i n circumstances, samples were not heated to analyse t h e i r heat-unmodified behaviour. Gels were run at a constant v o l t a g e of 150 V. Molecular weight standards used were E . c o l i b e t a - g a l a c t o s i d a s e (mol. wt. 116,000 ), r a b b i t muscle phosphorylase B ( mol. wt. 97,400 ), bovine serum albumen ( mol. wt. 66,000 ), ovalbumen ( mol. wt. 45,000 ), c a r b o n i c anhydrase ( mol. wt. 29,000 ), soybean t r y p s i n i n h i b i t o r ( mol. wt. 20,100 ) and lysozyme (mol. wt. 14,300), a l l obtained from Sigma Chemical Co. Gels were s t a i n e d with Coomassie B r i l l i a n t Blue dye or s i l v e r s t a i n (see below). Gels f o r the examination of l i p o p o l y s a c c h a r i d e were as 37 above but with 15$ acrylamide, and 4M urea was added ( T s a i and F r a s c h , 1982) to improve r e s o l u t i o n of the heterogeneous chain l e n g t h O-antigen bands. Tetrasodium-EDTA (40 mM) was a l s o added to the p r e v i o u s l y mentioned s o l u b i l i s a t i o n mix to improve LPS r e s o l u t i o n . 18. E l e c t r o p h o r e t i c b l o t t i n g of p r o t e i n s and LPS P r o t e i n s or LPS were t r a n s f e r r e d from polyacrylamide g e l s to n i t r o c e l l u l o s e using the method of Towbin e_t a_l. (1979) f o r SDS g e l s . The g e l s were placed i n c o n t a c t with 0.45 um pore s i z e n i t r o c e l l u l o s e membranes which had been pre-soaked i n 0.1$ (w/v) SDS at 60°C. These were then placed i n t o a Bio Rad b l o t t i n g apparatus and b l o t t e d e i t h e r o v e r n i g h t at a constant c u r r e n t of 1 mA or f o r 2 h at 400 mA. T r a n s f e r b u f f e r was composed of 192 mM g l y c i n e , 25 mM T r i s - H C l and 20$ (v/v) methanol pH 8.3. B l o t s were immuno-stained f o r s p e c i f i c antigens (see below). 19. Immunoenzymatic s t a i n i n g of p r o t e i n s or LPS on b l o t s The method of Mutharia e_t_ aJL. (1982) was used. B l o t s were incubated i n e i t h e r 3$ (v/v) f o e t a l c a l f serum (FCS) or 3$ (w/v) bovine serum albumen i n phosphate-buffered s a l i n e (PBS) f o r 30 min at 37°C to s a t u r a t e n o n - s p e c i f i c p r o t e i n b i n d i n g s i t e s . The b l o t s were then r i n s e d b r i e f l y i n PBS and 10 ml of antibody d i l u t e d i n 3$ FCS-PBS was added and incubated o v e r n i g h t at 4°C. The antibody d i l u t i o n used was s u f f i c i e n t 38 to g i v e an ELISA t i t r e of between 10 2 - 101*. The b l o t s were e x t e n s i v e l y washed with PBS and then 10 ml of goat anti-mouse IgG Fab 1 fragments conjugated to a l k a l i n e phosphatase were added, i n 3$ FCS/PBS and incubated 2 h at 37°C. The b l o t s were again washed e x t e n s i v e l y and s u b s t r a t e (1 mg/ml Naphthol AS-MX phosphoric a c i d , disodium s a l t + 2 mg/ml Fast Red TR s a l t [Sigma] i n 50 mM T r i s - H C l pH 8.5) was added. Colour development u s u a l l y took place w i t h i n 5 min. 20. Gel s t a i n i n g procedures a) P r o t e i n Coomassie s t a i n was composed of Coomassie B r i l l i a n t Blue R-250 dye at 0.5 mg/ml i n 20$ (v/v) methanol - 7.5$ (v/v) a c e t i c a c i d . P r o t e i n s were s t a i n e d with s i l v e r by the method of Wray e_t a l . (1981). Gels were incubated o v e r n i g h t with shaking i n 50% methanol, s t a i n e d with s i l v e r s o l u t i o n [0.5$ (w/v) i n 0.75$ (w/v) NaOH + 1.4$ (v/v) ammonium hy d r o x i d e ] , r i n s e d e x t e n s i v e l y and developed i n 0.005$ (w/v) c i t r i c a c i d , 0.019$ (v/v) formaldehyde i n d i s t i l l e d water. Gels were s t o r e d i n 50$ methanol. b) L i p o p o l y s a c c h a r i d e S i l v e r s t a i n f o r LPS employed a m o d i f i c a t i o n of the method of T s a i and Fra s c h (1982) and was performed s i m i l a r l y to the p r o t e i n s t a i n above, but with a p e r i o d a t e pre-treatment step to allow s t a i n i n g of the sugars. The g e l s were soaked 39 o v e r n i g h t i n 40$ (v/v) methanol with a g i t a t i o n then r i n s e d and soaked 5 min i n 0 . 7 $ (w/v) p e r i o d i c a c i d i n 40$ methanol. At t h i s p o i n t the s t a i n i n g c o n t a i n e r was changed and the pe r i o d a t e and methanol were washed o f f under running d e i o n i s e d water. S t a i n i n g reagent (as f o r p r o t e i n s t a i n ) was added and a g i t a t e d f o r 10 min, then r i n s e d w e l l . Developing s o l u t i o n ( a l s o as f o r p r o t e i n s t a i n ) was added and the s t a i n i n g was h a l t e d with 50$ methanol. Densitometer scanning of g e l s s t a i n e d f o r carbohydrate, by the method of S c h i f f (as modified by Zacharius e_t a_l. 1 9 6 9 ) , or f o r p r o t e i n by Coomassie blue, was performed with a Quick Scan J r . g e l scanner (Helena L a b o r a t o r i e s Corp., Beaumont, Tex.) . 21 . Protease d i g e s t i o n of outer membranes Outer membrane samples were resuspended to a c o n c e n t r a t i o n of 0 . 5 mg/ml i n 0.1 ml sample b u f f e r : 0 . 1 2 5 M T r i s - H C l pH 6 . 8 , 0 . 5 $ SDS, 10$ (v/v) g l y c e r o l and 0 . 0 0 0 1 $ (w/v) Bromphenol blue dye. This was heated at 100°C f o r 2 min. and cooled to 37°C f o r protease d i g e s t i o n . Protease was added i n 1 u l amounts at the c o n c e n t r a t i o n s i n d i c a t e d and the outer membrane samples were d i g e s t e d f o r 1 h at 37°C. Each sample was then d i v i d e d i n t o two p o r t i o n s , to one of which was added SDS, at a f i n a l c o n c e n t r a t i o n of 2$ (w/v), and to the other was added SDS and 2-ME (10$ - v/v) to help i d e n t i f y 2-ME-modifiable fragments of p r o t e i n F. These were f u r t h e r heated at 8 8 ° c and then loaded onto a 14$ (w/v) 40 acrylamide g e l . The i n i t i a l s o l u b i l i s a t i o n step i n 0.5% SDS was found to g r e a t l y a i d d i g e s t i o n of the f a i r l y protease-r e s i s t a n t outer membrane p r o t e i n s . 22. Assay f o r LPS carbohydrate LPS carbohydrate was assayed using a m o d i f i c a t i o n by Wright and Rebers (1972) of the c y s t e i n e - s u l p h u r i c a c i d assay. A 0.1 ml sample of p u r i f i e d LPS ( s t a n d a r d i s e d f o r keto-deoxyoctonate [KDO] content of 4.5 ug/ml) was cooled to 4°c and then 0.9 ml of 15 M H ^ Q ^ w a s a d d e d . T h e s a m p l e was warmed to room temperature and 0.02 ml of 3% (w/v) c y s t e i n e was added, then heated to 100°C f o r 20 min. The samples were l e f t to develop c o l o u r f o r 3 h i n the dark and were then scanned s p e c t r o p h o t o m e t r i c a l l y between the wavelengths 600 nm and 350 nm f o r coloured products t y p i c a l of c e r t a i n sugars. D-glycero-L-mannoheptose and D-glucose (and L-rhamnose) were used as standards f o r LPS heptose and hexose, r e s p e c t i v e l y . 23. Gas l i q u i d chromatography of whole c e l l f a t t y a c i d s For whole c e l l f a t t y a c i d a n a l y s i s , 10 mg of l y o p h i l i s e d c e l l s were premixed with i n t e r n a l standard (0.103 mg of pentadecanoic a c i d ) before h y d r o l y s i s with methanolic HC1. Samples were hydrolysed i n screw-capped tubes i n the presence of 1 ml of 2M methanolic HC1, prepared from a c e t y l c h l o r i d e ( I n s t a n t Methanolic HC1 K i t ; A p p l i e d Science L a b o r a t o r i e s Inc., State C o l l e g e , Pa.). H y d r o l y s i s was c a r r i e d out under 41 n i t r o g e n f o r 16 h at lOOOrj. A f t e r t h i s p e r i o d , the samples were cooled to room temperature and n e u t r a l i s e d with s i l v e r carbonate or Dowex AG1-X8 (HCC^-) (ca. 0.031 g ) . I n t e r n a l standard (methyltetradecanoate; 829 ug) was added to each tube, and these were then c e n t r i f u g e d to remove the i n s o l u b l e s a l t s or Dowex. P o r t i o n s (1 to 2 u l ) of the supernatant were analysed i n a Perkin-Elmer Sigma 3 gas chromatograph equipped with a g l a s s column (ca. 305 cm by 2 mm ID) c o n t a i n i n g 3% Sp-2100 D0H on 100- to 200-mesh Supelcoport (Supelco Inc., B e l l e f o n t e , Pa.). The f o l l o w i n g programme was used: i n i t i a l temperature, 160°C; f i n a l temperature, 200°C; ramp r a t e , 2°C/min; f i n a l time, 3 min. C a r r i e r gas (helium) flow was maintained at 18 ml/min. 24. C r o s s l i n k i n g of outer membranes and p u r i f i e d p r o t e i n s C e l l s f o r outer membrane c r o s s l i n k i n g experiments were grown i n BM2 minimal medium plus g l u c o s e . P u r i f i e d p r o t e i n F was obtained by L. Chan from c e l l s grown on PP2 medium. P r o t e i n s PhoE and P were i s o l a t e d (by R. Darveau and K. Poole, r e s p e c t i v e l y ) from c e l l s grown on HEPES-based phosphate-l i m i t i n g minimal medium. P r o t e i n s were p u r i f i e d as s t a t e d i n previous p u b l i c a t i o n s (F - Hancock, Decad and N i k a i d o , 1979; P - Hancock et al_. , 1982; PhoE - Benz e_t a l . , 1984). Outer membrane and p u r i f i e d p r o t e i n p r e p a r a t i o n s were c r o s s l i n k e d i n 0.015 ml volumes of 0.2M t r i e t h a n o l a m i n e b u f f e r pH 8.5, f o l l o w i n g the s p e c i f i c a t i o n s of Reithmeier and Bragg 42 (1977). The c r o s s l i n k e r , DSP, was d i s s o l v e d i n dimethylsulphoxide and added to the samples as a 1/15 d i l u t i o n to give the optimum f i n a l c o n c e n t r a t i o n of DSP as i n d i c a t e d f o r each experiment. A f t e r two minutes r e a c t i o n time, excess 1M T r i s - H C l pH 8.5 was added to stop the c r o s s l i n k i n g r e a c t i o n and the p r e p a r a t i o n s were d i l u t e d 1:1 i n t o sample b u f f e r c o n t a i n i n g 4$ (w/v) SDS, 0.5M T r i s - H C l pH 6.8 and 20$ (v/v) g l y c e r o l without reducing agent and el e c t r o p h o r e s e d as d e s c r i b e d above. Two-dimensional g e l e l e c t r o p h o r e s i s was performed by soaking the f i r s t dimension g e l s t r i p s i n 10$ 2-mercaptoethanol (v/v) or 10mM d i t h i o e r y t h r i t o l f o r 30 min at room temperature, s e a l i n g the s t r i p s atop the second dimension SDS-polyacrylamide g e l with 0.8$ (w/v) agarose and e l e c t r o p h o r e s i n g as d e s c r i b e d p r e v i o u s l y . Whole c e l l c r o s s l i n k i n g experiments were performed as de s c r i b e d by Palva and Rand a l l (1976) by c o n c e n t r a t i n g l o g a r i t h m i c phase c e l l s 100-fold to give approximately 1 X 10^0 c e l l s / m l i n a volume of one ml. A f t e r c e n t r i f u g a t i o n , outer membrane p e r m e a b i l i t y was not a l t e r e d (by the c r i t e r i o n of whole c e l l beta-lactamase assay) and c e l l s were m o t i l e , suggesting no a p p r e c i a b l e d i s t u r b a n c e of the c e l l s u r f a c e . C e l l s were c r o s s l i n k e d by the a d d i t i o n of 0.01 - 0.1 mg/ml of c r o s s l i n k i n g reagent, re a c t e d f o r two minutes, then the r e a c t i o n was terminated with excess 1M T r i s - H C l pH 8.5. Outer membranes were i s o l a t e d as d e s c r i b e d p r e v i o u s l y and analysed by two-dimensional g e l e l e c t r o p h o r e s i s . 43 CHAPTER ONE CHARACTERISATION OF A HYPERSUSCEPTIBLE MUTANT OF PSEUDOMONAS AERUGINOSA INTRODUCTION The high i n t r i n s i c a n t i b i o t i c r e s i s t a n c e of P.  aeruginosa has been proposed to be due to pos s e s s i o n of a h i g h l y impermeable outer membrane. I t has been suggested (Benz and Hancock, 1981) that t h i s i m p e r m e a b i l i t y i s due to poor a c t i v i t y of Pseudomonas p o r i n channels as compared to those of E s c h e r i c h i a c o l i . Although the P_j_ aeruginosa major p o r i n p r o t e i n was found to permit passage of l a r g e r molecules (6000 d a l t o n e x c l u s i o n l i m i t - Hancock e_t aJL. , 1979) than the major p o r i n of E_;_ c o l i (OmpF - 600 d a l t o n e x c l u s i o n l i m i t ) , the t o t a l p o r i n a c t i v i t y , as measured by black l i p i d b i l a y e r conductance assay, was much lower (Benz and Hancock, 1981). In a d d i t i o n , p e r m e a b i l i t y could a l s o be a f f e c t e d by some c h a r a c t e r i s t i c ( s ) of the l i p o p o l y s a c c h a r i d e , which i s the other major outer membrane c o n s t i t u e n t . The l i p o p o l y s a c c h a r i d e of P_^  aeruginosa i s c o n s i d e r a b l y d i f f e r e n t from that of c o l i , both with r e s p e c t to average le n g t h of LPS molecules and i n the types of c o n s t i t u e n t sugars. Other organisms such as N e i s s e r i a (Hitchcock, 1984) and Hemophilus spp. (Parr and Bryan, 1984), which possess LPS of short average chain l e n g t h , were found to be much more s u s c e p t i b l e to c e r t a i n types of a n t i b i o t i c s and de t e r g e n t s , e s p e c i a l l y to 44 hydrophobic agents (Nikaido, 1979). By studying a mutant, such as P_;_ aeruginosa s t r a i n M1 , with s i g n i f i c a n t s u s c e p t i b i l i t y to s e v e r a l a n t i b i o t i c s i t was hoped that the s t r u c t u r e ( s ) i n the outer membrane which are r e s p o n s i b l e f o r a n t i b i o t i c r e s i s t a n c e / s u s c e p t i b i l i t y i n Pseudomonas aeruginosa could be p i n p o i n t e d . The mutant M1 was d e r i v e d by m u l t i p l e mutagenesis steps from a w i l d type s t r a i n of P_;_ aeruginosa, WT1 , and s e l e c t e d as h y p e r s u s c e p t i b l e to a v a r i e t y of a n t i b i o t i c s (Zimmermann, 1979). Two spontaneous r e v e r t a n t s were i s o l a t e d i n t h i s study (designated P1-4 and P1-6, f o r £artially s u s c e p t i b l e phenotype) which had recovered p a r t i a l r e s i s t a n c e to a l l a n t i b i o t i c s . In a d d i t i o n , Dr. J . Fyfe (Univ. of Edinburgh) i s o l a t e d two transconjugant s t r a i n s prepared by c r o s s i n g an R68.45-carrying s t r a i n of the mutant M1 with a PA0222-derived, m u l t i p l y auxotrophic r e c i p i e n t . Two g e n e t i c l o c i were found to be a s s o c i a t e d with a change to a n t i b i o t i c s u s c e p t i b i l i t y . One of these ( i n the transconjugant P2-6), l i n k e d to the proA l o c u s (40 min on the FP2 - PAO chromosomal map; Royle et a l . , 1981), caused p a r t i a l h y p e r s u s c e p t i b i l i t y to a l l a n t i b i o t i c s . The other ( i n the transconjugant P2-8), was l i n k e d to the met-28 (30 min) and t_rp_C,D,E (35 min) l o c i , and caused h y p e r s u s c e p t i b i l i t y to B-lactam and aminoglycoside a n t i b i o t i c s o n l y . A t r a n s d u c t a n t c o n s t r u c t e d using the above two p a r t i a l l y s u s c e p t i b l e s t r a i n s (P2-6,8) e x h i b i t e d p a r t i a l s u s c e p t i b i l i t y to a l l a n t i b i o t i c s s i m i l a r l y to that of s t r a i n P2 - 6 , except that i t was more s u s c e p t i b l e to gentamicin, 45 s i m i l a r l y to P2-8 (Table I I ) . Due to the wide spectrum of a n t i b i o t i c s to which mutant M1 and a l l i t s d e r i v a t i v e s t r a i n s were s u s c e p t i b l e , our i n v e s t i g a t i v e e f f o r t s were l a r g e l y d i v e r t e d from mechanisms such as s p e c i f i c t a r g e t a l t e r a t i o n s (eg. p e n i c i l l i n b i n d i n g p r o t e i n s , which were i n f a c t u n a l t e r e d i n M1 - Zimmermann, 1980) and d e s t r u c t i v e enzymes (eg, beta-lactamase), to concentrate on p e r m e a b i l i t y of the outer membrane as a p o s s i b l e mechanism f o r t h i s a n t i b i o t i c h y p e r s u s c e p t i b i l i t y . The study of a n t i b i o t i c s u s c e p t i b i l i t y i n the aeruginosa mutant M1 covered three major areas: i ) the p a t t e r n of s u s c e p t i b i l i t y to a n t i b i o t i c s and the i s o l a t i o n of a n t i b i o t i c p a r t i a l l y r e s i s t a n t r e v e r t a n t s , i i ) e s t a b l i s h i n g the r e l a t i o n s h i p between s u s c e p t i b i l i t y to c e r t a i n a n t i b i o t i c s and a l t e r a t i o n s i n p e r m e a b i l i t y of the outer membrane, i i i ) c h a r a c t e r i s a t i o n of the a l t e r e d s t r u c t u r e s i n the outer membrane which caused i n c r e a s e d outer membrane p e r m e a b i l i t y and, consequently, a n t i b i o t i c hyper-s u s c e p t i b i l i t y . These s e c t i o n s are presented and summarised s e p a r a t e l y i n Chapter One. 46 RESULTS PART I. ANTIBIOTIC SUSCEPTIBILITY OF WILD TYPE AND MUTANT  STRAINS OF PSEUDOMONAS AERUGINOSA 1. D e r i v a t i o n of b a c t e r i a l s t r a i n s used i n c h a r a c t e r i s a t i o n of  a n t i b i o t i c s u s c e p t i b i l i t y and outer membrane a l t e r a t i o n s . Since d e t e r m i n a t i o n of the mechanism of a n t i b i o t i c r e s i s t a n c e i n P_^  aeruginosa was the u l t i m a t e g o a l of t h i s study, an a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n Pae K799/61, (designated M1 i n t h i s study) was used. T h i s s t r a i n had been i s o l a t e d as a n t i b i o t i c h y p e r s u s c e p t i b l e by using 4 c y c l e s of ethylmethane sulphonate (EMS) mutagenesis f o l l o w e d by one c y c l e of N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis. Dr. Zimmermann had shown that t h i s mutant was between 8- and 10,000-fold more s u s c e p t i b l e to a s e l e c t e d group of d i f f e r e n t beta-lactam a n t i b i o t i c s , and t h e r e f o r e i t was of g r e a t i n t e r e s t i n t h i s study of a n t i b i o t i c r e s i s t a n c e . In a d d i t i o n , he showed (Zimmermann, 1980) that there were no d i s c e r n i b l e d i f f e r e n c e s between mutant and w i l d type parent i n the a f f i n i t y of p e n i c i l l i n b i n d i n g p r o t e i n s f o r a v a r i e t y of p e n i c i l l i n s . These r e s u l t s suggested that a n t i b i o t i c s u s c e p t i b i l i t y might be due to an outer membrane a l t e r a t i o n . Due to the a p p a r e n t l y complex phenotype of mutant M1 and the number of mutagenesis steps i n v o l v e d i n i t s i s o l a t i o n , i t was necessary to attempt to segregate i n d i v i d u a l mutations i n t o separate s t r a i n s f o r c h a r a c t e r i s a t i o n . To move w i l d type 47 genes i n t o M1, the methods o f c o n j u g a t i o n and t r a n s d u c t i o n were attempted. These attempts were u n s u c c e s s f u l because the frequency of t r a n s f e r i n t o s t r a i n M1 by e i t h e r method was lower than the r e v e r s i o n frequency (10-7/CFU). Apparently, t r a n s f e r i n the o p p o s i t e d i r e c t i o n (that i s , from mutant M1 i n t o w i l d type) was not e q u a l l y i n h i b i t e d (see l a t e r experiments by J . F y f e , below). I t was t h e r e f o r e decided that r e v e r t a n t s to w i l d type a n t i b i o t i c r e s i s t a n c e would be i s o l a t e d , so that the number and nature of mutations i n t h i s s t r a i n might be determined. Revertants were s e l e c t e d d i r e c t l y by p l a t i n g c e l l s of s t r a i n Ml on n u t r i e n t medium c o n t a i n i n g e i t h e r gentamicin or c a r b e n i c i l l i n . Of f i f t e e n r e v e r t a n t s s e l e c t e d f o r g e n t a m i c i n r e s i s t a n c e , eleven were shown to be p a r t i a l r e v e r t a n t s i n that they had a n t i b i o t i c r e s i s t a n c e p a t t e r n s i n t e r m e d i a t e between those of the w i l d type s t r a i n WT1 and i t s a n t i D i o c i c -h y p e r s u s c e p t i b l e mutant, M1. S t r a i n P1-6 (see Tables I and I I ) was r e p r e s e n t a t i v e of t h i s pnenotypic c l a s s of r e v e r t a n t . The other four r e v e r t a n t s subsequently i s o l a t e d were as a n t i b i o t i c s u s c e p t i b l e as was mutant M1, and presumably r e s u l t e d from unstable r e v e r s i o n s . D e t a i l e d c h a r a c t e r i s a t i o n of a r e p r e s e n t a t i v e from these four s t r a i n s confirmed i t s i d e n t i t y with the mutant M1 (data not shown). Of 23 r e v e r t a n t s s e l e c t e d as c a r b e n i c i l l i n r e s i s t a n t , 14 were p a r t i a l r e v e r t a n t s (.represented by s t r a i n P1-4, see Tables I and I I ) and nine had f u l l y r e v e r t e d to w i l d type a n t i b i o t i c r e s i s t a n c e (represented by s t r a i n WT1-1). 48 A s e r i e s of transconjugants and t r a n s d u c t a n t s of s t r a i n M1 was prepared by Dr. J . Fyfe (Edinburgh U n i v e r s i t y , Edinburgh, UK;. Conjugation was performed using a d e r i v a t i v e of s t r a i n M1 which had spontaneously mutated to l e u c i n e auxotrophy and which c a r r i e d the c o n j u g a t i v e plasmic R68 .45. T h i s s t r a i n was mated with a m u l t i p l y auxotrophic PA0222 r e c i p i e n t s t r a i n (WT2). P u t a t i v e transconjugants were s e l e c t e d as having been converted to prototrophy at one of the auxotrophic marker s i t e s at v a r i o u s p o s i t i o n s on the chromosomal map. Transconjugants a r i s i n g from r e c e i p t of the d e s i r e d genes from M1 were s e l e c t e d by v i r t u e of possessing an a l t e r e d a n t i b i o t i c r e s i s t a n c e p a t t e r n which had been t r a n s f e r r e d along with the n u t r i e n t p r o t o t r o p h y . In t h i s way, the genes a p p a r e n t l y r e s p o n s i b l e f o r a n t i b i o t i c s u s c e p t i b i l i t y i n mutant M1 were t r a n s f e r r e d i n t o the w e l l c h a r a c t e r i s e d PAO g e n e t i c background. Two t r a n s f e r r e d genes at separate map l o c a t i o n s were found, by Dr. F y f e , to i n c r e a s e s u s c e p t i b i l i t y to e a r b e n i c i l l i n and trimethoprim i n the w i l d type PA0222 to values i n t e r m e d i a t e between those of PA0222 and M1 (see s t r a i n s P2-6 and P2-8, Tables I and I I I ) . Another s t r a i n , WT2-6, r e c e i v e d p r o + prototrophy s i m i l a r l y to s t r a i n P2-6 but r e t a i n e d w i l d type a n t i b i o t i c s u s c e p t i b i l i t y . Dr. Fyfe then prepared t r a n s d u c t a n t s of the newly cre a t e d y t r a n s c o n j u g a n t s , using phage F116C, again i n t o a PA0222 background (see s t r a i n s P2-6t and P2-8t, Tables I and I I I ) . The t r a n s d u c t a n t s r e t a i n e d the a n t i b i o t i c s u s c e p t i b i l i t y of t h e i r transconjugant parents, t h e r e f o r e the genes r e s p o n s i b l e f o r these phenotypes 49 Table I. Pseudomonas aeruginosa s t r a i n s . S t r a i n D e r i v a t i o n WT1 Pae K799/WT (ATCC 12055); pr o t o t r o p h M1 mutant 61 i s o l a t e d by repeated mutagenesis of WT1 using EMS and NTG ; pro t o t r o p h WT1-1 Spontaneous f u l l r e v e r t a n t of M1 i s o l a t e d on 0.5 ug/ml c a r b e n i c i l l i n j p r o t o t r o p h P1-4 Spontaneous p a r t i a l r e v e r t a n t of M1 i s o l a t e d on 0.25 ug/ml c a r b e n i c i l l i n j p r o t o t r o p h P1-6 Spontaneous p a r t i a l r e v e r t a n t of M1 i s o l a t e d on 0.4 ug/ml gentamicin ; pr o t o t r o p h WT3 P.aeruginosa PA01 ; pr o t o t r o p h AK 43 arg_C54, c h l - 2 , phage E79 r rough LPS phenotype WT2 met-28. trp-6 . lys-12 , his-4 . pro-82 . i l v - 2 2 6 ;wild type a n t i b i o t i c r e s i s t a n c e , auxotroph (PA0222) P2-6 PAZ3 ; pro+ recombinant from M1 l e u R68.45 X PA0222 ; a n t i b i o t i c p a r t i a l l y h y p e r s u s c e p t i b l e phenotype WT2-6 as above, but a n t i b i o t i c r e s i s t a n c e phenotype remained w i l d type P2-6t pro+ t r a n s d u c t a n t of PA0222 us i n g F116C grown on P2-6; p a r t i a l l y h y p e r s u s c e p t i b l e to a n t i b i o t i c s P2-8 PAZ1 ; met*, trp_ + recombinant from M1 l e u R68.45 X PA0222 ; B-lactam s u s c e p t i b l e P2-8t met+ tr a n s d u c t a n t of PA0222 using F116C grown on P2-8 P2-6,8 met+, p r o + t r a n s d u c t a n t prepared from s t r a i n s P2-6 and P2-8 ; p a r t i a l l y h y p e r s u s c e p t i b l e to a n t i b i o t i c s S t r a i n WT1 and i t s a n t i b i o t i c - s u p e r s u s c e p t i b l e mutant M1 were a g i f t from W. Zimmermann. S t r a i n s P2-6 , P2-6t, WT2-6, P2-8, P2-8t and P2-6.8 were a g i f t from J . F y f e . S t r a i n s WT3 and AK 43 were a g i f t from A. K r o p i n s k i . Other s t r a i n s were i s o l a t e d as d e s c r i b e d i n Methods. EMS, ethylmethane sulphonate; NTG, N-methyl-N'-nitro-N-nitrosoguanidine. 50 a p p e a r e d t o be c l o s e l y a s s o c i a t e d w i t h e i t h e r t h e met-28 ( P 2 -8) o r p r o - 8 2 (P2 -6) l o c i . A n o t h e r s t r a i n (P2-6 ,8 ) was p r e p a r e d by t r a n s d u c i n g t h e met p r o t o t r o p h y f r o m s t r a i n P2-8 i n t o s t r a i n P 2 - 6 , f o r m i n g a s t r a i n p o s s e s s i n g b o t h p u t a t i v e a n t i b i o t i c s u s c e p t i b i l i t y - e n c o d i n g s i t e s f r o m t h e chromosome. 2. S u s c e p t i b i l i t y o f w i l d t y p e and m u t a n t s t r a i n s t o a v a r i e t y  o f a n t i b i o t i c s w i t h d i f f e r e n t m e c h a n i s m s o f a c t i o n . MICs were d e t e r m i n e d f o r c e l l s grown i n PP2 b r o t h i n t o s t a t i o n a r y p h a s e , d i l u t e d 1/100 and t h e n a p p l i e d i n 10 u l v o l u m e s t o P P 2 - a g a r p l a t e s ( s e e T a b l e I I ) . The a n t i b i o t i c s were i n c o r p o r a t e d a t v a r i o u s d i l u t i o n s i n t o t h e a g a r p l a t e s . MICs were d e t e r m i n e d f o r a w i d e v a r i e t y o f a n t i b i o t i c s r e p r e s e n t i n g h y d r o p h i l i c , p o l y c a t i o n i c and h y d r o p h o b i c compounds, and a n t i b i o t i c s w i t h v a r y i n g t a r g e t s i t e s i n t h e o r g a n i s m . F o r e x a m p l e , h y d r o p h i l i c b e t a - l a c t a m s b i n d t o and i n h i b i t s p e c i f i c p e p t i d o g l y c a n s y n t h e t i c enzymes on t h e o u t e r s u r f a c e o f t h e c y t o p l a s m i c membrane, p o l y c a t i o n i c a m i n o g l y c o s i d e s and p o l y m y x i n have a v a r i e t y o f e f f e c t s on t h e c e l l i n c l u d i n g i n h i b i t i o n o f p r o t e i n s y n t h e s i s , and t h e m o d e r a t e l y h y d r o p h o b i c a n t i b i o t i c t r i m e t h o p r i m ( T a b l e I I I ) i n h i b i t s d i h y d r o f o l a t e r e d u c t a s e . A g e n t s u s e d i n t h i s s t u d y a r e r e f e r r e d t o as " h y d r o p h o b i c " f o l l o w i n g t h e n o m e n c l a t u r e o f N i k a i d o ( 1 9 7 6 ) , by v i r t u e o f t h e i r t e n d e n c y t o p e r m e a t e p h o p h o l i p i d b i l a y e r s . M I C s were d e t e r m i n e d f o r WT3 (PA01 w i l d t y p e ) , WT1 ( w i l d .type p a r e n t o f M1 ) , M1 ( a n t i b i o t i c h y p e r s u s c e p t i b l e m u t a n t ) 51 Table I I . Minimal i n h i b i t o r y c o n c e n t r a t i o n s (MIC) of a v a r i e t y of a n t i b i o t i c s f o r parent and a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s . S t r a i n M I C (ug/ml) B-lactam BP AP TC CB CF MX Aminoglycoside GM SM TM Other CM PX TET WT1 >2000 >500 20 WT1-1 >2000 >500 20 WT3 >2000 >500 20 P1-4 10 5 P1-6 10 5 M1 1.0 0.5 25 2 20 25 2 20 25 2 20 0.5 0.5 0.3 5 0.5 0.5 0.3 0.2 0.05 0.05 0.1 0.2 10 1 0 8 1 2 0 . 1 50 50 20 5 5 2 >200 20 >200 15 0.8 >200 10 0.5 >200 10 0.5 50 5 0.5 25 5 100 1 00 1 00 5 50 2 Minimal i n h i b i t o r y c o n c e n t r a t i o n s were performed as d e s c r i b e d i n Methods. BP, b e n z y l p e n i c i l l i n ; AP, a m p i c i l l i n ; TC, t i c a r c i l l i n ; CB, c a r b e n i c i l l i n ; CF, c e f s u l o d i n ; MX, moxalactam; GM, gentamicin; SM, streptomycin; TM, tobramycin; CM, chloramphenicol; PX, polymyxin B; TET, t e t r a c y c l i n e . and spontaneous r e v e r t a n t s to f u l l (WT1-1) and p a r t i a l (P1-4 and P1-6) a n t i b i o t i c r e s i s t a n c e . The r e s i s t a n c e of w i l d type s t r a i n s of P_;_ aeruginosa (WT3, WT1 and WT1-1) to the c o n v e n t i o n a l p e n i c i l l i n s , represented by b e n z y l p e n i c i l l i n and a m p i c i l l i n , was high. T h i s r e s i s t a n c e was more than 4 0 - f o l d that found i n E . c o l i (data not shown). For newer c a r b o x y p e n i c i l l i n s , such as t i c a r c i l l i n and c a r b e n i c i l l i n , as w e l l as f o r the t h i r d g e n e r a t i o n c e p h a l o s p o r i n , c e f s u l o d i n , and the oxycephalosporin, moxalactam, w i l d type r e s i s t a n c e was lower, mostly due to the i n c r e a s e d s t a b i l i t y to beta-lactamases of these agents (0'Callaghan, 1 9 7 9 ; Murakami and Yoshida, 1981). The mutant s t r a i n M1 was much more s u s c e p t i b l e to a l l beta-lactam-type a n t i b i o t i c s than i t s parent s t r a i n WT1. Both p a r t i a l r e v e r t a n t s P1-4 ( s e l e c t e d f o r c a r b e n i c i l l i n r e s i s t a n c e ) and P1-6 ( s e l e c t e d f o r gentamicin r e s i s t a n c e ) had s i m i l a r r e s i s t a n c e l e v e l s f o r a l l beta-lactams, which were in t e r m e d i a t e between w i l d type and mutant. Resi s t a n c e s to most beta-lactams t e s t e d were i d e n t i c a l f o r the two types of p a r t i a l r e v e r t a n t s . S u s c e p t i b i l i t y to the aminoglycosides gentamicin, streptomycin and tobramycin was i n c r e a s e d i n the mutant, although l e s s d r a s t i c a l l y than f o r beta-lactams, perhaps i n p a r t because of the g r e a t e r e f f e c t i v e n e s s of t h i s c l a s s of a n t i b i o t i c s on w i l d type P_j_ aeruginosa. Tobramycin, i n p a r t i c u l a r , was very e f f e c t i v e even on w i l d type s t r a i n s . There a l s o appeared to be a minor d i f f e r e n c e i n r e s i s t a n c e to 53 aminoglycosides between s t r a i n WT3 and the parent s t r a i n WT1, although the f u l l r e v e r t a n t s t r a i n WT1-1 was i d e n t i c a l to WT1, i t s parent. This d i f f e r e n c e was t h e r e f o r e presumably s t r a i n -s p e c i f i c and could be due to minor c e l l s u r f a c e or ribosomal d i f f e r e n c e s . Once again, p a r t i a l r e v e r t a n t s P1-4 and P1-6 had i n t e r m e d i a t e l e v e l s of r e s i s t a n c e to aminoglycosides, except i n the case of tobramycin where the margin between f u l l s u s c e p t i b i l i t y and r e s i s t a n c e was not l a r g e enough to d i s t i n g u i s h an i n t e r m e d i a t e l e v e l . For chloramphenicol and t e t r a c y c l i n e , which are moderately hydrophobic (Nikaido, 1976) p r o t e i n s y n t h e s i s i n h i b i t o r s , mutant s t r a i n M1 was a l s o more s u s c e p t i b l e than w i l d type. The two c l a s s e s of p a r t i a l r e v e r t a n t s d i s p l a y e d d i f f e r e n c e s i n s u s c e p t i b i l i t y to these two a n t i b i o t i c s , however. The s t r a i n i s o l a t e d f o r c a r b e n i c i l l i n r e s i s t a n c e (P1-4) was a p p a r e n t l y more r e s i s t a n t to chloramphenicol but l e s s r e s i s t a n t to t e t r a c y c l i n e than s t r a i n P1-6. Subsequent s t u d i e s have a l s o shown that the mutant M1 i s between 50- and 400-fold more s u s c e p t i b l e to the a d d i t i o n a l hydrophobic agents r i f a m y c i n SV, c r y s t a l v i o l e t , n a l i d i x i c a c i d and n o v o b i o c i n (data not shown). S t r a i n M1 was only two- to f o u r - f o l d more s u s c e p t i b l e to polymyxin B than the w i l d type. The d i f f e r e n c e i n a n t i b i o t i c r e s i s t a n c e between WT1 and i t s a n t i b i o t i c s u s c e p t i b l e mutant, M1, f o r a l l a n t i b i o t i c s was dramatic and ranged from 2- to >2000-fold, depending on the a n t i b i o t i c used. In c o n t r a s t , the d i f f e r e n c e between WT1 and WT3 was never g r e a t e r than 2 . 5 - f o l d . The f a c t t h a t M1 was 54 Table I I I . Minimal i n h i b i t o r y c o n c e n t r a t i o n s (MIC) of three a n t i b i o t i c s i n spontaneous r e v e r t a n t s , transconjugants and t r a n s d u c t a n t s of M1. S t r a i n Phenotype M I C ( u g / m l ) CB GM TP WT1 20 5 100 WT1-1 20 5 1 00 P1-4 AbsA, ? ,AbsC 0.5 0.5 1 0 P1-6 AbsA, ? ,AbsC 0.5 1 .0 10 M1 AbsA,AbsB,AbsC <0 .25 0 .05 2 WT2 20 5 100 P2-6 AbsA 0.25 0.5 20 WT2-6 20 5 200 P2-6t AbsA 0.5 1 100 P2-8 AbsB 1 0 .1 200 P2-8t AbsB 1 0 .1 200 P2-6 ,8 AbsA,AbsB 0.25 0 .1 20 Overnight c u l t u r e s were d i l u t e d 1/100 then 10 u l spots (approximately 2 X 10 5 c e l l s ) were a p p l i e d to PP2-agar p l a t e s c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s of a n t i b i o t i c . Minimal i n h i b i t o r y c o n c e n t r a t i o n s were the lowest c o n c e n t r a t i o n s of a n t i b i o t i c at which growth was i n h i b i t e d . CB, c a r b e n i c i l l i n ; GM, gentamicin; TP, trimethoprim. A l l data i n t h i s t a b l e were taken from a s i n g l e experiment f o r comparative purposes, although very s i m i l a r numbers have been obtained i n many t r i a l s . The phenotypic d e s i g n a t i o n s Abs ( a n t i b i o t i c s u s c e p t i b i l i t y ) A, B, and C are d i s c u s s e d i n the t e x t . 55 more s u s c e p t i b l e to a l l a n t i b i o t i c s t e s t e d i n d i c a t e d that an i n c r e a s e i n outer membrane p e r m e a b i l i t y was a more probable e x p l a n a t i o n f o r i t s a n t i b i o t i c s u s c e p t i b i l i t y than were the a l t e r n a t i v e e x p l a n a t i o n s of s p e c i f i c t a r g e t a l t e r a t i o n s or enzyme-mediated r e s i s t a n c e . 3. A n t i b i o t i c s u s c e p t i b i l i t y of transconjugant and  tran s d u c t a n t s t r a i n s d e r i v e d from mutant s t r a i n M1. MIC determinations were made f o r transconjugant and tra n s d u c t a n t s t r a i n s on three a n t i b i o t i c s , c a r b e n i c i l l i n , gentamicin and trimethoprim, considered to be r e p r e s e n t a t i v e of h y d r o p h i l i c beta-lactams, p o l y c a t i o n i c aminoglycosides and hydrophobic a n t i b i o t i c s , r e s p e c t i v e l y . The r e s u l t s , i n c l u d i n g p a r a l l e l data f o r w i l d type, mutant and r e v e r t a n t s t r a i n s , are shown i n Table I I I . For r e v e r t a n t s , a l t e r a t i o n s i n the a n t i b i o t i c s u s c e p t i b i l i t y p a t t e r n s always i n v o l v e d a l l three a n t i b i o t i c s t e s t e d , i n that the p a r t i a l r e v e r t a n t s showed in t e r m e d i a t e r e s i s t a n c e to a l l beta-lactams, aminoglycosides and other a n t i b i o t i c s t e s t e d (see a l s o Table I I ) . The two types of tra n s c o n j u g a n t s separated by J. Fyfe (P2-6 and P2-8) could be i d e n t i f i e d by t h e i r a n t i b i o t i c r e s i s t a n c e p a t t e r n s on these three a n t i b i o t i c s . P2-6 (pro+) was more s u s c e p t i b l e to c a r b e n i c i l l i n , gentamicin and trimethoprim than the wi l d type parent (WT2) or the p r o + , n o n - s u s c e p t i b l e transconjugant " s i b l i n g " (WT2-6), while P2-8 (met+ trp+) was more s u s c e p t i b l e to c a r b e n i c i l l i n and gentamicin but had approximately w i l d type r e s i s t a n c e to trimethoprim. These r e s u l t s showed that 56 Table IV. S u s c e p t i b i l i t y to v a r i o u s a n t i b i o t i c s of a l l P. aeruginosa s t r a i n s by d i s c i n h i b i t i o n diameter assay. S t r a i n Disk diameter (mm) B-lactams Other H i f i H i f o CB CF CX MX BP CZ AZ NT GM TC NR TP CM 500 50 100 50 5000 2 5 5 5 200 5 200 200 WT 1 - 1 0 7 - - - - - - - - - -M1 48 32 40 30 50 21 38 14 - 25 18 28 28 WT1-1 7 10 - - - - - - - - - - -P1-4 35 26 29 17 43 10 24 14 - 23 - - 18 P1-6 38 34 30 30 39 18 33 1 0 - 1 1 9 10 17 WT2 1 1 16 - - - - - - - 7 - - -P2-6 36 30 19 28 - 14 29 - - 9 - - -WT2-6 9 9 - - - - - - - - - - -P2-6t 36 28 44 29 7 13 26 - - 12 - - -P2-8 26 15 14 9 - - 1 1 - - 8 - - -P2-8t 22 15 13 9 7 - 1 1 - - 8 - - -A n t i b i o t i c s were a p p l i e d i n 10 u l volumes of the i n d i c a t e d c o n c e n t r a t i o n s ( i n ug/ml) to s t e r i l e f i l t e r d i s c s on PP2-agar p l a t e s onto which a lawn of b a c t e r i a had been swabbed. -, r e s i s t a n t ( i n h i b i t i o n zone diameter _< 6 mm); CB, c a r b e n i c i l l i n ; CF, c e f s u l o d i n ; CX, cefotaxime; MX, moxalactam; BP, b e n z y l p e n i c i l l i n ; CZ, c e f t a z i d i m e ; AZ, azthreonam; NT, N-formimidoylthienamycin (Imipenem); GM, gentamicin; TC, t e t r a c y c l i n e ; NR, n o r f l o x a c i n ; TP, trimethoprim; CM, chloramphenicol; H i f i , h y d r o p h i l i c ; H i f o , hydrophobic. The above data were taken from a s i n g l e experiment but separate t r i a l s performed on a l l s t r a i n s provided s i m i l a r numbers. 57 the two s e p a r a t e l y mapping mutations (or s e t s of mutations) produced d i f f e r e n t a n t i b i o t i c s u s c e p t i b i l i t y phenotypes. By surveying a g r e a t e r v a r i e t y of a n t i b i o t i c s using the d i s c assay (Table IV) , f o r which r e s i s t a n c e i s p r o p o r t i o n a l to the i n h i b i t i o n zone ( f o r most a n t i b i o t i c s ) , the p a r t i a l l y s u s c e p t i b l e phenotypes of s t r a i n s P2-6 and P2-8 were e v i d e n t . U n f o r t u n a t e l y , t h i s type of assay could not be used f o r other than comparitive purposes because c e r t a i n a n t i b i o t i c s , due to e i t h e r h y d r o p h o b i c i t y or l a r g e s i z e , do not d i f f u s e outward from the d i s c as w e l l as o t h e r s . For example, even though the l e v e l s of gentamicin, chloramphenicol, trimethoprim, N-formimidoylthienamycin and n o r f l o x a c i n were subsequently r a i s e d two- or f o u r - f o l d , the i n h i b i t i o n zone diameters were not s u b s t a n t i a l l y i n c r e a s e d . Consequently, r e s u l t s from the d i s c assay were used only to compare s t r a i n s , and the g r e a t e r trimethoprim s u s c e p t i b i l i t y of P2-6 and P2-6t was not observed i n t h i s assay as i t had been i n the p l a t e MIC d e t e r m i n a t i o n s . What was evident from the r e s u l t s i n Table IV was that the two transconjugant s t r a i n s appeared to be l e s s s u s c e p t i b l e than the mutant M1 or p a r t i a l r e v e r t a n t s to hydrophobic agents i n g e n e r a l and to s e l e c t e d beta-lactams. In p a r t i c u l a r , growth of both s t r a i n s was l e s s a f f e c t e d by the t h i r d g e n e r a t i o n c e p h a l o s p o r i n s (cefotaxime and c e f t a z i d i m e ) , as w e l l as p e n i c i l l i n G, N-formimidoylthienamycin (Imipenem) and azthreonam (a monobactam a n t i b i o t i c - see a l s o F i g . 1). S t r a i n P2-8 was a l s o l e s s s u s c e p t i b l e to the oxycephalosporin, moxalactam, and the t h i r d g e n e r a t i o n c e p h a l o s p o r i n , 58 c e f s u l o d i n . S t r a i n P2-6t was l e s s s u s c e p t i b l e to cefotaxime and trimethoprim (Table I I I ) t h e r e f o r e i t was p o s s i b l e that t h i s t r a n s d u c t a n t was not p h e n o t y p i c a l l y i d e n t i c a l to i t s parent, P2-6. For t h i s reason, only s t r a i n s P2-6 and P2-8 were c h a r a c t e r i s e d f u r t h e r . 4. Bacteriophage s e n s i t i v i t y of wild type and mutant s t r a i n s Bacteriophage s e n s i t i v i t y t e s t i n g i s a v a l u a b l e assay f o r comparison of c e l l s u r f a c e s t r u c t u r e s . Bacteriophages must bind to a c e l l s u r f a c e r e c e p t o r s i t e on the bacterium before t h e i r g e n e t i c m a t e r i a l can be i n j e c t e d i n t o the c e l l . The re c e p t o r s i t e must be a c o n s t i t u e n t of the outer membrane, e i t h e r a p r o t e i n such as p i l i n , a s p e c i f i c l i p o p o l y s a c c h a r i d e s t r u c t u r e or a r e c e p t o r composed of more than one type of molecule i n a c h a r a c t e r i s t i c conformation (Hancock and Reeves, 1976; Datta et a l , , 1977; Bradley, 1980). To c r u d e l y assess r e l a t e d n e s s of the c e l l s u r f a c e s of a l l the wi l d type and a n t i b i o t i c s u s c e p t i b l e s t r a i n s from each l i n e a g e , s e v e r a l phages known to have d i f f e r e n t outer membrane r e c e p t o r s i t e s were t e s t e d f o r t h e i r a b i l i t y to l y s e b a c t e r i a on the su r f a c e of an agar p l a t e (Table V). A l l of the phages used i n t h i s study had u n s p e c i f i e d p r o t e i n or LPS r e c e p t o r s (Mutharia e_t al., 1982) except phages D8 and pRD1. Phage D8 was i s o l a t e d as a rough L P S - s p e c i f i c phage by J a r r e l l and K r o p i n s k i (1981) and was used to determine whether the mutant s t r a i n M1 d i s p l a y e d the same rough LPS phenotype as s t r a i n AK 43 (on which the phage had 59 been i s o l a t e d ) . Phage pRD1 (which was s p e c i f i c f o r c e l l s c a r r y i n g the a n t i b i o t i c r e s i s t a n c e plasmid RP1) was used as a negative c o n t r o l f o r phage r e s i s t a n c e , s i n c e none of these s t r a i n s c a r r i e d the plasmid. A l l of the phages used, with the exception of phage D8, were t y p i n g phages i s o l a t e d f o r t h e i r a b i l i t y to grow on a PA01 type s t r a i n of P_;_ aeruginosa (Nicas and Hancock, 1980). S t r a i n WT2 (PA0222) was c l e a r l y r e l a t e d to s t r a i n WT3 (a PA01 w i l d type s t r a i n ) , by v i r t u e of i t s s e n s i t i v i t y to a l l phages with p r o t e i n and LPS r e c e p t o r s . S t r a i n WT1 and the f u l l r e v e r t a n t s t r a i n WT1-1, on the other hand, were r e s i s t a n t to s e v e r a l of the phages with p r o t e i n r e c e p t o r s . F u r t h e r comparisons of phage t y p i n g were t h e r e f o r e made between each mutant and i t s own w i l d type parent s t r a i n . Mutant M1 was s u s c e p t i b l e to a l l p r o t e i n and L P S - s p e c i f i c phages except f o r one p i l u s - s p e c i f i c phage, i n c o n t r a s t to i t s parent s t r a i n , WT1 , which was r e s i s t a n t to the four a d d i t i o n a l p r o t e i n - s p e c i f i c phages. This i n d i c a t e d that the mutant c e l l s u r f a c e was d i f f e r e n t to that of WT1, and seemed to have more exposed bacteriophage b i n d i n g s i t e s . However, the mutant s t r a i n was r e s i s t a n t to phage D8 and t h e r e f o r e d i d not d i s p l a y the same rough LPS phenotype as s t r a i n AK 43. This r e s u l t d i d not e l i m i n a t e the p o s s i b i l i t y that s t r a i n M1 was a rough LPS mutant, but simply demonstrated that the D8 r e c e p t o r (as yet undetermined) was e i t h e r not present or not exposed. The phage s e n s i t i v i t y p a t t e r n s of the p a r t i a l r e v e r t a n t s t r a i n s P1-4 and P1-6 were i d e n t i c a l to that of the mutant. 6 0 Table V. Bacteriophage s e n s i t i v i t y of w i l d type and a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s . S t r a i n Bacteriophage and r e c e p t o r type P r o t e i n P i l u s LPS R-LPS RP1 A2 A4 A8 B9A B9B C3 D3 C9 A7 B2 B4 D8 pRD1 WT1 R S WT1-1 R S P1-4 S S P1-6 S S M1 S S WT3 S S WT2 S S WT2-6 S S P2-6 S S P2-8 S S S R R R S R R R S S S S S S S S S S S S S S S S S S S S S S S S s s s s s s s s S R S S R S S R S S R S S R S S S S S S S S S S S S S s s s S S R R S S R R S S R R S S R R S S R R S S R R S S R R S S R R S S R R S S R R Bacteriophage suspensions were spotted onto b a c t e r i a l lawns using a m u l t i s y r i n g e i n o c u l a t o r . P o s i t i v e r e s u l t s f o r s e n s i t i v i t y (a c l e a r e d zone of b a c t e r i a l l y s i s due to phage) was determined a f t e r i n c u b a t i o n at 37°C f o r 20 h. R, r e s i s t a n t ; S, s e n s i t i v e . Phages are grouped a c c o r d i n g to p u t a t i v e c e l l s u r f a c e r e c e p t o r . P r o t e i n , undetermined outer membrane p r o t e i n r e c e p t o r ; LPS, undetermined l i p o p o l y s a c c h a r i d e r e c e p t o r ; R-LPS, rough LPS-s p e c i f i c ; RP1, s p e c i f i c f o r c e l l s c a r r y i n g RP1 plasmid. 61 The transconjugant d e r i v a t i v e s of WT2 were s e n s i t i v e to a l l phages t h e r e f o r e no c o n c l u s i o n s as to c e l l s u r f a c e a l t e r a t i o n s i n these s t r a i n s could be made. The f a c t that the f u l l r e v e r t a n t s t r a i n WT1-1 d i s p l a y e d a phage t y p i n g p a t t e r n s i m i l a r to the w i l d type parent WT1 i n d i c a t e d that the c e l l s u r f a c e changes i n the mutant were somehow r e l a t e d to a n t i b i o t i c s u s c e p t i b i l i t y . 5. Other p r o p e r t i e s of the a n t i b i o t i c h y p e r s u s c e p t i b l e mutant  s t r a i n . a) Frequency of r e v e r s i o n As d i s c u s s e d e a r l i e r i n the s e c t i o n d e a l i n g with the d e r i v a t i o n of s t r a i n s , the frequency of r e v e r s i o n to e i t h e r p a r t i a l or f u l l a n t i b i o t i c r e s i s t a n c e phenotype was g r e a t e r than the frequency of c o n j u g a t i o n or t r a n s d u c t i o n obtained f o r t r a n s f e r of w i l d type a n t i b i o t i c r e s i s t a n c e from WT1 i n t o the mutant s t r a i n M1. T h i s frequency should be d i s t i n g u i s h e d from the c o n j u g a t i o n experiments performed by J . Fyfe to segregate a n t i b i o t i c s u s c e p t i b i l i t y l o c i from M1 i n t o the w i l d type s t r a i n . Despite the complex procedure used to i s o l a t e mutant M1, the s i n g l e step s e l e c t i o n of f u l l r e v e r t a n t s which were p h e n o t y p i c a l l y i d e n t i c a l to w i l d type WT1 (see other s e c t i o n s , t h i s chapter) s t r o n g l y suggested that there was a s i n g l e major mutation i n mutant M1. One p o s s i b l e e x p l a n a t i o n f o r s i n g l e step r e v e r s i o n i s that there e x i s t s , i n mutant M1, more than one mutation causing a n t i b i o t i c s u s c e p t i b i l i t y , but that one mutation may s t a b i l i s e another. Another e x p l a n a t i o n i s that 62 the o r i g i n a l mutation may not have r e v e r t e d , but i n s t e a d , an a d d i t i o n a l mutation, which could d i s g u i s e the e f f e c t of the o r i g i n a l mutation, may have occurred (phenotypic s u p p r e s s i o n ) . Upon s e l e c t i o n of r e v e r t a n t s using c a r b e n i c i l l i n , r e v e r s i o n of one type of major mutation may have favoured r e v e r s i o n of another i n the M1 g e n e t i c background. However, two separate m u t a t i o n - c o n t a i n i n g l o c i from the M1 g e n e t i c background were s u c c e s s f u l l y t r a n s f e r r e d i n t o the PA0222 background. b) Growth r a t e of the mutant Mutant M1 grew only s l i g h t l y slower than the w i l d type s t r a i n WT1 i n r i c h medium (generation time of 50 min as opposed to 45 min i n PP2 - data not shown) and maintained the same growth r a t e as WT1 i n minimal medium with glucose as s o l e carbon source (50 min). I t was assumed from these o b s e r v a t i o n s that s t r a i n M1 had not s u s t a i n e d e x t e n s i v e damage to gross c e l l u l a r f u n c t i o n s which could perhaps i n f l u e n c e i t s response to a n t i b i o t i c c h a l l e n g e . Mutant M1 was more s e n s i t i v e to low temperature k i l l i n g , however (B. Angus, unpublished o b s e r v a t i o n ) . c) Colony morphology C o l o n i e s of mutant M1, P1-4, P1-6, P2-6 and P2-6,8 were n o t i c e a b l y g l o s s i e r i n appearance than P2-8 and the w i l d type s t r a i n s and a l s o had a smooth, rounded edge. The w i l d type P.  aeruginosa s t r a i n used i n t h i s study had a c h a r a c t e r i s t i c a l l y r e f r a c t i v e s u r f a c e . The mutant colony type was probably due to an a l t e r a t i o n i n LPS (see l a t e r s e c t i o n s , t h i s c h a p t e r ) . 63 6. SUMMARY The major c o n c l u s i o n s from chapter one, part one were: a) Mutant s t r a i n M1 was found to be h y p e r s u s c e p t i b l e to a l l c l a s s e s of a n t i b i o i c s t e s t e d , and possessed a mutant phage s e n s i t i v i t y p a t t e r n . b) Spontaneous p a r t i a l r e v e r t a n t s had in t e r m e d i a t e r e s i s t a n c e between that of M1 and WT1 f o r a l l a n t i b i o t i c s t e s t e d , yet r e t a i n e d the i d e n t i c a l phage t y p i n g p a t t e r n of mutant M1 . c) Spontaneous f u l l r e v e r t a n t s had i d e n t i c a l a n t i b i o t i c r e s i s t a n c e and phage s e n s i t i v i t y p a t t e r n s to w i l d type WT1. d) Both transconjugants P2-6 and P2-8 (and t h e i r r e s p e c t i v e t r a n s d u c t a n t s P2 - 6 t and P2 - 8 t ) had p a r t i a l l y a n t i b i o t i c r e s i s t a n t phenotypes, however these were e v i d e n t l y not the same phenotype as e i t h e r p a r t i a l r e v e r t a n t s or each other, based on d i f f e r e n c e s i n s u s c e p t i b i l i t y to c e r t a i n beta-lactam a n t i b i o t i c s as w e l l as trimethoprim, and on the s e p a r a t e l y mapping auxotrophic markers with which the mutations could be m o b i l i s e d . From the wide range of a n t i b i o t i c types i n v o l v e d , h y p e r s u s c e p t i b i l i t y i n s t r a i n M1 was suggested to be caused by enhanced outer membrane p e r m e a b i l i t y r a t h e r than by a n t i b i o t i c t a r g e t s i t e a l t e r a t i o n or plasmid-mediated r e s i s t a n c e i n the parent s t r a i n WT1. 6k Based on the MIC data, phenotypic d e s i g n a t i o n s were assigned to each s t r a i n i n an attempt to d e s c r i b e the number of separate mutations i n v o l v e d i n a n t i b i o t i c s u s c e p t i b i l i t y of the mutant s t r a i n M1. I t i s p o s t u l a t e d that at l e a s t three separate mutations are r e s p o n s i b l e f o r the mutant phenotype of M1 . The AbsA phenotype r e p r e s e n t s the p a r t i a l l y a n t i b i o t i c s u s c e p t i b l e phenotype l i n k e d to pro-82 i n s t r a i n P2-6. This phenotype was l a t e r found to be a s s o c i a t e d with a s t r u c t u r a l change i n LPS (part three of chapter one). The AbsB phenotype r e p r e s e n t s the p a r t i a l l y a n t i b i o t i c s u s c e p t i b l e phenotype l i n k e d to met-28 i n s t r a i n P2-8. The AbsC phenotype r e p r e s e n t s the p a r t i a l l y a n t i b i o t i c s u s c e p t i b l e phenotype of the p a r t i a l r e v e r t a n t s t r a i n s P1-4 and P1-6. AbsC was a l s o found to be a s s o c i a t e d with an LPS s t r u c t u r a l a l t e r a t i o n , but was p h e n o t y p i c a l l y d i s t i n c t from AbsA (see part t h r e e , chapter one) . 6 5 PART I I . CHARACTERISATION OF ALTERATIONS IN OUTER MEMBRANE PERMEABILITY OF MUTANT STRAINS The r e s u l t s of the previous s e c t i o n i n d i c a t e d that an i n c r e a s e i n outer membrane p e r m e a b i l i t y was p r i m a r i l y r e s p o n s i b l e f o r the i n c r e a s e i n a n t i b i o t i c s u s c e p t i b i l i t y of the P_^  aeruginosa mutant s t r a i n M1 . The data could not be expla i n e d by a l t e r a t i o n s e i t h e r i n enzymes r e s p o n s i b l e f o r degrading these a n t i b i o t i c s or i n t a r g e t s i t e s , s i n c e these would only a f f e c t s p e c i f i c groups of a n t i b i o t i c s and a l l mutant s t r a i n s had i n c r e a s e d s u s c e p t i b i l i t y to a wide range of a n t i b i o t i c s . T h e r e f o r e , the f o l l o w i n g experiments were designed to s p e c i f i c a l l y measure outer membrane p e r m e a b i l i t y i n each s t r a i n . D i f f e r e n t methods were used to s e p a r a t e l y examine porin-mediated, hydrophobic and self-promoted outer membrane uptake. In a d d i t i o n , the p r o p e r t i e s of the P.  aeruginosa type Id chromosomal beta-lactamase were examined to assess the p o t e n t i a l c o n t r i b u t i o n of t h i s enzyme i n each s t r a i n . 66 1 . H y d r o p h i l i c (porin-mediated) p e r m e a b i l i t y of whole c e l l s to a ohromogenic c e p h a l o s p o r i n a n t i b i o t i c . A l l P_j_ aeruginosa s t r a i n s c o n t a i n an i n d u c i b l e , chromosomally encoded type Id beta-lactamase (Sabath and Abraham, 1964). Since the apparent beta-lactamase a c t i v i t y of whole c e l l s of aeruginosa can be i n c r e a s e d by EDTA and gentamicin, agents which d i s r u p t the outer membrane p e r m e a b i l i t y b a r r i e r (Hancock ejt aJL. , 1 981 b ; Hancock and Wong, 1984), i t was p o s t u l a t e d that t h i s enzyme was l o c a t e d i n the per i p l a s m and that the outer membrane represented a s i g n i f i c a n t b a r r i e r to a n t i b i o t i c uptake i n w i l d type P.  aeruginosa. In view of t h i s , both the r e l a t i o n s h i p between a n t i b i o t i c s u s c e p t i b i l i t y and outer membrane b a r r i e r p r o p e r t i e s and the i n d u c i b i l i t y of the chromosomal beta-lactamase were measured. The t o t a l amount of chromosomal beta-lactamase enzyme produced by w i l d type, mutant and r e v e r t a n t s t r a i n s was measured by n i t r o c e f i n assay of French-pressed c e l l s uspensions. The a c t i v i t y of a l l s t r a i n s d e r i v e d from WT1 was between 6.2 and 9.6 nmol n i t r o c e f i n hydrolysed/min-mg. c e l l s (Table V I ) , t h e r e f o r e the non-induced p r o d u c t i o n of beta-lactamase appeared to be approximately the same and so none of the AbsA, B or C phenotypes could be produced by a mutation i n the s t r u c t u r a l gene f o r beta-lactamase. The i n d u c i b i l i t y of chromosomal beta-lactamase w i l l be d i s c u s s e d i n another s e c t i o n of t h i s chapter. Permeation of n i t r o c e f i n was measured as a f u n c t i o n a l 67 demonstration of the d i f f e r e n c e i n outer membrane p e r m e a b i l i t y between s t r a i n s WT1 and M1. Because of the p e r i p l a s m i c l o c a t i o n of aeruginosa beta-lactamase, the outer membrane t h e r e f o r e forms the major b a r r i e r between beta-lactam s u b s t r a t e s and the h y d r o l y t i c enzyme. The technique of Zimmermann and Ross e l e t (1977) was used to measure outer membrane p e r m e a b i l i t y . The b a s i s of t h i s technique i s that the beta-lactam h y d r o l y s i s r a t e i n the periplasm of whole c e l l s w i l l be l i m i t e d by the d i f f u s i o n r a t e of beta-lactams across the outer membrane b a r r i e r , assuming that a s u f f i c i e n t l y high l e v e l of beta-lactamase a c t i v i t y e x i s t s i n the p e r i p l a s m . The e q u i l i b r i u m h y d r o l y s i s r a t e by whole c e l l s w i l l t h e r e f o r e be p r o p o r t i o n a l to outer membrane p e r m e a b i l i t y f o r each s t r a i n . The chromogenic c e p h a l o s p o r i n n i t r o c e f i n was used to provide a h y d r o l y s i s product which could be s p e c t r o p h o t o m e t r i c a l l y measured. The p a r t i t i o n r a t i o f o r n i t r o c e f i n i n a p h a s e - p a r t i t i o n i n g system of 1-octanol/water was equal to 1.0 (legend, Table V I), t h e r e f o r e t h i s compound was s l i g h t l y hydrophobic i n comparison with other beta-lactam molecules. The a c t i v i t y of chromosomal beta-lactamase i n broken and whole c e l l s was measured and the r e s u l t s were used to c a l c u l a t e a value f o r the p e r m e a b i l i t y parameter "C" of Zimmermann and Ross e l e t (1977) and f o r c r y p t i c i t y (Table V I ) . The p e r m e a b i l i t y parameter was c a l c u l a t e d from the equation v = 0 ( S q _ s e ) , where v re p r e s e n t s the h y d r o l y s i s r a t e of n i t r o c e f i n i n whole c e l l s , S Q ±S the c o n c e n t r a t i o n of 68 n i t r o c e f i n added and S G i s t h e c a l c u l a t e d p e r i p l a s m i c n i t r o c e f i n c o n c e n t r a t i o n . Assuming that the p e r i p l a s m i c h y d r o l y s i s r a t e i s s u f f i c i e n t l y high at e q u i l i b r i u m , S G should t h e r e f o r e be n e g l i g i b l e compared to S q ( a s determined e x p e r i m e n t a l l y ) , and so C = v / S Q ) S U c h that the h y d r o l y s i s r a t e i s d i r e c t l y p r o p o r t i o n a l to outer membrane p e r m e a b i l i t y at a s p e c i f i e d s u b s t r a t e c o n c e n t r a t i o n . The p e r m e a b i l i t y parameter C ranged between 1.1 to 1.6 X 10~3 mi / min-mg c e l l s f o r w i l d type s t r a i n s WT3, WT1 and the f u l l r e v e r t a n t WT1-1. Mutant M1, on the other hand, had an approximately 4 . 5 - f o l d higher p e r m e a b i l i t y value than i t s w i l d type parent WT1. The p a r t i a l r e v e r t a n t P1-6 was a l s o approximately two-fold more permeable than w i l d type however P1-4 could not be d i s t i n g u i s h e d from WT1 by t h i s c r i t e r i o n . C r y p t i c i t y a l s o appeared to vary with the a n t i b i o t i c r e s i s t a n c e phenotype demonstrated by the MIC determinations i n Table I I (part I of t h i s c h a p t e r ) . C r y p t i c i t y was d e f i n e d as the beta-lactamase a c t i v i t y of broken c e l l s over that of whole c e l l s and, although the p e r m e a b i l i t y parameter C provided an e x c e l l e n t c a l c u l a t i o n of outer membrane p e r m e a b i l i t y , c r y p t i c i t y a l s o takes i n t o account the t o t a l beta-lactamase a c t i v i t y i n c e l l s . C r y p t i c i t y t h e r e f o r e can provide a c o n t r o l f o r p o s s e s s i o n of d i f f e r i n g amounts of beta-lactamase between s t r a i n s . T h i s v a r i a b l e i s u s u a l l y only important at low enzyme l e v e l s , where S G m a y approach the l e v e l of S 0 , or at high enzyme l e v e l s where beta-lactamase that may leak from c e l l s c o n t r i b u t e s s u b s t a n t i a l l y to the h y d r o l y s i s r a t e . 69 Table VI. Chromosomal beta-lactamase a c t i v i t y and n i t r o c e f i n permeation through the outer membrane of w i l d type and a n t i b i o t i c h y p e r s u s c e p t i b l e P_;_ aeruginosa s t r a i n s . S t r a i n Beta-lactamase a c t i v i t y C r y p t i c i t y "C" (ml/min-mg c e l l s Broken c e l l s Whole c e l l s X 10~3) WT3 11.1 1 .3 9.3 1 .5 WT1 9.6 1 .4 6.9 1 .6 WT1-1 9.4 0.92 10.2 1 .1 P1 -4 7.0 1 .5 4.7 1 .8 P1-6 7.8 2 .1 3.7 2.5 M1 6.2 4.7 1.3 5.6 The beta-lactamase a c t i v i t y i s the mean of three to f i v e separate t r i a l s and i s expressed as nmol of n i t r o c e f i n hydrolysed/mg of c e l l dry weight per minute. The c r y p t i c i t y r e p r e s e n t s the r a t i o of beta-lactamase a c t i v i t y of broken c e l l s / a c t i v i t y of whole c e l l s . The p e r m e a b i l i t y parameter "C" was c a l c u l a t e d from F i c k ' s Law of d i f f u s i o n and i s equal to the beta-lactamase a c t i v i t y i n whole c e l l s / n i t r o c e f i n c o n c e n t r a t i o n . The beta-lactamase a c t i v i t y of whole c e l l s of s t r a i n s M1 , P1-4 and P1-6 were shown to be s t a t i s t i c a l l y d i f f e r e n t from that of s t r a i n WT1 by Student t - t e s t (p < 0.02, < 0.01 and < 0.001, r e s p e c t i v e l y ) . Standard d e v i a t i o n s f o r the same s t r a i n s were 40$, 20$ and 7$ of the average whole c e l l beta-lactamase a c t i v i t y , and f o r s t r a i n WT1 was 18$. The 1-octanol/water p a r t i t i o n c o e f f i c i e n t of n i t r o c e f i n ( r a t i o of n i t r o c e f i n p a r t i t i o n i n g i n t o o c t a n o l to that p a r t i t i o n i n g i n t o water) was equal to 1.0. 70 The mutant M1 was not very c r y p t i c ( r a t i o of broken c e l l a c t i v i t y / whole c e l l a c t i v i t y was only s l i g h t l y > 1.0) i n comparison with the w i l d type s t r a i n s WT3 and WT1 (Table V I ) . In f a c t , the r a t i o of c r y p t i c i t y between the two s t r a i n s showed that s t r a i n M1 was f i v e - f o l d l e s s c r y p t i c toward n i t r o c e f i n . T h i s agreed w e l l with the c a l c u l a t e d value f o r "C" i n t h i s mutant. The c r y p t i c i t y ( b a r r i e r ) values f o r both p a r t i a l r e v e r t a n t s t e s t e d were higher than those of M1 but lower than WT1 and the f u l l r e v e r t a n t s t r a i n WT1-1, although the p e r m e a b i l i t y of P1-4 was again c l o s e r to w i l d type v a l u e s , as c a l c u l a t e d f o r "C". The n i t r o c e f i n permeation data were c o n s i s t e n t with the hypothesis that the outer membrane was more permeable i n the mutant and p a r t i a l r e v e r t a n t s than i n WT1 and WT1-1, and was a l s o c o n s i s t e n t with t h e i r a n t i b i o t i c r e s i s t a n c e p r o f i l e s (Part I, t h i s c h a p t e r ) . The f u l l and p a r t i a l r e v e r t a n t s had co r r e s p o n d i n g l y f u l l and p a r t i a l r e s t o r a t i o n of the b a r r i e r f u n c t i o n of the outer membrane. Outer membrane p e r m e a b i l i t y was a l s o measured using RP1-encoded, TEM-2 beta-lactamase (Sykes and Matthew, 1976) i n an attempt to i n c r e a s e enzyme a c t i v i t y and, i n t u r n , the accuracy of the assay. T h i s assay i n v o l v e d h y d r o l y s i s , by another p e r i p l a s m i c a l l y l o c a t e d beta-lactamase, of n i t r o c e f i n added to the o u t s i d e of whole c e l l s . C e l l s of the transconjugant s t r a i n s , P2-6 and P2-8, as w e l l as t h e i r w i l d type parent, WT2, were a l s o i n c l u d e d i n t h i s study. The values f o r C were approximately f i v e - f o l d higher i n 71 Table VII. Outer membrane p e r m e a b i l i t y of whole c e l l s to n i t r o c e f i n u s ing TEM-2 beta-lactamase. S t r a i n Beta-lactamase a c t i v i t y C r y p t i c i t y "C" Ratio (ml/mg c e l l s - m i n Broken c e l l s Whole c e l l s x 10"3) WT1 371 1.60 2 3 3 9.44 P1-4 257 1.43 180 8.56 M1 210 4.13 50 . 9 24.4 WT2 387 1 . 9 3 201 11.4 P2-6 408 1.60 256 9.44 P2-8 447 0.44 1015 2.60 The beta-lactamase a c t i v i t y i s expressed as nmol of n i t r o c e f i n hydrolysed/mg c e l l dry weight per minute. The c r y p t i c i t y r a t i o i s d e f i n e d as the r a t i o of h y d r o l y s i s of n i t r o c e f i n i n broken c e l l s over that i n whole c e l l s . The p e r m e a b i l i t y parameter " C was c a l c u l a t e d as d e s c r i b e d i n Table VI. Assays were performed on c e l l s c o n t a i n i n g the plasmid RP1, which expresses a high l e v e l of beta-lactamase. R e s u l t s r e p r e s e n t the means of four to f i v e t r i a l s . Standard d e v i a t i o n s f o r whole c e l l beta-lactamase a c t i v i t y of s t r a i n s WT1 and M1 were 65$ and 3%, r e s p e c t i v e l y , of the t o t a l . Whole c e l l a c t i v i t y r e s u l t s from s t r a i n M1 were shown to be s t a t i s t i c a l l y d i f f e r e n t from those of s t r a i n WT1 by Student t -t e s t (p < 0.02 ) . 72 t h i s assay than i n the previous set of data (Table V I ) . Two p o s s i b l e reasons f o r t h i s d i s c r e p a n c y are i ) that the higher h y d r o l y s i s r a t e s which could be generated by the TEM-2 beta-lactamase enhanced accuracy of the measurements, or i i ) that the l a r g e amount of beta-lactamase produced by the RP1 p l a s m i d - c a r r y i n g c e l l s caused some leakage of enzyme which remained c e l l - a s s o c i a t e d upon c e n t r i f u g a t i o n . In s p i t e of the higher v a l u e s , the same trends with r e s p e c t to p e r m e a b i l i t y of d i f f e r e n t s t r a i n s to n i t r o c e f i n were maintained. When the p e r m e a b i l i t y parameter C (Table VII) was c a l c u l a t e d from TEM-2 beta-lactamase-derived data, mutant M1 was found to be s i g n i f i c a n t l y (approximately 3 - f o l d ) more permeable than WT1 (p < 0.02 by Student t t e s t ) . S t r a i n s P1-H, WT2 and P2-6 d i s p l a y e d s i m i l a r values f o r C as WT1. S t r a i n P2-8 appeared to be l e s s permeable to n i t r o c e f i n than w i l d type, but the reason f o r t h i s r e s u l t was not pursued. RP1 d e r i v a t i v e s of s t r a i n s P1-6 and P2-6,8 were c o n s t r u c t e d at a l a t e r date than the s t r a i n s i n Table VII and e x h i b i t e d leakage of beta-lactamase, which could not be adequately c o n t r o l l e d f o r by s u b t r a c t i o n of supernatant h y d r o l y s i s , t h e r e f o r e these s t r a i n s were not i n c l u d e d . The c r y p t i c i t y r e s u l t s a l s o showed that w i l d type c e l l s had very low p e r m e a b i l i t y to the s u b s t r a t e n i t r o c e f i n . The c r y p t i c i t y values i n Table VII were much higher f o r RP1-c a r r y i n g s t r a i n s ( c f . Table VI) due to the much higher amounts of enzyme which could be detected i n broken c e l l s . The mutant M1 had approximately f i v e - f o l d higher p e r m e a b i l i t y than w i l d 73 type WT1 as measured by t h i s c a l c u l a t i o n , which agreed w e l l with r e s u l t s using chromosomal beta-lactamase ( f i v e - f o l d - see Table VI) but was somewhat higher than the value c a l c u l a t e d f o r "C" ( t h r e e - f o l d ) . When the data f o r a l l r e v e r t a n t and transconjugant s t r a i n s were analysed, the mutant M1 was found to be s i g n i f i c a n t l y more permeable than w i l d type c e l l s (p < 0.01 by Student t t e s t ) . Using the RP1 beta-lactamase, the p a r t i a l r e v e r t a n t and transconjugants could not be shown to be s i g n i f i c a n t l y more permeable than WT1 (p > 0.05). Although the change i n outer membrane p e r m e a b i l i t y seen i n the mutant M1 was only f i v e - f o l d g r e a t e r than that i n WT1 , a p o t e n t i a l l y f i v e - f o l d higher p e r i p l a s m i c c o n c e n t r a t i o n c a l c u l a t e d using the Michaelis-Menten equation from data i n Table VII - not shown) of B-lactam i n mutant M1 may rep r e s e n t an insurmountable c h a l l e n g e f o r the B-lactamase, a l l o w i n g f a r g r e a t e r access to the t a r g e t p r o t e i n s . A l s o , f o r E_;_ c o l i , which i s f a r more s u s c e p t i b l e than P_;_ aeruginosa to most a n t i b i o t i c s , outer membrane p e r m e a b i l i t y measured by t h i s method (Nicas and Hancock, 1983) was only t w e l v e - f o l d g r e a t e r than that of w i l d type Pseudomonas aeruginosa. 2. I n d u c t i o n p r o p e r t i e s of chromosomal beta-lactamase from  w i l d type and mutant s t r a i n s The chromosomal type Id beta-lactamase of P_;_ aeruginosa i s i n d u c i b l e by a v a r i e t y of beta-lactam a n t i b i o t i c s (Sykes and Matthew, 1976). This property of the beta-lactamase gene, i n concert with the b a r r i e r e f f e c t of the outer membrane, may 74 Table V I I I . I n d u c t i o n of chromosomal beta-lactamase i n P. aeruginosa w i l d type and a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s . S t r a i n C o n c e n t r a t i o n of Inducer (ug/ml) In d u c t i o n Ratio WT3 200 21 WT1 200 13 WT1-1 200 18 P1-6 2.0 2.4 M1 0.2 0.7 C e l l s were grown f o r two hours i n the presence of the given c o n c e n t r a t i o n of b e n z y l p e n i c i l l i n as i n d u c e r . N i t r o c e f i n assays were performed as p r e v i o u s l y d e s c r i b e d on French pressed c e l l s . The above i n d u c t i o n r a t i o s (beta-lactamase a c t i v i t y i n induced broken c e l l s over that i n uninduced broken c e l l s ) were obtained. 75 play an important r o l e i n r e s i s t a n c e to beta-lactams. I t was shown that the beta-lactamase l e v e l s of the w i l d type s t r a i n WT1 and f u l l r e v e r t a n t WT1-1 were i n c r e a s e d 13- to 18-fold by growth of the c e l l s f o r two hours i n the presence of b e n z y l p e n i c i l l i n (200 ug/ml) as an inducer (Table V I I I ) . S i m i l a r r e s u l t s were obtained f o r the w i l d type s t r a i n WT3 , although i n t h i s case the f u l l y induced l e v e l of beta-lactamase was between 50 to 100$ g r e a t e r than that f o r s t r a i n WT1. S i m i l a r l e v e l s of inducer could not be used f o r mutant M1 and a r e p r e s e n t a t i v e p a r t i a l r e v e r t a n t P1-6 s i n c e these s t r a i n s had MICs f o r b e n z y l p e n i c i l l i n of 1.0 and 10 ug/ml, r e s p e c t i v e l y (Table I I , part I ) . The a d d i t i o n of b e n z y l p e n i c i l l i n to a c o n c e n t r a t i o n o n e - f i f t h of the MIC, i n each case, caused a 2 . 4 - f o l d i n d u c t i o n f o r P1-6 but was i n s u f f i c i e n t to induce mutant M1. An attempt was then made to induce the beta-lactamase of mutant M1 using a non-beta-lactam in d u c e r . D a l h o f f and Cullmann (1984) had r e p o r t e d i n d u c t i o n of beta-lactamase i n P_;_ aeruginosa by tryptophan as w e l l as p h e n y l a l a n i n e , t y r o s i n e and h i s t i d i n e . Attempts to reproduce these experiments using the w i l d type s t r a i n WT1 f a i l e d t h e r e f o r e t h i s method could not be used. P o s s i b l y i n d u c t i o n by amino a c i d s was a s t r a i n - s p e c i f i c phenomenon, s i n c e these workers used a c l i n i c a l s t r a i n of undetermined g e n e t i c background. The s i g n i f i c a n c e of the l a c k of i n d u c t i o n of beta-lactamase i n the mutant w i l l be d i s c u s s e d . 76 3. Outer membrane p e r m e a b i l i t y to hydrophobic compounds. P e r m e a b i l i t y through the LPS-phospholipid b i l a y e r (hydrophobic outer membrane p e r m e a b i l i t y ) i s not normally a f a c t o r i n w i l d type c e l l s which possess smooth LPS. However, s i n c e the a n t i b i o t i c h y p e r s u s c e p t i b l e mutant M1 was shown to be q u i t e s u s c e p t i b l e to hydrophobic agents, i n part I of t h i s chapter, t h i s uptake pathway may play a more prominent r o l e . The f l u o r e s c e n t probe, NPN, was used as an i n d i c a t o r of p e r m e a b i l i t y through the hydrophobic p o r t i o n of the outer membrane, because i t f l u o r e s c e s weakly i n an aqueous environment but s t r o n g l y i n a hydrophobic environment, such as i n the c e l l envelope. NPN was added to whole, a z i d e - t r e a t e d c e l l s i n b u f f e r , then f l u o r e s c e n c e was measured at a s p e c i f i e d time p o i n t a f t e r a d d i t i o n . Azide pre-treatment prevented p o s s i b l e e nergised s e c r e t i o n of NPN, which had been found to i n t e r f e r e with f l u o r e s c e n c e measurements (Loh e_t al.. , 1984). An attempt was made to e s t a b l i s h a l i n k between hydrophobic p e r m e a b i l i t y and the a n t i b i o t i c r e s i s t a n c e (MIC) p a t t e r n s of the a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s . NPN was used as a t o o l f o r a s s e s s i n g both hydrophobic p e r m e a b i l i t y , due to i t s tendency to f l u o r e s c e only i n a hydrophobic environment such as a membrane, and to i d e n t i f y any a l t e r a t i o n s i n the s e l f -promoted uptake pathway. The l a t t e r was observed by n o t i n g changes i n NPN f l u o r e s c e n c e caused by a d d i t i o n of gentamicin ( d e s c r i b e d i n s e c t i o n 5 ). As can be seen i n Table IX, NPN re a c t e d only weakly with the w i l d type c e l l s , but demonstrated enhanced f l u o r e s c e n c e 77 Table IX. Fluorescence Increase due to uptake of NPN i n whole c e l l s . S t r a i n T o t a l f l u o r e s c e n c e i n c r e a s e Ratio of mutant/wild type ( a r b i t r a r y u n i t s ) WT1 1.43 1.0 P1-4 3.45 2.4 P1-6 4.88 3.4 M1 8.47 5.9 WT2 1.03 1.0 P2-6 4.43 4.3 P2-8 0.87 0.84 P2-6.8 2.80 2.7 T o t a l f l u o r e s c e n c e i n c r e a s e was measured two minutes a f t e r a d d i t i o n of NPN. R a t i o s of mutant/wild type f o r s t r a i n s P1-4, P1-6 and M1 were i n comparison with s t r a i n WT1 and f o r s t r a i n s P2-6, P2-8 and P2-6,8 were i n comparison with s t r a i n WT2. NPN, 1-N-phenylnaphthylamine. Res u l t s r e p r e s e n t the means of three to four t r i a l s . 78 Fig. 2. Gentamicin enhancement of NPN uptake Figure 2. Gentamicin enhancement of NPN uptake by whole c e l l s . C e l l s were suspended to a f i n a l o p t i c a l d e n s i t y at 600 nm of 0.5. NPN was added to g i v e a f i n a l c o n c e n t r a t i o n of 10 >iM then fluorescence emission was measured at 420 nm. Gentamicin was added to give a f i n a l c o n c e n t r a t i o n of 4 jug/ml. , WT1; , P1-4; M1 . 79 emission i n t e n s i t y when added to mutant M1 c e l l s (see a l s o F i g . 2 ) . A c o r r e l a t i o n between a n t i b i o t i c r e s i s t a n c e phenotype and i n t e n s i t y of NPN f l u o r e s c e n c e was a l s o observed f o r the r e v e r t a n t s of M1, i n that the p a r t i a l r e v e r t a n t s P1-4 and P1-6 e l i c i t e d 2.4- to 3 .4-fold higher NPN f l u o r e s c e n c e than s t r a i n WT1. F i g . 2 shows the " p a r t i a l " f l u o r e s c e n c e i n c r e a s e due to uptake of NPN i n t o s t r a i n P1-4. The transconjugants P2-6 and P2-6,8 a l s o caused i n c r e a s e d NPN f l u o r e s c e n c e (2 . 7 - to 4 .3-fold that of t h e i r r e s p e c t i v e w i l d type parent, WT2), whereas s t r a i n P2-8 e l i c i t e d m a r g i n a l l y l e s s f l u o r e s c e n c e than WT2. These r e s u l t s a l s o agreed w e l l with MIC r e s u l t s , where s t r a i n s P2-6 and P2-6,8 were more s u s c e p t i b l e to the r e l a t i v e l y hydrophobic a n t i b i o t i c , trimethoprim. Due to the v a r i a b i l i t y of r e s u l t s from experiments done on d i f f e r e n t days (even though comparitive r e s u l t s between s t r a i n s were c o n s i s t e n t on a given day), only NPN uptake by s t r a i n s P1-6, P2-6 and M1 was shown to be s i g n i f i c a n t l y d i f f e r e n t (by Student t t e s t ) from that of t h e i r r e s p e c t i v e w i l d type s t r a i n s , although s t r a i n s P1-4 and P2-6,8 a l s o appeared to take up more NPN than w i l d type. In c o n c l u s i o n , s t r a i n s M1 , P1-4, P1-6, P2-6 and P2-6,8 appeared to present a lower b a r r i e r to hydrophobic substances i n the environment, as judged by uptake of the hydrophobic probe and the c o r r e l a t e d h y p e r s u s c e p t i b i l i t y to hydrophobic substances. This hypothesis w i l l be d e a l t with f u r t h e r i n other s e c t i o n s of t h i s chapter. 80 4. Surface charge p r o p e r t i e s of w i l d type and mutant c e l l s From the previous experiments, i n v o l v i n g uptake of a hydrophobic probe, i t was apparent that the c e l l s u r f a c e of mutant M1 i n t e r a c t e d d i f f e r e n t l y with the aqueous environment than d i d that of WT1. For t h i s reason, the tendency of c e l l s to p a r t i t i o n i n t o one or the other phase of an aqueous, two-phase polymer system was examined. C e l l s were grown to mid-log phase i n r i c h medium, concentrated t e n - f o l d by c e n t r i f u g a t i o n , a p p l i e d to the polymer system, c a r e f u l l y mixed and l e f t to separate. Each phase was measured f o r ^^QQ due to the presence of c e l l s . The two-phase, p o l y e t h y l e n e g l y c o l (PEG) -dextran system separates m a t e r i a l on the bases of both charge and h y d r o p h o b i c i t y (Magnusson e_t al, 1977). The PEG-rich top phase provided a r e l a t i v e l y uncharged, h y d r o p h i l i c environment whereas the d e x t r a n - r i c h phase a t t r a c t e d molecules which were r e l a t i v e l y hydrophobic and/or charged. These c h a r a c t e r i s t i c s made i n t e r p r e t a t i o n of r e s u l t s d i f f i c u l t because s e p a r a t i o n would be achieved e i t h e r with r e s p e c t to charge or h y d r o p h o b i c i t y , depending which f o r c e had more i n f l u e n c e on a p a r t i c u l a r molecule. For i n s t a n c e , s e p a r a t i o n i n t o an uncharged phase might be caused by r e p u l s i o n of l i k e charges i n the dextran phase or by a l a c k of charged m o i e t i e s on the c e l l s u r f a c e . N e v e r t h e l e s s , t h i s method was shown to be u s e f u l f o r i d e n t i f y i n g changes i n the c e l l s u r f a c e of the mutant s t r a i n . Mutant s t r a i n M1 (Table X) separated p r e f e r e n t i a l l y i n t o 81 Table X. Phase p a r t i t i o n i n g of w i l d type and mutant c e l l s of P . aeruginosa. S t r a i n Ratio of A 600 i n top phase (PEG) btm phase (Dextran) WT1 0.175 M1 2.80 Phase p a r t i t i o n of whole c e l l s was performed i n an aqueous p o l y e t h y l e n e g l y c o l / d e x t r a n system as d e s c r i b e d i n the t e x t . R a t i o s were presented as the means of three separate t r i a l s . P a r t i t i o n of mutant s t r a i n M1 c e l l s i n t o the upper phase was found to be s i g n i f i c a n t l y d i f f e r e n t from w i l d type by Student t t e s t (p < 0.05). 82 the uncharged, PEG-rich top phase, whereas the w i l d type s t r a i n WT1 p a r t i t i o n e d i n t o the somewhat n e g a t i v e l y charged d e x t r a n - r i c h bottom phase. These r e s u l t s i n d i c a t e d that the mutant c e l l s u r f a c e was a l t e r e d i n i t s p r e s e n t a t i o n to the environment, although the exact nature of the a l t e r a t i o n could not be determined by t h i s method. 5. Self-promoted outer membrane p e r m e a b i l i t y j_ Aminoglycoside enhancement of outer membrane p e r m e a b i l i t y  to a hydrophobic compound Having e s t a b l i s h e d that the c e l l s u r f a c e of mutant s t r a i n M1 was a l t e r e d , and that t h i s a l t e r a t i o n may be r e s p o n s i b l e f o r i n c r e a s e d p e r m e a b i l i t y to two d i f f e r e n t agents ( n i t r o c e f i n and a hydrophobic probe), a t h i r d uptake mechanism ( s e l f -promoted uptake through c e l l s u r f a c e LPS molecules) was then examined. To determine whether the self-promoted uptake pathway ( p o s t u l a t e d by Nicas and Hancock, 1980, to e x p l a i n the p e r m e a b i l i s i n g e f f e c t of aminoglycosides) was a l t e r e d i n the a n t i b i o t i c h y p e r s u s c e p t i b l e mutants, three d i f f e r e n t methods were used. The f i r s t i n v o l v e d aminoglycoside enhancement of outer membrane p e r m e a b i l i t y to hydrophobic compounds. The hydrophobic f l u o r e s c e n t compound NPN ( p r e v i o u s l y d e s c r i b e d , p.77) was added to c e l l s , then gentamicin was added to give a f i n a l c o n c e n t r a t i o n of 4 ug/ml i n the cuvette and the e f f e c t on f l u o r e s c e n c e was measured ( F i g . 2). I t had been found, by Loh e_t a_l. (1984), that gentamicin s t i m u l a t e d NPN uptake by c e l l s . The f i n a l i n t e n s i t y of f l u o r e s c e n c e emission 83 Table XI. Rate of f l u o r e s c e n c e i n c r e a s e due to uptake of NPN by whole c e l l s a f t e r a d d i t i o n of 4 ug/ml gentamicin. S t r a i n Rate of f l u o r e s c e n c e i n c r e a s e Ratio:mutant/wild type (arb. u n i t s / m i n . - 10^ c e l l s ) WT1 0 .65 1 .0 P1-4 0.36 0 .55 P1-6 1 .35 2.1 M1 a a WT2 0.58 1 .0 P2-6 1 .97 3.4 P2-8 1.10 1 .9 P2-6 ,8 2.23 3.8 R e s u l t s r e p r e s e n t the means of three separate t r i a l s , a: the e f f e c t of gentamicin p e r m e a b i l i s a t i o n on s t r a i n M1 could not be examined because NPN alone caused near-maximal f l u o r e s c e n c e i n t h i s s t r a i n (see F i g . 2 ) . 84 s t i m u l a t e d i n t h i s manner was s i m i l a r at a l l c o n c e n t r a t i o n s of gentamicin, however i t was the r a t e of f l u o r e s c e n c e i n c r e a s e which was most r e l i a b l y c o r r e l a t e d with MIC values (Loh e_t a l . , 1984), t h e r e f o r e r e s u l t s f o r r a t e s have been r e p o r t e d i n Table XI. The a d d i t i o n of gentamicin (4 ug/ml) to c e l l s of the w i l d type s t r a i n s WT1 and WT2 produced only a low r a t e of f l u o r e s c e n c e i n c r e a s e due to NPN uptake by c e l l s (0.65 a r b i t r a r y units/min-108 c e l l s ) . Gentamicin thus appeared to exert only a modest p e r m e a b i l i s i n g e f f e c t , at t h i s c o n c e n t r a t i o n , on w i l d type c e l l s . Gentamicin appeared to have the g r e a t e s t e f f e c t on the pro+-transconjugant, P2-6 (3.4-fold g r e a t e r than WT2) and the double t r a n s d u c t a n t , P2-6,8 (3.8-fold g r e a t e r than WT2), with i n t e r m e d i a t e i n f l u e n c e on the met+-transconjugant P2-8 (1 . 9-fold) and on one of the r e v e r t a n t s , P1-6 ( 2 . 1 - f o l d , see Table X I ) . Another r e v e r t a n t , s t r a i n P1-4, d i s p l a y e d l e s s p e r m e a b i l i s a t i o n by gentamicin than s t r a i n WT1. Only s t r a i n P2-6 was shown to be s i g n i f i c a n t l y d i f f e r e n t from w i l d type by Student t t e s t . F i g . 2 shows example curves of NPN f l u o r e s c e n c e measurements before and a f t e r a d d i t i o n of gentamicin. The e f f e c t of gentamicin on the mutant M1 could not be judged because NPN alone caused maximal f l u o r e s c e n c e i n t e n s i t y i n t h i s s t r a i n , due to i t s s u b s t a n t i a l p e r m e a b i l i t y to hydrophobic compounds, and thus gentamicin had no f u r t h e r e f f e c t ( F i g . 2 ) . These r e s u l t s i n d i c a t e d that there might be a d e f e c t i n the self-promoted uptake pathway, f o r the AbsA phenotype 85 s t r a i n s , which was e i t h e r r e l a t e d t o , or separate from, the more obvious d e f e c t found i n r e s i s t a n c e to hydrophobic permeation ( s e c t i o n 4 ) . 6. Self-promoted outer membrane p e r m e a b i l i t y : EDTA, aminoglycoside and polymyxin B enhancement of  1ysozyme-mediated c e l l l y s i s . The e f f e c t of gentamicin and two other known " p e r m e a b i l i s e r s " (Warren e_t aJL. , 1957; Hancock and Wong, 1984), e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA) and polymyxin B, on p e r m e a b i l i t y of the outer membrane to lysozyme was a l s o examined. A p r o t e i n such as lysozyme normally would not have access to i t s t a r g e t , p e p t i d o g l y c a n i n the p e r i p l a s m , due to the presence of the outer membrane. Experiments by Hancock e_t a l . , (1981b) and Hancock and Wong, (1984) showed that many compounds, p a r t i c u l a r l y p o l y c a t i o n i c compounds or d i v a l e n t c a t i o n c h e l a t o r s such as EDTA, could a i d permeation of n i t r o c e f i n or lysozyme across the outer membrane b a r r i e r by d i s r u p t i n g i t s i n t e g r i t y . T h e r e f o r e , t h i s method was a l s o used to assess a l t e r a t i o n s i n self-promoted uptake i n the a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s . A d d i t i o n of p e r m e a b i l i s i n g compounds to whole c e l l s , i n the presence of lysozyme, caused c e l l l y s i s (see F i g u r e 3 ) . The amount of l y s i s observed v a r i e d with the c o n c e n t r a t i o n of p e r m e a b i l i s e r from a lower t h r e s h o l d up to a p o i n t of s a t u r a t i o n . Both EDTA ( F i g . 3A) and polymyxin B ( F i g . 3C) caused lysozyme-mediated l y s i s of between 40 and 85? of a l l 86 A. EDTA B. Gentamicin C. Polymyxin B fPX] (>jg/ml) F i g u r e 3. P e r m e a b i l i s a t i o n of whole c e l l s to lysozyme by EDTA, gentamicin or polymyxin B. A l i q u o t s of p e r m e a b i l i s e r were added to lysozyme p r e - t r e a t e d c e l l suspensions. Lysozyme-mediated l y s i s was followed as decrease i n o p t i c a l d e n s i t y at 600 nm. o, WT1; •, M1. 87 Table X I I . P e r m e a b i l i s a t i o n of whole c e l l s to lysozyme by EDTA, gentamicin or polymyxin B. S t r a i n Maximal L y s i s (%) I ( UM) EDTA GM PX EDTA GM PX WT1 70.0 53.0 63.0 32.0 4.75 1 .45 M1 43.0 15.0 61 .0 77.0 8 . 8 6 1 .70 WT2 84.0 42 .0 8 3 . 0 15 . 9 14 . 3 1 .64 P2-6 74 .5 44.0 63 .5 10 . 9 11.0 1 .45 P2-8 85 .0 20.0 57 .0 15.0 35 .4 1 .82 P2-6 ,8 86 .0 28.0 55.0 9 . 9 22 .5 2.40 The e f f e c t of v a r i o u s p e r m e a b i l i s e r s on l y s i s of whole c e l l s by lysozyme was followed s p e c t r o p h o t o m e t r i c a l l y as i n F i g . 3 - R e s u l t s r e p r e s e n t the means of three t r i a l s . EDTA, e t h y l e n e d i a m i n e t e t r a a c e t a t e ; GM, gentamicin s u l p h a t e ; PX, polymyxin B sulphate; I c o n c e n t r a t i o n of p e r m e a b i l i s e r causing 50% maximal l y s i s . 88 c e l l s as measured by absorbance at 600 nm, whereas gentamicin ( F i g . 3B) was l e s s e f f e c t i v e , causing l y s i s of between 15 and 50? of c e l l s (Table X I I ) . K i n e t i c parameters could not be examined i n t h i s assay s i n c e the l y s i s d i d not f o l l o w f i r s t -order k i n e t i c s . T h i s i s compatible with the theory behind self-promoted uptake, where i n t e r a c t i o n of the p e r m e a b i l i s e r with the l i p o p o l y s a c c h a r i d e i s c o o p e r a t i v e . There was l i t t l e d i f f e r e n c e between any of the mutant s t r a i n s and w i l d type c e l l s f o r polymyxin B, e i t h e r i n the maximum amount of l y s i s a t t a i n e d or i n the observed c o n c e n t r a t i o n s at which 50? l y s i s occurred ( I ^ Q ) . Although data from the p a r t i c u l a r experiment i n F i g . 3C showed that polymyxin B promoted more l y s i s i n mutant M1 than i n s t r a i n WT1 , t h i s trend d i d not hold true over three separate t r i a l s (Table X I I ) . The r e s u l t s f o r polymyxin permeation c o r r e l a t e d w e l l with r e s u l t s obtained from minimal i n h i b i t o r y c o n c e n t r a t i o n experiments f o r aeruginosa grown on polymyxin-containing medium, where there was only a f o u r - f o l d d i f f e r e n c e i n MIC between s t r a i n s WT1 and M1 (Table I I ) . The amount of lysozyme l y s i s promoted by gentamicin and EDTA p e r m e a b i l i s a t i o n was lower i n the mutant s t r a i n M1 than i n the w i l d type s t r a i n WT1, and the c o n c e n t r a t i o n s of p e r m e a b i l i s e r necessary f o r 50? maximal l y s i s were two-fold higher ( F i g . 3, Table X I I ) . With r e s p e c t to gentamicin only, there was a l s o a marked d i f f e r e n c e between s t r a i n P2-6, which d i s p l a y e d percent l y s i s and I 5 Q values c l o s e to those of the w i l d type s t r a i n WT2, and s t r a i n P2-8, which had i n t e r m e d i a t e 89 s u s c e p t i b i l i t y to p e r m e a b i l i s a t i o n by gentamicin. In t h i s assay, i n c o n t r a s t to gentamicin enhancement of NPN uptake, s t r a i n P2-6 showed wi l d type p e r m e a b i l i s a t i o n by the s e l f -promoted pathway. I t i s p o s s i b l e that the NPN assay of s e l f -promoted uptake was l e s s accurate f o r s t r a i n P2-6 due to the al r e a d y high background NPN uptake by t h i s s t r a i n (Table I X ) . Despite t h i s apparent anomaly, the observed d i f f e r e n c e s between a n t i b i o t i c h y p e r s u s c e p t i b l e mutant M1 and the w i l d type s t r a i n s i n d i c a t e d that some d e f e c t i n the i n t e r a c t i o n of gentamicin and EDTA at the c e l l s u r f a c e d i d indeed e x i s t . Given the g r e a t e r s u s c e p t i b i l i t y of the a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s , one might have expected g r e a t e r l y s i s with l e s s p e r m e a b i l i s e r . However, the a p p a r e n t l y c o n t r a d i c t o r y r e s u l t s obtained may i n f a c t r e f l e c t a reduced b i n d i n g a f f i n i t y or a reduced number of b i n d i n g s i t e s on the l i p o p o l y s a c c h a r i d e f o r these compounds, producing a consequently l e s s s t a b l e outer membrane. These p o s s i b i l i t i e s were explored i n p a r t three of chapter one and i n the D i s c u s s i o n . 7. SUMMARY The outer membrane of the mutant s t r a i n M1 was observed to be more permeable than the w i l d type by three c r i t e r i a : i ) Beta-lactamase a c t i v i t y of whole c e l l s was used as an i n d i c a t o r of outer membrane p e r m e a b i l i t y to h y d r o p h i l i c compounds. The outer membrane of mutant M1 was approximately f i v e times more permeable than that of the parent s t r a i n WT1 90 using p e r m e a b i l i t y to n i t r o c e f i n as a c r i t e r i o n . A l s o , measurement of beta-lactamase a c t i v i t y i n whole and broken c e l l s i n d i c a t e d that the p a r t i a l r e v e r t a n t s t r a i n s had outer membrane p e r m e a b i l i t y which was in t e r m e d i a t e between that of the w i l d type s t r a i n WT1 and the mutant s t r a i n M1. P e r m e a b i l i t y to n i t r o c e f i n i n transconjugant s t r a i n s was not s i g n i f i c a n t l y d i f f e r e n t from w i l d type. i i ) Hydrophobic outer membrane p e r m e a b i l i t y was measured using a hydrophobic f l u o r e s c e n t probe, NPN. Mutant s t r a i n M1 c e l l s caused approximately s i x - f o l d more NPN f l u o r e s c e n c e than d i d the w i l d type s t r a i n , i n d i c a t i n g that the hydrophobic p o r t i o n s of the c e l l s u r f a c e ( w i t h i n which NPN demonstrated enhanced f l u o r e s c e n c e ) were more a c c e s s i b l e i n the mutant. The p a r t i a l r e v e r t a n t s and the trimethoprim s u s c e p t i b l e (AbsA) transconjugant, P2-6, a l s o d i s p l a y e d enhanced uptake of NPN, which c o r r e l a t e d with t h e i r s u s c e p t i b i l i t y to hydrophobic a n t i b i o t i c s . i i i ) Mutant M1 and the tr a n s d u c t a n t P2-8 (AbsB) were found to be l e s s s u s c e p t i b l e to the outer membrane p e r m e a b i l i s i n g e f f e c t s of gentamicin and EDTA, based on permeation to lysozyme. S t r a i n P2-6 (AbsA) d i s p l a y e d p r o p o r t i o n a t e l y g r e a t e r p e r m e a b i l i s a t i o n to NPN by gentamicin but l e s s p e r m e a b i l i s a t i o n to lysozyme by gentamicin or EDTA, however these two r e s u l t s are not n e c e s s a r i l y at odds with the s e l f -promoted uptake c h a r a c t e r i s t i c s of the AbsA phenotype. 91 PART I I I . CHARACTERISATION OF ALTERATIONS IN OUTER MEMBRANE COMPONENTS INVOLVED IN ANTIBIOTIC SUSCEPTIBILITY Having e s t a b l i s h e d that the a n t i b i o t i c s u s c e p t i b l e phenotype of mutant M1 could be c o r r e l a t e d with c o n s i s t e n t i n c r e a s e s i n outer membrane p e r m e a b i l i t y , c h a r a c t e r i s a t i o n of outer membrane components was c a r r i e d out to i d e n t i f y any a l t e r e d s t r u c t u r e s . Outer membrane p r o t e i n s were f i r s t examined, then v a r i o u s s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s of LPS were t e s t e d . S t r a i n s M1 and WT1 were compared both to each other and to transconjugant s t r a i n s to i d e n t i f y mutations r e s p o n s i b l e f o r each s t r u c u r a l a l t e r a t i o n . 1. Outer membrane p r o t e i n p a t t e r n s . The f i r s t outer membrane c o n s t i t u e n t s to be examined were major p r o t e i n s . Outer membranes were prepared from l o g a r i t h m i c phase c e l l s and examined by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . As can be seen i n F i g . 4, there appeared to be no s i g n i f i c a n t d i f f e r e n c e between the outer membrane p r o t e i n p a t t e r n s of WT1 and M1 except f o r the absence of p r o t e i n I s t a i n i n g i n the w i l d type s t r a i n s WT1, WT2 and a l l transconjugant s t r a i n s . The outer membrane p r o t e i n p a t t e r n of s t r a i n WT3 (PA01 - data not shown) a l s o resembled that of s t r a i n WT1. A l t e r a t i o n s i n temperature of s o l u b i l i s a t i o n and non-reducing c o n d i t i o n s demonstrated no d i f f e r e n c e between s t r a i n s WT1 and M1 i n these a d d i t i o n a l outer membrane p r o t e i n p r o p e r t i e s (experiments performed by A. Carey, 1980; not 92 A B C D E F G H I F i g u r e 4. SDS-PAGE o f o u t e r membranes and c e l l e n v e l o p e s from w i l d type and a n t i b i o t i c h y p e r s u s c e p t i b l e mutant s t r a i n s o f P_j_ a e r u g i n o s a . Outer membranes or c e l l e n v e l o p e s were i s o l a t e d as d e s c r i b e d i n M e t h o d s , s o l u b i l i s e d by h e a t i n g a t 88°C, and r u n on a 14$ polyacry l a m i d e g e l . Lane A , WT1; l a n e B, M1; l a n e C, WT2; l a n e D, P 2 - 6 ; l a n e E , P 2 - 8 ; l a n e F , P 2 - 6 . 8 ; l a n e G, WT1-1: l a n e H, P 1 - 4 ; l a n e I , P 1 - 6 . Lanes A - F , o u t e r membrane samples; l a n e s G - I , c e l l envelope s a m p l e s . The b a n d i n g p o s i t i o n s of p r o t e i n F and l i p o p r o t e i n I a r e shown. 93 shown). Most i m p o r t a n t l y , there was no d i f f e r e n c e i n the amount or banding c h a r a c t e r i s t i c s of the major outer membrane p o r i n p r o t e i n F between mutant and wi l d type s t r a i n s when examined i n SDS-PAGE. The apparent presence or absence of l i p o p r o t e i n I was considered to be due to a d i f f e r e n c e i n the s t a i n i n g of the p r o t e i n caused by l i p o p o l y s a c c h a r i d e (LPS) mi g r a t i n g to a c o i n c i d e n t p o s i t i o n i n the g e l . P r e l i m i n a r y experiments (R.E.W. Hancock, data not shown), i n d i c a t e d that p r o t e i n I d i d not s t a i n i n whole c e l l , c e l l envelope or outer membrane pr e p a r a t i o n s of w i l d type s t r a i n s (see a l s o F i g . 4, lane A vs. B) unless 20 mM MgCl 2 w a s present i n the s o l u b i l i s a t i o n / r e d u c t i o n mix. Because the a l t e r a t i o n i n band s t a i n i n g of p r o t e i n I could be e f f e c t e d by Mg + + (which, i n ap p r o p r i a t e c o n c e n t r a t i o n s , causes aggregation of LPS) i t was proposed that rough core LPS was i n v o l v e d i n the s t a i n i n g phenomenon ( c f . s e c t i o n 2 ) . The major outer membrane p r o t e i n s of the r e v e r t a n t s and transconjugants were a l s o compared i n SDS-polyacrylamide g e l s ( F i g . 4 ) . As noted f o r s t r a i n s WT1 and M1, there were no n o t i c e a b l e d i f f e r e n c e s between p r o t e i n s i n any of the s t r a i n s with the exce p t i o n of the l i p o p r o t e i n I. The l i p o p r o t e i n I of the f u l l r e v e r t a n t WT1-1 d i d not s t a i n with Coomassie Blue on SDS-PA g e l s , s i m i l a r to the behaviour of p r o t e i n I of the wi l d type s t r a i n WT1. In c o n t r a s t , the p a r t i a l r e v e r t a n t s t r a i n s P1-4 and P1-6 had Coomassie B l u e - s t a i n a b l e p r o t e i n I, s i m i l a r to mutant M1. The c o n c l u s i o n from these data was that 94 r e v e r s i o n of the cause of t h i s s t a i n i n g phenomenon appeared to be concomitant with the r e v e r s i o n to f u l l a n t i b i o t i c r e s i s t a n c e phenotype. For the transconjugant s t r a i n s , no d i f f e r e n c e s could be seen between the w i l d type r e c i p i e n t s t r a i n WT2 and any of the progeny s t r a i n s . I t thus appeared that n e i t h e r the met*- l i n k e d mutation of s t r a i n P2-8 nor the p_ro +-linked mutation of s t r a i n P2-6 was r e s p o n s i b l e f o r any changes i n p r o t e i n banding or i n banding p o s i t i o n of rough core LPS. 2. Densitometry scans of e l e c t r o p h o r e s e d outer membrane  samples s t a i n e d f o r p r o t e i n or carbohydrate The p r o t e i n I s t a i n i n g phenomenon was probed using two s t a i n s : e i t h e r the c a r b o h y d r a t e - s e l e c t i v e S c h i f f s t a i n , Coomassie B r i l l i a n t Blue f o r p r o t e i n , or both, as shown i n F i g . 5. Panels A and D were e q u i v a l e n t to scans of F i g u r e k, lanes A and B. Only the bottom p o r t i o n s of the g e l s (where rough core LPS would be expected to band) were scanned. In the absence of Mg++, the LPS of WT1 ( s t a i n e d by the S c h i f f procedure) appeared i n a broad band ( F i g . 5D) with lower m o b i l i t y than that of p a r t i a l l y p u r i f i e d l i p o p r o t e i n I ( F i g . 5B). Coomassie p r o t e i n s t a i n i n g , subsequent to carbohydrate s t a i n i n g f o r s t r a i n WT1,revealed a l a r g e band of pu r p l e - b l u e m a t e r i a l c o - e l e c t r o p h o r e s i n g with the LPS-carbohydrate band ( F i g . 5D). This p u r p l e - b l u e - s t a i n i n g band appeared only when Coomassie Blue s t a i n i n g was performed a f t e r carbohydrate s t a i n i n g . P o s s i b l y the a c i d treatment i n v o l v e d i n the 95 F i g u r e 5. Densitometry scans of SDS-PA g e l s of P_;_ aeruginosa outer membranes s t a i n e d f o r carbohydrate and/or p r o t e i n . Outer membrane samples ( c o n t a i n i n g 20 ug of p r o t e i n and approximately 0.23 umol of l i p o p o l y s a c c h a r i d e were s o l u b i l i s e d and run on a 14$ acrylamide g e l as d e s c r i b e d i n Methods. EDTA (to improve LPS r e s o l u t i o n ) or MgCl 2 ( t o aggregate LPS) were added to the designated samples. Gels were s t a i n e d f o r carbohydrate using the S c h i f f - p e r i o d a t e method and subsequently f o r p r o t e i n with Coomassie blue. Densitometry scans were performed on the r e g i o n between p r o t e i n H2 (20,500 d a l t o n s ) and p r o t e i n I. The banding p o s i t i o n s of l i p o p r o t e i n s H2 and I are i n d i c a t e d . Densitometry of the carbohydrate-s t a i n e d g e l s was performed at a g a i n of 9-5, whereas p r o t e i n -s t a i n e d g e l s were scanned at a gain of 5.5 and thus these are not intended to be q u a n t i t a t i v e l y comparable. .... = p r o t e i n s t a i n alone; = carbohydrate s t a i n alone = carbohydrate s t a i n f o l l owed by p r o t e i n s t a i n . Where p r o t e i n s t a i n alone (....) i s not shown, i t followed the same curve as the carbohydrate plus p r o t e i n s t a i n ( ). F i g u r e 5A. Mutant s t r a i n M1 s o l u b i l i s e d i n the presence of 2mM EDTA. Fig u r e 5B. P a r t i a l l y p u r i f i e d l i p o p r o t e i n I of s t r a i n WT3 ( c o n t a i n i n g some p r o t e i n H2 but no LPS) s o l u b i l i s e d i n the presence of 2 mM EDTA. Fi g u r e 5C. L P S - d e f i c i e n t (rough) s t r a i n AK43 s o l u b i l i s e d i n the presence of 40 mM EDTA. Fig u r e 5D. Wild type s t r a i n WT1 s o l u b i l i s e d i n the presence of 2 mM EDTA. Fi g u r e 5E. Wild type s t r a i n WT1 s o l u b i l i s e d i n the presence of 20 mM MgCl 2 . 96 carbohydrate s t a i n allowed Coomassie s t a i n i n g of carbohydrate ( K r o p i n s k i e_t a_l. , 1982). The apparent absence of l i p o p r o t e i n I could be exp l a i n e d i f , i n wi l d type s t r a i n s l i k e WT1, the LPS c o - e l e c t r o p h o r e s e d with and thereby masked the l i p o p r o t e i n I. In d i r e c t agreement with t h i s , Mg + + treatment during s o l u b i l i s a t i o n prevented LPS from e n t e r i n g the g e l and l i p o p r o t e i n I c o - e l e c t r o p h o r e s e d with p a r t i a l l y p u r i f i e d p r o t e i n I ( F i g . 5E) and could be s t a i n e d by Coomassie Blue without pretreatment. Repeating these experiments with mutant M1 demonstrated that s i g n i f i c a n t l y l e s s c a r b o h y d r a t e - s t a i n i n g m a t e r i a l had entered the g e l s and that t h i s m a t e r i a l ran with a m o b i l i t y g r e a t e r than that of the l i p o p r o t e i n ( F i g . 5A). The rough core LPS of mutant M1 was thus a p p a r e n t l y unable to mask or i n f l u e n c e the e l e c t r o p h o r e t i c m o b i l i t y of l i p o p r o t e i n I, which c o - e l e c t r o p h o r e s e d with p a r t i a l l y p u r i f i e d l i p o p r o t e i n I and s t a i n e d without pretreatment. S i m i l a r r e s u l t s were obtained f o r M1 both i n the presence ( F i g . 5A) and absence ( a f t e r Mg + + treatment - data not shown) of LPS. There was no measurable c a r b o h y d r a t e - s t a i n i n g m a t e r i a l apparent i n t h i s r e g i o n of g e l s of a rough L P S - d e f i c i e n t mutant, AK 43 ( F i g . 5C), i n which, as with mutant M1, the l i p o p r o t e i n I s t a i n e d with Coomassie blue without Mg + + pre-treatment s i m i l a r l y to mutant M1. 3. Protease d i g e s t i o n p a t t e r n s of outer membrane A more ext e n s i v e examination of the major outer membrane p o r i n , p r o t e i n F, was performed by examination of peptide 98 A B C D E F G H I J K L Figure 6. SDS-PAGE of protease d i g e s t i o n and immunoreactivity of outer membrane samples from s t r a i n s WT1 and M1. Lanes A - J : outer membrane samples at a c o n c e n t r a t i o n of 0.5 mg/ml were d i g e s t e d f o r 1 h at 37°C with S_;_ g r i s e u s protease Type XIV at a r a t i o of 0.4 mg protease / mg outer membrane p r o t e i n . Digested samples were s o l u b i l i s e d and run on a 14$ acrylamide g e l and s t a i n e d f o r p r o t e i n . Lanes A - E were s o l u b i l i s e d i n the presence of 2-ME and lanes F - J were s o l u b i l i s e d i n the absence of 2-ME to help i d e n t i f y fragments of p r o t e i n F. Lanes A and F - WT1 c o n t r o l ; la n e s B and G - WT1 + S_^  g r i s e u s protease XIV (Pronase) ; lanes C and H - Pronase c o n t r o l ; lanes D and I - M1 + Pronase ; lanes E and J - M1 c o n t r o l . The banding p o s i t i o n s of unhydrolysed p r o t e i n F i n the presence (F^) and absence (F) of 2-mercaptoethanol as w e l l as of p r o t e o l y s i s fragments are noted with s m a l l arrows. Lanes K - L: outer membrane samples were run on an 11$ SDS-polyaerylamide g e l , b l o t t e d onto n i t r o c e l l u l o s e then reacted with monoclonal antibody MA5-8 a g a i n s t p r o t e i n F. Only s t r a i n s WT1 and M1 are shown, however a l l s t r a i n s r e a c t e d eq u a l l y w e l l with t h i s antibody. Lane K, WT1; lane L, M1. 99 fragments produced by protease d i g e s t i o n . F i g u r e 6 shows the protease d i g e s t i o n p a t t e r n s of WT1 and M1 outer membranes produced with Protease Type XIV (Pronase) from S_;_ g r i s e u s . There appeared to be no d i f f e r e n c e between the d i g e s t i o n p a t t e r n s produced by Pronase i n the w i l d type and mutant s t r a i n s or i n the l e n g t h of time r e q u i r e d f o r approximately complete d i g e s t i o n . Most i m p o r t a n t l y , there was no d i f f e r e n c e i n the d i g e s t i o n p a t t e r n s f o r the p o r i n p r o t e i n F. Pronase degradation products of approximately 34,000, 12,000 and 9,000 da l t o n s were observed i n 2-mercaptoethanol-treated samples. Without 2-ME treatment, only a s i n g l e , 20,000 d a l t o n product was v i s i b l e i n the g e l . P o s s i b l y t h i s was a fragment of p r o t e i n F which contained one or both of the two d i s u l p h i d e bonds which t h i s p r o t e i n possesses, and when 2-ME was added t h i s fragment was then cleaved to produce the 12,000 and 9,000 d a l t o n fragments. Protease d i g e s t i o n of whole c e l l s was a l s o attempted. Whole c e l l s of P_^  aeruginosa were very r e s i s t a n t to proteases i n g e n e r a l , and p r o t e o l y s i s was only observed when the LPS was d i s t u r b e d by a d d i t i o n of a high (200mM) c o n c e n t r a t i o n of Mg++ or EDTA (data not shown). Outer membrane samples were a l s o t e s t e d f o r r e a c t i v i t y with a monoclonal antibody s p e c i f i c f o r p r o t e i n F of s t r a i n WT3 (MA5-8 - L. Mutharia, Ph.D. T h e s i s , U.B.C., 1985). P r o t e i n F from a l l s t r a i n s r e a c t e d s t r o n g l y with t h i s antibody ( s t r a i n s WT1 and M1 only are shown i n F i g . 6), i n d i c a t i n g that the p a r t i c u l a r epitope was conserved i n h y p e r s u s c e p t i b l e 1 00 s t r a i n s . Since the epitope recognised by t h i s antibody was a c o n f o r m a t i o n a l epitope (L. Mutharia, Ph.D. T h e s i s ) , t h i s was considered to be good evidence that the n a t i v e c o n f i g u r a t i o n of p r o t e i n F was u n a l t e r e d i n mutant M1. 4. Self-promoted outer membrane p e r m e a b i l i t y ; I n t e r a c t i o n of polymyxin B_ with w i l d type and mutant c e l l  s u r f a c e s . Since the above data suggested that the mutant M1 had a LPS a l t e r a t i o n , and s i n c e the self-promoted pathway, which i n v o l v e s a LPS s i t e on the outer membrane (Moore e_t a_l. , 1986), was a l t e r e d ( F i g . 3), c h a r a c t e r i s a t i o n of the s e l f -promoted uptake pathway was extended using dansylated polymyxin B as a probe. Polymyxin B had been p o s t u l a t e d to promote i t s own uptake v i a i n t e r a c t i o n with s p e c i f i c Mg + + b i n d i n g s i t e s on LPS i n the outer membrane ( S c h i n d l e r and Teuber, 1975; Nicas and Hancock, 1980; Moore e_t a l . , 19 86 ). The dansyl group on dansyl polymyxin enabled measurement of polymyxin b i n d i n g to c e l l s , s i n c e b i n d i n g was accompanied by enhanced f l u o r e s c e n c e . Binding of d a n s y l polymyxin was f i r s t examined i n whole c e l l s to probe d i f f e r e n c e s i n the self-promoted uptake mechanism between w i l d type and mutant M1 (Table X I I I ) . C e l l s of both s t r a i n WT1 and M1 appeared to bind s i m i l a r amounts of the f l u o r e s c e n t compound (data not shown), and the c o n c e n t r a t i o n s of d a n s y l polymyxin which gave 50$ s a t u r a t e d b i n d i n g were the same. This c o r r e l a t e d with the MICs f o r 101 polymyxin, which were only f o u r - f o l d d i f f e r e n t between the two s t r a i n s . A f t e r near s a t u r a t i n g amounts of DPX were bound to c e l l s , a l i q u o t s of Mg + + were added to assess the r e l a t i v e a f f i n i t y of the c e l l s u r f a c e of each s t r a i n f o r Mg + + and dansyl polymyxin. A higher a f f i n i t y f o r the competing molecule would be re v e a l e d by a r a p i d decrease (as a f u n c t i o n of i n c r e a s i n g competitor molecule) i n f l u o r e s c e n c e i n t e n s i t y as dansyl polymyxin was removed. The c o n c e n t r a t i o n s of Mg + + which caused 50% of maximal f l u o r e s c e n c e i n h i b i t i o n ( I 5 Q ) w e r e moderately higher f o r the mutant than f o r w i l d type, which was s i m i l a r to the e f f e c t seen with lysozyme l y s i s i n whole c e l l s . T h i s i n d i c a t e d that the mutant M1 had a somewhat lower a f f i n i t y f o r Mg + + and presumably a l s o f o r gentamicin, s i n c e both i n t e r a c t at the same b i n d i n g s i t e (Moore e_t al_. , 1 986 ) 5. I n t e r a c t i o n of polymyxin B with l i p o p o l y s a c c h a r i d e :  Binding of Dansyl polymyxin Since l i p o p o l y s a c c h a r i d e has been proposed to be the major i n t e r a c t i o n s i t e of aminoglycosides d u r i n g self-promoted uptake (Nicas and Hancock, 1980), b i n d i n g of dansyl polymyxin B to l i p o p o l y s a c c h a r i d e and to one of i t s components, L i p i d A, was next examined. LPS was i s o l a t e d using the c o l d Mg + +-ethanol p r e c i p i t a t i o n method of Darveau and Hancock (1983). A l i q u o t s of dans y l polymyxin B were added to a f i x e d amount of LPS up to the p o i n t of s a t u r a t i o n . 102 Table X I I I . Binding of dansyl polymyxin B to whole c e l l s , LPS and L i p i d A, and competition of b i n d i n g by Mg++. S t r a i n Binding of DPX Whole c e l l s WT1 M1 LPS WT1 M1 L i p i d A WT 1 M1 (S 0.5) 1 -39 1 -39 0 .491 0.674 0.674 0.614 Competition by Mg + + Maximal Inhib'n (?) I 5 0 (mM) 75.0 83-3 67.0 61.0 68.0 63.0 1 .25 1 .67 2.06 2.26 0.83 1 .40 Dansyl polymyxin was bound to near the s a t u r a t i o n p o i n t (as measured by f l u o r e s c e n c e i n c r e a s e ) , then Mg + + was t i t r a t e d i n to assess e f f e c t i v e competition as de s c r i b e d f u l l y i n Methods. R e s u l t s were c a l c u l a t e d from H i l l p l o t s ( f o r DPX bin d i n g ) or Lineweaver-Burk p l o t s ( f o r Mg++ competition) of f l u o r e s c e n c e measurement data c o l l e c t e d from three separate t r i a l s . DPX, dansyl polymyxin B; S Q 5 > c o n c e n t r a t i o n of DPX at 50? maximal f l u o r e s c e n c e ; I 5 Q > c o n c e n t r a t i o n of p e r m e a b i l i s e r causing 50? maximal i n h i b i t i o n of f l u o r e s c e n c e . 103 Measurement of f l u o r e s c e n c e caused by b i n d i n g of dansyl polymyxin B (DPX) to w i l d type and mutant l i p o p o l y s a c c h a r i d e showed no d i f f e r e n c e between the s t r a i n s with r e s p e c t to maximal f l u o r e s c e n c e (DPX b i n d i n g ) per umol of LPS. Both w i l d type and mutant t h e r e f o r e seemed to bind s i m i l a r amounts of DPX to whole LPS molecules. The S Q ^ f o r s t r a i n WT1 (Table X I I I ) was 25$ lower than that f o r the mutant M1 , i n d i c a t i n g that the mutant LPS had a somewhat lower a f f i n i t y f o r dansyl polymyxin. The l o s s of b i n d i n g a f f i n i t y probably a f f e c t e d only a s p e c i f i c f r a c t i o n of the t o t a l number of s i t e s on the LPS, however. An attempt was t h e r e f o r e made to reduce the number of s i t e s being t e s t e d , by examining dansyl polymyxin b i n d i n g k i n e t i c s i n the innermost p o r t i o n of LPS, L i p i d A, i n the next s e c t i o n . 6. I n t e r a c t i o n of polymyxin B with l i p o p o l y s a c c h a r i d e : Binding of DPX to the i s o l a t e d L i p i d A f r a c t i o n of  LPS. The L i p i d A of gram negative b a c t e r i a c o n t a i n s phosphate mo i e t i e s which may be capable of b i n d i n g d i v a l e n t c a t i o n s . These phosphate groups are l o c a t e d i n the backbone of LPS and may be c r i t i c a l p o i n t s f o r d i v a l e n t c a t i o n s t a b i l i s a t i o n of the outer membrane. The phosphate groups are o f t e n s u b s t i t u t e d with other f u n c t i o n a l groups, which may or may not r e t a i n the phosphate charge c h a r a c t e r i s t i c s and can t h e r e f o r e i n f l u e n c e outer membrane s t a b i l i t y . Dansyl polymyxin b i n d i n g to L i p i d A was t h e r f o r e examined i n the w i l d type and mutant 104 s t r a i n s . L i p i d A was prepared by mild a c i d h y d r o l y s i s from LPS samples suspended to a known m o l a r i t y as d e s c r i b e d i n Methods. L i p i d A from the mutant s t r a i n bound approximately 25% l e s s of the Mg + +-competible p o r t i o n of DPX than d i d L i p i d A from the w i l d type s t r a i n ( F i g u r e 7 ) , although the r e l a t i v e a f f i n i t y of DPX f o r b i n d i n g s i t e s i n each s t r a i n (S. ._) w a s u . b approximately equal (Table X I I I ) . The amount of DPX bound to L i p i d A was determined from a standard curve of DPX b i n d i n g to excess L i p i d A as d e s c r i b e d i n Methods. I t appeared from these r e s u l t s that the mutant M1 might be missing a p a r t i c u l a r p o r t i o n of DPX b i n d i n g s i t e s on L i p i d A. This hypothesis was te s t e d f u r t h e r using competition f o r these s i t e s by Mg + +. 7. Competition by Mg±± of DPX b i n d i n g to i s o l a t e d  l i p o p o l y s a c c h a r i d e and L i p i d A Binding of Mg + + to s e v e r a l s i t e s on LPS has been proposed to s t a b i l i s e the outer membrane of P_^_ aeruginosa. Dansyl polymyxin was re p o r t e d (Moore e_t a.1. , 1986) to bind i n part to s i t e s on the LPS to which Mg + + a l s o binds. Therefore competition by Mg + + could provide meaningful i n f o r m a t i o n r e g a r d i n g the r o l e of Mg + + i n outer membrane s t a b i l i t y of the mutant s t r a i n . The e f f e c t of competition f o r LPS b i n d i n g s i t e s was examined by adding a l i q u o t s of Mg + + to u n f r a c t i o n a t e d LPS with near s a t u r a t i n g amounts of DPX bound. For Mg + + (Table X I I I ) , the maximum percent i n h i b i t i o n of 105 Figure 7. Binding of dans y l polymyxin to L i p i d A and competition of bi n d i n g by Mg++. Binding of DPX to L i p i d A was measured as inc r e a s e In flu o r e s c e n c e emission at 485 nm. Mg++ was added to DPX-saturated L i p i d A at the i n d i c a t e d c o n c e n t r a t i o n s , and competition f o r DPX bi n d i n g s i t e s was measured by l o s s of flu o r e s c e n c e emission i n t e n s i t y . o, WT1; • , M1. 106 f l u o r e s c e n c e seen f o r the w i l d type WT1 was q u i t e s i m i l a r to that f o r the mutant M1. A s l i g h t l y higher c o n c e n t r a t i o n of Mg + + was necessary to produce half-maximal i n h i b i t i o n ± n the mutant as compared to the w i l d type. T h i s was s i m i l a r to what was seen i n whole c e l l s . Mg++ competition experiments f o r L i p i d A were performed s i m i l a r l y to those of LPS, using the molar r a t i o s s t a t e d i n Methods. For L i p i d A, the I,_ 0 f o r Mg + + was almost doubled i n the mutant s t r a i n M1 (see Table X I I I ) . These e f f e c t s were c o n s i s t e n t l y obtained i n three independent measurements. Thus, the mutant s t r a i n appeared to be d e f i c i e n t i n a s p e c i f i c , Mg + +-competable DPX bi n d i n g s i t e l o c a t e d on the L i p i d A component of the l i p o p o l y s a c c h a r i d e . There are proposed to be two p o t e n t i a l b i n d i n g s i t e s f o r Mg + + on the L i p i d A, at the two phosphate groups. However, these p o t e n t i a l s i t e s may be p a r t i a l l y s u b s t i t u t e d with m o i e t i e s which preclude b i n d i n g of Mg + +. This could account f o r an apparent 25% r e d u c t i o n i n the a v a i l a b l e number of bin d i n g s i t e s f o r the mutant M1. An a l t e r n a t i v e e x p l a n a t i o n i s that the a f f i n i t y , r a t h e r than the number, of Mg + + b i n d i n g s i t e s on the L i p i d A was lower i n the mutant. This problem i s addressed i n s e c t i o n 14. In e i t h e r case, the r e l a t i v e i n a b i l i t y to bind Mg + + at t h i s s i t e may i n f l u e n c e s t a b i l i t y of the l i p o p o l y s a c c h a r i d e i n whole c e l l s , p o s s i b l y accounting f o r s u s c e p t i b i l i t y to a n t i b i o t i c s which use the self-promoted and hydrophobic uptake mechanisms as t h e i r means of passage across the outer membrane of P_^  aeruginosa. 1 07 o-0 10 20 30 AO 50 60 [GM] (ug/ml) F i g u r e 8. I n h i b i t i o n by gentamicin of dansyl polymyxin b i n d i n g to L i p i d A. Gentamicin was added to DPX-saturated L i p i d A at the i n d i c a t e d c o n c e n t r a t i o n s , and competition f o r DPX b i n d i n g s i t e s was measured by decrease of f l u o r e s c e n c e emission i n t e n s i t y at 485 nm. o, WT1; •, M1. 108 Gentamicin competition was a l s o examined f o r L i p i d A, using the same methods as f o r Mg + + ( F i g . 8). Gentamicin competed p o o r l y with DPX bound to mutant L i p i d A (only 20% i n h i b i t i o n of f l u o r e s c e n c e at 50 ug/ml gentamicin) as compared to w i l d type L i p i d A (35$ i n h i b i t i o n at the same c o n c e n t r a t i o n ) . This r e s u l t i s c o n s i s t e n t with the data i n Table XII and F i g . 3 i n which gentamicin demonstrated a reduced a b i l i t y to p e r m e a b i l i s e the mutant c e l l s to lysozyme. 8. Whole c e l l f a t t y a c i d composition Whole c e l l s were assayed f o r f a t t y a c i d methyl e s t e r s a f t e r h y d r o l y s i s i n 2 M methanolic HC1. LPS from P.  aeruginosa c o n t a i n s f i v e f a t t y a c i d s i n the L i p i d A f r a c t i o n : dodecanoic a c i d , 3-hydroxydecanoic a c i d , 2- and 3-hydroxydodecanoic a c i d and hexadecanoic a c i d (Meadow, 1975). These were i d e n t i f i e d i n Table XIV on the b a s i s of t h e i r r e t e n t i o n times and q u a n t i t a t e d by using response f a c t o r s c a l c u l a t e d f o r each, using commercial standards. These analyses demonstrated no d i f f e r e n c e s i n four of the seven major f a t t y a c i d s but s i g n i f i c a n t (P<0.1 by Student t t e s t ) r e d u c t i o n s i n the amounts of dodecanoic a c i d and hexadecanoic a c i d , and a s l i g h t i n c r e a s e i n 2-hydroxydodecanoic a c i d i n whole c e l l s of mutant M1 compared with the w i l d type, WT1. The l e v e l s of each of the seven major f a t t y a c i d peaks i n the f u l l r e v e r t a n t were not s i g n i f i c a n t l y d i f f e r e n t (P > 0.5) from the amounts i n WT1 109 Table XIV. Fa t t y a c i d composition of whole c e l l s of P. aeruginosa s t r a i n s WT1 , P1-1, P1-4 and M1. S t r a i n F a t t y a c i d composition T o t a l f a t t y a c i d content LPS/PL (% t o t a l f a t t y a c i d s ) (nmol/mg c e l l dry wt.) r a t i o 30H10:0 12 :0 20H12:0 30H12:0 16 : 1 16 :0 18 : 1 WT1 5.5 4 .3 5.9 6 . 9 15 . 1 33 . 1 29 .7 315.8 0.290 WT1-1 6.5 4 .0 5.6 7 . 3 13 .6 33 . 1 29 .8 300.6 0 .306 P1-4 7.7 3 . 3 6.7 7.7 13 .7 30 .4 29 .5 290.4 0.345 M1 5.9 2 .2 6 . 3 7.1 14 .7 29 .9 32 .5 285.7 0 .279 For f a t t y a c i d nomenclature, the number to the l e f t of the colon r e p r e s e n t s the number of carbon atoms, and the number to the r i g h t r e p r e s e n t s the number of double bonds. OH r e f e r s to hydroxy f a t t y a c i d s . The r e s u l t s r epresent the mean of three separate d e t e r m i n a t i o n s . LPS/PL, r a t i o of L P S - s p e c i f i c f a t t y a c i d s (30H10:0, 12:0, 20H12:0 and 30H12:0) to p h o s p h o l i p i d - l o c a t e d f a t t y a c i d s (16:1, 16:0 and 18:1). (Table XIV), whereas the p a r t i a l r e v e r t a n t , P1-4, showed an i n t e r m e d i a t e phenotype. LPS:phospholipid r a t i o s showed that the t o t a l amount of LPS was approximately i d e n t i c a l . The d i f f e r e n c e s i n whole c e l l f a t t y a c i d s were a l s o r e f l e c t e d i n analyses of LPS and L i p i d A f a t t y a c i d s ( K r o p i n s k i et^ al.. , 1982). In f a c t , a 40 to 50$ decrease i n the l e v e l of dodecanoate i n the mutant, and a 20$ i n c r e a s e i n the l e v e l of 2-hydroxydodecanoate was observed f o r L i p i d A. The l e v e l of 3-hydroxydodecanoate remained approximately constant f o r whole c e l l and L i p i d A. I t could not be d i s t i n g u i s h e d whether changes i n LPS f a t t y a c i d content were a primary mutation causing a l t e r a t i o n s i n a n t i b i o t i c s u s c e p t i b i l i t y or were an a d a p t a t i o n to another c e l l s u r f a c e a l t e r a t i o n . 9. L i p o p o l y s a c c h a r i d e rough core n e u t r a l sugar composition I s o l a t e d LPS of the w i l d type and mutant s t r a i n s was f i r s t examined by carbohydrate assay to look f o r a l t e r a t i o n s i n LPS sugar content which might r e f l e c t d i f f e r e n c e s i n LPS s t r u c t u r e or f u n c t i o n . Since heptoses and glucose/rhamnose are major components of the inner and outer core o l i g o s a c c h a r i d e p o r t i o n s of LPS, and amino-substituted carbohydrates are the main c o n s t i t u e n t s of the 0-antigen p o r t i o n (Meadow, 1975), absorbance scans of the c y s t e i n e -s u l p h u r i c a c i d assays f o r n e u t r a l sugars were considered to r e f l e c t the r e l a t i v e amounts of inner and outer core sugars, r e s p e c t i v e l y . The w i l d type s t r a i n s WT3 and WT1 gave almost i d e n t i c a l 111 I 1 1 I r — 550 500 450 400 350 Absorbance (nm) F i g u r e 9. Spectrophotometeric s c a n s o f c a r b o h y d r a t e assay on p u r i f i e d l i p o p o l y s a c c h a r i d e from a e r u g i n o s a s t r a i n s WT1, WT3 and M1. A Wright and Rebers c y s t e i n e - s u l p h u r i c a c i d a s s a y was performed as s t a t e d i n Methods on p u r i f i e d l i p o p o l y s a c c h a r i d e from w i l d type and mutant s t r a i n s . Scans were performed w i t h n o n - c a r b o h y d r a t e - c o n t a i n i n g assay f l u i d as a r e f e r e n c e . Heptose s u g a r s produced c o l o u r e d p r o d u c t s o f maximum a b s o r b a n c e a t 505nm. Hexoses and m e t h y l p e n t o s e s produced a b s o r b a n c e maxima at 410 and 396 nm, r e s p e c t i v e l y . 112 products as r e v e a l e d by wavelength scans between o p t i c a l d e n s i t i e s at 550 and 350 nm (see F i g . 9 ) . T h i s s t r o n g l y suggested that these s t r a i n s had extremely s i m i l a r n e u t r a l sugar compositions. In c o n t r a s t , the p r o f i l e s of the wavelength scan were s u b s t a n t i a l l y a l t e r e d f o r the a n t i b i o t i c h y p e r s u s c e p t i b l e mutant M1. Another observed d i f f e r e n c e was that the amount of background c o l o u r produced by HgSOjj h y d r o l y s i s of the LPS of mutant M1 was c o n s i d e r a b l y l e s s than that produced from the w i l d type s t r a i n s . The d i f f e r e n c e between the two types of LPS i n t h i s assay was obvious enough that the produced c o l o u r s could be d i s t i n g u i s h e d by eye. Wild type were orange, due to absorbance of products i n both the 500 and 400 nm range, while M1 LPS products absorbed mainly i n the 500 nm range and appeared pink. Standards L-glycero-D-mannoheptose, D-glucose and L-rhamnose had peak a b s o r p t i o n s of 505 nm ( p i n k ) , 415 nm ( y e l l o w ) , and 396 nm ( y e l l o w ) , r e s p e c t i v e l y . A l l s t r a i n s were subsequently t e s t e d f o r rough core l i p o p o l y s a c c h a r i d e ( n e u t r a l ) sugars, and these r e s u l t s are presented i n Table XV. The r a t i o of hexose (glucose) plus methylpentose (rhamnose) sugars/heptose sugars was two to t h r e e - f o l d lower than w i l d type l e v e l s f o r the mutant s t r a i n M1 and the two p a r t i a l r e v e r t a n t s P1-4 and P1-6. A l l s t r a i n s d e r i v e d from s t r a i n WT2 had w i l d type l e v e l s of hexose and methylpentose. L e v e l s of heptose were the same f o r a l l s t r a i n s . These r e s u l t s were c o n s i s t e n t with data of K r o p i n s k i et a l . (1982) using gas l i q u i d chromatography to q u a n t i t a t e 113 Table XV. A n a l y s i s of LPS n e u t r a l sugars. S t r a i n Measured absorbance at absorbance maxima Heptose U 5 0 5 ) WT1 P1-4 P1-6 M1 0 .24 0 .22 0.21 0 .20 Hexose ( A 1 J 0 5 ) 0 .76 0.31 0 .22 0.24 Ratio of hexose heptose 3-2 1 .4 1 .0 1 .2 WT2 P2-6 P2-8 0 .24 0.26 0.32 0.65 0.73 0.96 2.7 2.8 3-0 A modified c y s t e i n e - s u l p h u r i c a c i d assay was performed as s t a t e d i n Methods on p u r i f i e d l i p o p o l y s a c c h a r i d e which had been s t a n d a r d i s e d f o r ketodeoxyoctonate content. Values were de r i v e d from data analogous to that i n F i g . 9» using peak hei g h t s from each r e s p e c t i v e b a s e l i n e , and numbers repo r t e d i n t h i s t a b l e f o r hexose a c t u a l l y r e p r e s e n t hexose plus methylpentose, where the absorbance maxima A 3 9 6 > r e s p e c t i v e l y ) could not be separated. (A 410 and 114 LPS carbohydrates. These r e s u l t s seemed to I n d i c a t e that the mutant s t r a i n and the two p a r t i a l r e v e r t a n t s were d e f i c i e n t i n the outer r e g i o n of t h e i r l i p o p o l y s a c c h a r i d e rough co r e s , s i n c e the amounts of heptose and ketodeoxyoctonate (KDO) were normal. Transconjugant s t r a i n s appeared to have w i l d type LPS core, i n d i c a t i n g that the pro+- and me_t +-linked mutations (AbsA and B phenotypes) were not r e s p o n s i b l e f o r core LPS changes. 10. SDS-PAGE banding p a t t e r n of rough core l i p o p o l y s a c c h a r i d e  i n w i l d type and mutant s t r a i n s . The s t r u c t u r e of the l i p o p o l y s a c c h a r i d e (LPS) i n a l l s t r a i n s was f u r t h e r c h a r a c t e r i s e d using SDS-urea-polyacrylamide g e l e l e c t r o p h o r e s i s . LPS i n many b a c t e r i a (Palva and Makela, 1980; Peterson et a l . , 1986) has been found, by t h i s method, to be heterogeneous with r e s p e c t to p o l y s a c c h a r i d e chain l e n g t h , even w i t h i n p r e p a r a t i o n s from the same s t r a i n . The heterogeneous p o p u l a t i o n of LPS molecules separate roughly a c c o r d i n g to chain l e n g t h i n t h i s g e l system (Peterson e_t a_l. , 1 986 ) forming a " l a d d e r n - l i k e p a t t e r n , with the l a r g e s t molecules (rough core plus the l a r g e s t number of 0- antigen r e p e a t i n g sugar u n i t s ) m i g r a t i n g slowest, and rough core with no s u b s t i t u t i o n s m i g r a t i n g f a s t e s t . Two major d i f f e r e n c e s between w i l d type (WT1) and mutant (M1) P.  aeruginosa LPS were observed u s i n g t h i s method (Figure 10). F i r s t , the major band with highest m o b i l i t y , corresponding to rough core LPS, was observed i n a d i f f e r e n t 115 A B C D E F G H 1 J F i g u r e 10. SDS-PAGE o f LPS from w i l d type and a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n s . LPS was i s o l a t e d as d e s c r i b e d i n M e t h o d s , t h e n samples were s t a n d a r d i s e d f o r KDO c o n t e n t and s o l u b i l i s e d i n 4$ SDS, 10$ 2 - m e r c a p t o e t h a n o l , 40 mM EDTA f o r 5 min at 1 0 0 P C . Samples were run on a SDS-urea-15$ p o l y a c r y l a m i d e g e l and s t a i n e d f o r c a r b o h y d r a t e u s i n g p e r i o d a t e and s i l v e r n i t r a t e . Lanes A and E , WT1 ; l a n e s B and F , M1; l a n e C, P 1 - 4 ; l a n e D, P 1 - 6 ; l a n e G, WT2; l a n e H, P 2 - 6 ; l a n e I , P 2 - 8 ; l a n e J , P 2 - 6 , 8 . Lanes A - D were o v e r l o a d e d to show the 0 - a n t i g e n b a n d i n g p a t t e r n s more c l e a r l y i n s t r a i n s M1, P1-4 and P 1 - 6 . Banding p o s i t i o n s o f w i l d type (WR) and mutant (MR) rough c o r e LPS as w e l l as w i l d type (WS) and mutant (MS) smooth LPS are i n d i c a t e d on the m a r g i n . 116 p o s i t i o n on the g e l f o r the mutant s t r a i n M1. The higher m o b i l i t y seen f o r mutant rough core was c o n s i s t e n t with data from the carbohydrate assay of rough core, which had suggested that some sugar components from the outer core (hexoses) were mi s s i n g , which would cause a shortened core LPS s t r u c t u r e . The two p a r t i a l r e v e r t a n t s of mutant s t r a i n M1, P1-4 and P1-6, which had d i s p l a y e d s i m i l a r core carbohydrate assay r e s u l t s , a l s o showed a l t e r e d rough core banding on g e l s . These data were a l s o c o n s i s t e n t with the c o n c l u s i o n that p r o t e i n I was v i s i b l e i n the outer membrane p a t t e r n s of above s t r a i n s ( F i g . 4) because i t banded above the lower core LPS band ( F i g . 5) and there was thus no i n t e r f e r e n c e from LPS i n the p r o t e i n s t a i n i n g procedure. The rough core LPS of f u l l r e v e r t a n t WT1-1 migrated s i m i l a r l y to that of the wi l d type s t r a i n WT1 (data not shown). There was a l s o no d i f f e r e n c e i n rough core LPS mi g r a t i o n between a l l transconjugants and t r a n s d u c t a n t s and the w i l d type s t r a i n s WT2 or WT1. This confirmed that the rough core LPS a l t e r a t i o n was not due to e i t h e r the me_t+- or pr_o +- l i n k e d mutation, and was t h e r e f o r e given the "AbsC" phenotypic d e s i g n a t i o n . 11. SDS-PAGE banding p a t t e r n of major l i p o p o l y s a c c h a r i d e 0-a n t i g e n - c o n t a i n i n g s p e c i e s . The next major o b s e r v a t i o n was that there a l s o appeared to be a r e g i o n of a l t e r e d LPS banding i n SDS-PAGE corresponding to O - a n t i g e n - s u b s t i t u t e d rough core ( i e . 117 "smooth") LPS. Two h e a v i l y s t a i n i n g bands of lower m o b i l i t y ( F i g u r e 10) seen i n each s t r a i n were presumed to represent major ch a i n l e n g t h s p e c i e s of the heterogeneous LPS p o p u l a t i o n . I t was noted that these bands had a p a r t i c u l a r spacing between them i n s t r a i n s with w i l d type (WT1 and WT2) or the met*- l i n k e d mutation (P2-8) however, i n the mutant M1 , p a r t i a l r e v e r t a n t s P1-4 and P1-6, and i n the p_ro+- l i n k e d transcon jugant P2-6 and pro*, me_t+- transcon jugant P2-6,8, the band occupying the upper p o s i t i o n i n the g e l had a higher m o b i l i t y than the corresponding band i n w i l d type s t r a i n s . This phenomenon was r e p r o d u c i b l y observed i n m u l t i p l e t r i a l s , at v a r i o u s l o a d i n g c o n c e n t r a t i o n s of LPS and over more than one LPS i s o l a t i o n attempt (data not shown). Since the a l t e r e d chain l e n g t h LPS occurred i n s t r a i n s P2-6 and P2-6,8 (as w e l l as mutant M1), t h i s i n d i c a t e d that the mutation r e s p o n s i b l e f o r the s t r u c t u r a l property was l i n k e d to the "AbsA" phenotype. Mutant M1 (lane F) a l s o had fewer rough core molecules s u b s t i t u t e d with 0-antigen c h a i n s , c o n f i r m i n g r e s u l t s from e a r l i e r s t u d i e s (Darveau and Hancock, 1983). I t was concluded that the 0-antigen a l t e r a t i o n (AbsA) was not r e l a t e d to p o s s e s s i o n of a shortened rough core (the AbsC phenotype), s i n c e a l l of the transconjugant s t r a i n s were normal i n the l a t t e r r e s p e c t . Thus, there appeared to e x i s t two separate mutations causing s t r u c t u r a l a l t e r a t i o n of the LPS, l i n k e d to the AbsA and AbsC a n t i b i o t i c r e s i s t a n c e phenotypes. 118 12. Immunoreactivity of a l t e r e d outer membrane components with  monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t l i p o p o l y s a c c h a r i d e . Although the s i l v e r s t a i n method used f o r the g e l i n Fi g u r e 10 was purported to be s p e c i f i c f o r carbohydrate m o i e t i e s ( T s a i and Fr a s c h , 1982), i t was necessary to confirm the i d e n t i t y of the a l t e r e d components i n the mutant as LPS by another c r i t e r i o n . To do t h i s , an e l e c t r o p h o r e t i c b l o t of LPS from a SDS-urea-polyacrylamide g e l ( s i m i l a r to the g e l i n Fi g u r e 10) was made. This was then r e a c t e d with monoclonal a n t i b o d i e s r a i s e d a g a i n s t LPS determinants from a PA01 s t r a i n (WT3) and subsequently with a l k a l i n e phosphatase conjugated Goat-anti-mouse Fab' fragment as the secondary antibody. One of the monoclonals (MA1-8) was 0-antigen serotype s p e c i f i c (L. Mutharia, Ph. D. T h e s i s , 1985) and could only be used f o r the WT2-derived s t r a i n s , WT2, P2-6, P2-8 and P2-6,8, and not f o r the WT1-derived s t r a i n s , P1 -1 , P1-4, P1-6 or M1, to which i t had very low r e a c t i v i t y . The nature of the low m o b i l i t y , h e a v i l y s t a i n e d bands from WT2-derived s t r a i n s was determined to be LPS, due to t h e i r r e a c t i o n on Western b l o t s ( F i g u r e 11, lanes E - H) with the O - a n t i g e n - s p e c i f i c monoclonal antibody MA1-8. These bands repr e s e n t an e x t r a long chain p o p u l a t i o n of LPS molecules (E. McGroarty, p e r s o n a l communication). The antibody r e a c t i o n produced a s i m i l a r p a t t e r n to that seen by s i l v e r s t a i n i n g of 0-antigen i n F i g . 10, c o n f i r m i n g that a p o p u l a t i o n of smooth, 0-antigen c o n t a i n i n g LPS molecules was a l t e r e d i n s t r a i n s 119 w s W R M R B C D E F G H F i g u r e 11. Immunoreactivity of e l e c t r o p h o r e t i c a l l y b l o t t e d LPS from w i l d type and mutant s t r a i n s . LPS samples were t r e a t e d as i n F i g . 10, except that the g e l was not s t a i n e d , but was b l o t t e d onto n i t r o c e l l u l o s e and reacted with a monoclonal antibody s p e c i f i c f o r P^ . a eruginosa rough core LPS (MA3-5 - lanes A - D), or with a monoclonal antibody s p e c i f i c f o r P_j_ aeruginosa serotype 5 0-antigen (MA1-8 - lanes E - H). Lane A, WT1; lane B, M1 ; lane C, P1 - 4; lane D P1-6; lane E, WT2; lane F, P2-6; lane G, P2-8; lane H, P2-6,8. The banding p o s i t i o n s of rough and smooth LPS are provided f o r r e f e r e n c e from F i g . 10, which was run c o n c u r r e n t l y . 120 demonstrating the AbsA phenotype. The high m o b i l i t y s t a i n e d areas were a l s o confirmed to be rough core LPS by r e a c t i o n on b l o t s ( F igure 11, lanes A - D) with a c o r e - s p e c i f i c monoclonal antibody (MA3-5). Monoclonal antibody MA3-5 r e a c t e d with LPS core from WT1, WT2, P 2 - 6 , P2 - 8 and P2-6,8 but not with P1-4, P1-6 or M1 (only r e s u l t s f o r WT1-derived s t r a i n s are shown i n F i g u r e 11). Perhaps the reason that the core LPS from the mutant s t r a i n M1 and p a r t i a l r e v e r t a n t s d i d not r e a c t with t h i s monoclonal was that the a n t i g e n i c determinant was present i n the outer r e g i o n of the l i p o p o l y s a c c h a r i d e core, and was t h e r e f o r e not present i n the s u s c e p t i b l e s t r a i n s with shortened LPS core m o i e t i e s . 13. LPS phosphate content Because LPS phosphate m o i e t i e s were presumed to be important as Mg + + b i n d i n g s i t e s , the phosphate content was determined f o r s t r a i n s WT1 and M1, i n both whole LPS and the L i p i d A f r a c t i o n . Phosphate was assayed using the a s c o r b i c a c i d / molybdate method of Ames and Dubin (1960). I s o l a t e d mutant M1 LPS contained approximately 80? as much phosphate as w i l d type when both samples were s t a n d a r d i s e d f o r KDO (Table XVI). For the L i p i d A f r a c t i o n , there e x i s t e d an even g r e a t e r d i s p a r i t y between the two s t r a i n s . The mutant M1 had only 62% as much phosphate as d i d WT1. This d i s p a r i t y could be e x p l a i n e d i f the r e d u c t i o n i n amounts of assayable phosphate i n the mutant M1 was r e s t r i c t e d to the L i p i d A - a s s o c i a t e d phosphates and d i d not i n v o l v e LPS rough c o r e - a s s o c i a t e d 121 Table XVI. Phosphate content of LPS and L i p i d A from w i l d type and mutant s t r a i n M1. S t r a i n LPS Ratio mutant L i p i d A Ratio mutant phosphate w i l d type phosphate wild type (mg/ml) (mg/ml) WT1 1.39 N/A 1.38 N/A M1 1.10 0.79 0.85 0.616 Values f o r the amounts of phosphate were s p e c t r o p h o t o m e t r i c a l l y determined by absorbance at 820 nm. Samples had been p r e v i o u s l y s t a n d a r d i s e d f o r ketodeoxyoctonate content to ensure e q u i m o l a r i t y . 122 phosphate. Assuming that these r a t i o s were r e p r e s e n t a t i v e of the phosphate content of LPS i n whole c e l l s , r a t h e r than the r e s u l t of a r t i f a c t s from the i s o l a t i o n procedure, then the lower phosphate content of mutant L i p i d A could e x p l a i n i t s a l t e r e d dansyl polymyxin and Mg + + b i n d i n g c h a r a c t e r i s t i c s ( s e c t i o n s 6 and 7) as having been due to a reduced number of Mg + + b i n d i n g s i t e s , r a t h e r than reduced a f f i n i t y of a p a r t i c u l a r s i t e . 123 14. SUMMARY Examination of outer membrane p r o t e i n s from w i l d type s t r a i n WT1 and mutant M1 on SDS-polyaerylamide g e l s showed no a l t e r a t i o n s i n p o r i n p r o t e i n F or any other outer membrane p r o t e i n except f o r l i p o p r o t e i n I, which was not s t a i n e d by Coomassie Blue i n the wi l d type s t r a i n s but was s t a i n e d i n the a n t i b i o t i c - s u s c e p t i b l e mutant M1 and p a r t i a l r e v e r t a n t s t r a i n s . D i f f e r e n t i a l s t a i n i n g f o r l i p o p o l y s a c c h a r i d e and p r o t e i n i n SDS-polyacrylamide g e l s showed that rough core LPS from the wi l d type s t r a i n WT1 i n t e r f e r e d with the banding of p r o t e i n I i n the g e l . Protease treatment of outer membranes from w i l d type and mutant s t r a i n s showed no d i f f e r e n c e i n the a c c e s s i b i l i t y of p r o t e i n F to p r o t e o l y s i s or i n the fragments produced by p r o t e o l y s i s of p r o t e i n F, t h e r e f o r e the p r o t e i n i t s e l f was s t r u c t u r a l l y a l i k e i n the two s t r a i n s . Reaction of p r o t e i n F with monoclonal antibody a l s o i n d i c a t e d s t r u c t u r a l i d e n t i t y , between the two s t r a i n s , of p r o t e i n F i n i t s n a t i v e form . Crude chemical a n a l y s i s of l i p o p o l y s a c c h a r i d e carbohydrate content demonstrated that there were d i f f e r e n c e s between w i l d type and mutant. The mutant s t r a i n had a much lower hexose content as compared to heptose content, i n d i c a t i n g that M1 was a l t e r e d i n the outer r e g i o n of i t s rough core LPS. The two p a r t i a l r e v e r t a n t s , P1-4 and P1-6, r e t a i n e d t h i s a l t e r a t i o n . There appeared to be at l e a s t two mutations r e s p o n s i b l e f o r separate a l t e r a t i o n s i n LPS s t r u c t u r e i n the a n t i b i o t i c 124 h y p e r s u s c e p t i b l e mutant M1. One of the mutations, which caused higher m o b i l i t y of a s p e c i e s of O-antigen-containing LPS (the p_ro +- l i n k e d phenotype, designated AbsA), appeared to be d i r e c t l y r e s p o n s i b l e f o r p a r t i a l , wide spectrum a n t i b i o t i c s u s c e p t i b i l i t y , and i n c r e a s e d hydrophobic p e r m e a b i l i t y , as seen i n the transconjugant P2-6. The other mutation (causing the phenotype designated AbsC), appeared to r e s u l t i n a trun c a t e d rough core LPS, to be p a r t l y r e s p o n s i b l e f o r i n c r e a s e d hydrophobic p e r m e a b i l i t y across the outer membrane, and was not represented i n e i t h e r of the two transconjugant s t r a i n s . The s t r u c t u r a l nature of the me_t+- l i n k e d mutation i n the other transcon jugant s t r a i n P2-8, which caused beta-lactam and aminoglycoside s u s c e p t i b i l i t y (designated AbsB), was not apparent. Mutant M1 was a l s o found to be a l t e r e d with r e s p e c t to the a f f i n i t y of the L i p i d A f o r Mg + +. i t i s not known whether l o s s of a f f i n i t y f o r Mg + + of the mutant L i p i d A was caused by a separate mutation to that which caused the change(s) i n LPS s t r u c t u r e . 125 DISCUSSION To study the i n t r i n s i c a n t i b i o t i c r e s i s t a n c e of P.  aeruginosa, I have used a s t r a i n mutated such that i t has l o s t i n t r i n s i c ( w i l d type) r e s i s t a n c e (.ie. an a n t i b i o t i c h y p e r s u s c e p t i b l e s t r a i n ) i n an attempt to determine which outer membrane s t r u c t u r e s might be i n v o l v e d . Although there may be s e v e r a l outer membrane components r e s p o n s i b l e , and there i s no guarantee that the s t r u c t u r e s r e s p o n s i b l e f o r r e s i s t a n c e w i l l n e c e s s a r i l y be the same ones which mutate to cause s u s c e p t i b i l i t y , t h i s type of study n e v e r t h e l e s s g i v e s a good i n d i c a t i o n of what kinds of changes are able to cause s u s c e p t i b i l i t y . The mutant I chose to c h a r a c t e r i s e , M1 , was most i n t e r e s t i n g because of both i t s extreme a n t i b i o t i c s e n s i t i v i t y and the l a r g e v a r i e t y of a n t i b i o t i c s to which i t was s u s c e p t i b l e . These two f a c t o r s made i t an i d e a l candidate to be a mutant that was a l t e r e d i n outer membrane p e r m e a b i l i t y . Because the mutant M1 had been mutagenised with both e t h y l methanesulphonate and N-methyl-N'-nitro-N-n i t r o s o g u a n i d i n e , i t was l i k e l y that more than one mutation to s u s c e p t i b i l i t y had taken p l a c e . For t h i s reason, transconjugant s t r a i n s were used to i s o l a t e s i n g l e mutations and t h e r e f o r e make t h e i r i d e n t i f i c a t i o n e a s i e r . A plan of study was undertaken to answer the q u e s t i o n s : i ) i s an i n c r e a s e i n outer membrane p e r m e a b i l i t y r e s p o n s i b l e f o r a n t i b i o t i c h y p e r s u s c e p t i b i l i t y and, i f so, i i ) which outer membrane components are r e s p o n s i b l e f o r such a p e r m e a b i l i t y 126 a l t e r a t i o n ? In t h i s p u r s u i t , an e x t e n s i v e c h a r a c t e r i s a t i o n of the a n t i b i o t i c h y p e r s u s c e p t i b l e mutant has been presented. A l t e r a t i o n s i n both hydrophobic and self-promoted uptake were observed. Another property of mutant M1 was that i n d u c i b i l i t y of the Type Id Pseudomonas chromosomal beta-lactamase could not be demonstrated. Although the mutant was shown to possess a f u n c t i o n a l beta-lactamase, from n i t r o c e f i n p e r m e a b i l i t y data (Table V I ) , the l e v e l of enzyme a c t i v i t y could not be induced to i n c r e a s e above b a s a l l e v e l (Table V I I I ) . Lack of i n d u c t i o n of the beta-lactamase, i f t h i s were the case, could mean c o n s i d e r a b l e l o s s of r e s i s t a n c e to beta-lactam a n t i b i o t i c s , because the uninduced l e v e l of p e r i p l a s m i c enzyme could not keep up with permeation of beta-lactam molecules through the outer membrane. However, the apparent l a c k of i n d u c i b i l i t y may w e l l be an a r t i f a c t caused by p o s s e s s i o n of a more permeable outer membrane. I t i s d i f f i c u l t to d i s t i n g u i s h , u s ing these experimental c o n d i t i o n s , between an u n i n d u c i b l e beta-lactamase and the s i t u a t i o n where the amount of beta-lactam necessary f o r i n d u c t i o n i s g r e a t e r than the amount which w i l l i n h i b i t the c e l l . As demonstrated by Nordstrom and Sykes (1974), i n d u c t i o n of beta-lactamase i n P_^  aeruginosa w i l d type s t r a i n s a p p a r e n t l y takes 80 min at an inducer c o n c e n t r a t i o n of 200 ug of b e n z y l p e n i c i l l i n per ml. The time r e q u i r e d f o r i n d u c t i o n presumably r e f l e c t s low l e v e l exposure to beta-lactam over t h i s 80 min p e r i o d . A combination of the low r a t e of permeation of b e n z y l p e n i c i l l i n across the outer 127 membrane, the n o n s p e c i f i c b i n d i n g of t h i s beta-lactam by c e l l s u r f a c e and p e r i p l a s m i c components, and the h y d r o l y s i s of b e n z y l p e n i c i l l i n by the low l e v e l of beta-lactamase i n uninduced c e l l s would r e s u l t i n only a smal l f r a c t i o n of the e x t e r n a l b e n z y l p e n i c i l l i n c o n c e n t r a t i o n being a v a i l a b l e to induce the beta-lactamase. In c o n t r a s t , the r e l a t i v e l y high p e r m e a b i l i t y of mutant M1 outer membranes might prevent t h i s p e r i o d of low l e v e l exposure, s i n c e an i n c r e a s e i n the amount of p e n i c i l l i n i n the periplasm, s u f f i c i e n t to s a t u r a t e the beta-lactamase and n o n s p e c i f i c b i n d i n g s i t e s , would q u i t e r a p i d l y r e s u l t i n b i n d i n g of b e n z y l p e n i c i l l i n to i t s t a r g e t p e n i c i l l i n b i n d i n g p r o t e i n s and consequent k i l l i n g of the c e l l s . Two th i n g s argue i n favour of t h i s view. F i r s t , the amount of b e n z y l p e n i c i l l i n r e q u i r e d to k i l l mutant M1 (1.0 ug/ml - Table I I ) could be hydrolysed by the uninduced l e v e l of beta-lactamase from 1 08 c e l l s i n 2 to 4 min (C. Soga and R.E.W. Hancock, unpublished r e s u l t s ) . Thus, although t h i s l e v e l might normally be s u f f i c i e n t f o r i n d u c t i o n , i n a h i g h l y permeable mutant the r a p i d i n f l u x of a n t i b i o t i c might r e s u l t i n c e l l u l a r i n a c t i v a t i o n before e i t h e r h y d r o l y s i s of p e r i p l a s m i c beta-lactam by the uninduced beta-lactamase or beta-lactamase i n d u c t i o n could occur. In c o n t r a s t , i n the w i l d type s t r a i n , the escape of a few molecules of beta-lactam from h y d r o l y s i s by the uninduced beta-lactamase c o u l d , over time, r e s u l t i n i n d u c t i o n of t h i s beta-lactamase. Second, a decrease i n the p e r m e a b i l i t y of outer membranes as seen f o r s t r a i n P1-6 (Table VI) allowed f o r treatment with a l e v e l 128 ( i . e . 2 ug/ml) of inducer higher than the MIC f o r mutant M1 and consequent s l i g h t i n d u c t i o n of the beta-lactamase (Table V I I I ) . Even so, t h i s l e v e l of inducer was s t i l l 1 0 - f o l d lower than the minimum e x t e r n a l inducer c o n c e n t r a t i o n r e q u i r e d to induce beta-lactamase f u l l y i n w i l d type s t r a i n s . I t should be noted that even i f the beta-lactamase of the mutant has been a l t e r e d such that i t i s no longer i n d u c i b l e , i t i s d o u b t f u l that such an a l t e r a t i o n plays a major r o l e i n causing l o s s of r e s i s t a n c e i n t h i s s i t u a t i o n , where h y p e r s u s c e p t i b i l i t y of mutant M1 to a wide range of non-beta-lactam a n t i b i o t i c s i s i n v o l v e d (Table I I ) . The t o t a l l o s s of P • aeruginosa beta-lactamase i n s t u d i e s by Ohmori e_t a l . (1976) or the l o s s of beta-lactamase i n d u c i b i l i t y i n s t u d i e s by R o s s e l e t and Zimmermann (1973) d i d not r e s u l t i n l o s s of r e s i s t a n c e to the extent o c c u r r i n g i n mutant M1. In another experiment, c e l l s of the mutant s t r a i n M1 were shown to take up more of a f l u o r e s c e n t hydrophobic probe than the w i l d type s t r a i n (Table IX and F i g . 2). This evidence i m p l i e d that there was some a l t e r a t i o n i n hydrophobic uptake of the outer membrane, which c o r r e l a t e d with MIC data (Tables I I and I I I ) showing t h a t the mutant was more s u s c e p t i b l e to hydrophobic a n t i b i o t i c s than the w i l d type s t r a i n WT1 . Since the amount of hydrophobic uptake through the outer membrane i s normally very low i n gram negative c e l l s with smooth LPS (Nikaido, 1976), some d e f e c t must t h e r e f o r e be present i n the a n t i b i o t i c h y p e r s u s c e p t i b l e mutant which allowed enhanced permeation of these types of compounds. The mechanism of 129 enhanced hydrophobic type p e r m e a b i l i t y i n mutant M1 was presumably through the LPS-phospholipid b i l a y e r and was assumed to be due to at l e a s t one of the a l t e r a t i o n s i n LPS s t r u c t u r e which were r e p o r t e d . The LPS/phospholipid r a t i o s (Table XIV) were u n a l t e r e d i n the mutant M1, t h e r e f o r e outer membrane p e r t u r b a t i o n of the type suggested by Smit e_t a l . (1975) f o r deep rough s t r a i n s of Salmonellae was not r e s p o n s i b l e f o r outer membrane p e r m e a b i l i t y i n s t r a i n M1. In t h e i r model, the p h o s p h o l i p i d content of the outer membrane (and the outer l e a f l e t i n p a r t i c u l a r ) was proposed to be i n c r e a s e d i n deep rough mutants. S e v e r a l p o s s i b l e e x p l a n a t i o n s f o r enhanced permeation of hydrophobic agents were shown to e x i s t . F i r s t l y , the c h a r a c t e r i s t i c 0-antigen banding p a t t e r n of the mutant could be c o r r e l a t e d with s u s c e p t i b i l i t y to hydrophobic agents. S t r a i n s with the higher m o b i l i t y 0-antigen s p e c i e s ( M1, the p a r t i a l r e v e r t a n t s t r a i n s P1-4 and P1-6, and the AbsA transconjugants P2-6 and P2-6,8) each took up 2.5- to 6 - f o l d more hydrophobic probe (NPN - Table I X ) , and were 10 - f o l d more s u s c e p t i b l e to trimethoprim (Table I I I ) , than other s t r a i n s . This would seem to suggest that p o s s e s s i o n of the higher m o b i l i t y LPS chain l e n g t h s p e c i e s of AbsA s t r a i n s allowed hydrophobic uptake by e i t h e r i ) d e s t a b i l i s i n g the outer membrane i n g e n e r a l or i i ) exposing a normally unexposed s i t e on the outer membrane which permitted p a r t i t i o n i n g of hydrophobic compounds. Such a s i t e might be l i p i d i n nature. Evidence from dansyl polymyxin b i n d i n g favoured the l a t t e r 1 30 hypothesis and w i l l be d i s c u s s e d f u r t h e r . To understand how a change i n 0-antigen banding on g e l s might i n f l u e n c e hydrophobic p e r m e a b i l i t y , i t i s necessary to know what caused the a l t e r e d banding of LPS on g e l s . Although P. aeruginosa LPS i s heterogeneous, u n l i k e Salmonella LPS i t does not d i s p l a y an even d i s t r i b u t i o n of a l l 0-antigen chain l e n g t h s p e c i e s (Darveau and Hancock, 1983). Instead, as i s confirmed i n t h i s study, two types of smooth LPS p o p u l a t i o n s predominate i n both w i l d type and mutant M1, along with s m a l l e r amounts of other LPS p o p u l a t i o n s . The d i f f e r e n c e between these two s p e c i e s appears, on the s u r f a c e , to be s i z e , as suggested by s e v e r a l r e s e a r c h e r s (Jann e_t a^l. , 1975; Goldman and L e i v e , 19 80), however other reasons f o r d i f f e r e n t i a l m o b i l i t y on g e l s have not been r u l e d out. These could i n c l u d e a l t e r e d sugar contents or d i f f e r e n t i a l capping of amino group charges on 0-antigen m o i e t i e s by a c e t y l groups, however r e a c t i o n of 0-antigen from AbsA s t r a i n s P2-6 and P2-6,8 with 0-antigen s e r o t y p e - s p e c i f i c monoclonal antibody MA1-8, r a i s e d a g a i n s t w i l d type LPS ( F i g . 11) makes t h i s seem l e s s l i k e l y , and f r a c t i o n a t i o n by column chromatography (E. M GGroarty, manuscript i n p r e p a r a t i o n ) has demonstrated two such smooth LPS s p e c i e s : "long c h a i n " and " e x t r a long c h a i n " 0-antigen. Furthermore, no d i f f e r e n c e s i n the sugar composition of the smooth LPS f r a c t i o n s of s t r a i n s WT1 and M1 were observed ( K r o p i n s k i e_t a_l. , 1982). In any case, the d i f f e r e n c e between the two s p e c i e s of LPS must be created dur i n g s y n t h e s i s . In t h i s process, long 0-antigen chains are 131 f i r s t assembled, then t r a n s f e r r e d i n t a c t to rough core acceptor molecules (Osborn, 1979). Since two major d i s t r i b u t i o n s of LPS types predominate, i t appears that P.  aeruginosa t r a n s f e r enzymes ( or membrane c a r r i e r m o l e c u l e s ) , which are i n v o l v e d i n t r a n s f e r r i n g completed 0-antigen u n i t s onto the rough LPS acceptor molecule, are more s t r i n g e n t with r e s p e c t to 0-antigen type than those i n S_j_ typhimurium. There may be two enzyme v a r i a n t s which r e c o g n i s e d i f f e r e n t 0-antigen chain l e n g t h s p e c i e s or two rough core s p e c i e s to which d i f f e r e n t O-antigens are p r e f e r e n t i a l l y segregated. T h i s would mean that the AbsA phenotype might be caused by a mutation i n one of these t r a n s f e r enzymes such that i t r e c o g n i s e d a s h o r t e r 0-antigen s p e c i e s or i n the rough core composition such that the a b i l i t y to accept completed 0-antigen u n i t s from one of the t r a n s f e r a s e s was d e f e c t i v e or m o d i f i e d . I f the higher m o b i l i t y seen on SDS g e l s of e x t r a long chain smooth LPS molecules of M1 i s indeed due to s h o r t e r c h a i n l e n g t h , the d i f f e r e n c e i n l e n g t h between the two s t r a i n s may be q u i t e s u b s t a n t i a l , s i n c e i t was observed i n the upper p o r t i o n of the g e l . A d i f f e r e n c e i n Mp a u c h a s t h a t observed i n F i g . 10 would correspond roughly to a d i f f e r e n c e i n molecular weight ( f o r p r o t e i n s ) of roughly 20,000, e q u i v a l e n t to approximately 100 s i n g l e sugar m o i e t i e s ( or 20 to 30 LPS 0-antigen u n i t s ) . In theory, t h e r e f o r e , the a l t e r a t i o n i n 0-ant i g e n observed f o r mutant s t r a i n M1 could e a s i l y be r e s p o n s i b l e f o r enhanced hydrophobic p e r m e a b i l i t y . The other LPS s t r u c t u r a l change, that of the truncated 132 rough core ( AbsC), was a l s o present i n p a r t i a l r e v e r t a n t s , but not i n e i t h e r the AbsA or AbsB recombinant s t r a i n s . This suggested that the two LPS mutations (0-antigen and rough core) were not r e l a t e d . The c o n t r i b u t i o n of the core mutation toward hydrophobic p e r m e a b i l i t y could t h e r e f o r e not be a s c e r t a i n e d because i t was i n combination with other mutations i n p a r t i a l r e v e r t a n t s . Since the p a r t i a l r e v e r t a n t s were only s l i g h t l y more permeable to NPN than the AbsA s t r a i n s , the AbsC mutation seemed to have l i t t l e a d d i t i o n a l e f f e c t beyond that of AbsA. The acute hydrophobic p e r m e a b i l i t y seen i n mutant M1 may have been the r e s u l t of the lower number of rough core molecules which were s u b s t i t u t e d with 0-antigen i n t h i s s t r a i n (Darveau and Hancock, 1983; F i g . 10, lane F ) . The AbsB phenotype d i d not appear to c o n t r i b u t e to enhanced hydrophobic p e r m e a b i l i t y , s i n c e the transconjugant bearing t h i s u n i d e n t i f i e d mutation was not s u s c e p t i b l e to hydrophobic a n t i b i o t i c s and d i d not take up NPN a p p r e c i a b l y . I t should be pointed out, however, that these r e s u l t s ( i e . s h o r t e r 0-antigen l e n g t h a f f e c t i n g outer membrane hydrophobic p e r m e a b i l i t y ) appear to be i n d i r e c t c o n t r a s t to r e s u l t s obtained by Meadow and Wells (1985). These workers s e l e c t e d f o r mutants of P_^  aeruginosa with v a r i o u s LPS chain lengths and could only observe enhanced p e r m e a b i l i t y to hydrophobic compounds when s i g n i f i c a n t p o r t i o n s of core LPS were removed along with 0-antigen. Stud i e s by K r o p i n s k i e_t a l . (1978) showed s i m i l a r r e s u l t s , whereas Bryan e_t a_l (1984) found LPS a l t e r a t i o n s i n s t r a i n s i s o l a t e d f o r r e s i s t a n c e to 133 aminoglycosides. P o s s i b l y the r e s u l t s f o r M1 may r e f l e c t the other u n d e r l y i n g mutations i n t h i s s t r a i n . Work done by Peterson e_t a_l. (1 986 ) i n d i c a t e d that 0-antigen l e n g t h i n E.  c o l i may a c t u a l l y a f f e c t c a t i o n b i n d i n g to L i p i d A. These s t u d i e s were done using a c a t i o n i c s p i n l a b e l probe, CAT-12, with LPS of v a r y i n g chain lengths i s o l a t e d from w i l d type c e l l s . LPS probably has an important r o l e i n outer membrane f l u i d i t y , and 0-antigen chain l e n g t h may a f f e c t t h i s property as w e l l . Membrane f l u i d i t y has been repo r t e d to i n f l u e n c e s u s c e p t i b i l i t y to complement ( T a y l o r , 1983) and s o - c a l l e d serum (complement) - s e n s i t i v e gram negative c e l l s tend to be those with s h o r t chain LPS or rough LPS mutations. Mutant M1 was found to be c o n s i d e r a b l y more serum s e n s i t i v e compared to WT1 (L. Mutharia, unpublished r e s u l t s ) . LPS changes i n the mutant could t h e r e f o r e e i t h e r cause p o s s i b l e outer membrane f l u i d i t y a l t e r a t i o n s themselves or could be adapt a t i o n s to f l u i d i t y changes caused by another mutation. The c o n t r i b u t i o n which enhanced hydrophobic p e r m e a b i l i t y made to g e n e r a l outer membrane p e r m e a b i l i t y i n the mutant can a l s o be assessed. From data f o r p e r m e a b i l i t y to n i t r o c e f i n , i t was observed that only mutant M1 (and to a l e s s e r degree p a r t i a l r e v e r t a n t s P1-4 and P1-6) was s i g n i f i c a n t l y more permeable to the beta-lactam. Since the transconjugant s t r a i n P2-6 had w i l d type p e r m e a b i l i t y to n i t r o c e f i n , the AbsA phenotype t h e r e f o r e d i d not appear to a f f e c t outer membrane p e r m e a b i l i t y v i a the p o r i n pathway, as measured by t h i s 134 method. The r e f o r e , AbsA-mediated enhanced hydrophobic p e r m e a b i l i t y could not be s o l e l y r e s p o n s i b l e f o r the o v e r a l l a n t i b i o t i c h y p e r s u s c e p t i b i l i t y of mutant M1. The f a c t t h a t outer membrane p e r m e a b i l i t y was not a f f e c t e d by enhanced hydrophobic p e r m e a b i l i t y alone argued that n i t r o c e f i n d i d not enter i n t o the perip l a s m by the hydrophobic pathway i n w i l d type c e l l s . T h i s o b s e r v a t i o n was c o n s i s t e n t with s t u d i e s done by Nikaido e_t al.. , ( 1 9 8 3 ) , who p o s t u l a t e d that beta-lactam a n t i b i o t i c s enter the perip l a s m through p o r i n p r o t e i n s . T h i s was f u r t h e r confirmed f o r n i t r o c e f i n i n aeruginosa by Nicas and Hancock, 1983). The enhanced n i t r o c e f i n p e r m e a b i l i t y of the mutant M1 may t h e r e f o r e r e f l e c t e i t h e r i ) higher p o r i n a c t i v i t y , or i i ) uptake of n i t r o c e f i n through the LPS-phospholipid b i l a y e r , due to a more extreme d i s r u p t i o n of outer membrane i n t e g r i t y i n t h i s s t r a i n . Although the major p o r i n p r o t e i n F appeared s t r u c t u r a l l y i d e n t i c a l i n both s t r a i n s , on the b a s i s of protease d i g e s t i o n fragment a n a l y s i s and monoclonal antibody r e a c t i v i t y , p o r i n a c t i v i t y could c o n c e i v a b l y be r e g u l a t e d by i n t e r a c t i o n with other molecules, f o r example LPS, pep t i d o g l y c a n or other p r o t e i n s . An a l t e r a t i o n i n i n t e r a c t i o n s with other outer membrane p r o t e i n s could not be r u l e d out, although no apparent changes i n the p a t t e r n of outer membrane p r o t e i n s i n s t r a i n s M1 and WT1 were observed on SDS-polyacrylamide g e l s ( F i g . 4 ) . The i n t e r a c t i o n of p r o t e i n F with p e p t i d o g l y c a n was a l s o s u p e r f i c i a l l y unchanged i n the mutant, based on separate i s o l a t i o n procedures performed by A. 135 Carey and R. Darveau (unpublished r e s u l t s ) wherein i s o l a t i o n depended on a s s o c i a t i o n with p e l l e t t e d p e p t i d o g l y c a n complexes. A two-dimensional g e l a n a l y s i s of LPS i n t e r a c t i o n with i s o l a t e d p r o t e i n F from w i l d type and mutant s t r a i n s was attempted (R. Darveau, unpublished r e s u l t s ) but r e s u l t s were v a r i a b l e . Two pore s i z e s f o r P_j_ aeruginosa p r o t e i n F have r e c e n t l y been i d e n t i f i e d i n black l i p i d b i l a y e r experiments (Woodruff ejt a_l. , i n p r e s s ) , and which may be s u b j e c t to s p e c i f i c r e g u l a t i o n . Mutation i n an as yet unknown p o r i n r e g u l a t o r y system, which could r e g u l a t e f u n c t i o n a l pore s i z e , i s another p o s s i b i l i t y which could not be t e s t e d although, from the data of hydrophobic and dansyl polymyxin p e r m e a b i l i t y , an i n c r e a s e i n p o r i n a c t i v i t y i s not b e l i e v e d to be the major mechanism of enhanced outer membrane p e r m e a b i l i t y i n mutant M1. Furthermore, black l i p i d b i l a y e r s t u d i e s (R.P. Darveau and R.E.W. Hancock, unpublished o b s e r v a t i o n s ) f a i l e d to show an a l t e r a t i o n i n the d i s t r i b u t i o n of channel s i z e s of p r o t e i n Fs from WT1 and M1. In summary, then, c i r c u m s t a n t i a l evidence p o i n t s to d i s r u p t i o n of the LPS/phospholipid b i l a y e r as the major f a c t o r r e s p o n s i b l e f o r higher permeation of n i t r o c e f i n i n t o the peri p l a s m i n s t r a i n M1. N i t r o c e f i n was determined to be moderately hydrophobic by 1-octanol-water p a r t i t i o n i n g i n t h i s study (legend, Table V I ) , t h e r e f o r e a d i s r u p t i o n of the w i l d type b a r r i e r to hydrophobic uptake could allow p a r t i t i o n i n g of n i t r o c e f i n i n t o the hydrophobic i n t e r i o r of the c e l l envelope. V e s i c l e s t u d i e s of Yamaguchi e_t a_l. (1982) showed that beta-136 lactam a n t i b i o t i c s which were able to permeate more e a s i l y through the w a l l s of p h o s p h o l i p i d v e s i c l e s were a l s o more e f f e c t i v e a g a i n s t p o r i n - d e f i c i e n t mutants (Sawai e_t a l . , 1977). These r e s u l t s appear to i n d i c a t e t h a t , although the primary path f o r beta-lactam uptake across the outer membrane i s through p o r i n s , these agents can a l s o pass through the LPS/phospholipid b i l a y e r with v a r y i n g degrees of success based on the p h y s i c a l c h a r a c t e r i s t i c s of both the a n t i b i o t i c and the c e l l s u r f a c e . An i n t e r e s t i n g problem which i s r a i s e d by the study of a n t i b i o t i c h y p e r s u s c e p t i b l e mutants i s t h a t , g i v e n the poor a c t i v i t y which i s a s c r i b e d to aeruginosa p o r i n s and the c e n t r a l r o l e which t h i s poor a c t i v i t y a p p a r e n t l y p l a y s i n a n t i b i o t i c r e s i s t a n c e (Nikaido and Hancock, 1986), i t i s odd that a l l a n t i b i o t i c h y p e r s e n s i t i v e mutants of P_^  aeruginosa so f a r d e s c r i b e d (Hancock, 1984) are thought to be LPS- r a t h e r than p o r i n - d e f e c t i v e . P o s s i b l y the wrong s e l e c t i o n methods have been employed f o r P_^  aeruginosa, i n attempting to d u p l i c a t e the r e s u l t s obtained f o r c o l i , or perhaps mutations i n LPS occur more e a s i l y or are l e s s damaging to c e l l v i a b i l i t y . The t h i r d a l t e r a t i o n which was observed i n mutant M1 was r e l a t e d to c a t i o n s t a b i l i s a t i o n of the outer membrane. As d i s c u s s e d p r e v i o u s l y , d i v a l e n t c a t i o n s are proposed to c r o s s l i n k LPS molecules i n gram negative b a c t e r i a . Mg++ c r o s s b r i d g i n g of adjacent LPS molecules appears to be e s p e c i a l l y important i n P_^  aeruginosa (Nicas and Hancock, 137 1980), where exposure to Mg++_deficient c o n d i t i o n s l e d to i n d u c t i o n of a p r o t e i n , H1, which was proposed to r e p l a c e the c r o s s b r i d g i n g f u n c t i o n of Mg++ i n the outer membrane. Polymyxin B, to which JP^ aeruginosa i s r e l a t i v e l y s u s c e p t i b l e , appears to bi n d , i n p a r t , to c a t i o n b i n d i n g s i t e s i n the outer membrane of gram negative b a c t e r i a (Newton, 1960; Moore e_t a l . . 1 986 ). A dansylated polymyxin probe of these LPS s i t e s (Moore e_t a_l. , 1986) was t h e r e f o r e used to examine the c a t i o n b i n d i n g s i t e s i n wi l d type and mutant c e l l s . No major d i f f e r e n c e s could be detected between w i l d type and mutant c e l l s or i s o l a t e d LPSs with r e s p e c t to the extent of dansyl polymyxin b i n d i n g or Mg++ i n h i b i t i o n of t h i s b i n d i n g . However, i t was l i k e l y that a l a r g e number of c a t i o n b i n d i n g s i t e s were being probed i n these samples because P.  aeruginosa LPS has at l e a s t four dansyl polymyxin b i n d i n g s i t e s per molecule (Moore e_t a_l. , 1 986 ) . In the mutant M1 , most of these s i t e s were presumed to be u n a l t e r e d , t h e r e f o r e a s i g n i f i c a n t change i n dansyl polymyxin b i n d i n g at one s i t e could be masked by complete b i n d i n g at the other s i t e s . The p o r t i o n of LPS most l i k e l y to have a c r i t i c a l r o l e i n outer membrane s t a b i l i s a t i o n was L i p i d A, s i n c e i t i s the backbone s t r u c t u r e of LPS and i s found at the i n t e r f a c e of the medium and the hydrophobic core of the outer membrane. Each L i p i d A diglucosamine head group has a phosphate moiety at each end (Osborn, 1979), both of which are capable of b i n d i n g d a n s y l polymyxin i n w i l d type P_^  aeruginosa (Moore e_t a l . , 1 986 ) . Thus, each of these would be a p o t e n t i a l Mg++ bi n d i n g s i t e 138 depending on whether or not i t was s u b s t i t u t e d during s y n t h e s i s with a c h a r g e - n e u t r a l i s i n g r e s i d u e . The L i p i d A p o r t i o n s of adjacent LPS molecules have been proposed to be cr o s s b r i d g e d v i a these phosphate r e s i d u e s by d i v a l e n t c a t i o n s (Nicas and Hancock, 19 80) . Dansyl polymyxin b i n d i n g to i s o l a t e d L i p i d A from w i l d type and mutant was t h e r e f o r e examined. I t appeared that L i p i d A from mutant c e l l s bound approximately 25% l e s s dansyl polymyxin than that from w i l d type c e l l s ( F i g . 7 ) . Competition experiments with Mg + + gave r i s e to the c o n c l u s i o n that the s i t e s m issing from mutant s t r a i n M1 L i p i d A were the highest a f f i n i t y Mg++ b i n d i n g s i t e s , because the Mg + + competition curve f o r s t r a i n WT1 f e l l r a p i d l y through the high a f f i n i t y r e g i o n to assume the same l e v e l as the curve f o r M1. Logic demands that one think of such a l o s s of a f f i n i t y f o r dansyl polymyxin as o c c u r r i n g due to some mutational change i n one of the L i p i d A b i n d i n g s i t e s , r a t h e r than a simultaneous 25% lowering of a f f i n i t y f o r dansyl polymyxin at both phosphate r e s i d u e s , and, i n f a c t , the S Q ^ f o r d a n s y l polymyxin was q u i t e s i m i l a r f o r both L i p i d As. Although only two p o t e n t i a l b i n d i n g s i t e s e x i s t per L i p i d A molecule, and one might t h e r e f o r e expect a 50% l o s s of dansyl polymyxin b i n d i n g c a p a c i t y from l o s s of one s i t e , i n r e a l i t y L i p i d A i s a heterogeneous s t r u c t u r e (Osborn, 1979), much l i k e whole LPS (Goldman and L e i v e , 1980). A p a r t i c u l a r phosphate r e s i d u e (eg_. at the 4' p o s i t i o n of one glucosamine) i s not n e c e s s a r i l y s t o i c h i o m e t r i c a l l y modified with other m o i e t i e s i n 139 each L i p i d A molecule. In the case of mutant M1, the L i p i d A may be s u b s t i t u t e d at one of the two attached phosphates on 50$ of the L i p i d A molecules, c r e a t i n g two d i s t i n c t p o p u l a t i o n s of L i p i d A. This would e f f e c t i v e l y block the necessary negative charge at 25$ of the t o t a l L i p i d A Mg + + b i n d i n g s i t e s . T h i s e x p l a n a t i o n i s c o n s i s t e n t with 31p_NMR data from Dr. M B a t e l y (McQuarie Univ., unpublished o b s e r v a t i o n s ) using L i p i d A from s t r a i n s WT1 and M1 which I pro v i d e d . These data suggested that mutant M1 does indeed have the phosphate r e s i d u e at the 1 p o s i t i o n of diglucosamine 25$ s u b s t i t u t e d with an as yet u n i d e n t i f i e d carbohydrate moiety. Furthermore, an assay of phosphate from LPS and L i p i d A of each s t r a i n (Table XVI) showed that the mutant had a lower amount of assayable phosphate (20$ l e s s i n whole LPS and 40$ l e s s i n L i p i d A). An a d d i t i o n a l anecdotal piece of evidence to support l o s s of a Mg + + b i n d i n g s i t e was observed duri n g i s o l a t i o n of LPS using a method which employs Mg + + c r o s s b r i d g i n g of LPS to cause aggregation f o r c e n t r i f u g a l c o l l e c t i o n (Darveau and Hancock, 1983). I t was n o t i c e d that more s t r i n g e n t temperature c o n d i t i o n s and longer c e n t r i f u g a t i o n times were necessary to p e l l e t LPS from the mutant s t r a i n . T h i s phenomenon was presumed to be due to formation of s m a l l e r LPS aggregates, a p p a r e n t l y caused by l o s s of a p o r t i o n of Mg++ bi n d i n g s i t e s . Data f o r lysozyme l y s i s , u s i ng the Mg + + c h e l a t o r EDTA as p e r m e a b i l i s e r (Table X I I ) , i n d i c a t e d a lessened s e n s i t i v i t y to removal of Mg + + i n mutant M1, which a l s o agreed with t h i s p r o p o s a l . 1 40 The t o t a l number of a l t e r e d Mg++ bi n d i n g s i t e s which could r e s u l t from a b o l i t i o n of 25$ of t o t a l L i p i d A s i t e s would be 1 X 1()6 / c e l l (based on an estimate of 2 X 10 6 LPS molecules (or 4 X 10& L i p i d A phosphate s i t e s ) / c e l l - Angus et a l . , 1982). In a d d i t i o n , s i n c e Mg + + i s a d i v a l e n t i o n and LPS c r o s s b r i d g i n g i s n e c e s s a r i l y c o o p e r a t i v e , between 25 and 50$ of the c r o s s b r i d g i n g s i t e s could p o t e n t i a l l y be destroyed by a 25$ l o s s of dansyl polymyxin b i n d i n g s i t e s on L i p i d A, depending on the o r g a n i s a t i o n of Mg++ b r i d g i n g between molecules i n the membrane. Such an a l t e r a t i o n would cause profound outer membrane d e s t a b i l i s a t i o n , as suggested by Vaara and Vaara (1983a and b) f o r other b a c t e r i a l s p e c i e s . The reason that these e f f e c t s were not n o t i c e d i n whole c e l l d a n s y l polymyxin b i n d i n g assays was presumably because a d d i t i o n a l dansyl polymyxin b i n d i n g s i t e s on LPS r e t a i n e d t h e i r b i n d i n g a b i l i t y . From a l l of the above evidence, i t i s t h e r e f o r e l i k e l y that the major mutations causing h y p e r s u s c e p t i b i l i t y i n the mutant M1 caused a l t e r a t i o n s i n the phosphate s u b s t i t u e n t s of L i p i d A. P o s s i b l y the other LPS s t r u c t u r a l a l t e r a t i o n s (AbsA phenotype, at l e a s t ) served to s t a b i l i s e the primary mutation and, i n doing so, a l s o c o n t r i b u t e d to enhanced outer membrane p e r m e a b i l i t y . C e r t a i n other p r o p e r t i e s of the mutant M1 and i t s transconjugants were d i s c o v e r e d from experiments probing i n  viv o (whole c e l l ) self-promoted outer membrane p e r m e a b i l i t y . F i r s t l y , although mutant M1 was more s u s c e p t i b l e than were 141 w i l d type c e l l s to k i l l i n g by the aminoglycoside a n t i b i o t i c , gentamicin (Table I I ) , i t was appa r e n t l y at l e a s t two-fold l e s s s e n s i t i v e to outer membrane p e r m e a b i l i s a t i o n by the same a n t i b i o t i c (and by EDTA) to lysozyme (see F i g . 3 and Table X I I ) . Although these two r e s u l t s appear to c o n f l i c t , the i n s e n s i t i v i t y of the mutant M1 to p e r m e a b i l i s a t i o n can be r e a d i l y e x plained w i t h i n the context of the missing Mg + + b i n d i n g s i t e . As mentioned p r e v i o u s l y , l o s s of t h i s s i t e l i k e l y caused profound i n s t a b i l i t y i n the outer membrane, which would ease passage of most molecules, i n c l u d i n g gentamicin, across the outer membrane b a r r i e r , r e s u l t i n g i n the heightened s u s c e p t i b i l i t y phenotype. However, l o s s of a p o r t i o n of Mg + + b i n d i n g s i t e s may a l s o mean l o s s of an e q u i v a l e n t number of s p e c i f i c gentamicin i n t e r a c t i o n s i t e s . Gentamicin i s proposed to compete s u c c e s s f u l l y with Mg + + at the s t a b i l i s i n g s i t e , causing severe d i s r u p t i o n of the outer membrane b a r r i e r . T h i s might be c o n c e p t u a l i s e d as p h y s i c a l s e p a r a t i o n of LPS molecules due to s t e r i c problems when gentamicin attempts to f i l l the same niche as Mg + +. The magnitude of t h i s d i s r u p t i o n i s p r o p o r t i o n a l to the l e v e l of gentamicin used (Loh e_t a_l. , 1984). Loss of the aforementioned b i n d i n g s i t e on mutant L i p i d A may prevent t h i s process from o c c u r r i n g so t h a t , whereas the mutant outer membrane i s more permeable to gentamicin i t s e l f , i t i s not a f f e c t e d by the more severe p e r m e a b i l i s i n g e f f e c t s of t h i s agent ( i . e . p e r m e a b i l i s a t i o n to the 14,000 d a l t o n p r o t e i n , lysozyme). 142 Another apparent anomaly which needed e x p l a n a t i o n was that the transconjugant P2-6 had high p e r m e a b i l i s a t i o n to NPN by gentamicin but d i s p l a y e d w i l d type p e r m e a b i l i s a t i o n by gentamicin to lysozyme. In c o n t r a s t , transconjugant P2-8 showed i n t e r m e d i a t e p e r m e a b i l i s a t i o n by gentamicin to both NPN (Table XI) and lysozyme (Table X I I ) . I t t h e r e f o r e seems that these two assays, which were both designed to measure s e l f -promoted uptake i n w i l d type s t r a i n s , were not i n f a c t measuring the same parameter i n s t r a i n P2-6. One e x p l a n a t i o n f o r the behaviour of s t r a i n P2-6 i s that gentamicin caused w i l d type p e r m e a b i l i s a t i o n ( p o s i t i v e d i s r u p t i o n ) of the outer membrane i n t h i s s t r a i n , but i n the case of NPN, the pre-e x i s t i n g high uptake of the hydrophobic compound (Table XI) r e s u l t e d i n a b e r r a n t l y high NPN uptake when gentamicin was added. S t r a i n P2-8's low p e r m e a b i l i s a t i o n by gentamicin to lysozyme would seem to i n d i c a t e that t h i s s t r a i n possessed an a l t e r a t i o n of the L i p i d A Mg + + b i n d i n g s i t e seen i n s t r a i n M1, however p r e l i m i n a r y s t u d i e s of dans y l polymyxin b i n d i n g to L i p i d A from the transconjugant (data not i n c l u d e d ) f a i l e d to s u b s t a n t i a t e t h i s theory. The reason f o r low gentamicin p e r m e a b i l i s a t i o n i n the AbsB s t r a i n P2-8, j u s t l i k e i t s a n t i b i o t i c s u s c e p t i b i l i t y p a t t e r n , could not be a s c r i b e d to a s p e c i f i c phenotypic a l t e r a t i o n . I t should be pointed out that even i n the extreme case a f f o r d e d by the mutant M1, a r e l a t i v e l y modest change was observed i n v o l v i n g only a p o r t i o n of the t o t a l L i p i d A Mg + + s i t e s . Therefore i t may be that the AbsB mutation causes a more s u b t l e a l t e r a t i o n i n these s i t e s 143 ( a f f e c t i n g eg. 1C-5 s i t e s per c e l l i n s t e a d of 10 6 s i t e s i n the case of M1) and that the dansyl polymyxin assays used proved inadequate f o r probing t h i s s m a l l change. The c o o p e r a t i v e nature of LPS - LPS i n t e r a c t i o n s means that an appa r e n t l y minor change, such as a 25% l o s s of Mg + + b i n d i n g c a p a c i t y , can be en v i s i o n e d to cause major d i s r u p t i o n s of the c e l l envelope. Beside the c o o p e r a t i v e Mg + + c r o s s l i n k i n g of LPS, other interdependent f u n c t i o n s have a l s o been a s c r i b e d to have a r o l e i n a n t i b i o t i c s u s c e p t i b i l i t y . For example, the r a t e of p e r m e a b i l i t y to a n t i b i o t i c s which face d e s t r u c t i v e p e r i p l a s m i c enzymes (eg_. beta-lactamase) p l a y s an important r o l e i n r e s i s t a n c e . Maintenance of a very slow beta-lactam permeation r a t e across the outer membrane w i l l g r e a t l y i n c r e a s e the chances f o r complete h y d r o l y s i s of the a n t i b i o t i c before i t can i n t e r a c t with b i o s y n t h e t i c t a r g e t p r o t e i n s (see I n t r o d u c t i o n - Richmond and C u r t i s , 1974). The combination, i n mutant M1, of both the major a n t i b i o t i c s u s c e p t i b i l i t y mutation ( l o s s of Mg++ bi n d i n g s i t e on L i p i d A) and the a d d i t i o n a l i n c r e a s e i n hydrophobic outer membrane p e r m e a b i l i t y (due to the AbsA phenotype) as w e l l as an undefined a l t e r a t i o n i n self-promoted uptake (due to the AbsB mutation) presumably combined to cause the observed moderate changes i n outer membrane p e r m e a b i l i t y . This enhancement of p e r m e a b i l i t y i s p o s t u l a t e d to have caused, i n t u r n , the extreme i n c r e a s e i n a n t i b i o t i c s u s c e p t i b i l i t y of M1 by three interdependent mechanisms: i ) d e s t a b i l i s a t i o n of the outer membrane v i a a lack of 144 i n t e r a c t i o n between LPS molecules, i i ) i n t e r a c t i o n between enhanced hydrophobic uptake (tr u n c a t e d 0-antigen) and d e s t a b i l i s e d L i p i d A backbone, and i i i ) l o s s of synergy between the outer membrane b a r r i e r and p e r i p l a s m i c beta-lactamase, due to the e f f e c t of enhanced d i f f u s i o n across the outer membrane as w e l l as on i n d u c i b i l i t y of beta-lactamase. These a l t e r a t i o n s are summarised i n the model i n F i g . 12. From study of an a n t i b i o t i c h y p e r s u s c e p t i b l e mutant one can draw c e r t a i n c o n c l u s i o n s about the s t r u c t u r e s r e s p o n s i b l e f o r a n t i b i o t i c r e s i s t a n c e i n w i l d type P_^  a eruginosa. As had been demonstrated p r e v i o u s l y , low p o r i n a c t i v i t y i s undoubtedly a major f a c t o r i n r e s i s t a n c e . I f there i s a gen e r a l c o n c l u s i o n one can draw from t h i s study, i t i s that LPS of P_^  aeruginosa a l s o appears to play a powerful r o l e i n a n t i b i o t i c r e s i s t a n c e and outer membrane p e r m e a b i l i t y i n g e n e r a l . In a d d i t i o n , Mg + + c r o s s b r i d g i n g of LPS to f u l l c a p a c i t y seems to be c r i t i c a l , and maintenance of at l e a s t one c l a s s of LPS molecules with e s p e c i a l l y long 0-antigen chain l e n g t h i s important f o r r e s i s t a n c e to hydrophobic agents. I t i s t h e r e f o r e apparent that even minor s t r u c t u r a l changes i n w i l d type LPS can produce major r e p e r c u s s i o n s i n the f u n c t i o n of the outer membrane as an e f f e c t i v e b a r r i e r to d e s t r u c t i v e agents. 145 Figure 12. Model suggesting how LPS a l t e r a t i o n s i n an a n t i b i o t i c h y p e r s u s c e p t i b l e mutant of P_^  aeruginosa (M1) a f f e c t p e r m e a b i l i t y . The outer l e a f l e t of the outer membrane i s diagrammed above, showing the two predominant 0-antigen chain length s p e c i e s of LPS molecules (L) (with r e p r e s e n t a t i v e Mg++ c r o s s b r i d g i n g ) , as w e l l as a p o r i n molecule (P). The p u t a t i v e s i t e s of entry or i n t e r a c t i o n of v a r i o u s types of agents with the outer membrane are a l s o shown f o r both wild type and mutant s t r a i n M1. A, wil d type; B, mutant M1; AG, aminoglycosides; HPHI, h y d r o p h i l i c agents; HPHO, hydrophobic agents. 146 CHAPTER TWO CHEMICAL CROSSLINKING OF PSEUDOMONAS AERUGINOSA AND  ESCHERICHIA COLI OUTER MEMBRANE PORIN PROTEINS TO REVEAL  NATIVE OLIGOMERS I n t r o d u c t i o n E a r l y s t u d i e s on the p o r i n s of E s c h e r i c h i a c o l i i n d i c a t e d that these p r o t e i n s e x i s t i n t h e i r n a t i v e form as oligomers. C e r t a i n p h y s i c a l evidence such as the low m o b i l i t y of p o r i n p r o t e i n OmpF a f t e r i n c u b a t i o n at moderate temperatures i n SDS p r i o r to a n a l y s i s by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (Nakae e_t a_l. , 1979 ) and, more d i r e c t l y , sedimentation e q u i l i b r i u m experiments (Yu et_ a_l. , 1 979 ) s t r o n g l y pointed to a n a t i v e t r i m e r i c a s s o c i a t i o n of t h i s major E_^ c o l i p o r i n , as w e l l as the major p o r i n of S.  typhimurium ( I s h i i and Nakae, 1980; Tokunaga et a l . , 1979). These data were confirmed by c r o s s l i n k i n g (Reithmeier and Bragg, 1977; Palva and R a n d a l l , 1978) and o p t i c a l d i f f r a c t i o n s t u d i e s (Dorset e_t a_l. , 1983). Other E^ _ c o l i p o r i n s (OmpC and LamB) were a l s o shown to be o l i g o m e r i c by v i r t u e of t h e i r apparent molecular weights on SDS-polyacrylamide g e l s a f t e r s o l u b i l i s a t i o n under non-denaturing c o n d i t i o n s ( i e . temperatures < 60°C). In c o n t r a s t , Pseudomonas aeruginosa p o r i n p r o t e i n F formed monomer bands on SDS-polyacrylamide g e l e l e c t r o p h o r e s i s even a f t e r s o l u b i l i s a t i o n i n SDS at room temperature (Hancock and Carey, 1979). One other P_;_ aeruginosa p o r i n , D1, was a l s o s e n s i t i v e to SDS, whereas another, p r o t e i n 147 P, showed oligomers a f t e r low temperature SDS s o l u b i l i s a t i o n . Sedimentation e q u i l i b r i u m a n a l y s i s was not attempted f o r the P. aeruginosa p o r i n p r o t e i n F due to the t i g h t a s s o c i a t i o n of l i p o p o l y s a c c h a r i d e seen duri n g i s o l a t i o n of the p r o t e i n (Hancock and Carey, 1 979 ; Parr e_t a_l. , 1 986 ) , which would have hampered i n t e r p r e t a t i o n of sedimentation r e s u l t s . C r o s s l i n k i n g t h e r e f o r e appeared to be a most v i a b l e a l t e r n a t i v e method f o r determining n a t i v e a s s o c i a t i o n s i n Pseudomonas p o r i n p r o t e i n s . Chemical c r o s s l i n k i n g has been widely used i n a v a r i e t y of systems to demonstrate n a t i v e a s s o c i a t i o n s of p r o t e i n s ( J i , 1979; Freedman, 1979). A v a r i e t y of agents have become a v a i l a b l e which allow a) choice of c r o s s l i n k a b l e f u n c t i o n a l groups on the p r o t e i n , b) d i f f e r e n t degrees of h y d r o p h o b i c i t y s u i t e d to the p r o t e i n ' s environment, c) c l e a v a b i l i t y and d) d i f f e r e n t span lengths of the c r o s s l i n k e r . Reagents l i n k i n g amino or s u l p h h y d r y l groups, and p h o t o a c t i v a t i b l e azido bonds have been employed • ( J i , 1979). A n a l y s i s of c r o s s l i n k e d products i n v o l v e s o b s e r v a t i o n s of s h i f t s i n m o b i l i t y on SDS-p o l yacrylamide g e l s due to a d d i t i o n of the reagent. A more s o p h i s t i c a t e d technique i n v o l v e s alignment of c r o s s l i n k e d products with t h e i r cleaved components on two-dimensional g e l s ( J i , 1979; Palva and R a n d a l l , 1976), where the r e l a t i v e i n t e n s i t y of each c r o s s l i n k e d s p e c i e s can a l s o give an i n d i c a t i o n of the most favoured o l i g o m e r i c a s s o c i a t i o n . C o n d i t i o n s of r e a c t i o n must be adjusted to minimise unproductive monofunctional r e a c t i o n of c r o s s l i n k e r , 148 spontaneous aggregation of i s o l a t e d p r o t e i n s , and c r o s s l i n k i n g due to chance con t a c t between p r o t e i n s , a l l of which are p o t e n t i a l hazards of the technique. Most of these can be c o r r e c t e d simply by a d j u s t i n g s o l u b i l i s i n g c o n d i t i o n s , r e l a t i v e amounts of reagent and p r o t e i n , and the time allowed f o r r e a c t i o n . With s t r i n g e n t l y a p p l i e d c o n d i t i o n s , c r o s s l i n k i n g i s a va l u a b l e technique f o r i n d i c a t i o n of n a t i v e oligomers, however i t cannot provide s o l e proof of such a s s o c i a t i o n s . For t h i s study, a moderately hydrophobic, amino group-r e a c t i v e , 1.2 nm long, c l e a v a b l e reagent, d i t h i o - b i s -( s u c c i n i m i d y l propionate) (DSP - Lomant and Fa i r b a n k s , 1976; Reithmeier and Bragg, 1977) was deemed to be most s u i t a b l e f o r i n t e g r a l outer membrane p r o t e i n s . The bridge l e n g t h and r e a c t i o n time were chosen to allow r e a c t i o n only with subunits i n c l o s e p r o x i m i t y , to t r y to e l i m i n a t e any r e a c t i o n which could be due to random c o l l i s i o n of p r o t e i n s . A cleavage s i t e comprised of a d i s u l p h i d e bond was a l s o present i n the middle of DSP a l l o w i n g d i s s o c i a t i o n of c r o s s l i n k e d product with 2-ME and thus a n a l y s i s i n a two-dimensional SDS-polyacrylamide g e l system. Chemical c r o s s l i n k i n g s t u d i e s on outer membrane p r o t e i n s of E_^_ c o l i (Reithmeier and Bragg, 1977) and S.  typhimurium ( P a l v a , 1980) were used to demonstrate o l i g o m e r i c a s s o c i a t i o n s f o r p r o t e i n s OmpF and OmpC (E_^ c o l i ) as w e l l as f o r the 45K p r o t e i n of JLi. typhimurium. Pseudomonas aeruginosa p o r i n p r o t e i n s possess c h a r a c t e r i s t i c s of s i m i l a r i t y and 1 49 d i s s i m i l a r i t y with r e s p e c t to the above E_^  c o l i and Salmonella p o r i n s . For example, although aeruginosa p r o t e i n F appears to be of s i m i l a r molecular weight as E_^  c o l i p r o t e i n OmpF ( 3 8 K ) , i t has been shown to form both l a r g e r (5nS) and smaller (O.36 nS) h y d r o p h i l i c channels i n an a r t i f i c i a l b i l a y e r membrane system (Woodruff e_t a_l. , i n press) and (as mentioned above) i s l e s s s t a b l e to SDS d e n a t u r a t i o n to the monomer form. P r o t e i n P, induced under phosphate s t a r v a t i o n c o n d i t i o n s and SDS-stable (Hancock e_t a_l. , 1 982), i s t h e r e f o r e analogous to E^ _ c o l i p r o t e i n PhoE (Overbeeke and Lugtenberg, 19 8 0; Tommassen and Lugtenberg, 1 9 8 0 ) but has been shown to form s m a l l e r (0.2nS), more a n i o n - s p e c i f i c pores than PhoE (Hancock et a l . , 1982) and i s of a l a r g e r apparent molecular weight on SDS-polyacrylamide g e l s (48K vs. 3 8 K ) . I t was t h e r e f o r e of i n t e r e s t to compare the n a t i v e a s s o c i a t i o n s of these p r o t e i n s using the c r o s s l i n k i n g technique. Evidence s u p p o r t i n g an o l i g o m e r i c a s s o c i a t i o n of each of these p r o t e i n s was obtained i n t h i s study. 150 RESULTS 1 . C r o s s l i n k i n g of E. c o l i outer membrane p r o t e i n PhoE As a c o n t r o l f o r s t u d i e s with the P_^_ aeruginosa p o r i n s , and to demonstrate the v a l i d i t y of my techniques, I f i r s t undertook c r o s s l i n k i n g s t u d i e s of p o r i n p r o t e i n PhoE. P r o t e i n PhoE of E_^  c o l i has r e c e n t l y been shown to be a pore-forming p r o t e i n which i s thr e e - to f o u r - f o l d s e l e c t i v e f o r anions over c a t i o n s ( K o r t e l a n d et^ a_l. , 1982; Benz et; a^l. , 1984). In the unheated form on SDS poly a c r y l a m i d e g e l s , i t forms an oligomer with a s i m i l a r e l e c t r o p h o r e t i c behaviour to other E_j_ c o l i p o r i n s (Benz e_t a^l. , 1984). In a d d i t i o n , the s t r u c t u r a l gene has been i s o l a t e d by Tommassen and c o l l e a g u e s and shown to demonstrate strong n u c l e i c a c i d and primary amino a c i d sequence (64$) homology with the OmpF p o r i n of E_;_ c o l i (Tommassen e_t a_l, , 1982). T h e r e f o r e , i t may be expected that t h i s p r o t e i n would be a tr i m e r l i k e the OmpF p o r i n . T h i s was confirmed by c r o s s l i n k i n g and e l e c t r o p h o r e t i c a n a l y s i s i n both one and two dimensions (Figure 13). When c r o s s l i n k e d p u r i f i e d PhoE p r e p a r a t i o n s run on f i r s t dimension SDS polyacrylamide g e l s were t r e a t e d with 2 -mercaptoethanol to break the c r o s s l i n k b r i d g e s and then run i n the second dimension, evidence of multimers was observed i n s i l v e r s t a i n e d g e l s ( F i g u r e 13). The major spots seen by s i l v e r s t a i n i n g were confirmed to be proteinaceous using 35s-m e t h i o n i n e - l a b e l l e d outer membranes ( f o r p r o t e i n F) and Coomassie b l u e - s t a i n e d g e l s ( f o r a l l p r o t e i n s ) . A r t i f a c t 151 F i g u r e 13- One- and two-dimensional c r o s s - l i n k i n g a n a l y s i s of p u r i f i e d E. c o l i p r o t e i n Pho E. P u r i f i e d p r o t e i n Pho E was t r e a t e d with 500 ug DSP/mg p r o t e i n and run on an 11$ pol y a c r y l a m i d e g e l . The f i r s t dimension g e l was cut out, soaked i n r e d u c t i o n mix c o n t a i n i n g 2-mercaptoethanol to break DSP c r o s s l i n k s and then the lane l a i d on top of another 11$ acrylamide g e l and run i n the second dimension as d e s c r i b e d i n Methods. The f i r s t dimension g e l showed c r o s s l i n k e d products: Lane A - Pho E untreated c o n t r o l ; lane B - Pho E + DSP. Products observed i n the f i r s t and/or second dimension g e l s were an i n t e r n a l l y c r o s s l i n k e d monomer ( E i 1 ) , dimers (E2), t r i m e r s (E3)» and a small amount of higher molecular weight p r o d u c t ( s ) of unknown d e r i v a t i o n . The running p o s i t i o n s of the monomer of the Pho E p r o t e i n i n the f i r s t dimension and the monomer and spots d e r i v e d from c r o s s l i n k e d oligomers i n the second dimension are l a b e l l e d on the l e f t hand s i d e . 152 P h o E 3 -P h o E 2 -P h o E 67 K 45 K 30 K s t r e a k s seen across g e l s at apparent molecular weights of 60,000 and 67,000 d a l t o n s were enhanced by s i l v e r s t a i n i n g and were p o s s i b l y due to i m p u r i t i e s i n the reducing agent (Tasheva and Dessev, 1983). These blemishes of two-dimensional p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s have a l s o been r e p o r t e d by other authors (Guevara e_t ajL. , 1982; M a r s h a l l and W i l l i a m s , 1984). Multimers appeared as spots below the d i a g o n a l l i n e of u n c r o s s l i n k e d p r o t e i n s i n F i g u r e 13, due to t h e i r higher m o b i l i t y a f t e r 2-mercaptoethanol cleavage between the f i r s t and second dimensions. The cleaved spots were h o r i z o n t a l l y a l i g n e d to the p r o t e i n from which they were o r i g i n a l l y d e r i v e d . This confirmed that the higher molecular weight products seen i n the Coomassie-stained one dimensional g e l s ( F i g . 13, lane A) were d e r i v e d from the PhoE p r o t e i n and were not a r t i f a c t s . The p u r i f i e d 38,000 d a l t o n monomer was f r e e l y c r o s s l i n k e d to form a 34,000 d a l t o n band, which was probably due to an i n t r a c h a i n bridge s i n c e t h i s could r e s u l t i n higher g e l m o b i l i t y and s i n c e PhoE was demonstrated to possess s e v e r a l (14) f r e e amino groups (Hancock e_t a_l. , i n p r e s s ) . A high p r o p o r t i o n of dimer of 73,000 d a l t o n s and a t r i m e r of 100,000 d a l t o n s were a l s o observed. In a d d i t i o n , a band of estimated molecular weight 140,000 d a l t o n s was seen i n two-dimensional a n a l y s i s . Since l a r g e amounts of a s i m i l a r product ( c a l l e d band C) were seen i n c r o s s l i n k i n g experiments using p u r i f i e d OmpF p r o t e i n , and ex p l a i n e d by the authors as due to aberrant m o b i l i t y i n g e l s or as an aberrant 154 conformation of an oligomer i n SDS (Palva and R a n d a l l , 1978), t h i s product may not n e c e s s a r i l y r e p r e s e n t a tetramer. T h e r e f o r e , these s t u d i e s demonstrated that our b a s i c c r o s s l i n k i n g techniques were sound and suggested that PhoE, l i k e other E_j_ c o l i p o r i n s , was a t r i m e r . 2. C r o s s l i n k i n g of P. aeruginosa p r o t e i n P_ P r o t e i n P, l i k e the PhoE p r o t e i n , i s induced by phosphate s t a r v a t i o n (Hancock e_t .al. , 1982). Unlike other P_^  aeruginosa p o r i n s (but l i k e the PhoE p r o t e i n ) , i t forms s t a b l e oligomers on SDS polyacrylamide g e l s a f t e r s o l u b i l i s a t i o n at low temperatures (Hancock e_t aJL. , 1982). I t d i f f e r s from the PhoE p r o t e i n i n that i t has a higher apparent monomer molecular weight of 48,000 d a l t o n s ( F i g s . 13 and 14), a s m a l l e r channel s i z e and a higher s e l e c t i v i t y f o r anions i n that i t i s 100-f o l d s e l e c t i v e f o r anions over c a t i o n s i n black l i p i d b i l a y e r s (Hancock e_t a_l. , 1982; Benz e_t aJL. , 1983). Four c r o s s l i n k e d products of the 48,000 d a l t o n monomer of p r o t e i n P (of estimated molecular weights 44,000, 80,000, 90,000 and 145,000 da l t o n s could be v i s u a l i s e d ( F i g u r e 14). The 44,000 d a l t o n spot was presumably due to an i n t r a c h a i n c r o s s l i n k which caused t i g h t e r f o l d i n g of the p r o t e i n and thus a lower apparent molecular weight. S i m i l a r l y , the 80,000 d a l t o n band was p o s s i b l y a dimer of subun i t s with an i n t r a c h a i n c r o s s l i n k . Based on molecular weight, the 90,000 d a l t o n band was most l i k e l y a dimer of the 48,000 d a l t o n p r o t e i n P monomer. Another spot seen on both the one and two-155 F i g u r e 14. One- and two-dimensional c r o s s - l i n k i n g a n a l y s i s of p u r i f i e d P. aeruginosa p r o t e i n P. P u r i f i e d p r o t e i n P was t r e a t e d as i n F i g . 13 except that a 9% polyacrylamide g e l was used. C r o s s l i n k e d products were observed i n both the f i r s t amd second dimension g e l s : Lane A, u n c r o s s l i n k e d c o n t r o l ; lane B, f i r s t dimension DSP c r o s s l i n k e d p r o t e i n P . Products i n c l u d e d a monomer of p r o t e i n P^  with an i n t e r n a l c r o s s l i n k r e s u l t i n g i n lower apparent molecular weight ( P ± 1 ) , a dimer of p r o t e i n P., ( P 2 ) , a dimer with at l e a s t one i n t e r n a l c r o s s l i n k ( p i 2 ) a n d a t r i m e r of p r o t e i n P. ( p , ) . The running p o s i t i o n s of the monomer of p r o t e i n P i n the f i r s t dimension and the monomer and spots d e r i v e d from c r o s s l i n k e d products i n the two-dimensional g e l are i n d i c a t e d on the l e f t hand s i d e . 156 / 5 7 dimensional g e l s probably corresponded to a t r i m e r of p r o t e i n P, with an estimated molecular weight of 145,000 d a l t o n s . This product was present at lower amounts i n the g e l than the t r i m e r of the PhoE p r o t e i n , p o s s i b l y due to e i t h e r a) i n s u f f i c i e n t c r o s s l i n k e r , b) a higher tendency to form i n t r a c h a i n c r o s s l i n k s (as evidenced by the l a r g e amount of the 80,000 d a l t o n band f o r which no corresponding product was observed a f t e r c r o s s l i n k i n g of the PhoE p r o t e i n ) or c) c r o s s l i n k i n g to l i p o p o l y s a c c h a r i d e (LPS) which c o - p u r i f i e d with p r o t e i n P and which has f r e e amino groups i n i t s 0-antigen (Koval and Meadow, 1975). C r o s s l i n k e r was used at an approximate molar r a t i o of DSP : p r o t e i n P of 60. In order to r u l e out i n s u f f i c i e n t c r o s s l i n k e r as the reason, two-fold more c r o s s l i n k e r was added and s t i l l only l i m i t e d amounts of t r i m e r were observed on one dimensional g e l s . At s t i l l higher c o n c e n t r a t i o n s of c r o s s l i n k e r most of p r o t e i n P (and p r o t e i n F, see below) no longer entered the g e l as monomers or oligomers, presumably due to the formation of SDS-insoluble complexes with c r o s s l i n k e r (the p r o t e i n was probably not simply i n the form of l a r g e c r o s s l i n k e d complexes s i n c e we d i d not observe p r o t e i n s t a i n i n g i n the s t a c k i n g g e l ) . This confirms the data of other workers who have shown that higher c o n c e n t r a t i o n s of c r o s s l i n k e r (up to 500-fold molar excess over p r o t e i n c o n c e n t r a t i o n was used f o r p r o t e i n P i n the above case) can r e s u l t i n n o n - s p e c i f i c c r o s s l i n k i n g (Palva and R a n d a l l , 1978; Freedman, 1979; J i , 1979) and presumably s a t u r a t i o n of amino groups on the p r o t e i n . 158 3. C r o s s l i n k i n g of p u r i f i e d P. aeruginosa p r o t e i n F_ The p u r i f i c a t i o n procedure f o r p r o t e i n F ( i n v o l v i n g s o l u b i l i s a t i o n i n T r i t o n X-100 - EDTA; Hancock e_t a_l. , 1979) r e t a i n e d the p r o t e i n ' s pore-forming a c t i v i t y i n black l i p i d b i l a y e r s and p r o t e i n F was t h e r e f o r e presumed to be s t i l l i n i t s n a t i v e or a c t i v e conformation. C r o s s l i n k i n g of p u r i f i e d p r o t e i n F c l e a r l y r e v e a l e d a dimer product of 70,000 d a l t o n s ( F i g u r e 15). In a d d i t i o n , a smear of p r o t e i n with an average molecular weight of 120,000 was observed. These oligomers were only seen i n two dimensional a n a l y s e s , although broad areas of s t a i n i n g corresponding to dimer and t r i m e r products were observed i n s i l v e r s t a i n e d one dimensional g e l s ( i e . p r i o r to cleavage of c r o s s l i n k s ) . C r o s s l i n k e r c o n c e n t r a t i o n s were v a r i e d over a 500-fold range (4 ug to 2 mg/ mg p r o t e i n f o r both p u r i f i e d p r o t e i n F (not shown) and outer membranes (see F i g u r e 16 where a 150-fold c o n c e n t r a t i o n range i s demonstrated), and the time of treatment with c r o s s l i n k e r v a r i e d f r o n 30 sec to 30 min. At no c r o s s l i n k e r c o n c e n t r a t i o n were sharp oligomer bands seen, although the p r o t e i n F monomer band disappeared at higher c r o s s l i n k e r c o n c e n t r a t i o n s (0.2 - 1 mg DSP per mg p r o t e i n depending on the treatment t i m e s ) . At the same time, when the c r o s s l i n k e d products were run i n the f i r s t dimension then cleaved and run i n the second dimension, broad spots with average molecular weights of 70,000 and 100,000 d a l t o n s appeared as d i s c u s s e d above. The experiment demonstrated i n F i g u r e 15 was performed at a c o n c e n t r a t i o n at 159 F i g u r e 15. T w o - d i m e n s i o n a l c r o s s l i n k i n g a n a l y s i s o f p u r i f i e d P. a e r u g i n o s a p r o t e i n F . P u r i f i e d p r o t e i n F was t r e a t e d as i n F i g . 13* C r o s s l i n k e d p r o d u c t s i n c l u d e d d i n e r s (F2) and t r i m e r s (F3) o f p r o t e i n F as w e l l as h i g h e r m o l e c u l a r weight c r o s s l i n k e d p r o d u c t s o f p r o t e i n F (F ) . n 160 which 60$ of the p r o t e i n F disappeared from the monomer p o s i t i o n . I t was estimated that approximately 40$ appeared at a molecular weight e q u i v a l e n t to dimer ( F 2 ) and 20$ appeared as higher oligomers i n c l u d i n g apparent t r i m e r s ( F 3 ) . The higher oligomers were not f u r t h e r c h a r a c t e r i s e d but may be r e l a t e d to the oligomers of high molecular weight seen f o r PhoE ( F i g u r e 13) and other E_^  c o l i p o r i n s (Palva and R a n d a l l , 1978) . In these and a l l other p r o t e i n F c r o s s l i n k i n g experiments, the 38,000 d a l t o n monomer of p r o t e i n F appeared s l i g h t l y above the di a g o n a l due to the e f f e c t of 2-mercaptoethanol, which broke n a t i v e i n t r a c h a i n d i s u l p h i d e b r i d g e s , causing the p r o t e i n to u n f o l d f u r t h e r and r a i s i n g i t s apparent molecular weight from 35,000 to 38,000 d a l t o n s i n the second dimension, as d e s c r i b e d p r e v i o u s l y (Hancock and Carey, 1979) . 4. C r o s s l i n k i n g of p r o t e i n s i n P. aeruginosa outer  membranes with v a r i o u s c o n c e n t r a t i o n s of c r o s s l i n k e r Because the data obtained f o r p u r i f i e d p r o t e i n F were more d i f f i c u l t to i n t e r p r e t than those obtained f o r p r o t e i n s PhoE and P, there was a p o s s i b i l i t y t h a t the apparent o l i g o m e r i c a s s o c i a t i o n of p r o t e i n F was due to an a r t i f a c t u a l a s s o c i a t i o n of p r o t e i n F monomers induced during p u r i f i c a t i o n . The use of outer membranes f o r c r o s s l i n k i n g experiments allowed lower molar r a t i o s of c r o s s l i n k e r to be used (compare F i g u r e 15 and F i g u r e 17) than those used with p u r i f i e d p r o t e i n F, presumably due to the higher c o n c e n t r a t i o n of p r o t e i n F i n 161 Figure 16. One-dimensional SDS-polyacrylamide g e l e l e c t r o -p h o r e s i s of outer membrane samples t r e a t e d with v a r i o u s c o n c e n t r a t i o n s of DSP f o r 2 min. C r o s s l i n k e d outer membranes were run on an 11$ polyacrylamide g e l as d e s c r i b e d i n Methods. Concentrations of DSP i n d i c a t e d beneath the i n d i v i d u a l l a n e s are expressed as jug/mg p r o t e i n . C r o s s l i n k i n g was v i s u a l i s e d i n the f i r s t dimension as a decrease i n s t a i n i n g i n t e n s i t y of p r o t e i n bands and a concomitant i n c r e a s e i n amount of high molecular weight m a t e r i a l unable to enter the s e p a r a t i n g g e l . 162 outer membrane c r o s s l i n k i n g experiments. At c o n c e n t r a t i o n s of c r o s s l i n k e r (40 ug DSP/mg p r o t e i n ) causing minimal a l t e r a t i o n of outer membrane p r o t e i n p a t t e r n s (see F i g u r e 16), the only c r o s s l i n k e d product observed i n two dimensional analyses was a dimer of 70,000 d a l t o n s (Figure 17). Using another c l e a v a b l e c r o s s l i n k e r (dimethyl 3,3'-d i t h i o b i s - p r o p i o n i m i d a t e ) at higher c o n c e n t r a t i o n s , up to an estimated 20% of the monomer could be converted to dimer without the appearance of other c r o s s l i n k e d products i n the a n a l y s i s (data not shown). At a r e l a t i v e l y high c o n c e n t r a t i o n of DSP (200 ug/mg p r o t e i n ; N.B. the treatment time used here was l e s s than that i n Fi g u r e 17), p r o t e i n F was c r o s s l i n k e d to form v i s i b l e dimers of estimated molecular weight 70,000 dalt o n s and a streak of higher molecular weight products (F i g u r e 18), some due to multimers and some ap p a r e n t l y due to a s s o c i a t i o n s with other p r o t e i n s or l i p o p o l y s a c c h a r i d e (LPS). In order to confirm that p r o t e i n F could be c r o s s l i n k e d to LPS, a two-dimensional g e l of c r o s s l i n k e d outer membranes was s t a i n e d f o r LPS (Figure 19) by the p e r i o d a t e / s i l v e r s t a i n method of T s a i and Frasch (1982), which appeared to block s t a i n i n g of a l l major p r o t e i n s . S e v e r a l c r o s s l i n k e d spots not p r e v i o u s l y seen on g e l s s i l v e r - s t a i n e d f o r p r o t e i n were observed at lower apparent molecular weights than the p r o t e i n F oligomers seen i n F i g u r e s 15 and 17. I t thus appeared that c e r t a i n s p e c i e s of LPS could a l s o be c r o s s l i n k e d by DSP, e i t h e r to other LPS molecules or to p r o t e i n s . By v e r t i c a l l y a l i g n i n g the c r o s s l i n k e d products of g e l s s t a i n e d f o r p r o t e i n 163 Figure 17. Two-dimensional c r o s s l i n k i n g of outer membranes usi n g 40 jjg DSP/mg p r o t e i n . Outer membranes were t r e a t e d with 40 jug DSP/mg p r o t e i n f o r 30 sec and run on an 11$ pol y a c r y l a m i d e g e l . the lane was cut out , soaked i n r e d u c t i o n mix c o n t a i n i n g 2-mercaptoethanol to break c r o s s l i n k s and then the lane l a i d on top of another 11$ acrylamide g e l . The lowered c r o s s l i n k e r c o n c e n t r a t i o n produced only one product, a dimer of F ( F 2 ) . An unknown, high molecular weight p r o t e i n (X) was 2-ME-modified but not c r o s s l i n k e d (see a l s o F i g . 20). 164 F 2 F L F| Figure 18. Two-dimensional c r o s s l i n k i n g of outer membranes usi n g 200 DSP/mg p r o t e i n . Outer membranes were c r o s s l i n k e d with 200 ,ug DSP/mg p r o t e i n f o r 30 seconds, run on a 12.5$ p o l y a e r y l a m i d e g e l , and the lane cut out , soaked i n r e d u c t i o n mix c o n t a i n i n g 2-mercaptoethanol to break DSP c r o s s l i n k s and then the lane l a i d on top of another 12.5$ acrylamide running g e l , and run i n the second dimension as described i n Methods. C r o s s l i n k e d products r e v e a l e d and l a b e l l e d on the F i g u r e i n c l u d e d a dimer of p r o t e i n F (F2), a proposed c r o s s l i n k between p r o t e i n F and a p a r t i c u l a r s p e c i e s of LPS (FL -see a l s o F i g . 5D) as w e l l as higher molecular weight m a t e r i a l unable to enter the f i r s t dimension g e l . The monomer of F (F1) was 2-mercaptoethanol-m o d i f i a b l e , a c c o u n t i n g f o r i t s s l i g h t l y o f f - d i a g o n a l p o s i t i o n . 165 and LPS we were able to examine whether any of the p r o t e i n F spots seen i n F i g u r e 18 were due to LPS:protein F c r o s s l i n k i n g . At l e a s t one spot due to LPS:protein F i n t e r a c t i o n ( l a b e l l e d FL) was seen i n t h i s way, but none of the p r o t e i n F multimer spots noted above could be accounted f o r by p r o t e i n F:LPS c r o s s l i n k s . I t should be noted that both the 0-antigen and rough core LPS of P_j_ aeruginosa c o n t a i n amino groups (Koval and Meadow, 1975; K r o p i n s k i e_t a^l. , 1982). Two dimensional a n a l y s i s of u n c r o s s l i n k e d outer membrane p r e p a r a t i o n s was done as a c o n t r o l to r e v e a l any d i s u l p h i d e -l i n k e d multimers ( F i g u r e 20). Only one high molecular weight p r o t e i n ( l a b e l l e d X) was observed which c o n s i s t e n t l y appeared below the d i a g o n a l . Although the i d e n t i t y of t h i s p r o t e i n was not determined, i t i s l i k e l y that i t forms an i n t e r c h a i n d i s u l p h i d e - l i n k e d dimer which was cleaved by 2-mercaptoethanol between the f i r s t and second dimensions. No other d i s u l p h i d e -l i n k e d or S D S - r e s i s t a n t multimers of outer membrane p r o t e i n s were d e t e c t e d . Whole P^ aeruginosa c e l l s were c r o s s l i n k e d to f u r t h e r confirm that the c r o s s l i n k e d products seen i n outer membrane experiments were a c t u a l l y n a t i v e s t r u c t u r e s and not the r e s u l t of rearrangement o c c u r r i n g during outer membrane i s o l a t i o n . The r e s u l t s showed more ext e n s i v e c r o s s l i n k i n g of c e l l s u r f a c e molecules i n whole c e l l s than i n outer membranes but the 70,000 d a l t o n dimer of p r o t e i n F and 95,000 d a l t o n dimer of another, glucose-induced p r o t e i n , D1, were i d e n t i f i e d (Table XVII). 166 I I T ~ I F 2 F L L L F i g u r e 19. T w o - d i m e n s i o n a l SDS-PAGE o f c r o s s l i n k e d o u t e r membrane samples s t a i n e d f o r c a r b o h y d r a t e . Outer membranes were t r e a t e d as i n F i g . 17 e x c e p t t h a t the g e l was s t a i n e d f o r LPS (see M e t h o d s ) . C r o s s l i n k e d p r o d u c t s which appeared to be due to LPSrLPS i n t e r a c t i o n (L) as w e l l as one p r o p o s e d L P S : p r o t e i n F i n t e r a c t i o n ( F L ) were o b s e r v e d . The spot l a b e l l e d FL was i d e n t i f i e d by i t s v e r t i c a l a l i g n m e n t w i t h a p r o t e i n F spot which d i d not s t a i n by t h i s p r o c e d u r e but i s c l e a r l y seen i n F i g . 18, s t a i n e d f o r p r o t e i n . The p o s i t i o n o f the dimer o f p r o t e i n F ( F 2 ) , which was not s t a i n e d by s i l v e r / p e r i o d a t e , i s marked. 167 F i g u r e 20. Two-dimensional SDS-PAGE of u n c r o s s l i n k e d c o n t r o l outer membrane samples. Un c r o s s l i n k e d c o n t r o l outer membranes were run on an 11$ polyacrylamide g e l as i n F i g . 17. No v i s i b l e o f f - d i a g o n a l spots other than p r o t e i n X ( p o s s i b l y a disu l p h i d e - b o n d e d dimer cleaved by 2-mercaptoethanol treatment) were observed. 168 To c o nfirm that the observed p r o t e i n F multimers were the r e s u l t of chemical b r i d g i n g between proximal subunits i n t h e i r n a t i v e conformation and not due to random contact between f r e e monomers, a c o n t r o l p r o t e i n F sample was p r e t r e a t e d with SDS p r i o r to c r o s s l i n k i n g . I t was p r e v i o u s l y observed that p r o t e i n F formed almost e x c l u s i v e l y monomers on SDS-polyacrylamide g e l s a f t e r SDS-heat treatment (Hancock and Carey, 1979). No c r o s s l i n k e d p r o t e i n products were observed i n the SDS-pretreated c o n t r o l (data not shown). Another P.  aeruginosa outer membrane p r o t e i n , H1 (Nicas and Hancock, 1980), which was not suspected to be a p o r i n , was subjected to the same c r o s s l i n k i n g c o n d i t i o n s . No multimers of t h i s p r o t e i n were observed (data not shown), demonstrating that not a l l outer membrane p r o t e i n s could be c r o s s l i n k e d , i n agreement with outer membrane (Figure 17) and whole c e l l c r o s s l i n k i n g experiments. 169 DISCUSSION The r e s u l t s of t h i s study are summarised i n Table XVII. When a n a l y s i n g the c r o s s l i n k e d products produced i n each experiment, i t was necessary to designate oligomers as dimers or t r i m e r s , e t c . based on molecular weights, alignment i n the second dimension with monomers of the p r o t e i n of i n t e r e s t and frequency of appearance ( i n t e n s i t y of s t a i n i n g ) . For i n s t a n c e , one would expect multimers r e q u i r i n g only one c r o s s l i n k i n g molecule to appear more f r e q u e n t l y than multimers r e q u i r i n g two c r o s s l i n k i n g molecules, t h e r e f o r e dimers would appear much more f r e q u e n t l y than t r i m e r s , even i f the n a t i v e s t r u c t u r e were a t r i m e r . The exact frequency of each s p e c i e s cannot be p r e d i c t e d i n the absence of s p e c i f i c i n f o r m a t i o n about the number of amino groups which could be i n v o l v e d i n c r o s s l i n k i n g r e a c t i o n s . For p r o t e i n F, the molecular weight and frequency of appearance of c r o s s l i n k e d products were c o n s i s t e n t with dimer and t r i m e r forms. Since the molecular weights of the c r o s s l i n k e d products were measured i n the absence of 2-mercaptoethanol treatment, the 70,000 d a l t o n dimer of p r o t e i n F would be composed of two 35,000 d a l t o n (2-mercaptoethanol-unmodified) s u b u n i t s . For p r o t e i n s P and PhoE, however, i n t e r p r e t a t i o n of spots formed was made d i f f i c u l t by i n t r a c h a i n c r o s s l i n k s . I t was concluded, based on the molecular weight of c r o s s l i n k e d products, that spots seen f o r p r o t e i n P were most l i k e l y dimer (90,000 d a l t o n ) , dimer with at l e a s t one i n t r a c h a i n c r o s s l i n k (80,000) and 170 Table XVII. Summary of the c r o s s l i n k e d multimers of p o r i n p r o t e i n s observed i n t h i s study. P r o t e i n Assignment and M"r Native SDS-— r e s i s t a n t Monomer Dimer Trimer Other oligomer P.aeruginosa 38,000 70,000 120,000 35,000 No p r o t e i n F (minus 2-ME) P.aeruginosa 47,000 95,000 145,000 29,000 No p r o t e i n D1 (unheated) P.aeruginosa 48,000 90,000 145,000 44,000 Yes p r o t e i n P ( i n t e r n a l c r o s s l i n k ) 80 ,000 E- c o l i 38,000 73,000 100,000 34,000 Yes p r o t e i n PhoE ( i n t e r n a l c r o s s l i n k ) Mr ( r e l a t i v e molecular weight) values were determined by comparison with the molecular weight standards s t a t e d i n Methods. 171 t r i m e r (145,000) products. For p r o t e i n PhoE, the 73,000 d a l t o n spot was assumed to be a dimer and the 100,000 d a l t o n spot a t r i m e r . I f there was a spot corresponding to dimers with i n t r a c h a i n c r o s s l i n k s , i t may have been obscured by the 73,000 d a l t o n dimer spot, however such a spot was not seen even i n two dimensional a n a l y s e s . The presence of a dimer with an i n t r a c h a i n c r o s s l i n k would n e c e s s i t a t e possession of at l e a s t three a c c e s s i b l e amino groups on each p r o t e i n molecule, and at l e a s t 14 t r i n i t r o b e n z e n e s u l p h o n a t e - a c c e s s i b l e l y s i n e epsilon-amino groups were found i n each of the two above p r o t e i n s (Hancock e_t a^, 1986). P o s s i b l y the d i f f e r e n t conformations of PhoE and p r o t e i n P, as suggested by t h e i r d i f f e r e n t channel s i z e s (1.1nm and 0.6nm i n diameter, r e s p e c t i v e l y ) and molecular weights (Table XVII), and consequently the r e l a t i v e p o s i t i o n i n g of l y s i n e s i d e chains e x p l a i n s these o b s e r v a t i o n s . In a l l cases, heterologous c r o s s l i n k e d products due to the c r o s s l i n k i n g of p o r i n p r o t e i n s to other p o l y p e p t i d e s or to LPS were e l i m i n a t e d from c o n s i d e r a t i o n . Such products were i d e n t i f i e d by a p o r i n spot below the d i a g o n a l i n the second dimension and d i r e c t l y above or below a p r o t e i n or LPS spot with which i t had co-migrated (due to c r o s s l i n k i n g ) i n the f i r s t dimension. Although the heterologous c r o s s l i n k e d products seen i n the g e l s were not s t r i n g e n t l y analysed due to t h e i r complexity, c r o s s l i n k e d products commonly i n v o l v e d s e v e r a l protein-LPS i n t e r a c t i o n s . P r o t e i n s P and PhoE c r o s s l i n k e d multimers were more e a s i l y v i s u a l i s e d than those of p r o t e i n F. Since the ease of 172 c r o s s l i n k i n g r e f l e c t s the a v a i l a b i l i t y of two c r o s s l i n k e r -r e a c t i v e amino groups w i t h i n 1.2 nm (one on each s u b u n i t ) , i t may be that such amino groups are more a c c e s s i b l e on both p r o t e i n s P and PhoE. The s i m i l a r c r o s s l i n k i n g p a t t e r n s of these p r o t e i n s , d e s p i t e t h e i r d i f f e r e n c e s i n molecular weight, may r e f l e c t t h e i r analogous f u n c t i o n s i n phosphate uptake through the outer membrane (Hancock e_t al^. , 1982; K o r t e l a n d e_t a l • , 1982). I t has been shown (Benz et; a_l. , 1984) that the s e l e c t i v i t y f i l t e r s (presumably the mouths of p o r i n channels) of p r o t e i n s P and PhoE have amino groups which appear to be important i n t h e i r f u n c t i o n as a n i o n - s e l e c t i v e channels. A c e t y l a t i o n of these groups p a r t l y n e u t r a l i s e d the a n i o n i c s e l e c t i v i t y of the channels i n black l i p i d b i l a y e r s but, at l e a s t f o r the PhoE p r o t e i n , d i d not change the apparent s i z e of the pore (Darveau e_t a_l. , 1984). Given the above-mentioned s i m i l a r i t i e s between p r o t e i n s P of P_^  aeruginosa and PhoE of E. c o l i , perhaps these two p r o t e i n s are developmentally r e l a t e d . The d i f f e r e n c e i n pore s i z e could be e x p l a i n e d i f some of the e x t r a 10,000 d a l t o n p o r t i o n of the l a r g e r p r o t e i n P was present toward the i n t e r i o r of the channel. This d i f f e r e n t channel s t r u c t u r e might a l s o e x p l a i n the f a c t that p r o t e i n P more e a s i l y y i e l d e d dimeric a s s o c i a t i o n s with i n t e r n a l c r o s s l i n k s while the PhoE p r o t e i n c r o s s l i n k e d more r e a d i l y to t r i m e r s . The c r o s s l i n k e d bands of p r o t e i n F were harder to v i s u a l i s e than those of p r o t e i n s P and PhoE. This was p r i m a r i l y due to the f a c t that p r o t e i n F d i d not r e s u l t i n 173 d i s t i n c t bands of c r o s s l i n k e d p o l y p e p t i d e s i n the f i r s t dimension even when the c r o s s l i n k e r c o n c e n t r a t i o n was v a r i e d over a 500-fold range (Figure 16) using e i t h e r whole outer membranes or p u r i f i e d p r o t e i n F. S i m i l a r d i f f i c u l t i e s can be observed from examination of c r o s s l i n k i n g p a t t e r n s of the Chromatium vinosum 42,000 d a l t o n p r o t e i n (Lane and H u r l b e r t , 1980). A n a l y s i s of the products of p r o t e i n F c r o s s l i n k i n g a f t e r cleavage and e l e c t r o p h o r e s i s i n the second dimension demonstrated that the dimer and t r i m e r c r o s s l i n k e d products formed smeared spots which covered r e g i o n s of the g e l s corresponding to about 60,000 to 80,000 d a l t o n s f o r the dimer and about 110,000 to 130,000 d a l t o n s f o r the t r i m e r . These spots were not a r t i f a c t s s i n c e the dimer was observed using low c o n c e n t r a t i o n s of c r o s s l i n k e r and a l s o using e i t h e r 35$_ methionine, Coomassie blue or s i l v e r s t a i n to r e v e a l i t . Furthermore, the products i n these molecular weight regions corresponded to homologous c r o s s l i n k e d products r a t h e r than heterologous products s i n c e they d i d not v e r t i c a l l y a l i g n with other p o l y p e p t i d e or LPS s p o t s . Although the spots were spread over a range of molecular weights i n the second dimension (as seen f o r other c r o s s l i n k e d p o r i n oligomers L e i t h and Morse, 1980; Palva and R a n d a l l , 1978), we estimated t h e i r molecular weights using e i t h e r l i m i t i n g amounts of c r o s s l i n k e r , such that s m a l l e r spots were seen (e.g. F i g u r e 17) or by t a k i n g the centre of the spots as the apparent r e l a t i v e m o b i l i t y of the multimers. Using e i t h e r method of a n a l y s i s , the molecular weights of the multimers were 174 approximately two- ( f o r the dimer) and t h r e e - ( f o r the t r i m e r ) f o l d m u l t i p l e s of the monomeric p r o t e i n F molecular weight. Although the amount of t r i m e r s p e c i e s f o r p r o t e i n F (and f o r p r o t e i n P, see F i g u r e 14) was c o n s i d e r a b l y l e s s than seen f o r the dimer s p e c i e s , i t should be noted t h a t , d e s p i t e the f a c t that the lambda r e c e p t o r (maltose p o r i n ) of c o l i i s r e c o g n i s e d to be a t r i m e r , c r o s s l i n k i n g with DSP r e s u l t s only i n d i m e r i c aggregates, with no t r i m e r s p e c i e s demonstrated (Palva and Westermann, 1979). This can be simply understood s i n c e chemical c r o s s l i n k i n g w i l l only occur i f p r o p e r l y o r i e n t e d amino groups spaced at 1.2 nm d i s t a n c e are a v a i l a b l e on adjacent s u b u n i t s . Thus, i f each monomer has only a s i n g l e such group, the p r o t e i n can never be c r o s s l i n k e d to a t r i m e r ( s i n c e a d i m e r i c product would use up two of the three a c c e s s i b l e amino groups i n the o l i g o m e r ) . S i m i l a r l y , the p r o b a b i l i t y of seeing a t r i m e r w i l l i n c r e a s e as the number of a c c e s s i b l e , p r o p e r l y o r i e n t e d amino groups i n c r e a s e s . The smearing of the dimer and t r i m e r bands i n the f i r s t dimension could have a v a r i e t y of e x p l a n a t i o n s i n c l u d i n g : a) non-c o v a l e n t i n t e r a c t i o n of p r o t e i n F with LPS which forms a v a r i e t y of bands i n t h i s area of the g e l ( K r o p i n s k i e_t al.. , 1982), b) i n t r i n s i c p h y s i c a l h e t e r o g e n e i t y of p r o t e i n F molecules, s i n c e f u n c t i o n a l h e t e r o g e n e i t y has been demonstrated (Benz and Hancock, 1981; Nicas and Hancock, 1983 ; Woodruff e_t al^. , 1 986 ), c) monofunctional r e a c t i o n of amino groups which are a c c e s s i b l e to DSP but are improperly o r i e n t e d f o r c r o s s l i n k i n g , and d) p r o t e i n F i s known to be 175 a b l e t o o c c u p y a t l e a s t s i x d i f f e r e n t m o l e c u l a r w e i g h t p o s i t i o n s on SDS p o l y a c r y l a m i d e g e l s a c c o r d i n g t o t e m p e r a t u r e o f s o l u b i l i s a t i o n and 2 - m e r c a p t o e t h a n o l c o n c e n t r a t i o n s ( H a n c o c k and C a r e y , 1979). A l t h o u g h i t c a n n o t d e f i n i t e l y be c o n c l u d e d t h a t any o f t h e p r o t e i n s s t u d i e d h e r e f o r m s a s p e c i f i c t r i m e r , I have c l e a r l y d e m o n s t r a t e d t h a t t h e s e p r o t e i n s a r e o l i g o m e r s , a t l e a s t d i m e r s , i n t h e i r n a t i v e s t a t e s . By a n a l o g y w i t h o t h e r p o r i n s i n o t h e r o r g a n i s m s ( P a l v a and West e r m a n n , 1979; L e i t h and M o r s e , 1980; P a l v a , 1980) , I f e e l t h a t t h e most l i k e l y s t r u c t u r e i s a t r i m e r and t h e r e s u l t s , w h i l e n o t p r o v i n g t h i s , a r e a t l e a s t c o n s i s t e n t w i t h t h i s p r o p o s a l . I f t h i s i s s o , i t i s i n t e r e s t i n g t h a t t h e t r i m e r i c f o r m may be c h a r a c t e r i s t i c o f a l l p o r i n s e x a m i n e d so f a r d e s p i t e d i f f e r e n c e s i n c h a n n e l s i z e , s e l e c t i v i t y and p h y s i c o c h e m i c a l p r o p e r t i e s . L. M u t h a r i a ( P h . D. t h e s i s , U.B.C., 1985) d e m o n s t r a t e d t h a t p r o t e i n F, e v e n a f t e r t r e a t m e n t w i t h SDS, c o n t a i n e d some o l i g o m e r i c p r o d u c t s w h i c h c o u l d o n l y be v i s u a l i s e d u s i n g W e s t e r n i m m u n o b l o t s w i t h m o n o c l o n a l a n t i b o d i e s t o p r o t e i n F. T h i s s m a l l l e v e l o f o l i g o m e r s a f t e r SDS t r e a t m e n t p r o v i d e d a d d i t i o n a l e v i d e n c e f o r t h e e x i s t e n c e o f an o l i g o m e r i c f o r m o f p r o t e i n F. 176 LITERATURE CITED Abraham, E.P. 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