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Use of the fluorescent probe 1-N-phenyl napthylamine to study the interactions of aminoglycoside antibiotics… Loh, Bernadette Anne 1984

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Use of the fluorescent probe 1-N-phenyl napthylamine to study the interactions of aminoglycoside a n t i b i o t i c s with the outer membrane of Pseudomonas aeruginosa by Bernadette Anne jLoh B.Sc, U n i v e r s i t y of B.C., 1980 A thesis submitted i n p a r t i a l f u l f i l l m e n t of the requirements f o r the degree of Master of Science r in The Faculty of Graduate Studies Department of Microbiology We accept t h i s thesis as conforming to the required standard The U n i v e r s i t y of B r i t i s h Columbia September 1984 © Bernadette Anne Loh, 1984 »6 I n 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 o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 a n d s t u d y . I f u r t h e r a g r e e 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f 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 n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . i D e p a r t m e n t o f M I C R O B I O L O G Y T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main M a l l V a n c o u v e r , C a n a d a V6T 1Y3 D a t e October 11, 1984 D E - 6 (3/81) ABSTRACT The mode of i n t e r a c t i o n of the po l y c a t i o n i c aminoglycoside a n t i b i o t i c s with the surface of Pseudomonas aeruginosa c e l l s was studied using the hydrophobic fluorescent probe 1-N-phenyl napthylamine (NPN). Addition of the aminoglycoside gentamicin to in t a c t c e l l s in the presence of NPN l e d to a s h i f t in the fluorescence emission maximum from 460 to 420 nm. At the same time the NPN fluorescence i n t e n s i t y increased four f o l d . Gentamicin caused no such e f f e c t s when added to outer membrane v e s i c l e s suggesting that the increased fluorescence resulted from the i n t e r a c t i o n of gentamicin with i n t a c t c e l l s . Gentamicin-promoted NPN uptake was 2+ 2+ i n h i b i t e d by the dival e n t cations Mg and Ca , but occurred in the absence of gentamicin transport across the inner membrane. Low concentrations of gentamicin (2 ug/ml) caused NPN fluorescence to increase over a period of 4 minutes i n a sigmoidal fashion. At higher concentrations (50 ug/ml) the increase occurred within a few seconds. The f i n a l fluorescence i n t e n s i t y was almost independent of the gentamicin concentration. A c e n t r i f u g a t i o n technique was used to demonstrate that gentamicin caused actual uptake of NPN from the supernatant. The i n i t i a l rate of NPN uptake varied according to the gentamicin concentration i n a sigmoidal fashion. Similar data were obtained f o r seven other aminoglycoside a n t i b i o t i c s . The data when reanalysed as a H i l l - p l o t gave a series of l i n e s with a mean slope (the H i l l number) of 2.26 ± 0.26, suggesting that the i n t e r a c t i o n of aminoglycosides with the c e l l surface to permeabilize i t to NPN involved at l e a s t 3 s i t e s and demonstrated p o s i t i v e c o o p e r a t i v i t y . There was a s t a t i s t i c a l l y s i g n i f i c a n t r e l a t i o n s h i p between the pseudo-association constant K from the H i l l s plots and the minimal i n h i b i t o r y concentrations f o r the 8 a n t i b i o t i c s These r e s u l t s are consistent with the concept that aminoglycosides i n t e r a c t at a div a l e n t cation binding s i t e on the P. aeruginosa outer membrane and permeabilize i t to the hydrophobic probe NPN. i v Table of Contents Page ABSTRACT i i TABLE OF CONTENTS . . i v L i s t of Figures v i L i s t of Tables ix ACKNOWLEDGEMENTS X INTRODUCTION 1 MATERIALS AND METHODS B a c t e r i a l s t r a i n s and growth conditions A Preparation of c e l l suspension 4 A n t i b i o t i c s and chemicals 4 Fluorescence and p o l a r i z a t i o n measurements . . . . 5 Measurement of cell-bound NPN 5 Preparation of outer membrane v e s i c l e s and liposomes 6 RESULTS Selec t i o n of a fluorescent probe 7 E f f e c t s of growth media and buffers 27 M i c r o s t i r r e r Apparatus and Aeration E f f e c t 27 Cyanide pretreatment of c e l l s . . . . 28 Gentamicin enhancement of NPN fluorescence and i n h i b i t i o n by d i v a l e n t cations 35 Uptake of NPN 40 Kin e t i c s of gentamicin-promoted NPN uptake . 47 P o l a r i z a t i o n studies . .' 52 V Table of Contents, continued Page DISCUSSION 57 LITERATURE CITED 62 v i L i s t of Figures Page 1. E f f e c t s of gentamicin and magnesium on the fluorescence 8 i n t e n s i t y of ANS in the presence of P. aeruginosa i n t a c t c e l l s or outer membrane v e s i c l e s . 2. I n t r i n s i c emission and e x c i t a t i o n spectra of outer 10 membrane v e s i c l e s of P. aeruginosa H103 3. E x c i t a t i o n and emission spectra of ANS fluorescence in the 12 presence of outer membrane v e s i c l e s . 4. E x c i t a t i o n and emission fluorescence spectra of ANS added to 14 outer membrane v e s i c l e s and the e f f e c t of added gentamicin. 5. E f f e c t of magnesium and gentamicin addition on ANS fluorescence 16 in phospholipid v e s i c l e s . 6. Fluorescence of phospholipid v e s i c l e s i n the presence of 19 pyrene, and the e f f e c t of gentamicin addition. 7. E f f e c t of gentamicin and magnesium on NPN fluorescence in 21 membrane v e s i c l e s . 8. Time course of increase in NPN fluorescence i n t e n s i t y in the 23 presence of i n t a c t P. aeruginosa c e l l s and d i f f e r e n t 2+ concentrations of gentamicin or Mg 9. E f f e c t of sequential addition of NPN on fluorescence of 25 outer membrane v e s i c l e s in the presence of gentamicin. 10. E f f e c t of potassium succinate (or glucose) addition and 29 aeration of the c e l l suspension on NPN fluorescence. 11. E f f e c t of KCN treatment on the gentamicin promoted r i s e 31 in NPN fluorescence. v i i L i s t of Figures, continued Page 12. (A) Gentamicin-promoted increase in NPN fluorescence a f t e r 33 addition of NPN to i n t a c t c e l l s of P. aeruginosa i n the presence of sodium azide. (B) I n h i b i t i o n by the addition of diva l e n t cations of the gentamicin-promoted increase i n NPN fluorescence of i n t a c t c e l l s of P. aeruginosa. 13. E x c i t a t i o n and emission spectra of NPN fluorescence i n 36 aqueous s o l u t i o n . 14. Emission and e x c i t a t i o n spectra of NPN fluorescence a f t e r 38 addition of NPN to a suspension of P. aeruginosa H103 in the presence of gentamicin. 15. I n h i b i t i o n of the gentamicin-promoted enhancement of NPN 41 fluorescence i n i n t a c t c e l l s of P. aeruginosa H103 by divalent cations. 16. E f f e c t of diva l e n t cations on the i n i t i a l rate of the 43 gentamicin-promoted increase i n NPN fluorescence of P. aeruginosa c e l l s . 17. Kinetics of gentamicin-promoted increase in NPN 47 fluorescence of i n t a c t c e l l s . 18. Relationship between the minimal i n h i b i t o r y concentrations 50 (MIC) of the d i f f e r e n t aminoglycoside a n t i b i o t i c s and the l o g 1 Q of the pseudoassociation constants Ks, extrapolated from H i l l p lots such as those shown in F i g . 17. v i i i L i s t of Figures, continued 19. The e f f e c t of gentamicin addition on the polarized fluorescence 54 i n t e n s i t y of DPH added to i n t a c t c e l l s of P. aeruginosa. ix L i s t of Tables Page I. Influence of gentamicin concentration on the t o t a l uptake of 45 NPN and enhancement of NPN fluorescence in P. aeruginosa c e l l s . I I . Fluorescence p o l a r i z a t i o n of the DPH taken up a f t e r the 55 i n t e r a c t i o n of gentamicin and polymyxin B with P. aeruginosa c e l l s . ACKNOWLEDGEMENTS I owe many thanks to Dr. R.E.W. Hancock for his generous advice, support and encouragement throughout t h i s project. I would also l i k e to thank the members of my supervisory committee, Dr. D. Syeklocha and Dr. R.A.J. Warren f o r t h e i r time and patience, and Susan Heming for her s k i l l f u l typing of t h i s manuscript. x i DEDICATION This thesis i s dedicated with gratitude to my parents who have given me great support, i n s p i r a t i o n and encouragement over the years. 1 INTRODUCTION The aminoglycosides are a group of po l y c a t i o n i c a n t i b i o t i c s f i r s t recognized in the 1940s with the discovery of streptomycin. Since then t h i s family of a n t i b i o t i c s e.g. gentamycin, tobramycin, kanamycin, has proved very e f f e c t i v e i n the treatment of many b a c t e r i a l i n f e c t i o n s . Aminoglycoside uptake into b a c t e r i a l c e l l s follows a sigmoidal r e l a t i o n s h i p with time and can be divided into three phases (1,6). F i r s t , an i o n i c binding i n t e r a c t i o n occurs between the aminoglycoside molecules and the c e l l , which i s followed by two energy dependent phases c a l l e d EDPI and EDPII. The i n i t i a l binding at the c e l l surface i s rapid and r e v e r s i b l e and tends to ne u t r a l i z e the c e l l ' s net negative surface charge (7). The f i r s t energy dependent phase, EDPI, involves gradual uptake of aminoglycosides (1,6,7). EDPII begins when accumulation of aminoglycoside becomes l i n e a r and rapid. I t has been suggested that the actual l e t h a l event to the c e l l s precedes or i s coincident with the onset of EDPII (7,10). Both the EDPI and EDPII phases of uptake have been assumed to represent uptake across the energized cytoplasmic membrane. The uptake of po l y c a t i o n i c a n t i b i o t i c s , such as aminoglycosides, across the outer membrane of Pseudomonas aeruginosa has been postulated to occur v i a the s e l f promoted uptake pathway (7,15). This uptake mechanism involves the displacement of divalent cations e.g. Mg + + or C a + + from the LPS by the p o l y c a t i o n i c aminoglycosides. Thus, the dival e n t cations crossbridging of lipopolysaccharide (LPS) i s disrupted causing l o c a l i z e d d e s t a b i l i z a t i o n or d i s t o r t i o n of the membrane (7). In support of t h i s 2 hypothesis the i n t e r a c t i o n of streptomycin or gentamicin with P. aeruginosa causes enhancement of outer membrane permeability to lysozyme and increased uptake of the fi-lactam, n i t r o c e f i n (8). In addition, ethylenediaminetetraacetate (EDTA), which disrupts Mg^+ crossbridges by c h e l a t i o n rather than displacement causes s i m i l a r enhancement of uptake of lysozyme and n i t r o c e f i n (8) as well as an increased rate of k i l l i n g by aminoglycosides (18). Another l i n e of evidence i s that outer membrane-altered mutants of P. aeruginosa, with an apparent decrease in the number of Mg-binding s i t e s show cross-resistance to EDTA, polymyxin and aminoglycosides (14). However, the self-promoted uptake pathway f o r aminoglycosides has not yet been demonstrated in other Gram-negative b a c t e r i a and aminoglycoside uptake may occur in these organisms v i a the porin protein mediated pathway (13). To obtain further information about the int e r a c t i o n of aminoglycosides with P. aeruginosa, we decided to u t i l i z e fluorescent probes as t o o l s . The use of fluorescent probes to study the structure and function of b i o l o g i c a l membranes i s well documented (4,5,9,11). Commonly used probes in biomembrane studies are 1,8-anilino-l-napthalene sulphonic acid (ANS) and 1-N-phenyl naphthylamine (NPN). These probes are p a r t i c u l a r l y useful because they fluoresce weakly in aqueous environments but become very strongly fluorescent in non-polar or hydrophobic environments. Furthermore, they are extremely s e n s i t i v e to environmental factors such as solvent, pH and temperature, and very l i t t l e material i s required in a given assay. In t h i s t h e s i s , the r e s u l t s of a study using fluorescent probes to look at the i n t e r a c t i o n of aminoglycosides with P. aeruginosa i s reported. I have obtained further evidence that aminoglycosides permeabilize the c e l l at the outer membrane and that the s i t e of i n t e r a c t i o n i s probably a div a l e n t cation binding s i t e . 4 MATERIALS AND METHODS B a c t e r i a l s t r a i n s and growth conditions. Pseudomonas aeruginosa PA01 s t r a i n H103 (14) was used in a l l experiments. I t was grown in 1% (wt/vol) proteose peptone No. 2 medium (D i f c o ) . Experimental cultures were started from an overnight broth culture and grown at 37°C with vigorous shaking to an o p t i c a l density at 600 nm (0D.rt/.) of 0.4 - 0.6. Preparation of c e l l suspension. F i f t y ml of mid-log phase c e l l s were centrifuged down at 3000 x g f o r 10 min, and resuspended i n 5 mM Na Hepes buffer pH 7.2, with or without 1 mM KCN, at an 0D.nn of 0.5. This c e l l 600 o suspension was l e f t at 23 C for 30-60 min before adding any reagents. Control experiments using P. aeruginosa s t r a i n H103 (RPl) containing a plasmid-encoded B-lactamase i n i t s periplasm, demonstrated that during the time course of the experiments no B-lactamase leaked out of the c e l l , suggesting that the outer membrane remained i n t a c t . A n t i b i o t i c s and chemicals. Gentamicin s u l f a t e , neomycin s u l f a t e , kanamycin s u l f a t e and streptomycin s u l f a t e were purchased from the Sigma Chemical Co. (St. Louis, Mo.). Tobramycin and netilmycin s u l f a t e were received from E l i L i l l y , Inc. Canada (Scarborough, Ontario). Amikacin was a g i f t from Bristol-Myers Canada (Ottawa, Ontario), while sisomycin s u l f a t e was obtained from the Schering Corporation (Pointe C l a i r e , Quebec). NPN; 1,6-diphenylhexatriene (DPH); and ANS were purchased from Sigma Chemical Co. 5 Fluorescence and p o l a r i z a t i o n measurements. ANS was made as a 60mM stock so l u t i o n in 0.9% (w/v) NaCl and used at a f i n a l concentration of 60 yM. Ex c i t a t i o n and emission wavelengths for ANS were set at 375 and 475 nm, res p e c t i v e l y with s l i t widths of 5nm. NPN was dissolved i n acetone at a concentration of 500 yM and used at a f i n a l concentration ( i n c e l l suspension) of 10 yM. Control experiments showed no s i g n i f i c a n t e f f e c t of the added acetone on the r e s u l t s reported here. Fluorescence spectra and emission i n t e n s i t i e s were measured using a Perkin-Elmer 650-10S fluorescence spectrophotometer equipped with a Haake c i r c u l a t i n g water bath to maintain the cuvette holding chamber at 30°C. E x c i t a t i o n and emission wavelengths for NPN were usually set at 350 nm and 420nm res p e c t i v e l y , with s l i t widths of 5nm. Fluorescence i n t e n s i t i e s are given in a r b i t r a r y u n i t s . Measurement of cell-bound NPN. The amount of NPN bound to c e l l s before and a f t e r gentamicin treatment was determined by a modification of the cen t r i f u g a t i o n technique described by Nieva-Gomez et a l . (16). Samples (4 ml) were prepared containing cyanide-treated c e l l s (resuspended to OD 6 0 0 = 0.5) and 10 yM NPN. Aliquots (1 ml) of each sample were taken before and a f t e r the addition of gentamicin at varying concentrations, and at the end of each experiment. These aliquots were centrifuged at 9,000 rpm for 1 min using an Eppendorf minifuge. Control experiments without added c e l l s or gentamicin were also performed. The NPN concentration of the supernatant was determined by measuring the fluorescence in the presence 6 of 3% (v/v) T r i t o n X-100 and comparison with a standard curve. Standard curves were l i n e a r over the range of NPN concentrations used (0 - 10 uM). Preparation of outer membrane v e s i c l e s and liposomes. Outer membranes from s t r a i n H103 were prepared as previously described (14) and resuspended at a f i n a l protein concentration of 5 mg/ml. Twently y l of outer membrane suspension was d i l u t e d i n 2 ml Hepes buffer and sonicated f o r 15-30 sec. 2+ Mg , gentamicin and fluorescent probe were added as required. V e s i c l e s were also prepared from commercially obtained l i p i d s such as phosphatidylcholine and phosphalidylethanolamine and from H103 lipopolysaccharide (LPS) [prepared according to method by Darveau, 1983 (3)] . 7 RESULTS Selection of a fluorescent probe. Preliminary experiments were c a r r i e d out with 1,8-ANS, a probe commonly used in membrane studies. Gentamicin (GM) addition to i n t a c t P. aeruginosa c e l l s i n the presence of ANS caused a s u b s t a n t i a l increase in fluorescence; however, the k i n e t i c s of t h i s increase were complex and included an i n i t i a l immediate increase followed by a second, slower rate of increase ( F i g . 1, curve A). These complex k i n e t i c s l e d me to look at a simpler system using membrane or LPS v e s i c l e s and liposomes. I t was found that both inner and outer membrane v e s i c l e s had i n t r i n s i c e x c i t a t i o n and emission wavelengths of 350 and 430 nm r e s p e c t i v e l y ( F i g . 2). In the presence of ANS, e x c i t a t i o n was shown as a broad band between 330-390 nm while the emission wavelength was r e d - s h i f t e d to 520 nm ( F i g . 3). With the addition of 50 ug/ml GM, there was an immediate increase in fluorescence i n t e n s i t y ( F i g . 1, curve C), with a s h i f t in emission wavelength back to 470 nm ( F i g . 4). V e s i c l e s prepared from phosphatidylcholine (PC) and phosphatidyl ethanolamine (PE) as well as those prepared from H103-lipopolysaccharide (LPS) showed a s i m i l a r response ( F i g . 5). That i s , gentamicin addition caused an immediate enhancement of ANS fluorescence and no secondary increase. 2+ 2+ Furthermore, diva l e n t cations l i k e Mg and Ca caused a s i m i l a r increase in ANS fluorescence ( F i g . 1, curve B; 5). 8 Figure 1. E f f e c t s of gentamicin and magnesium on the fluorescence i n t e n s i t y of ANS in the presence of P. aeruginosa i n t a c t c e l l s or outer membrane v e s i c l e s . At 0 min 5 yM ANS was added to the c e l l s (curves A and D) or v e s i c l e s (B and C). Shortly thereafter (as indicated by the arrow) the following additions were made: Curve A - 20 yg/ml gentamicin added to i n t a c t c e l l s ; Curve B - 10 yM MgCl^ added to outer membrane v e s i c l e s ; Curve C - 20 yg/ml gentamicin added to outer membrane v e s i c l e s ; Curve D - no furth e r additions to i n t a c t c e l l s ( s i m i l a r r e s u l t s to curve D were obtained when no addition was made to outer membrane v e s i c l e s ) . 10 Figure 2. I n t r i n s i c emission and e x c i t a t i o n spectra of outer membrane v e s i c l e s of P. aeruginosa H103. Outer membrane v e s i c l e s were resuspended to a f i n a l concentration of 0.1 mg/ml protein i n 2 ml of 5 mM Hepes and sonicated f o r 30 sec. E x c i t a t i o n and emission wavelengths were set at 370 nm and 478 nm r e s p e c t i v e l y f o r emission and e x c i t a t i o n scans. In the absence of added probe, the emission and e x c i t a t i o n maxima were 430 nm (A) and 350 nm (B) r e s p e c t i v e l y . Similar values were obtained with inner membrane v e s i c l e s . 11 370 nm 478 nm 12 Figure 3. E x c i t a t i o n and emission spectra of ANS fluorescence in the presence of outer membrane V e s i c l e s . Outer membrane v e s i c l e s were resuspended to a f i n a l concentration of 100 yg/ml in 2 ml of 5 mM Hepes. ANS was added to 60 yM and e x c i t a t i o n and emission wavelengths were set at 375 and 480 nm r e s p e c t i v e l y . Emission and e x c i t a t i o n maxima were 520 nm (A) and a broad band of 330-390 nm (B) re s p e c t i v e l y . S i m i l a r scans were obtained with inner membranes. 13 480 nm 1 375 nm * 520 nm 330 nm A B 14 Figure 4. E x c i t a t i o n (A) and emission (B) fluorescence spectra of ANS added to outer membrane v e s i c l e s and the e f f e c t of added gentamicin. E x c i t a t i o n and emission wavelengths were set at 375 and 475 nm r e s p e c t i v e l y . Outer membranes were resuspended to a f i n a l concentration of 100 ug/ml in 2 ml of 5 mM Hepes. ANS was added to a concentration of 60 yM and gentamicin to 50 yg/ml. Emission and e x c i t a t i o n maxima were 470 and 385 nm r e s p e c t i v e l y . Inner membrane v e s i c l e s gave s i m i l a r r e s u l t s . 375nm 740 nm i 475 nm 385 nm 16 Figure 5. E f f e c t of magnesium and gentamicin addition on ANS fluorescence in phospholipid v e s i c l e s . Phosphatidylcholine v e s i c l e s (PCy) were resuspended to 100 yg/ml in 2 ml of 5 mM Hepes buffer and sonicated f o r 30 sec. ANS was added at 60 yM. Subsequently Mg 2 + (MgCl2> was added to 0.1 mM and gentamicin to 20 yg/ml f i n a l concentration. Similar values of fluorescence i n t e n s i t y were seen with phosphatidylethanolamine and LPS v e s i c l e s . 17 0 2 A 6 8 10 12 U TIME (min) 18 Presumably, the e f f e c t s of gentamicin enhancement of fluorescence i n inta c t c e l l s were complicated by charge n e u t r a l i z a t i o n of negatively-charged ANS by p o l y c a t i o n i c aminoglycosides allowing further uptake of ANS. Such e f f e c t s were previously demonstrated f o r di-and t r i v a l e n t cations (5,11). A second fluorescent probe, pyrene, was found to be r a p i d l y taken up by phospholipid v e s i c l e s but t h i s fluorescence was not stable and almost instantaneously started to decay ( F i g . 6). This suggested that pyrene probably had a very short fluorescence l i f e t i m e in t h i s system and was therefore not s u i t a b l e . Subsequent gentamicin addition d i d not enhance fluorescence. In contrast to these data, the fluorescence of the neutral probe NPN, was enhanced upon the i n t e r a c t i o n of i n t a c t c e l l s with gentamicin ( F i g . 8, curve A) and a f t e r plateauing remained stable f o r 10-20 min. However, although NPN i t s e l f was r a p i d l y taken up by outer membrane and phospholipid v e s i c l e s , very l i t t l e or no fluorescence enhancement was 2+ 2+ observed upon addition of gentamicin, Mg or Ca (F i g . 7). This suggested that NPN was s p e c i f i c a l l y reporting on an i n t e r a c t i o n of gentamicin with the surface of i n t a c t c e l l s . Therefore, NPN was chosen for a l l subsequent studies. In addition, the sequential addition of NPN to outer membrane v e s i c l e s caused an increase i n fluorescence from 1-5 ym NPN, a peak or plateau at 7-8 ym NPN, and s l i g h t lower fluorescence emission at higher NPN concentrations to 10 ym (F i g . 9). 19 Figure 6. Fluorescence of phospholipid v e s i c l e s in the presence of pyrene, and the e f f e c t of gentamicin addition. Phosphatidylcholine v e s i c l e s (PC^) were resuspended to 100 ug/ml in 2 ml of 5 mM Hepes buffer and sonicated for 30 sec. Pyrene was added at 2.5 um and subsequently gentamicin was added at 20 yg/ml. Phosphatidylethanolamine v e s i c l e s (PE ) gave s i m i l a r r e s u l t s . 20 ' 1 — i 1 1 1 r -0 2 k 6 8 10 TIME (min) 21 Figure 7. E f f e c t of gentamicin or magnesium addition on NPN fluorescence i n membrane v e s i c l e s . Inner or outer membranes were resuspended i n 2 ml of 5 mM Hepes buffer in the presence of 10 yM NPN. Magnesium was added to 1 mM and gentamicin to 20 yg/ml as required. 22 8 IM 1 GM i IM 1 Mg GM i OM i Mg GM NPN NPN • NPN 1 TIME 23 Figure 8. Time course of increase in NPN fluorescence i n t e n s i t y in the presence of i n t a c t P. aeruginosa c e l l s and d i f f e r e n t concentrations of gentamicin or Mg^+. At the arrow l a b e l l e d GM the following additions were made: Curve A - 20 yg/ml of gentamicin; Curve B - 2 yg/ml of gentamicin; Curve C - 2 yg/ml of gentamicin and 100 yM MgCl 2; Curve D - no gentamicin added ( r e s u l t s were i d e n t i c a l whether or not MgCl^ was added in the absence of gentamicin). C e l l s were pretreated with 1 mM KCN as described in the below (see F i g . 11). t-O 25 Figure 9. E f f e c t of sequential addition of NPN on fluorescence of outer membrane v e s i c l e s i n the presence of gentamicin. Outer membranes were resuspended i n 2 ml of 5 mM Hepes buffer (arrow A). Gentamicin was added at 20 yg/ml (arrow B). NPN was then added sequentially, s t a r t i n g at 0.5 yM (arrow C) and continuing from 1-10 yM. F l u o r e s c e n c e to 27 E f f e c t s of d i f f e r e n t growth media and b u f f e r s . Resuspension of an overnight culture of s t r a i n H103 in PP2 broth resulted in a very high l e v e l of i n t r i n s i c fluorescence (presumably due to the production of endogeneous fluorophores). No increase in fluorescence was observed with addition of NPN 20 um or gentamicin at 20 ug/ml. C e l l s grown and resuspended in nutrient broth or BM2 (minimal media with low Mg^+) showed a f a i r l y high but unstable fluorescence in the presence of NPN. Gentamicin addition r e s u l t e d in a moderate NPN fluorescence increase in NB-grown c e l l s , but a decrease in fluorescence of BM2 grown c e l l s . Of the d i f f e r e n t media used f o r growing and resuspending c e l l s , growth in PP2 both overnight and resuspension in 5 mM Hepes buffer? a f t e r centrifugation proved to be the best combination for convenience and consistency. Control "Experiments gave comparable r e s u l t s from day to day and allowed repeated r e s u l t s to be analyzed. M i c r o s t i r r e r Apparatus and Aeration E f f e c t . The fluorescence spectrophotometer was equipped with a m i c r o s t i r r e r apparatus (Lawrence Instruments, Ltd.) which consisted of a t i n y magnetic s t i r r e r , u nit attached under the cuvette holder. Our objective was to f i n d out i f continuous s t i r r i n g of the sample would ensure more e f f e c t i v e contact between probe molecules and b a c t e r i a l c e l l s and produce more consistent results among d i f f e r e n t experiments. Unexpectedly, t h i s caused prolonged fluctuations in fluorescence l e v e l and a generally higher baseline fluorescence in any p a r t i c u l a r sample. Many factors could be responsible 28 for t h i s including aeration of the sample and l i g h t scatter from the magnetic f l e a bar in the cuvette. In addition, manual a g i t a t i o n / a e r a t i o n (shaking) of the c e l l suspension a f t e r reaching the maximum fluorescence emission caused the fluorescence to decrease to i t s base l e v e l . The same e f f e c t was observed i f a carbon source such as potassium succinate or glucose (1 mM) was added ( F i g . 10). Again, these phenomena were reminiscent of s i m i l a r studies in E. c o l i (2) . Cyanide pretreatment of c e l l s . In order to ensure that the observed r e s u l t s were not complicated by e i t h e r the e f f e c t s of gentamicin on the cytoplasmic membrane during transport or by post-uptake e f f e c t s on. c e l l metabolism (7), we pretreated c e l l s with 1 mM potassium cyanide which blocks (both EDPI and EDPII) gentamicin transport and k i l l i n g (1,5). As shown in F i g . 11, the i n i t i a l rate of increase of fluorescence was i d e n t i c a l in the presence or absence of cyanide. This was confirmed under a v a r i e t y of the conditions tested below. However, cyanide-treated c e l l s demonstrated a continuing fluorescence increase u n t i l a steady state was achieved, whereas in non-treated c e l l s , the increase was followed by a decline in fluorescence to baseline l e v e l s . Another energy i n h i b i t o r , sodium azide (1 mM) also prevented the decline in fluorescence without i n f l u e n c i n g the i n i t i a l rates of the gentamicin-promoted increase in NPN fluorescence ( F i g . 12A). S i m i l a r phenomena ( i . e . a fluorescence increase followed by a steady decrease) have been observed in E. c o l i . and an energized secretion of NPN, which would be blocked by cyanide or azide in 29 Figure 10. E f f e c t of potassium succinate (or glucose) addition and aeration of the c e l l suspension on NPN fluorescence. At the arrow l a b e l l e d 1, i n t a c t c e l l s of P. aeruginosa H103 were resuspended in 2 ml of 5 mM Hepes buffer with 10 yM NPN. At arrow 2, potassium succinate (or D-glucose) was added to 2 mM. At arrow 3, the cuvette was shaken manually to aerate the c e l l suspensions. At l a t e r times the fluorescence rose again u n t i l i t achieved a pre-aeration l e v e l of fluorescence. 31 Figure 11. E f f e c t of KCN treatment on the gentamicin promoted r i s e in NPN fluorescence. At 0 min 5 yM NPN was added to i n t a c t P. aeruginosa c e l l s . At the arrow 20 yg/ml gentamicin was added. Curve A - c e l l s resuspended in 1 mM KCN in Hepes buffer and held for 10 min p r i o r to NPN addition. Curve B - c e l l s resuspended in Hepes buffer. 33 Figure 12. (A) Gentamicin-promoted increase i n NPN fluorescence a f t e r addition of NPN to i n t a c t c e l l s of P. aeruginosa in the presence of sodium azide. At the arrow l a b e l l e d 1, H103 c e l l s were resuspended in 2 ml of 5 mM Hepes buffer with 10 yM NPN and 1 mM Na azide. At arrow 2, gentamicin was added to 10 yg/ml. (B) I n h i b i t i o n by the addition of diva l e n t cations of the gentamicin-promoted increase in NPN fluorescence of i n t a c t c e l l s P. aeruginosa. At the arrow l a b e l l e d 1, H103 i n t a c t c e l l s were resuspended in 2 ml of 5 mM 2+ Hepes buffer with 10 yM NPN and 1 mM Na azide. At arrow 2, Mn , 2+ 2+ Mg or Ba were added to 5 mM. At arrow 3, gentamicin was added to a f i n a l concentration of 10 yg/ml. 35 t h i s case, was postulated to be responsible f o r these e f f e c t s (2). A l l subsequent experiments were performed wth cyanide due to the simpler k i n e t i c s and i d e n t i c a l i n i t i a l rates i n the presence or absence of cyanide. Gentamicin enhancement of NPN fluorescence and i n h i b i t i o n by divalent  cations. Control experiments were performed to demonstrate that 10 uM NPN was the i d e a l non-limiting concentration for v i s u a l i z a t i o n of the ef f e c t s of gentamicin on c e l l s . The addition of 10 yM NPN to cyanide-treated P. aeruginosa s t r a i n H103 c e l l s caused an immediate small increase i n fluorescence i n t e n s i t y above the background l e v e l of the c e l l s ( F i g . 8, curve D). At t h i s stage the e x c i t a t i o n wavelength maximum was 340 nm and the emission wavelength maximum was 460 nm, s i m i l a r to the maxima observed with NPN added to aqueous solution (Fig. 13). When gentamicin was added, the emission maximum s h i f t e d to 420 nm and the fluorescence i n t e n s i t y at t h i s wavelength increased in a time dependent process. The e x c i t a t i o n wavelength maximum s h i f t e d to 350 nm (F i g . 14). When c e l l s in the presence of 10 yM NPN were excited by l i g h t of 350 nm wavelength and the emission at 420 nm followed over time, the k i n e t i c s of fluorescence increase varied with the concentration of gentamicin added. At low gentamicin concentrations (2 yg/ml-below the minimal i n h i b i t o r y concentration f o r s t r a i n H103 c e l l s grown under these conditions), the enhancement of NPN fluorescence was biphasic (Fig. 8, curve B). At higher concentrations of gentamicin eg. 20 yg/ml, the increase of NPN fluorescence was rapid and plateaued within 20 sec a f t e r a 36 Figure 13. E x c i t a t i o n and emission spectra of NPN fluorescence in aqueous sol u t i o n . A s o l u t i o n of 5 yM NPN in 5 mM Hepes was made. Emission and e x c i t a t i o n maxima were observed at 460 (A) and 340 nm (B) r e s p e c t i v e l y . 38 Figure 14. Emission and e x c i t a t i o n spectra of NPN fluorescence a f t e r addition of NPN to a suspension of P. aeruginosa H103 in the presence of gentamicin. H103 c e l l s were resuspended in 5 mM Hepes buffer with 10 yM NPN and 20 yg/ml gentamicin. Emission and e x c i t a t i o n maxima were observed at 420 nm (A) and 350 nm (B) r e s p e c t i v e l y . 40 4 f o l d increase in fluorescence emission ( F i g . 8, curve A). The fluorescence increase in the presence of 100 ug/ml gentamicin was almost instantaneous and fluorescence d i d not decay over time (data not shown). The concentration of gentamicin added influenced only the k i n e t i c s of fluorescence increase and not the steady state l e v e l achieved (see F i g . 8, curves A and B). 2+ 2+ 2+ 2+ The addition of the d i v a l e n t cations, Mg , Ca , Ba , Mn 2+ 2+ and Sr at low l e v e l s (eg. 50 yM Mg , F i g . 8, curve C; F i g . 15) i n h i b i t e d the enhancement, by gentamicin, of NPN fluorescence. This e f f e c t was observed in both cyanide and Na azide-pretreated c e l l s . 2+ 2+ Increasing concentrations of 0 - 50 yM Mg or Ca produced corresponding decreases in the i n i t i a l rate of NPN fluorescence enhancement ( F i g . 16A). Increasing the gentamicin concentration in the 2+ 2+ presence of a f i x e d amount of Ca or Mg l e d to a gradual increase in the i n i t i a l rate of fluorescence ( F i g . 16B). As mentioned above, 2+ 2+ Ca and Mg or any of the other cations used, did not themselves cause enhancement of NPN fluorescence i n whole c e l l s or phospholipid v e s i c l e s . Uptake of NPN. The increase of NPN fluorescence upon gentamicin addition could have two explanations; i t could represent NPN being taken up into the c e l l s from the supernatant or i t could be due to an a l t e r a t i o n in the environment of the NPN r e s u l t i n g i n fluorescence enhancement. Our data (Table I) suggested that a combination of these two e f f e c t s was responsible for the observed fluorescence increase. The amount of NPN 41 Figure 15. I n h i b i t i o n of the gentamicin-promoted enhancement of NPN . fluorescence i n i n t a c t c e l l s of P. aeruginosa H103 by divalent cations. At the arrow l a b e l l e d 1, H103 c e l l s were resuspended in 5 mM Hepes buffer 2+ with 1 mM KCN. At arrow 2, Mg was added to 5 mM. At arrow 3, NPN was added to 10 yM and at arrow 4, gentamicin was added to a f i n a l concentration of 20 yg/ml. Similar r e s u l t s were obtained with addition 2+ 2+ 2+ 2+ of Mn , Ca , Ba or Sr to 5 mM. Fluorescence o oo •-NO m 43 Figure 16. E f f e c t of diva l e n t cations on the i n i t i a l rate of the gentamicin-promoted increase in NPN fluorescence of P. aeruginosa c e l l s . ' 2+ 2+ In panel A increasing concentrations of Mg ( A ) and Ca (•) were added to separate cuvettes containing c e l l s and NPN as described in Methods. Immediately afterwards 20 yg/ml gentamicin was added to each cuvette and the k i n e t i c s of fluorescence increase followed over time. In 2+ 2+ panel B e i t h e r 50 yM Mg ( A ) , 40 yM Ca (•) or no cation (0) were added p r i o r to the addition of varying amounts of gentamicin to the f i n a l concentration given on the X axis. A l l experiments were performed on cyanide-treated c e l l s . 44 45 Table I. Influence of gentamicin concentration on the t o t a l uptake of KPN and enhancement of NPN fluorescence in P. aeruginosa c e l l s . Gentamicin Concentration (ug/ml) C e l l bound NPN i n A r b i t r a r y Units a (umol NPN taken up) Tot a l fluorescence of c e l l bound NPN in A r b i t r a r y units' 5 Calculated fluoresence enhancement of c e l l bound NPN c 2 10.3 ± 0.6 (1.7) 17.7 ± 4.6 1.7 5 12.4 ± 4.5 (2.0) 23.3 ± 4.4 1.9 10 12.0 ± 0.8 (1.9) 26.3 ± 2.4 2.2 20 14.0 + 1.8 (2.1) 23.4 ± 2.4 1.7 50 17.7 + 4.6 (2.5) 29.7 ± 2.0 1.7 a C e l l bound NPN was c a l c u l a t e d by determining the free NPN i n the absence of gentamicin ( a f t e r removal of c e l l s by c e n t r i f u g a t i o n and addition of 3% T r i t o n X-100) and subtracting the determination of the NPN remaining in the supernatant 10 min a f t e r the addition of gentamicin. The r e s u l t s (means + standard deviations) in a r b i t r a r y fluorescent units were converted to umol NPN taken up by reference to a standard curve constructed by a d d i t i o n of 3% T r i t o n X-100 to d i f f e r e n t amounts of NPN. ^T o t a l fluorescence was the actual increase in fluorescence measured in the 10 min a f t e r a d d i t i o n of gentamicin to c e l l s and i s the fluorescence i n t e n s i t y of the c e l l sample a f t e r gentamicin addition minus the fluorescence i n t e n s i t y p r i o r to gentamicin a d d i t i o n . [ P r i o r to gentamicin addition, 4 fluorescent units (0.3 umol of NPN) were associated with c e l l s on average]. Calculated as the r a t i o of t o t a l fluorescence (column 3) to c e l l bound NPN (column 2). This i s the increase in the fluorescence of c e l l bound NPN over and above the expected fluorescence in 3% T r i t o n X-100. 46 bound to c e l l s before and a f t e r gentamicin treatment was determined by a c e n t r i f u g a t i o n technique using T r i t o n X-100. T r i t o n X-100 was previously demonstrated to enhance only the fluorescence of c e l l - f r e e NPN but had no e f f e c t on c e l l bound NPN (16). In the absence of added gentamicin, approximately 4 fluorescent units of NPN were associated with c e l l s . A f t e r addition of gentamicin and s u f f i c i e n t time to allow fluorescence to reach a steady state l e v e l , c e l l - a s s o c i a t e d fluorescence had increased four to seven f o l d . The maximum l e v e l of c e l l bound NPN was r e l a t i v e l y independent of the gentamicin concentration since over a 25 f o l d range of gentamicin concentrations, c e l l bound fluorescence changed only 1.7 f o l d . The t o t a l increase in fluorescence a f t e r gentamicin addition could not be accounted for by the c e l l bound NPN (compare columns 2 and 3 of Table I ) . Thus, part of the increase must have been due to the NPN being incorporated into an environment in which i t was more highly fluorescent than in T r i t o n X-100 solutions (see column 4, Table I; N.B. t h i s fluorescence enhancement apparently only occurred as a consequence of uptake - thus the t o t a l fluorescence increase r e f l e c t s NPN uptake). The increase in fluorescence might have been caused by an increased l i f e t i m e of the probe (9). The increase in fluorescent y i e l d of the c e l l bound NPN was almost independent of the gentamicin concentration, suggesting that the NPN p a r t i t i o n e d into a s i m i l a r environment in a l l experiments. Kin e t i c s of gentamicin-promoted NPN uptake. A series of experiments, using gentamicin concentrations between 1 and 20 yg/ml, were performed to evaluate how the rate of NPN uptake was affected by the gentamicin 47 Figure 17. K i n e t i c s of gentamicin promoted increase in NPN fluorescence of i n t a c t c e l l s . In panel A the i n i t i a l rate of fluorescence increase (V expressed in a r b i t r a r y fluorescence units per min per mg c e l l dry weight), taken from a s e r i e s of experiments l i k e those depicted in curves A and B of F i g . 8, was p l o t t e d against the concentration of gentamicin (GM) in yg/ml. A l l experiments were performed with cyanide-treated c e l l s . This data was reanalyzed according to a H i l l p l o t (panel B) i n which the gentamicin concentration was converted to molar concentrations and <(> i s the r a t i o of the rate of fluorescence increase at the given gentamicin concentration to the maximal rate of fluorescence increase extrapolated from panel A. The H i l l number (n = the slope of the H i l l plot) i s given in panel B. The Y axis intercept i s equal to - l n K g. The c o r r e l a t i o n c o e f f i c i e n t f or the l i n e a r regression of the data f o r t h i s and a l l other H i l l p l o t s was greater than 0.99. 49 concentration present. We obtained a family of sigmoidal curves of fluorescence increase over time a f t e r gentamicin addition (eg. F i g . 8, curves A and B). Due to the complexity of these in t e r a c t i o n s , we attempted only to analyze the i n i t i a l rates of the NPN fluorescence increase. A pl o t of these i n i t i a l rates against gentamicin concentration produced a sigmoidal plot ( F i g . 17A). From t h i s , i t appeared that a cooperative i n t e r a c t i o n had occurred between gentamicin and c e l l s giving r i s e to the uptake of NPN, i e . the i n t e r a c t i o n of one molecule of gentamicin with the c e l l surface enhanced subsequent i n t e r a c t i o n s . The data were replotted as a H i l l p l o t ( F i g . 17B) which allows one to d i s t i n g u i s h simple, multiple or cooperative i n t e r a c t i o n s . The slope of the H i l l p l o t i s referred to as the H i l l number. This number i s usually interpreted as the approximate minimum number of binding s i t e s . For gentamicin, the H i l l number was 1.95 ± 0.3 (average of 5 experiments) i n d i c a t i n g a cooperative i n t e r a c t i o n with a minimum of 2 - 3 i n t e r a c t i o n s i t e s . Similar l i n e a r H i l l plots were obtained with each of eight d i f f e r e n t aminoglycoside a n t i b i o t i c s . I n t e r e s t i n g l y , the H i l l numbers derived from H i l l p l o t analyses of these data were very s i m i l a r among the eight aminoglycosides. A mean and standard deviation of 2.26 ± 0.26 f o r the 30 analyses was calculated. In addition to t h i s i n t e r a c t i o n c o e f f i c i e n t , one can c a l c u l a t e from the H i l l plot a K g value (pseudo-association constant) f o r the i n t e r a c t i o n of gentamicin with c e l l s , since the Y-axis intercept i s - l n Ks. The ca l c u l a t e d K values were used f o r r e l a t i v e comparisons of s the d i f f e r e n t aminoglycosides since they varied over 4 orders of 50 Figure 18. Relationship between the minimal i n h i b i t o r y concentrations (MIC) of the d i f f e r e n t aminoglycoside a n t i b i o t i c s and the 1°8^Q of the pseudo-association constants K g, extrapolated from H i l l plots such as those shown in F i g . 17. The abbreviations f o r the aminoglycoside a n t i b i o t i c s and t h e i r symbols were: tobramycin = TM (A); amikacin = AK ( 0 ) ; sisomycin = SI (x); gentamicin = GM (•); streptomycin = SM (•); netil-ymycin = NT ( A ) ; neomycin = NM ( f ) ; kanamycin = KM (!) . 6H 4l 2H KM • SM • oNM • • o o SI * • • A • • o • • o • X T AK NT • • GM TB — i — 1 — i — 10 MIC (um) 100 52 magnitude. A pl o t of log K g against the logarithm of the minimal i n h i b i t o r y concentrations (MIC) f o r the d i f f e r e n t aminoglycosides i s shown in Figure 18. There were s i g n i f i c a n t v a r i a t i o n s for each a n t i b i o t i c in i n d i v i d u a l determinations of the K despite the fac t that these numbers s derived from H i l l p lots i n which the c o r r e l a t i o n c o e f f i c i e n t s (r) were greater than 0.99 and, as noted above, the slopes of the i n d i v i d u a l l i n e s (the H i l l number) were remarkably constant. One possible reason f o r t h i s might be d i f f e r e n t i a l contamination of i n d i v i d u a l batches of c e l l s by divalent cations since the i n i t i a l rate of NPN uptake was strongly affected by diva l e n t cations ( F i g . 8, curve C; F i g . 15). Nevertheless, a highly s i g n i f i c a n t c o r r e l a t i o n between log MIC and log K was s demonstrated (r = 0.68, df = 29, p <0.001 f o r a str a i g h t l i n e with a slope of 1.33 by l e a s t squares a n a l y s i s ) . The a f f i n i t y of a substrate f o r i t s i n t e r a c t i o n s i t e i n a cooperative i n t e r a c t i o n i s sometimes expressed as the SQ or substrate concentration at h a l f maximal v e l o c i t y (given by the X axis intercept of the H i l l p l o t = log S Q ^. We were also able to demonstrate a s i g n i f i c a n t c o r r e l a t i o n between and MIC for d i f f e r e n t aminoglycosides. P o l a r i z a t i o n Studies. Addition of the neutral probe DPH to c e l l s followed by gentamicin addition caused s i m i l a r k i n e t i c s of fluorescence increase as those shown in F i g . 8 f o r NPN ( F i g . 19). The enhancement of NPN (Table I) and DPH fluorescence a f t e r gentamicin-promoted uptake suggested that these probes were p a r t i t i o n i n g into a more f l u i d environment. To confirm t h i s , we performed fluorescence p o l a r i z a t i o n studies with DPH, which has 53 frequently been used f o r such studies. DPH in acetone gave a low p o l a r i z a t i o n value (Table II) since the probe i s highly mobile in t h i s solvent ( F r e i f e l d e r , 1982). Decreasing mobility or binding of the probe r e s u l t s i n increased p o l a r i z a t i o n ( F r e i f e l d e r , 1982) as demonstrated in Table II f o r DPH in water and c e l l - a s s o c i a t e d DPH in the absence of added a n t i b i o t i c . In the presence of gentamicin, or another p o l y c a t i o n i c a n t i b i o t i c polymyxin 8, a decrease in p o l a r i z a t i o n was observed. This suggested that these a n t i b i o t i c s caused DPH to move into a more mobile environment within the c e l l s . 54 Figure 19. The e f f e c t of gentamicin addition on the p o l a r i z e d fluorescence i n t e n s i t y of DPH added to i n t a c t c e l l s of P. aeruginosa. S t r a i n H103 c e l l s were resuspended in 2 ml of 5 mM Hepes buffer with 2 yM DPH. a - fluorescence i n t e n s i t y with the input p o l a r i z e r set at 90° and the output p o l a r i z e r set at 0 ° . b - fluorescence i n t e n s i t y with th input p o l a r i z e r set at 90° and the o output p o l a r i z e r set at 90 . c - fluorescence i n t e n s i t y with the input p o l a r i z e r set at 0° and the o output p o l a r i z e r set at 90 . d - fluorescence i n t e n s i t y with the input p o l a r i z e r set at 0° and the output p o l a r i z e r set at 0°. At the arrow, 25 yg/ml gentamicin was added with the p o l a r i z e r s set as f o r d and the fluorescence increase followed over 12 min. F l u o r e s c e n c e Ln Ln Table I I : Fluorescence p o l a r i z a t i o n of the DPH taken up af t e r the i n t e r a c t i o n of gentamicin and polymyxin B with P. aeruginosa c e l l s . Experimental Conditions P o l a r i z a t i o n DPH in water 0.16 DPH in acetone 0.05 DPH c e l l s 0.36 DPH + c e l l s + gentamicin (25 yg/ml) 0.27 DPH + c e l l s + polymyxin B (20 yg/ml) 0.28 57 DISCUSSION In t h i s t h e s i s , the effectiveness of fluorescent probes in studying the interactions of AG's with P. aeruginosa c e l l s has been demonstrated. Since experiments with the anionic probe ANS produced rather complex k i n e t i c s , such that the probe d i d not s p e c i f i c a l l y indicate what was happening at the outer surface of P. aeruginosa c e l l s , a l l subsequent experiments were c a r r i e d out with NPN. The uptake of NPN that was measured probably r e f l e c t s outer membrane permeabilization to NPN by amino glycosides. As evidence for t h i s , enhancement of NPN uptake by gentamicin could only be demonstrated in i n t a c t b a c t e r i a l c e l l s . We found that gentamicin did not stimulate uptake of NPN into b a c t e r i a l outer membrane v e s i c l e s , inner membrane v e s i c l e s or phospholipid liposomes prepared s y n t h e t i c a l l y . In other words, the increase i n NPN fluorescence upon addition of GM was independent of gentamicin concentration. These v e s i c l e s became immediately saturated with NPN, which had spe c t r a l properties s i m i l a r to the NPN taken up by gentamicin-treated whole c e l l s . This suggests that gentamicin i s disorganizing the c e l l surface in such a way that NPN can p a r t i t i o n into the outer (and probably also the inner) membrane. Presumably the outer membrane structure was disorganized by the French Press treatment used to make outer membrane v e s i c l e s , and synthetic phospholipid v e s i c l e s are simple l i p i d b i l a y e r liposomes. In further agreement with t h i s concept, the rate of NPN fluorescence increase was re l a t e d to the amount of gentamicin added (Figs. 8, 17), although the f i n a l amount of NPN associated with c e l l s was r e l a t i v e l y 58 constant. This suggests that NPN enters the b a c t e r i a l c e l l v i a a l i m i t e d number of access areas, but that NPN uptake, once i n i t i a t e d , proceeds u n t i l the s i t e s with which NPN associates are saturated. Presumably the number of access s i t e s for NPN i s determined by the concentration of gentamicin in the system. I t was previously proposed in t h i s lab, that the i n t e r a c t i o n s i t e s f o r aminoglycosides on the outer membrane of P. aeruginosa are those s i t e s where cations noncovalently crossbridge adjacent LPS molecules (14,15). Presumably, aminoglycosides displace Mg^+ from these s i t e s thus d i s t o r t i n g outer membrane structure. In agreement with t h i s , we could 2+ 2+ demonstrate that Mg or Ca i n h i b i t e d gentamycin-promoted uptake of NPN (Fig. 8,16). This r e s u l t could be explained by competition of the polyca t i o n i c a n t i b i o t i c gentamicin and the divalent cations f o r a dival e n t cation binding s i t e on the outer membrane. Evidence has been published which suggests that both the p o l y c a t i o n i c a n t i b i o t i c polymyxin B and the dival e n t cation chelator EDTA i n t e r a c t with the same divalent cation binding s i t e as aminoglycosides l i k e gentamicin. In further agreement with t h i s both polymyxin B (12) and EDTA (9) cause an increase in the fluorescent i n t e n s i t y of hydrophobic fluorescent probes added to c e l l s . Helgerson and Cramer (9) postulated that EDTA treatment removed the permeability b a r r i e r of the outer membrane of E. c o l i to NPN. This would allow the NPN molecules greater access to binding s i t e s on the c e l l surface. More recently, i t has been shown that polymyxin B interacts with a di v a l e n t cation binding s i t e on LPS (17). Schindler and Osborn (17) used fluorescence analysis of dansylated derivatives of LPS to show that LPS contains one and possibly two types of binding s i t e s f o r Mg + + and C a + + , in the KDO and phosphate groups. Furthermore, i t has been shown that polymyxin B and gentamicin each compete with a c a t i o n i c spin l a b e l probe, CAT.^, for a divalent cation binding s i t e on P. aeruginosa LPS (AA Paterson, REW Hancock, EJ McGroarty, manuscript in preparation). One of the major observations of t h i s paper i s that the i n i t i a l rates of fluorescence increase vary according to the gentamicin concentration i n a fashion that i s amenable to k i n e t i c a n a l y s i s . A plot of i n i t i a l rates of fluorescence increase as a function of gentamicin concentrations gave a sigmoidal plot suggesting p o s i t i v e c o o p e r a t i v i t y . To confirm t h i s , we replotted the data as a H i l l p l o t . The advantage of th i s treatment i s that the ordinate term [log (l-o))/o), where <J> i s the r a t i o of the rate of fluorescence increase at a given gentamicin concentration], has no units and thus i s independent of the a r b i t r a r y fluorescent u n i t s . Thus, assuming that the rate of NPN uptake d i r e c t l y r e f l e c t s the i n t e r a c t i o n of gentamicin with outer membranes, which seems l i k e l y , the H i l l p l o t provides k i n e t i c data which r e f l e c t only t h i s i n t e r a c t i o n . S i milar arguments are v a l i d for each of the eight aminoglycosides subjected to k i n e t i c analysis by t h i s method. In H i l l p l o t analyses, of a l l eight aminiglycosides incorporating 30 separate sets of data, the H i l l number (the slope of the H i l l plot) was 2.26 ± 0.26. Since t h i s number provides an estimation of the minimum number of in t e r a c t i o n s i t e s involved in the cooperative permaeabilization of outer membranes to NPN, we can make the assumption that at l e a s t three or more s i t e s are involved. While the H i l l numbers were remarkably s i m i l a r for each of the 8 aminoglycosides, i t was i n t e r e s t i n g that the pseudoassociat ion constant Ks varied s u b s t a n t i a l l y . The highly s i g n i f i c a n t (p < 0.001) l i n e a r r e l a t i o n s h i p between the Ks value and the MIC value for the d i f f e r e n t aminoglycosides suggests that the measured i n t e r a c t i o n at the surface of the outer membrane may be an important determinant of the e f f i c i e n c y and/or rate of k i l l i n g . In agreement with t h i s , Mg + + and Ca^ + which strongly antagonize the transport of and k i l l i n g by aminoglycosides (1) also s i g n i f i c a n t l y i n h i b i t e d the enhancement of NPN uptake by aminoglycosides ( F i g . 8, curve C, 16). I t should be noted, however, that our experiments were performed in the presence of cyanide which blocks energy-dependent transport and k i l l i n g , suggesting that i n t e r a c t i o n at the outer membrane precedes these energy dependent events. In t h i s regard, i t is i n t r i g u i n g that the time course of NPN uptake following the treatment of c e l l s with aminoglycosides strongly mimicked the time course of aminoglycoside uptake (1,7). Our experiments also suggested that the increase in NPN fluorescence af t e r gentamicin addition to c e l l s was due to a combination of NPN being taken up into the c e l l s and (subsequently) an a l t e r a t i o n in the environment of NPN molecules which enhanced i t s fluorescence y i e l d . Furthermore, p o l a r i z a t i o n studies with DPH, another neutral fluorescence probe, confirmed that the presence of gentamicin in a c e l l suspension caused the probe molecules to p a r t i t i o n into a more highly mobile or f l u i d environment within the c e l l s , as shown by a decrease in p o l a r i z a t i o n value. A l t e r n a t i v e l y t h i s decrease in p o l a r i z a t i o n might be explained by 61 an a l t e r a t i o n in fluorescence l i f e t i m e although we could not measure t h i s on our apparatus. Although t h i s does not formally demonstrate that aminoglycosides i n t e r a c t with outer membranes to promote t h e i r own uptake, we have described the enhancement a f t e r gentamicin treatment of the uptake of hydrophobic probes NPN and DPH, and previously of the protein lysozyme and B-lactam a n t i b i o t i c n i t r o c e f i n (8). This data i s thus consistent with the self-promoted uptake hypothesis (7,15). To further test and prove t h i s hypothesis, i t w i l l be necessary to learn more about the events following the i n i t i a l i n t e r a c t i o n . Attempts to f l u o r e s c e n t l y tag the aminoglycoside a n t i b i o t i c s d i r e c t l y are cu r r e n t l y underway for t h i s reason. 62 LITERATURE CITED 1. Bryan, L.E. and H.M. van den Elzen. 1976. Streptomycin accumulation in susceptible and r e s i s t a n t strains of Escherichia c o l i and Pseudomonas aeruginosa. Antimicrob. Agents and Chemother. 9:928-938. 2. Cramer, W.A., P.W. Postma. and S.L. Helgerson. 1976. An evaluation of N-phenyl-l-napthylamine as a probe of membrane energy state i n Escherichia c o l i . Biochim. Biophys. Acta 449:401-411. 3. Darveau, R.P. , Hancock, R.E.W. 1983. Procedure f o r I s o l a t i o n of Ba c t e r i a l Lipopolysaccharides from both Smooth and Rough Pseudomonas  aeruginosa and Salmonella typhimurium s t r a i n s . J. B a c t e r i o l . 155:831-838. 4. F r e i f e l d e r , D. 1982. "Physical Biochemistry", 2nd ed. W.H. Freeman and Co. San Francisco, 562p. 5. Gomperts, B., F. Lantelme, and R. Stock. 1970. Ion assoc i a t i o n reactions with b i o l o g i c a l membranes, studied with the fluorescent Dye 1,8-ANS. J. Membr. B i o l . 3:241-246. 6. Hancock, R.E.W. 1981. Aminoglycoside uptake and mode of action -with s p e c i a l reference to streptomycin and gentamicin. I. Antagonists and mutants. Antimicrob. Chemother. 8:249-276. 7. Hancock, R.E.W. 1981. Aminoglycoside uptake and mode of action -with s p e c i a l reference to streptomycin and gentamicin. I I . E f f e c t s of aminoglycosides n c e l l s . Antimicrob. Chemother. 8:429-445. 63 8. Hancock, R.E.W., V.J. R a f f l e , and T.I. Nicas. 1981. Involvement of the outer membrane in gentamicin and streptomycin uptake and k i l l i n g in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 19:7 7 7-783. 9. Helgerson, S.L., and Cramer, W.A. 1977. Changes in Es c h e r i c h i a c o l i c e l l envelope structure and the s i t e s of fluorescence probe binding caused by carbonyl cyanide p-trifluoromethoxypahneylhydrazone. Biochem. 16:4109-4117. 10. Hurwitz, C , and C.L. Rosano. 1965. Evidence for a streptomycin permease. J. B a c t e r i o l . 90:1233-1237. 11. Madeira, V.M.C., and M.C. Antunes-Madeira. 1973. Inte r a c t i o n of Ca^ + and Mg^+ with synaptic plasma membranes. Biochim. Biophys. Acta 323:396-407. 12. Newton, B.A. 1954. Si t e of action of polymyxin on Pseudomonas  aeruginosa: Antagonism by cations. J. Gen. M i c r o b i o l . 10:491-499. 13. Nakae, R., and T. Nakae. 1982. Di f f u s i o n of aminoglycoside a n t i b i o t i c s across the outer membrane of Escherichia c o l i • Antimicrob. Agents Chemother. 22:554-559. 14. Nicas, T.I., and R.E.W. Hancock. 1980. Outer membrane protein HI of Pseudomonas aeruginosa: Involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin. J. B a c t e r i o l . 143:872-878. 15. Nicas, T.I. and R.E.W. Hancock. 1983. A l t e r a t i o n of s u s c e p t i b i l i t y to EDTA, polymyxin B and gentamicin in Pseudomonas aeruginosa by divalent cation regulation of outer membrane protein HI. J. Gen. Micr o b i o l . 129:509-517. 6 4 16. Nieva-Gomez, D. , J. Konisky, and R.B. Gennis. 1976. Membrane changes in Escherichia c o l i induced by c o l i c i n Ia and agents known to disrupt energy transduction. Biochem. 15:2747-2753. 17. Schindler, M., and M.J. Osborn. 1979. Interaction of dival e n t cations and polymyxin B with lipopolysaccharide. Biochem. 18:4425-4430. 18. Sykes, R., and A. Morris. 1975. Resistance of Pseudomonas aeruginosa to antimicrobial drugs. Progr. Med. Chem. 333:393. 19. Uratani, V. 1982. Dansyl chloride l a b e l l i n g of Pseudomonas  aeruginosa treated with Pyocin RI: Change in permeability of the c e l l envelope. J. B a c t e r i o l . 149:523-528. 

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