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Immunochemistry of Pseudomonas aeruginosa outer membrane proteins Mutharia, Lucy Muthoni 1984

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IMMUNOCHEMISTRY  OF PSEUDOMONAS AERUGINOSA OUTER MEMBRANE PROTEINS  by  Lucy Muthoni M u t h a r i a B.Sc.  (Hons), 1976, U n i v e r s i t y  M.Sc,  of Nairobi,  1980, U n i v e r s i t y o f N a i r o b i ,  Kenya  Kenya  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Faculty  o f Graduate  Department University  We a c c e p t t h i s  Studies  of M i c r o b i o l o g y  of B r i t i s h  Columbia  t h e s i s as conforming to the r e q u i r e d  / The U n i v e r s i t y  of B r i t i s h  © Lucy Muthoni September,  Columbia  Mutharia,/Y&J. 1984  standard  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  requirements f o r an advanced degree a t the  the  University  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 it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  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 of 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  department or by h i s or her  the head o f  representatives.  my  It is  understood t h a t copying or 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 gain  s h a l l not be  allowed without my  permission.  Department of  Hyr.R-Q  The U n i v e r s i t y of B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3  E-6  (3/81)  &IQ  L-OQH  Columbia  '  written  ABSTRACT  The  immunochemistry and c o n s e r v a t i o n o f Pseudomonas a e r u g i n o s a  membrane components was s t u d i e d .  Conservation  outer  o f the major outer membrane  p r o t e i n s o f P. a e r u g i n o s a was demonstrated by the s t r o n g s i m i l a r i t i e s i n the SDS-polyacrylamide g e l p r o f i l e s o f o u t e r membrane p o l y p e p t i d e s different  s t r a i n s o f P. a e r u g i n o s a .  Immunological c r o s s - r e a c t i v i t y was  demonstrated f o r p r o t e i n s F, H2 and I among 17 serotype  s t r a i n s of  P. a e r u g i n o s a ,  u s i n g a r a b b i t p o l y c l o n a l a n t i - o u t e r membrane serum.  order t o study  the c o n s e r v a t i o n o f s p e c i f i c  P. a e r u g i n o s a  from 47  In  a n t i g e n i c s i t e s among  s t r a i n s and other gram-negative b a c t e r i a , monoclonal  antibodies s p e c i f i c  f o r p r o t e i n s F, H2 and l i p o p o l y s a c c h a r i d e (LPS) were  isolated. Monoclonal a n t i b o d i e s were used t o study of the d i f f e r e n t  r e g i o n s o f LPS.  the a n t i g e n i c c o n s e r v a t i o n  These a n t i b o d i e s demonstrated  strong  c o n s e r v a t i o n o f the l i p i d A r e g i o n o f LPS among a l l t e s t e d gram-negative b a c t e r i a , l e s s c o n s e r v a t i o n o f the P. a e r u g i n o s a heterogeneity  o f the LPS O-antigen.  be h i g h l y conserved  p r o t e i n F showed two d i s t i n c t two d i s t i n c t  In c o n t r a s t , p r o t e i n H2 was shown t o  among the f l u o r e s c e n t Pseudomonads by the r e a c t i v i t y  of the monoclonal antibody MA1-6.  least  rough core and g r e a t  Monoclonal a n t i b o d i e s s p e c i f i c f o r  specificities  suggesting  a n t i g e n i c s i t e s on the p r o t e i n .  MA4-2, MA2-10 and MA4-10 i n t e r a c t e d w i t h  t h a t there were a t  A n t i b o d i e s MA4-4,  p r o t e i n F i n the o u t e r membranes  of a l l t e s t e d P. strains.  a e r u g i n o s a s t r a i n s as w e l l as P. s y r i n g a e and P. p u t i d a  These a n t i b o d i e s a l s o i n t e r a c t e d with d e f i n e d p r o t e o l y t i c  p e p t i d e s of p r o t e i n F.  The e p i t o p e r e c o g n i z e d by these a n t i b o d i e s was  d e s t r o y e d by 2-mercaptoethanol and cyanogen bromide treatment.  In  c o n t r a s t , antibody MA5-8 i n t e r a c t e d o n l y with P. a e r u g i n o s a s t r a i n s , two  cyanogen b r o m i d e - d e r i v e d  p e p t i d e s of the p r o t e i n and w i t h  2-mercaptpethanol t r e a t e d or u n t r e a t e d p r o t e i n .  with  the  None o f these a n t i b o d i e s  i n t e r a c t e d w i t h a p r o t e i n F d e f i c i e n t P. a e r u g i n o s a  strain.  iv  TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  iv  L i s t of Tables  x  L i s t of F i g u r e s  . .  xii  ACKNOWLEDGEMENTS  .  . . . . . . . . .  xv  DEDICATION  xvi  INTRODUCTION . . . . . . . . . 1.  The m e d i c a l  2.  The  3.  Components and  4.  5.  importance of Pseudomonas a e r u g i n o s a  1  c e l l envelope of gram-negative b a c t e r i a  3  s t r u c t u r e of the o u t e r membrane  (A)  Lipopolysaccharide  (B)  Proteins  5  .  6 •  C e l l envelope and  the  immune response  7 9  (A)  Lipopolysaccharide  10  (B)  Proteins  13  Aims of t h i s  MATERIALS AND 1.  1  study  16  METHODS  Bacteria  . . . . . . .  (A)  Media and  (B)  Bacterial strains  (C)  C e l l envelope and  18  growth c o n d i t i o n s  18 18  o u t e r membrane i s o l a t i o n  20  TABLE OF CONTENTS, c o n t i n u e d Page  2.  3.  (D)  Protein  and LPS p u r i f i c a t i o n  .  . . . . . . .  (E)  Sodium d o d e c y l s u l p h a t e ( S D S ) - p o l y a c r y l a m i d e g e l  21  electrophoresis  21  (i)  protein  22  (ii)  LPS  staining  staining  .  (F)  Protein  and Keto-deoxyoctonate  (KDO)  assays  . . . . .  (G)  Enzymatic d i g e s t i o n of p r o t e i n F . . . . . . . . . .  (H)  Chemical c l e a v a g e of p r o t e i n F .  22 22  .  23 23  Animals (A)  Animals used  (B)  Immunizing  Tissue  24  protocol  . . .  24  Culture  (A)  Myeloma c e l l  (B)  Medium, maintenance  (C)  G e n e r a t i o n o f monoclonal (i)  cell  (i i)  cloning  lines  25 and f r e e z i n g of c e l l  lines  . . . .  antibodies  28  fusion  (D)  Ascites  production  (E)  Antibody p u r i f i c a t i o n  26  29 .  31 31 32  vi  TABLE OF CONTENTS, c o n t i n u e d  Page 4.  5.  Antibody  characterization  (A)  Enzyme-linked immunosorbent assays-ELISA  33  (B)  Western e l e c t r o p h o r e t i c  36  (C)  Double immunodiffusion assay  (D)  Agglutination  (E)  Indirect  (F)  Colony b l o t t i n g  blot transfers  . . . . . . . .  37  assays  .  38  immunofluorescence  Serum s e n s i t i v i t y t e s t i n g  .  40 . . . . . .  41  . . . . . . . . . . .  42  RESULTS CHAPTER I .  Outer membrane a n t i g e n s o f P. a e r u g i n o s a  1.  Outer membrane p a t t e r n s  2.  Cystic f i b r o s i s  of serotyping  strains  .  44  i s o l a t e s - c h a r a c t e r i z a t i o n and  outer membrane p a t t e r n s 3.  I s o l a t i o n and c h a r a c t e r i z a t i o n against  4.  5.  of a p o l y c l o n a l  antisera  o u t e r membranes o f P. a e r u g i n o s a  Interaction  of p o l y c l o n a l  54  s e r a w i t h the o u t e r  membranes o f P. a e r u g i n o s a s t r a i n s  56  Summary  63  CHAPTER I I . 1.  47  .  Monoclonal a n t i b o d i e s  t o o u t e r membrane a n t i g e n s  I s o l a t i o n and c h a r a c t e r i z a t i o n  o f monoclonal a n t i b o d i e s  (A)  An LPS 0-Antigen s p e c i f i c monoclonal a n t i b o d y  (B)  Rough LPS s p e c i f i c monoclonal a n t i b o d i e s  (C)  L i p i d A s p e c i f i c monoclonal a n t i b o d i e s  . .  . . . . .  65 65 66 67  vii  TABLE OF CONTENTS, c o n t i n u e d Page (D) - A l i p o p r o t e i n H2 s p e c i f i c  2.  (E)  Protein F specific  (F)  Monoclonal  monoclonal antibody  . . . .  monoclonal a n t i b o d i e s  a n t i b o d y MA1-3  .  67  . .  68  . .  68  Use o f monoclonal a n t i b o d i e s t o study o u t e r membrane a n t i g e n h e t e r o g e n e i t y o f P. a e r u g i n o s a  . . .  (A)  monoclonal  Specificity  of p r o t e i n F s p e c i f i c  73  antibodies (B)  73  C r o s s - r e a c t i v i t y o f a l i p o p r o t e i n H2  specific  monoclonal a n t i b o d y  3.  83  (C)  H e t e r o g e n e i t y o f LPS-rough c o r e i n P. a e r u g i n o s a  . .  (D)  H e t e r o g e n e i t y o f LPS O-antigen  96  (E)  Conservation of L i p i d A  98  Summary  CHAPTER I I I .  93  104  C h a r a c t e r i z a t i o n o f o u t e r membrane e p i t o p e s u s i n g  monoclonal a n t i b o d i e s 1.  C e l l surface l o c a l i z a t i o n  o f the a n t i g e n i c s i t e s  r e c o g n i z e d by monoclonal a n t i b o d i e s . . ' (A)  Surface l o c a l i z a t i o n  s t u d i e s by immunofluorescence  (B)  Surface l o c a l i z a t i o n  s t u d i e s by colony  immunoblotting (C)  . . .  I n t e r a c t i o n o f monoclonal a n t i b o d i e s with b a c t e r i a by c o l o n y immunoblotting  .  106  . .  106  I l l other 122  vi i i  TABLE OF CONTENTS, c o n t i n u e d Page 2.  S t u d i e s on p r o t e i n F e p i t o p e s r e c o g n i z e d by monoclonal antibodies (A)  123  E f f e c t o f 2-mercaptoethanol on b i n d i n g o f p r o t e i n F s p e c i f i c monoclonal a n t i b o d i e s  (B)  I n t e r a c t i o n o f monoclonal a n t i b o d i e s with cyanogen bromide  (C)  123  fragments o f p r o t e i n F  126  I n t e r a c t i o n o f monoclonal a n t i b o d i e s w i t h p r o t e o l y t i c peptides of p r o t e i n F  3.  129  Summary  137  DISCUSSION 1.  Conservation (A)  and h e t e r o g e n e i t y o f LPS e p i t o p e s  LPS o f P. a e r u g i n o s a patients  2.  isolates  from c y s t i c  139 fibrosis  . . . . . . . . . . . . .  142  A n t i g e n i c c o n s e r v a t i o n o f P. a e r u g i n o s a o u t e r membrane proteins  . . . . . . .  143  (A)  Conservation  o f o u t e r membrane p r o t e i n p a t t e r n s  (B)  Antigenic conservation revealed using p o l y c l o n a l  . . .  antiserum (C)  Conservation  145 o f s p e c i f i c e p i t o p e s on P.  aeruginosa  outer membrane p r o t e i n s (D)  144  147  D i s t r i b u t i o n o f s p e c i f i c e p i t o p e s among o t h e r species of b a c t e r i a  .  149  ix  TABLE OF CONTENTS, c o n t i n u e d Page 3.  Surface a c c e s s i b i l i t y antibodies  s t u d i e s u s i n g monoclonal  . . . . .  4.  P a r t i a l c h a r a c t e r i z a t i o n of two p r o t e i n F e p i t o p e s  5.  Prospectives  LITERATURE CITED  154 . . . .  156 158 160  X  LIST OF TABLES Table I.  Page Colony morphology, LPS phenotype, s e r o t y p a b i l i t y , and s e n s i t i v i t y t o normal human serum o f s e l e c t e d P. a e r u g i n o s a i s o l a t e s from p a t i e n t s with c y s t i c  fibrosis  and rough  L P S - a l t e r e d mutants. II.  51  C r o s s - r e a c t i o n s o f a n t i s e r a to o u t e r membrane and p u r i f i e d o u t e r membrane components with o u t e r membranes from v a r i o u s serotype s t r a i n s o f P. a e r u g i n o s a  III.  I n t e r a c t i o n o f monoclonal a n t i b o d i e s w i t h p a r t i a l l y  55 purified  major o u t e r membrane p r o t e i n s o f P. a e r u g i n o s a PA01. . . . . . IV.  I n t e r a c t i o n o f monoclonal a n t i b o d i e s w i t h the o u t e r membranes of LPS a l t e r e d  V.  69  s t r a i n s o f P. a e r u g i n o s a .  70  I n t e r a c t i o n o f monoclonal a n t i b o d i e s MA2-10 and MA4-4 w i t h o u t e r membranes o f P. a e r u g i n o s a s e r o t y p e s t r a i n s and c y s t i c fibrosis  VI.  isolates  74  C r o s s - r e a c t i o n o f monoclonal a n t i b o d i e s s p e c i f i c f o r P. a e r u g i n o s a o u t e r membranes a s s e s s e d by ELISA  VII.  .  84  I n t e r a c t i o n o f monoclonal a n t i b o d i e s MAi-3, MA1-6, and MA1-8 with the o u t e r membrane antigens from 17 s e r o t y p e s t r a i n s o f P. a e r u g i n o s a  V I I I . Demonstration  97 of c e l l  s u r f a c e l o c a l i z a t i o n o f o u t e r membrane  components o f P. a e r u g i n o s a by immunofluorescence.  110  xi  LIST OF TABLES, c o n t i n u e d Table IX.  Page I n t e r a c t i o n o f p r o t e i n F s p e c i f i c monoclonal a n t i b o d i e s w i t h p u r i f i e d p r o t e i n - F, o u t e r membranes and whole c e l l s o f s t r a i n H103 a s s e s s e d by ELISA. .  X.  112  B i n d i n g o f 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 P. a e r u g i n o s a o u t e r membrane a n t i g e n s to c o l o n y b l o t s  XI.  R e a c t i o n o f LPS s p e c i f i c monoclonal a n t i b o d i e s w i t h membranes from P. a e r u g i n o s a s t r a i n s  XII.  114  outer  i n ELISA assays  P o t e n t i a l taxonomic v a l u e o f monoclonal a n t i b o d i e s  140 151  X I I I . D i f f e r e n t i a t i o n o f two c l a s s e s o f monoclonal a n t i b o d i e s specific  f o r P. a e r u g i n o s a p r o t e i n F .  157  xi i  LIST OF FIGURES Figure 1.  Page  SDS-polyacrylamide  g e l electrophoretogram  o f o u t e r membranes  of P. a e r u g i n o s a s t r a i n H103 and s e r o t y p i n g s t r a i n s 2.  SDS-gel e l e c t r o p h o r e t o g r a m P. aeruginosa  3.  o f o u t e r membrane p r o t e i n s o f  from p a t i e n t s w i t h c y s t i c f i b r o s i s  SDS-gel e l e c t r o p h o r e t o g r a m s isolates  4.  isolates  . . .  f i b r o s i s s t a i n e d f o r LPS . .  of e l e c t r o p h o r e t i c b l o t  p r o t e i n s of P. a e r u g i n o s a a f t e r  Densitometer  53  o f o u t e r membrane  interaction  with r a d i o i o d i n a t e d  a n t i b o d i e s t o o u t e r membrane p r o t e i n s 5. A. and B.  49  o f o u t e r membranes o f P. a e r u g i n o s a  from p a t i e n t s w i t h c y s t i c  Autoradiogram  46  58  t r a c i n g s o f autoradiograms  of separated  outermembrane p r o t e i n s from P. a e r u g i n o s a s t r a i n H103, s e r o t y p e strains  6.  and c l i n i c a l  i s o l a t e PI  61, 62  Western immunoblots o f P. a e r u g i n o s a s t r a i n H103 interaction  after  w i t h monoclonal a n t i b o d i e s MAl-8, MA3-5, MA1-6,  MA4-4 and 5E4 . . . . 7.  .  Western immunoblots o f o u t e r membranes o f the s e r o t y p i n g strains  o f P. a e r u g i n o s a a f t e r  interaction  w i t h monoclonal  antibody MA5-8 8.  72  78  I n t e r a c t i o n o f monoclonal a n t i b o d y MA2-10 w i t h o u t e r membranes from c y s t i c  f i b r o s i s P. a e r u g i n o s a  isolates  .  80  xi i i  LIST OF FIGURES, c o n t i n u e d Figure 9.  Page  Cross-reactivity  of protein  F s p e c i f i c monoclonal a n t i b o d y  MA4-4 with o u t e r membranes from other b a c t e r i a l s p e c i e s 10.  Western immunoblots o f o u t e r membranes o f s e r o t y p e of P. a e r u g i n o s a a f t e r  . . . .  strains  i n t e r a c t i o n with monoclonal a n t i b o d y  MA1-6 11.  88  Western immunoblot P. a e r u g i n o s a  o f o u t e r membranes o f mucoid and non-mucoid  isolates  interacted  with monoclonal a n t i b o d i e s  MA4-4 and MA1-6 12.  90  Western immunoblots o f o u t e r membranes o f d i f f e r e n t Pseudomonads i n t e r a c t e d  13.  after reaction  Western immunoblot serotype s t r a i n s  15.  with monoclonal antibody MA1-6  with monoclonal a n t i b o d y MA3-5.  after reaction  with monoclonal a n t i b o d y 5E4  . 101  R e a c t i o n o f monoclonal antibody 5E4 w i t h a Western b l o t o f  Indirect  immunofluorescerit  i s o l a t e s o f P. a e r u g i n o s a  Colony immunoblot  103  l a b e l l i n g o f i n t a c t P. a e r u g i n o s a  tagged w i t h monoclonal a n t i b o d i e s t o o u t e r membrane components. 17.  95  o f o u t e r membranes o f P. a e r u g i n o s a  outer membranes o f c l i n i c a l 16.  92  Western immunoblot o f o u t e r membranes o f P. a e r u g i n o s a serotype s t r a i n s  14.  82  109  showing i n t e r a c t i o n o f monoclonal antibody  MA4-10 w i t h d i f f e r e n t b a c t e r i a l s t r a i n s  117  xiv  LIST OF  FIGURES, c o n t i n u e d  Figure 18.  Page  Colony  immunoblot showing i n t e r a c t i o n  MAl-8 with serotype s t r a i n s P. aeruginosa 19.  Colony MA1-6  20.  of monoclonal  and c l i n i c a l  isolates  antibody  of  . . . . . . . . . . . . .  immunoblots showing i n t e r a c t i o n with d i f f e r e n t  bacterial  119 of monoclonal  antibody  strains  Western immunoblots of p u r i f i e d p r o t e i n  121 F:  effect  of  2-  mercaptoethanol 21.  125  SDS-gel e l e c t r o p h o r e t o g r a m p u r i f i e d protein  and Western immunoblots of  F b e f o r e and a f t e r d e g r a d a t i o n w i t h cyanogen  bromide 22.  128  Western immunoblot of n a t i v e and p r o t e o l y t i c a l l y protein  F, from a p u r i f i e d p r o t e i n  and whole c e l l s 23.  treated  F sample, o u t e r membranes  .  132  SDS-gel electrophoretograms course p r o t e o l y s i s  of p r o t e i n  of p u r i f i e d p r o t e i n  F p e p t i d e s from a time F w i t h the enzyme  trypsin 24.  Interaction  134 of p r o t e i n  with p r o t e o l y t i c a l l y  F s p e c i f i c monoclonal antibody MA2-10  t r e a t e d whole c e l l s of P.  CF C46nm and P. p u t i d a ATCC 12633  aeruginosa 136  ACKNOWLEDGEMENTS  I am s i n c e r e l y constant  indebted  to my s u p e r v i s o r , Bob Hancock., f o r h i s  a d v i c e , guidance, and f r i e n d s h i p d u r i n g the course o f my  studies. I am g r a t e f u l to the Department o f M i c r o b i o l o g y Commonwealth S c h o l a r s h i p Plan f o r f i n a n c i a l  and the Canadian  support.  I wish t o thank members o f the M i c r o b i o l o g y Department, and members of my s u p e r v i s o r y committee f o r t h e i r h e l p and a d v i c e .  In p a r t i c u l a r ,  many thanks to the v a r i e d members o f Bob's l a b f o r t h e i r f r i e n d s h i p and support.  Thanks t o Susan Heming f o r h e r p a t i e n c e  i n typing this  thesis.  xv i  DEDICATION  This  thesis  i s d e d i c a t e d to my  have always supported  p a r e n t s , E. N j e r i and T. M u t h a r i a ,  our endeavours and  aspirations.  \  who  1  INTRODUCTION  The  gram-negative  proliferates  bacterium Pseudomonas a e r u g i n o s a e x i s t s  i n a wide range  of n a t u r a l environments  n u t r i t i o n a l v e r s a t i l i t y which allows i t to u t i l i z e carbon  sources.  During the l a s t 20-30 years P.  due  to i t s  a l a r g e v a r i e t y of  a e r u g i n o s a has emerged as  a major o p p o r t u n i s t i c pathogen a s s o c i a t e d with high m o r t a l i t y acquired  1.  importance  o f P.  aeruginosa  i m p r e s s i v e i n c r e a s e i n the i n c i d e n c e of gram-negative  i n f e c t i o n s o c c u r r e d w i t h the i n t r o d u c t i o n of p e n i c i l l i n the subsequent  broad spectrum  infections.  a n t i b i o t i c s has  more a n t i b i o t i c  antibiotics  and  S i n c e then, the i n t e n s i v e a p p l i c a t i o n o f  resulted  in bacterial substitution,  resistant strains replacing highly antibiotic  with  susceptible  In p a r t i c u l a r , P. a e r u g i n o s a which i s w e l l known f o r i t s h i g h  intrinsic 1979;  bacterial  e l i m i n a t i o n of S t r e p t o c o c c i and S t a p h y l o c o c c i as the major  causes of nosocomial  ones.  in hospital  infections.  The m e d i c a l An  and  r e s i s t a n c e to a n t i b i o t i c s , d e t e r g e n t s and a n t i s e p t i c s  Hancock, 1981)  (Bryan,,  has been a s s o c i a t e d with every aspect of the  hospital  environment. Although  o n l y 4-10%  of the normal human a d u l t p o p u l a t i o n are f e c a l or  pharyngeal c a r r i e r s of P. a e r u g i n o s a (Bodey §_t a l . , 1983), the r a t e i s r e p o r t e d to r i s e to 43% among p a t i e n t s h o s p i t a l i z e d 15 days (Bodey et a l . , 1983). P. aeruginosa caused  carrier  f o r more than  Young et a l . (1982) r e p o r t e d t h a t  o n l y 20% o f the t o t a l gram-negative  although  infections, i t  2  was a s s o c i a t e d w i t h 84% m o r t a l i t y .  The high  antibiotic  r e s i s t a n c e o f P.  aeruginosa as w e l l as the l a r g e r e p e r t o i r e o f v i r u l e n c e f a c t o r s by t h i s organism presumably c o n t r i b u t e  elaborated  t o i t s p r e v a l e n c e and v i r u l e n c e  (Young, 1980; L i u , 1974). Among the gram-negative b a c t e r i a c a u s i n g P. aeruginosa i s e x c e p t i o n a l  in its ability  nosocomial i n f e c t i o n s ,  to i n f e c t  specific  sub-populations o f p a t i e n t s due e i t h e r to the u n d e r l y i n g or as a r e s u l t o f t h e r a p e u t i c  measures.  disease  condition  Among those most s u s c e p t i b l e are  immunocompromised p a t i e n t s , e s p e c i a l l y those with acute leukemia o r neoplastic diseases  which r e s u l t i n n e u t r o p e n i a (Young, 1979; Young and  P o l l a c k , 1980) as w e l l as p a t i e n t s r e c e i v i n g immunosuppressive  therapy,  such as c y t o t o x i c and c o r t i c o s t e r o i d therapy f o r c a n c e r , t i s s u e or organ transplantations due  (Schimpff ejt a l . , 1970).  Among these p a t i e n t s ,  to P. a e r u g i n o s a b a c t e r e m i a has been r e p o r t e d  t o be as h i g h  fatality as 50-807.  (Rodriquez and Bodey, 1979). P. a e r u g i n o s a i s a l s o the predominant gram-negative b a c t e r i u m o f burn wound i n f e c t i o n s .  Pruitt  and Lindenberg  (1979) r e p o r t e d  v i c t i m s were c o l o n i s e d by the b a c t e r i u m w i t h i n hospitalization.  t h a t 50% o f burn  48 hours o f  The f r e q u e n c y o f P. a e r u g i n o s a i n f e c t i o n s i s a l s o  high  among p a t i e n t s r e q u i r i n g i n v a s i v e s u r g i c a l procedures o r i n - d w e l l i n g urinary or vascular  catheters,  and among d i a b e t i c s and intravenous  a d d i c t s , where the b a c t e r i u m gains  drug  access to s t e r i l e t i s s u e .  P. a e r u g i n o s a i s w e l l known f o r c h r o n i c  infections in cystic  p a t i e n t s where i t i s o f t e n a s s o c i a t e d with t e r m i n a l these p a t i e n t s , the b a c t e r i u m e l a b o r a t e s  fibrosis  pulmonary i l l n e s s .  massive amounts o f a mucoid  In  3  exopolysaccharide.  The f a i l u r e o f pulmonary c l e a r a n c e o f P.  may be due to the copious  amounts o f mucoid m a t e r i a l which i s p r e c i p i t a t e d  by the high amounts o f c a l c i u m p r e s e n t p a t i e n t s , as w e l l as the presence f i b r o s i s f a c t o r " which prevents 1975).  aeruginosa  i n the lung f l u i d s  o f these  i n serum o f a p o s t u l a t e d " c y s t i c  normal c i l i a r y  movement (Bowman e t a l . ,  Thomassen e_t a l . (1979) r e p o r t e d the presence  of a factor  serum o f these p a t i e n t s t h a t impaired normal p h a g o c y t i c  i n the  f u n c t i o n of r a b b i t  and human a l v e o l a r macrophages. In a d d i t i o n , P. a e r u g i n o s a pneumonias, u r i n a r y t r a c t  i s a common cause o f h o s p i t a l - a c q u i r e d  i n f e c t i o n s and e n d o c a r d i t i s and sometimes causes  f a t a l meningitis.  2.  The c e l l  envelope  o f gram-negative b a c t e r i a  The  envelope  o f P. a e r u g i n o s a ,  cell  like  that of other  b a c t e r i a , c o n s i s t s o f three l a y e r s , the c y t o p l a s m i c peptidoglycan The  c y t o p l a s m i c membrane i s a p h o s p h o l i p i d b i l a y e r c o n t a i n i n g 40-60%  energy-coupled  L o c a l i z e d i n t h i s membrane are s p e c i f i c  t r a n s p o r t systems and components o f the c e l l u l a r  energy-generating  systems, as w e l l as enzymes f o r the s y n t h e s i s and export  outer membrane and p e p t i d o g l y c a n The  ( i n n e r ) membrane, a  (murein) l a y e r , and the o u t e r membrane.  by weight o f p r o t e i n s .  of  gram-negative  components.  p e r i p l a s m , l o c a t e d between the o u t e r and i n n e r membranes, i s r i c h  in o l i g o s a c c h a r i d e s , c a t a b o l i c enzymes, b i n d i n g p r o t e i n s and enzymes involved i n degradation  of toxic  substances  (Benveniste  and Davies,  1973) .  4  The  cell  shape and osmotic  stability  s t r u c t u r e of the p e p t i d o g l y c a n l a y e r .  c o n v i n c i n g evidence  by the r i g i d  The c h e m i c a l composition and  s t r u c t u r e of the p e p t i d o g l y c a n i s s i m i l a r (Meadow e_t a l . , 1978).  i s maintained  i n a l l gram-negative b a c t e r i a  In P. a e r u g i n o s a PA01, u n l i k e E. c o l i .  t h e r e i s no  o f c o v a l e n t - l i n k a g e o f t h i s l a y e r to the outer  membrane components, although  strong non-covalent  l i n k a g e has been  demonstrated (Hancock e_t a l . , 1981) . The  cell  s u r f a c e ( o u t e r ) membrane o f gram-negative b a c t e r i a forms the  i n t e r f a c e between the c e l l  and i t s environment.  p r o p e r t i e s , the o u t e r membrane prevents and  i n h i b i t s the e n t r y o f t o x i c  leakage  substances  like  Among  i t s functional  of periplasmic proteins s a l t s , d e t e r g e n t s and  a n t i b i o t i c s while allowing entry of e s s e n t i a l n u t r i e n t s .  Thus, i t  c o n s t i t u t e s a major p h y s i c a l and f u n c t i o n a l b a r r i e r f o r c e l l s .  In  P. aeruginosa, the o u t e r membrane has been shown to be the main b a r r i e r r e s p o n s i b l e f o r the h i g h i n t r i n s i c antibiotics importance  r e s i s t a n c e of t h i s bacterium to  (Nicas and Hancock, 1983; Angus e t a l . , 1982).  Of g r e a t e r  to the p r e s e n t study, the o u t e r membrane r e p r e s e n t s the  c e l l u l a r component i n v o l v e d i n the primary  i n t e r a c t i o n with the hosts*  immune system and t h e r e f o r e c o n t r i b u t e s t o the a b i l i t y o f the organism t o invade, p e r s i s t , evade and r e s i s t therefore manifest involved  the h o s t ' s defence mechanisms and  the d i s e a s e syndrome.  The o u t e r membrane components  i n each o f the above f u n c t i o n a l , p h y s i c a l and p r o t e c t i v e  f u n c t i o n s have not a l l been d e f i n e d , but a b r i e f overview  o f the s t r u c t u r e  and components o f the o u t e r membrane as r e l a t e d t o f u n c t i o n i s p r e s e n t e d below.  5  In a d d i t i o n t o these components o f the c e l l all  gram-negative b a c t e r i a c o n t a i n  structures.  additional c e l l  In many b a c t e r i a , the c e l l  amorphous or a h i g h l y o r d e r e d  envelopes, some but not  capsular  surface  envelope  i s c o v e r e d by e i t h e r an  l a y e r t h a t can be o f p r o t e i n (Kay  et  a l . , 1981, Winter §_t a l . , 1978) o r p o l y s a c c h a r i d e  et  a l . , 1977).  strains  P. a e r u g i n o s a  mannuronic and g u l u r o n i c  acid residues.  Anchored i n the c e l l and f i m b r i a e .  cell  envelope and are r e s p o n s i b l e pili  formotility  ( e s s e n t i a l f o r conjugation)  composed m a i n l y o f p r o t e i n .  rigid  cell  envelope i s a common  T h i s common a n t i g e n  exhibits  a n t i g e n i c c r o s s - r e a c t i v i t y among a wide v a r i e t y o f gram-negative (Sompolinsky e t a l . , 1980a).  membrane  s t r u c t u r e and f u n c t i o n s o f the o u t e r membrane o f e n t e r i c b a c t e r i a  have been e x t e n s i v e l y s t u d i e d Alphen, 1983).  (Osborn and Wu, 1980; Lugtenberg and Van  The o u t e r membrane, as determined by e l e c t r o n microscopy,  i s a b i l a y e r composed o f p h o s p h o l i p i d s , (LPS).  (Craven and Montie, 1981),  i n adhesion (Woods e t a l . , 1980a, b ) .  Components and s t r u c t u r e o f the o u t e r The  flagella,  a l l three l a y e r s of the  and the much s m a l l e r ,  Another component o f the Pseudomonas aeruginosa  extensive  appendages l i k e  F l a g e l l a are a s s o c i a t e d w i t h  f i m b r i a e have been i m p l i c a t e d  3.  but those  seaweed a l g i n a t e and composed o f  envelope are c e l l u l a r  pili  bacteria  (Moorhouse  i s o l a t e d from c y s t i c f i b r o s i s p a t i e n t s are o f t e n surrounded by a resembling  antigen  material  s t r a i n s do not have a t r u e c a p s u l e  mucoid e x o p o l y s a c c h a r i d e  while  associated  p r o t e i n s and l i p o p o l y s a c c h a r i d e  The o u t e r membrane o f gram-negative b a c t e r i a i s a v e r y  unusual  6  b i o l o g i c a l membrane i n t h a t i t has i t s components.  A l l the LPS  the major l i p i d i c inner l e a f l e t and  a h i g h l y asymmetrical d i s t r i b u t i o n of  i s present  i n the o u t e r  monolayer where i t i s  molecule, w h i l e v i r t u a l l y a l l the p h o s p h o l i p i d  (with  the p o s s i b l e e x c e p t i o n  S a l m o n e l l a typhimurium); p r o t e i n s  of some LPS  are p r e s e n t  i s i n the  mutants of E.  coli  i n both l a y e r s and  in  some-cases are membrane-spanning.  (A)  Lipopolysaccharide LPS  very  (LPS)  i s an a m p h i p h i l i c  molecule and  has  been shown to be s t r u c t u r a l l y  s i m i l a r i n a l l gram-negative b a c t e r i a s t u d i e d  R i e t s c h e l et a l . , 1983). structure  (Nikaido  and  hydrophobic r e g i o n , attached  Structural studies  Nakae, 1979;  indicate a  Wilkinson,  1983)  to a rough core  r e g i o n which i s o f t e n , but  More d e t a i l e d s t u d i e s on LPS  g l y c o l i p i d usually containing  c o n s i s t i n g of a  and  l i n k e d to the d i g l u c o s a m i n e .  a diglucosamine residue  i n c l u d e B-OH  and  cell  that  a  is substituted  5 to 6 f a t t y a c i d s , The  f a t t y acids  fatty  acids  t h a t are  two  are  amide  These hydroxy f a t t y a c i d s are unique to  b a c t e r i a l LPS.  The  rough core t y p i c a l l y c o n t a i n s  octosaccharide,  Z-keto-3-deoxyoctonate (KDO)  a highly  (Nikaido  t h i s octose i s a l s o h i g h l y s p e c i f i c  a d d i t i o n , the rough core  always, capped  have shown the l i p i d A to be  of which are amide l i n k e d to the d i g l u c o s a m i n e . almost always s a t u r a t e d ,  not  covalently  c h a i n e x t e n d i n g from the  with phosphate and/or pyrophosphate r e s i d u e s  i t s proximal end;  tripartite  the L i p i d A ( a l s o known as e n d o t o x i n ) ,  with a h y d r o p h i l i c 0 - polysaccharide surface.  (Orskov et a l . , 1977 ;  contains  and  unusual Nakae, 1979)  to LPS.  a v a r i e t y of hexoses and  at  In  heptoses which  7  may be s u b s t i t u t e d with phosphate and ethanolamine phosphate.  The rough  core r e g i o n o f LPS i s s t r u c t u r a l l y h i g h l y conserved  among the i n d i v i d u a l  E n t e r o b a c t e r i a l s p e c i e s (Lugtenberg  1983).  and Van Alphen,  The L i p i d  A-rough core r e g i o n c o n t a i n s a high d e n s i t y o f n e g a t i v e l y - c h a r g e d  residues  and has been shown t o be the d i v a l e n t c a t i o n b i n d i n g s i t e o f the outer membrane The  ( S c h i n d l e r and Osborn, 1979). h y d r o p h i l i c o l i g o s a c c h a r i d e p o r t i o n o f LPS c o n s i s t s o f a h i g h l y  v a r i a b l e number o f r e p e a t e d substantial v a r i a b i l i t y within a single  tri-  to p e n t a s a c c h a r i d e  i n composition  units.  The  o f t h i s r e g i o n , which occurs  s p e c i e s (or genus) allows f o r tremendous v a r i a t i o n  m o l e c u l a r make-up o f the c e l l  surface.  1979;  (B)  i n the  T h i s v a r i a t i o n o f the  p o l y s a c c h a r i d e c h a i n has been used as a f i n e s e r o l o g i c a l t y p i n g resulting  even  tool,  i n the name "0" a n t i g e n f o r t h i s r e g i o n (Brokopp and Farmer,  L a n y i and Bergan, 1979).  Proteins SDS-polyacrylamide  g e l e l e c t r o p h o r e s i s techniques  have c o n t r i b u t e d  g r e a t l y to the r e s o l u t i o n and i d e n t i f i c a t i o n o f b a c t e r i a l o u t e r  membrane  proteins.  aeruginosa  SDS-gel e l e c t r o p h o r e t o g r a m s  o f o u t e r membranes o f P.  (Hancock and Carey, 1979), l i k e those o f o t h e r gram-negative b a c t e r i a , show the presence  o f a few, very prominent "major" p o l y p e p t i d e bands and  numerous other "minor" p o l y p e p t i d e s 1979;  Osborn and Wu, The  reason  ( F o r reviews  1980; Lugtenberg  f o r the presence  and Van Alphen,  1983).  o f a r e s t r i c t e d number o f major p r o t e i n s  4 i n high copy number (2 x 10  see N i k a i d o and Nakae,  5 t o 3 x 10  c o p i e s per c e l l )  i s uncertain  8  although  i n some cases  demonstrated. the E. c o l i  important  s t r u c t u r a l and f u n c t i o n a l r o l e s have been  For example i n P. a e r u g i n o s a  p e p t i d o g l y c a n bound l i p o p r o t e i n  l i p o p r o t e i n I , analogous to (Braun,  1975), l i p o p r o t e i n H2  and p r o t e i n F a l l have s t r u c t u r a l r o l e s , being n o n c o v a l e n t l y a s s o c i a t e d with the p e p t i d o g l y c a n  (Mizuno, 1979; Hancock, e t a l . , 1981).  A major f u n c t i o n o f the outer membrane i s as a s i z e dependent permeability b a r r i e r .  T h i s b a r r i e r f u n c t i o n i s l a r g e l y determined  f a m i l y o f transmembrane p r o t e i n s named p o r i n s . present  These p o r i n p r o t e i n s are  i n a l l gram-negative b a c t e r i a examined t o date, and t h e i r f u n c t i o n  has been e x t e n s i v e l y s t u d i e d (Reviews by Lugtenberg N i k a i d o and Nakae, 1979).  and V a l Alphen 1983;  They have been shown to form q u i t e  n o n - s p e c i f i c , w a t e r - f i l l e d h y d r o p h i l i c channels  a c r o s s the o u t e r membrane. 4  P o r i n p r o t e i n s are p r e s e n t c o p i e s per c e l l ) ,  have apparent  i n h i g h copy numbers (10  t o 10  (DiRienzo e_t a l . ,  and are o r g a n i z e d as t r i m e r s i n the o u t e r membrane (Angus and  Hancock, 1983; Yu e t a l . , 1979; I s h i i channels  with a f a i r l y  the E. c o l i  In P. a e r u g i n o s a  o f up t o 6,000 d a l t o n s  P. a e r u g i n o s a e x h i b i t s lower (Nicas and Hancock, 1983). aeruginosa  They form  and thus determine the m o l e c u l a r  F o r example, the e x c l u s i o n l i m i t o f  and S. typhimurium p o r i n s i s about 500 - 650 d a l t o n s  1976b).  molecules  and Nakae, 1980).  c o n s t a n t diameter  s i z e o f compounds e n t e r i n g the c e l l .  1975;  5  m o l e c u l a r weights o f 32 - 42,000 d a l t o n s ,  are n o n - c o v a l e n t l y a s s o c i a t e d w i t h the p e p t i d o g l y c a n 1978)  by a  (Nakae,  the o u t e r membrane i s permeable t o (Hancock, §_t a l . , 1979), y e t  p e r m e a b i l i t y to a n t i b i o t i c s  than E. c o l i  T h i s p r o p e r t y has been a t t r i b u t e d to P.  having l e s s than 1% o f i t s p o r i n p r o t e i n molecules  forming  9  f u n c t i o n a l open pores (Benz and Hancock, 1981; Nicas In P. aeruginosa.  p r o t e i n F i s the main n o n - s p e c i f i c h y d r o p h i l i c  pore-forming p r o t e i n c o n t r i b u t i n g t o the g e n e r a l membrane (Nicas and Hancock, 1983). P are induced respectively  4.  and Hancock, 1983).  by growth i n g l u c o s e  p e r m e a b i l i t y o f the o u t e r  The a l t e r n a t i v e p o r i n p r o t e i n s DI and and under phosphate l i m i t i n g  (Hancock and Carey, 1980; Hancock e t a l . , 1982).  C e l l envelope and the Immune Response Bacterial c e l l  s u r f a c e components  i n t e r a c t i o n o f the b a c t e r i u m w i t h contribute  considered  the h o s t ' s  In t h i s r e s p e c t , t h e c e l l  as v i r u l e n c e f a c t o r s .  the g e n e r a t i o n foreign  are i n v o l v e d  i n the primary  immune system and, t h e r e f o r e ,  i n p a r t to the a b i l i t y o f the b a c t e r i a t o i n f e c t the  s u s c e p t i b l e host.  of s p e c i f i c  surface  components  One i n d i c a t i o n o f these i n t e r a c t i o n s i s  antibodies  i n the i n f e c t e d h o s t ,  a g a i n s t the  s t u d i e s on the a n t i g e n i c i t y and r o l e i n v i r u l e n c e o f  b a c t e r i a l s u r f a c e s were i n v o l v e d with clinical  surface  appendages.  i s o l a t e s possessed appendages l i k e p i l i ,  components has t h e r e f o r e been a s c r i b e d a r o l e i n b a c t e r i a l and f i m b r i a e  epithelial cells  are i n v o l v e d  (Woods, 1980a, b ) .  e_t aJL. , 1981) .  Aeromonas s a l m o n i c i d a .  Each o f these pathogenesis.  i n b a c t e r i a l adhesion t o s p e c i f i c Flagella  schemes) have been c o r r e l a t e d t o i n v a s i v e n e s s (Holder  Most o f the  f i m b r i a e , f l a g e l l a or  c a p s u l e s , which were o f t e n l a c k i n g i n a v i r u l e n t s t r a i n s .  tissue  can be  ( b a c t e r i a l ) antigens.  The e a r l i e s t  Pili  conditions  ( t h e b a s i s o f H-antigen o f P. a e r u g i n o s a  i n burn  Capsules i n c r e a s e the v i r u l e n c e of  Neisseria meningitidis  and Haemophilus  influenzae  10  type b (Frasch and Robbins, 1978;  Munn e t a l . , 1982)  i n p e r s i s t e n c e and r e s i s t a n c e t o p h a g o c y t o s i s and Pennington,  1981;  o f E. c o l i  C o s t e r t o n e_t a l . , 1983).  a n t i g e n i c v a r i a t i o n o f these components ( P i t t ,  heterologous  and hence t h e i r  to s t r a i n .  1980;  The  B r i n t o n , 1982;  i n a b i l i t y to provide active  Virji  immunity a g a i n s t  s u r f a c e components.  outer membrane c o n t a i n s two immunologically  the LPS and p r o t e i n s .  important  molecules,  Most o f the s t u d i e s on the immune response  membrane components have been performed on LPS. been shown t o a g g l u t i n a t e , and promote opsonic bacteria  (Blackwood  organisms o f t h e same s p e c i e s , has f o c u s s e d a t t e n t i o n on  other b a c t e r i a l c e l l The  cells  However, where s t u d i e d ,  these components can vary s u b s t a n t i a l l y from s t r a i n  et a l . , 1983)  and may be i n v o l v e d  (Ka'ss and W o l f f , 1973).  to outer  A n t i b o d i e s t o LPS have phagocytosis  o f homologous  In c o n t r a s t , outer membrane p r o t e i n s  have been s u b j e c t e d t o i n t e n s i v e r e s e a r c h o n l y i n r e c e n t y e a r s , w i t h the search f o r n o n - t o x i c , L P S - f r e e  (A)  vaccines.  LPS B a c t e r i a l LPS has been the s u b j e c t o f e x t e n s i v e s t r u c t u r a l and  immunological  s t u d i e s (Kass  W i l k i n s o n , 1983). LPS  L a n y i and Bergan, 1979;  The b i o l o g i c a l o r immunological  depend on i t s source  purify  and W o l f f , 1973,  a c t i v i t i e s of isolated  and t o some e x t e n t on the procedures  i t . A major b i o l o g i c a l p r o p e r t y o f P. a e r u g i n o s a  o t h e r gram-negative b a c t e r i a , factor i n infections and c o l l e a g u e s  (Cho  l i k e that of  i s e n d o t o x i c i t y which i s a major v i r u l e n c e  (Owen, 1981). et al.,  LPS,  used t o  1979;  In a series of d e t a i l e d Tanamoto and Homma 1982)  s t u d i e s , Homma i s o l a t e d and  11  f r a c t i o n a t e d LPS  from E. c o l i ,  P. a e r u g i n o s a . S. typhimurium  c h o l e r a e and showed t h a t the i n t a c t L i p i d A r e g i o n was component of LPS  and was  the  V.  endotoxin  also e s s e n t i a l f o r i t s pyrogenicity in rabbits  and f o r i t s m i t o g e n i c i t y towards mouse B - c e l l lymphocytes. these  and  important b i o l o g i c a l p r o p e r t i e s , LPS  e f f e c t s on i n f e c t e d hosts ( B r a d l e y , 1979;  demonstrates  In a d d i t i o n to  a p l e t h o r a of  Tanamoto and Homma,  1982;  Tanamoto e t a l . , 1979). The  s t r o n g immunogenic a c t i v i t y of LPS  i s a f u n c t i o n of the  p o l y s a c c h a r i d e O - a n t i g e n i c s i d e c h a i n when l i n k e d t o i t s own adjuvant  natural  ( L i p i d A).. As mentioned above, the s u b s t a n t i a l v a r i a t i o n  c h e m i c a l c o m p o s i t i o n and consequently O-antigen  of d i f f e r e n t  the a n t i g e n i c i t y o f the  i n the  LPS  s t r a i n s o f b a c t e r i a w i t h i n a s p e c i e s i s the b a s i s  of the f i n e t y p i n g schemes of gram n e g a t i v e b a c t e r i a Brokopp and Farmer, 1979)  (Orskov et_ a l . ,  1977;  *  In P. a e r u g i n o s a a v a r i e t y o f s e r o t y p i n g schemes have been d e s c r i b e d (Brokopp  and Farmer, 1979;  L a n y i and Bergan, 1979) .  I n t e r n a t i o n a l A n t i g e n Typing Scheme comprises  One  o f t h e s e , the  17 d i s t i n c t  serotype  and c o n t a i n s , as s u b s e t s , the serotypes o f a l l o t h e r schemes. problem  of these s e r o t y p i n g schemes i s the h i g h frequency  d i s e a s e i s o l a t e s o f non-typable now  specific  s t u d i e s o f P. a e r u g i n o s a . LPS  i n the O-antigen  T h i s o b s e r v a t i o n b r i n g s i n t o q u e s t i o n the broad  these O-antigen  a n t i s e r a i n s e r o t y p i n g and The  major  aeruginosa  or p o l y a g g l u t i n a b l e s t r a i n s , a p r o p e r t y  shown t o c o r r e l a t e t o d e f i c i e n c e s  1983).  i n P.  One  strains  (Hancock e t a l . , a p p l i c a t i o n of  in epidemiological  s t r o n g a n t i g e n i c i t y o f the O-side c h a i n of  has made i t a p o p u l a r candidate f o r v a c c i n e s .  However, the  antigenic  12  v a r i a t i o n and e n d o t o x i c i t y noted (IgG) response a g a i n s t LPS  w i t h the  above as we'll as the l a c k of a memory  '0' a n t i g e n , are f a c t o r s which tend to argue  as a good v a c c i n e .  Structural similarities rough form LPS  i n the i n n e r core r e g i o n of both  among i n d i v i d u a l s p e c i e s of gram-negative b a c t e r i a have  been shown ( Z i e g l e r e t a l . , 1975; 1984)  Braude e t a l . , 1983;  using a n t i s e r a s p e c i f i c f o r that region.  core r e g i o n of the E. c o l i animals  J5 (LPS mutant) LPS  a g a i n s t gram-negative bacteremia  a e r u g i n o s a bacteremia  i n f o r m a t i o n was  important  by serum f a c t o r s and  has  1978;  a v a i l a b l e on the s p e c i f i c  to the rough  been shown to p r o t e c t  Chedid  P.  e t a l . , 1968;  e p i t o p e s of  no this  LPS.  r o l e s of b a c t e r i a l LPS  are i n r e s i s t a n c e to  i n r e s i s t a n c e to phagocytosis  as i n r e s i s t a n c e to d e t e r g e n t s and a n t i b i o t i c s The  Antiserum  However, p r i o r t o the p r e s e n t study,  s t r u c t u r a l l y complex r e g i o n of the Other  Galanos et a l . ,  and endotoxemia i n c l u d i n g  (Braude et a l . , 1977,  P o l l a c k and Young, 1979). specific  smooth and  killing  ( T a y l o r 1983), as w e l l  (Kropinski et a l . ,  p r o p e r t y of serum b a c t e r i c i d a l r e s i s t a n c e (Rowley, 1968)  c o r r e l a t e d to the l e n g t h of t h e 0 - a n t i g e n i c s i d e c h a i n s  1978).  was  (smooth  strains  w i t h long 0-side c h a i n s being serum r e s i s t a n t and rough s t r a i n s with 0 - s i d e chains b e i n g serum s e n s i t i v e ) . to  The  l o n g s i d e c h a i n s were  no  proposed  b l o c k the b i n d i n g of a c t i v a t e d serum complement complexes t o the  b a c t e r i a l s u r f a c e (reviewed by T a y l o r , 1983). 0-antigen was  The  composition  of the  LPS  a l s o c o r r e l a t e d to r e s i s t a n c e t o p h a g o c y t o s i s by mouse  macrophage c e l l s  i n S a l m o n e l l a typhimurium ( L i a n g - T a k a s a k i e t a l . , 1982).  The mechanism of r e s i s t a n c e was  proposed  to be of b a c t e r i a l - p h a g o c y t e  13  affinity,  because the r a t e o f k i l l i n g  o f phagocytosed b a c t e r i a was the  same f o r a l l LPS O-antigen v a r i a n t s . The  importance o f the '0' a n t i g e n  the o b s e r v a t i o n  t h a t g e n e r a l l y only  i n pathogenesis can be summed up by  smooth r a t h e r than rough (0  a n t i g e n - d e f i c i e n t ) gram-negative b a c t e r i a are i s o l a t e d from bacteremic i n f e c t i o n s , where r e s i s t a n c e to serum b a c t e r i c i d a l a c t i v i t y and p h a g o c y t o s i s would be major f a c t o r s i n b a c t e r i a l p e r s i s t e n c e .  (B)  Proteins Studies  on the a n t i g e n i c i t y o f b a c t e r i a l outer membrane p r o t e i n s  p r o g r e s s e d r a p i d l y i n recent LPS-free vaccines of a g i v e n has  providing  bacterium.  years,  due t o the need to d e v e l o p  heterologous p r o t e c t i o n against  Surface  been shown by b a c t e r i o p h a g e b i n d i n g  receptors  (Braun and K r i e g e r - B r a u e r  non-toxic,  all  a c c e s s i b i l i t y o f outer membrane  have  serotypes proteins  t o outer membrane p r o t e i n  1977; D a t t a e t a l . , 1977),  surface  125 l a b e l l i n g w i t h " n o n - p e n e t r a t i n g " r a d i o l a b e l l e d dextrans o r and  Nikaido,  1977), and i n d i r e c t  immunofluorescence u s i n g  I (Kamio  polyclonal  sera  ( H o f s t r a e t al'.', 1979). The has  mitogenicity  o f outer membrane p r o t e i n s  lymphocytes  been shown f o r P. a e r u g i n o s a p r o t e i n F, and l i p o p r o t e i n s H2 and I  (Chen e_t a l . , 1980), f o r N. m e n i n g i t i d i s et  forB cell  a l . , 1983) and f o r the E. c o l i  Henning, 1979).  surface  antigens  (Melancon  OmpF, OmpA and Lpo p r o t e i n s  ( B e s s l e r and  In p r i n c i p l e , the a n t i g e n i c i t y o f outer membrane  was shown by the a b i l i t y of a c t i v e immunization with outer p r o t e i n s , t o l e a d t o p r o t e c t i o n o f mice a g a i n s t  proteins  membrane  experimental  salmonellosis  14  i n s u c k l i n g mice (Kuusi shigellosa  (Adamus §_t a l . ; 1980).  used i n t r i a l v a c c i n e et  e t a l , , 1979) and a g a i n s t k e r a t o c o n j u c t i v i t i s  studies  Outer membrane p r o t e i n s  i n H. i n f l u e n z a e  a l . , 1982), and N e i s s e r i a sp.  have a l s o been  (Lam e t a l . , 1980b; Hansen  ( Z o l l i n g e r et. a l . , 1978, Buchanan,  1977) . In P. a e r u g i n o s a s t r o n g  evidence f o r the immunogenicity o f o u t e r  membrane p r o t e i n s has been demonstrated i n c y s t i c  fibrosis  patients  c o l o n i z e d w i t h t h i s b a c t e r i u m (Hancock e t a l . , 1984) and i n c o n v a l e s c e n t patients recovering  from Pseudomonas b a c t e r e m i a (Lam e t a l . , 1983,  Fernandes e t a l . , 1981) as w e l l as i n r a t s with  induced c h r o n i c P.  aeruginosa lung  i n f e c t i o n s (Lam e t a l . , 1983).  In a l l these  antibodies  i n t e r a c t w i t h P. a e r u g i n o s a o u t e r membrane p r o t e i n s were  that  cases,  identified. H o f s t r a and c o l l e a g u e s 1980)  ( H o f s t r a and Dankert, 1979; H o f s t r a e_t a l . ,  demonstrated t h a t p o l y c l o n a l a n t i s e r a r a i s e d a g a i n s t  p o r i n p r o t e i n s OmpF and OmpC showed e x t e n s i v e c r o s s - r e a c t i v i t y with proteins membranes o f other  immunological  o f 32-42,000 d a l t o n s  Enterobacteriaceae.  from the  from other  Enterobacteriaceae  outer  S i m i l a r c r o s s - r e a c t i v i t y was  demonstrated f o r t h e 33,000 d a l t o n OmpA p r o t e i n o f E. c o l i proteins  t h e E. c o l i K12  with s i m i l a r  s t r a i n s ( H o f s t r a e_t a l . , 1980) .  In  s i m i l a r s t u d i e s , a n t i g e n i c c r o s s - r e a c t i o n s were shown by Braun e t a l . , (1976) f o r the p e p t i d o g l y c a n - b o u n d l i p o p r o t e i n o f E. c o l i enterobacterial strains.  In i n d i r e c t  a l . , (1976) a l s o showed t h a t t h i s a c c e s s i b l e i n i n t a c t organisms.  and other  immunofluorescent s t u d i e s , Braun e t  l i p o p r o t e i n was n o t s u r f a c e In c o n t r a s t  to t h e e x t e n s i v e  localised or  15  immunological c r o s s - r e a c t i v i t y Enterobacteriaceae,  among the major p o r i n s o f  a n t i s e r a to the E. c o l i  LamB p r o t e i n d i d not r e a c t  with any other p r o t e i n . Monoclonal a n t i b o d i e s s p e c i f i c  f o r outer membrane p r o t e i n s have been  used to show the s u r f a c e a c c e s s i b i l i t y o f those organisms o f E. c o l i  (Gabay and Schwartz, 1982) and N e i s s e r i a gonorrhoeae  (Nachamkin e_t a l . , 1981). tentatively  proteins in intact  identified  In a d d i t i o n , s u r f a c e exposed a n t i g e n s  have been  i n N. gonorrhoeae (Swanson e t a l . , 1982) and H.  i n f l u e n z a e type b (Robertson  e t a l . , 1982; G u l i g e t a l . , 1982) by 125  i n t e r a c t i o n o f monoclonal a n t i b o d i e s w i t h f o l l o w e d by d e t e r g e n t complexes.  lysis  However, these  intact  I-labelled bacteria,  and p r e c i p i t a t i o n o f the a n t i g e n - a n t i b o d y s t u d i e s are not r e a l l y d e f i n i t i v e  r a d i o l a b e l l i n g has been shown t o p e r t u r b o u t e r membrane ( S u l l i v a n and W i l l i a m s ,  since  integrity  1982).  In r e c e n t y e a r s , g r e a t  i n t e r e s t has developed  i n the a p p l i c a t i o n o f  outer membrane p r o t e i n s i n c l a s s i f i c a t i o n o f some b a c t e r i a l s p e c i e s , as an a l t e r n a t i v e to serogrouping  based on LPS O-antigen.  Substantial variation  i n the outer membrane p r o t e i n p a t t e r n s o f i n d i v i d u a l s t r a i n s have been demonstrated f o r E. c o l i  (Overbeeke and Lugtenberg, 1980), V. c h o l e r a e  (Kabir and Mann, 1980), N. m e n i g i t i d i s ( T s a i e t a l . , 1981), N. gonorrhoeae (Buchanan and H i l d e b r a n d t ,  1981), H. i n f l u e n z a e (Loeb and Smith, 1982;  Barenkamp, 1981), and V. a n g u i l l a r u m of  these  (Nakajima e t a l . , 1983).  s p e c i e s , a b i o t y p i n g scheme based on grouping  F o r many  a c c o r d i n g t o the  s p e c i f i c outer membrane p r o t e i n p a t t e r n s o f i n d i v i d u a l s t r a i n s has been proposed.  The h e t e r o g e n e i t y  of protein patterns  i s o f t e n r e f l e c t e d by  16  t h e i r a n t i g e n i c i t y , and  a n t i s e r a to s p e c i f i c o u t e r membrane p r o t e i n s  been used to s e r o l o g i c a l l y c l a s s i f y N.  gonorrhoeae (Buchanan  Hildebrandt,  H.  1980), and  1981;  to LPS,  e_t a l . , 1981), and  a c t i v i t y was  due  TraT) p r o t e i n  5.  and P.  Johnson,  s u r v i v a l of a b a c t e r i u m iri  gonorrhoeae (Lambden et a l . , 1979), A. E. c o l i  vivo  salmonicida  [where r e s i s t a n c e to serum b a c t e r i c i d a l  i n v e s t i g a t i o n was  to l o o k at the a n t i g e n i c i t y  Studies  using  of  p o l y c l o n a l a n t i s e r u m to s t r a i n H103  outer  membranes demonstrated a n t i g e n i c c r o s s - r e a c t i v i t y at the p o l y c l o n a l of the major o u t e r membrane p r o t e i n s .  However, the e x p r e s s i o n  a n t i g e n i c d e t e r m i n a n t s on these p r o t e i n s was demonstrate the aim,  study the  studied  were developed.  specific  These monoclonal a n t i b o d i e s  s t r a i n s and  clinical  i s o l a t e s of P.  aeruginosa.  proteins  on  i n t a c t b a c t e r i a was  indirect  immunofluorescent assays and  studied using  i n both  The  level  specific  to  To  for porin protein F  a n t i g e n i c d i s t r i b u t i o n of these p r o t e i n s  of  i n order  a n t i g e n i c r e l a t i o n s h i p s of these a n t i g e n s .  monoclonal a n t i b o d i e s  l i p o p r o t e i n H2  (the  study  immunochemietry of major outer membrane p r o t e i n s  aeruginosa.  this  has  ( M o l l 1980)].  o b j e c t i v e of t h i s  surface  membrane  to the presence of an outer membrane plasmid-encoded  Aims of t h i s The  (McDade and  the d i r e c t c o n t r i b u t i o n of o u t e r  to v i r u l e n c e by enhancing the  o n l y been shown f o r N. (Kay  influenzae  and  L e g i o n e l l a pneumophila ( J o l y , et a l . , 1983).  In c o n t r a s t proteins  Knapp et a l . , 1984)  has  assist  in  and were used to  laboratory  l o c a l i z a t i o n of  the  the monoclonal a n t i b o d i e s  in d i r e c t antiserum absorption  in  with  17  i n t a c t organisms.  The  r e s u l t s from these  o b t a i n e d with a monoclonal antibody s e r o t y p e of P. a e r u g i n o s a  and  s t u d i e s were compared to  specific  of  one  5 other monoclonal a n t i b o d i e s r e c o g n i z i n g  e i t h e r the rough core or L i p i d A r e g i o n s of The  f o r the O-antigen  those  LPS.  p r o t e i n and L i p i d A - s p e c i f i c monoclonal a n t i b o d i e s were used i n a  taxonomic study of a n t i g e n i c c o n s e r v a t i o n i n the o u t e r membrane of gram-negative b a c t e r i a from d i f f e r e n t  taxonomic groups.  In a d d i t i o n , the  i n t e r a c t i o n of the p r o t e i n F - s p e c i f i c monoclonal a n t i b o d i e s with p r o t e i n F p e p t i d e s o b t a i n e d by cyanogen bromide c l e a v a g e  and enzyme p r o t e o l y s i s  p u r i f i e d p r o t e i n F, p r o t e i n F i n o u t e r membranes and whole c e l l s were studied.  of  18  MATERIALS AND  I (A)  METHODS  Bacteria Media and growth  conditions  One % (wt/vol) Proteose-peptone Mi.) was except  used  no.  2 (PP2)  i n s p e c i f i e d cases where 1% (wt/vol) T r y p t i c a s e supplemented with 1 mM  2  2  used.  (BBL,  Liquid  at 37°C or 30°C.  Bacterial strains Pseudomonas a e r u g i n o s a PA01  r e f e r e n c e s t r a i n throughout b a c t e r i a l s t r a i n s used S t r a i n H283 was p r o t e i n F.  The  the study.  was  Sources  i n t h i s study are l i s t e d  a mutant of H103,  s t r a i n was  as d e s c r i b e d  s t r a i n H103  from A. K r o p i n s k i  as the w i l d type  and p r o p e r t i e s  and  o f the  below.  i s o l a t e d by random heavy mutagenesis of  by N i c a s and Hancock (1983).  strain  S t r a i n s AK1160, AK1188, a e r u g i n o s a PA01  (Queen's U n i v e r s i t y , K i n g s t o n , O n t a r i o ) .  o b t a i n e d as a mutant of H103  b a c t e r i o p h a g e 44  used  l a c k i n g the o u t e r membrane p o r i n  AK1012 and AK1121 were L P S - a l t e r e d mutants of P.  was  Soy b r o t h  M g C l . 6 H 0 was  c u l t u r e s were grown w i t h v i g o r o u s a e r a t i o n  H103  Detroit,  as a r i c h medium f o r the growth o f a l l b a c t e r i a l s t r a i n s ,  C o c k s e y v i l l e , Md)  (B)  ( D i f c o Labs,  r e s i s t a n t t o smooth  obtained  S t r a i n H223  LPS-specific  (Hancock e t a l . , 1982) .  A set of 17 s e r o t y p e - s p e c i f i c  strains representative  o f the  I n t e r n a t i o n a l A n t i g e n Typing Scheme (IATS, c o m m e r c i a l l y marketed by L t d . , D e t r o i t , Mi.) was  a kind g i f t  L o u i s v i l l e , Kentucky).  These s t r a i n s were named as f o l l o w s :  Difco  from P. L i u ( U n i v e r s i t y of L o u i s v i l l e , Type 1 (ATCC  19  33348), Type 2 (ATC.C 33349), Type 3 (ATCC 33350), Type 4 (ATCC 33351), Type 5 (ATCC 33352), Type 6 (ATCC 33354), Type 7 (ATCC 33353), Type 8 (ATCC 33355), Type 9 (ATCC 33356), Type 10 (ATCC 33357), Type 11 (ATCC 3358), Type 12 (ATCC 3359), Type 13 (ATCC 33360), Type 14 (ATCC 33361), Type 15 (ATCC 33362), Type 16 (ATCC 33363), and Type 17 (ATCC 33364). . A s e t o f 12 P. a e r u g i n o s a c l i n i c a l f i b r o s i s were o b t a i n e d from G.B.  Pier  isolates  from p a t i e n t s w i t h  cystic  (Harvard M e d i c a l S c h o o l , Mass.).  These s t r a i n s were named CF 221, CF 284, CF 832, CF 1278, CF 1452, CF 2314, CF 3790, CF 4349, CF 4522, CF 6094, CF 9490, and CF 3660-1; L,  a l s o o b t a i n e d from G.B.  P i e r , was  a blood i s o l a t e .  p a i r s of mucoid P. a e r u g i n o s a c l i n i c a l  isolates  strain  In a d d i t i o n ,  from c y s t i c  12  fibrosis  p a t i e n t s , and t h e i r spontaneous non-mucoid r e v e r t a n t s , were o b t a i n e d from D.P. Speert  ( C h i l d r e n ' s H o s p i t a l , Vancouver, B.C.).  named Pirn and Plnm; Clm and nm; and  nm; C47m and nm;  and  C96m and nm.  C2lm and nm;  C46m and nm;  C4m  and nm;  Other Pseudomonadaceae  These s t r a i n s  C20m and nm; C91m  and nm;  C6m  and nm;  C81m  s t r a i n s used i n t h i s  were  and  C81m  nm;  study were,  T two s t r a i n s of P. p u t i d a ATCC 4359 and ATCC 12633  ; two P.  fluorescens  T  s t r a i n s ATCC 949 and ATCC 13525 ; P. a e r u g i n o s a ATCC 9721, ATCC 8689 and T  T  T  ATCC 19305 ; P. s y r i n g a e ATCC 19310 ; P. s t u t z e r i ATCC 17588 ; T  T  P. solanacearum ATCC 11696 ; P. m a l t o p h i l i a ATCC 13639 ; P. T T c h l o r o r a p h i s ATCC 9446 ; P. c e p a c i a ATCC 25416 ; P. aureofacxens ATCC T T 13985 ; P. p s e u d o m a l l e i ATCC 23343 ; P. a n g u i l l i s e p t i c a ET2 and ET7601; and P. a c i d o v o r a n s ATCC 9353  ( s u p e r s c r i p t T denoted Type  C e l l envelopes or l i p o p o l y s a c c h a r i d e  (LPS) p r e p a r a t i o n s were a l s o  i n our l a b o r a t o r y from E s c h e r i c h i a c o l i  CGSC 6044 and PC0479, and  S a l m o n e l l a typhimurium LT2 SGSC205 and SGSC206.  strain). obtained  20  Outer membranes from E d w a r d s i e l l a t a r d a , E79054, V i b r i o c h o l e r a e PS V i b r i o a n g u i l l a r u m ET208 and HT Aeromonas h y d r o p h i l a ET2,  7910,  7602, Aeromonas s a l m o n i c i d a NCMB2020,  P. a n g u i l l i s e p t i c a ET2  and ET7601, and  P.  acidovorans ATCC 9353 were i s o l a t e d by K. Nakajima i n our l a b o r a t o r y , and LPS  from Agrobacterium  EV76 was  tumefaciens  i s o l a t e d by R.P.  PLT4 and PLT  Darveau i n our l a b o r a t o r y .  v i n e l a n d i i OP o u t e r membranes were a k i n d g i f t of  British  (C)  and o u t e r membrane  For both p r e p a r a t i o n s , c e l l s c o l l e c t e d and resuspended  Tris-HCl  i n a French Press at 15,000 p s i .  supernatant was  diluted  2 hr and the p e l l e t  (pH 7.4)  f o r 10 min.  in d i s t i l l e d resuspended  Hancock and Carey Tris-HCl  two-step  (pH 7.4)  and  the above  water and c e n t r i f u g e d a t 160,000 x g i n water. s t e p sucrose g r a d i e n t method  In t h i s method, the French P r e s s l y s a t e i n  and 20%  (wt/vol) sucrose was  l a y e r e d onto a  (wt/vol) s u c r o s e i n the bottom  (wt/vol) sucrose i n the top l a y e r , and c e n t r i f u g e d at 21,000  i n a Beckman SW27 r o t o r f o r 16 h r s .  membrane bands. fraction.  S t . L o u i s , Mo.)  For c e l l e n v e l o p e s ,  sucrose g r a d i e n t c o n s i s t i n g of 70%  l a y e r and 52% rpm  (1979).  Co.,  c o n t a i n i n g 10 ug/ml  Whole c e l l s were removed by  Outer membranes were i s o l a t e d by the one  10 mM  from R. Moore, U n i v e r s i t y  from l o g a r i t h m i c phase c u l t u r e s were  i n 10 mM  c e n t r i f u g a t i o n at 3,000 rpm  of  Azotobacter  isolation  p a n c r e a t i c d e o x y r i b o n u c l e a s e 1 (Sigma Chemical  for  pestis  Columbia.  C e l l envelope  broken  S-1005 and Y e r s i n i a  The  T h i s r a p i d method gave  h i g h e r d e n s i t y band was  two  the p u r i f i e d o u t e r membrane  21  (D)  P r o t e i n and LPS p u r i f i c a t i o n Outer membrane p r o t e i n F ( p o r i n ) , and l i p o p r o t e i n H2 were p u r i f i e d  from P. a e r u g i n o s a s t r a i n H103 by L. Chan ( M i c r o b i o l o g y , U.B.C.) u s i n g the method of Hancock e_t a l . (1979). LPS was i s o l a t e d  i n our l a b o r a t o r y by L. Chan and R. Darveau u s i n g  the method o f Darveau and Hancock (1983). result  T h i s method has been shown t o  i n h i g h y i e l d s of p r o t e i n - f r e e LPS from both rough  and smooth  strains.  (E)  Sodium d o d e c y l s u l p h a t e (SDS) - 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 The method used was as d e s c r i b e d by Hancock and Carey  a c r y l a m i d e c o n c e n t r a t i o n i n the lower  (1979).  The  s e p a r a t i n g g e l was 14% (wt/vol)  acrylamide and 0.12% ( ( w t / v o l ) N,N' methylene b i s a c r y l a m i d e  ( i n 12.5 ml  of g e l mix a l s o c o n t a i n i n g 3.125 ml o f 1.5 M T r i s - H C l pH 8.8 and 0.25 ml of 10% (wt/vol) SDS). The g e l was p o l y m e r i z e d by the a d d i t i o n o f 20 y l of TEMED (N, N, N, N ' - t e t r a m e t h y l e t h y l e n e d i a m i n e 1%  (wt/vol) ammonium p e r s u l p h a t e ( B i o r a d ) .  c o n t a i n e d 0.07 M NaCl proteins  - B i o r a d ) and 0.3 ml o f  The s e p a r a t i n g g e l a l s o  f o r b e t t e r r e s o l u t i o n o f the lower m o l e c u l a r  (Hancock and Carey,  1979).  weight  The upper s t a c k i n g g e l c o n t a i n e d 3%  (wt/vol) a c r y l a m i d e and 0.12% (wt/vol) b i s a c r y l a m i d e  ( i n 5 ml o f g e l mix  a l s o c o n t a i n i n g 50 y l o f 10% (wt/vol) SDS and 1.25 ml o f 0.5 M T r i s - H C l pH 6.8) and was p o l y m e r i z e d by the a d d i t i o n o f 10 y l TEMED and 0.12 ml of 1% (wt/vol) ammonium p e r s u l p h a t e . mM T r i s base,  The e l e c t r o d e b u f f e r c o n t a i n e d 25.6  191.82 mM g l y c i n e and 0.1% (wt/vol) SDS (pH 8.4).  22  Samples were s o l u b i l i z e d by s o l u b i l i z i n g mixture c o n t a i n i n g 0.25  M Tris-HCl  (pH  6.8)  and  2-Mercaptoethanol ( B i o r a d ) . minutes.  For e s t i m a t i o n  the 4%  a d d i t i o n of an equal volume of  (wt/vol) SDS,  where s p e c i f i e d 10% The  20%  (vol/vol) glycerol in  (vol/vol)  samples were heated at 88°C f o r  10  of m o l e c u l a r weights of p e p t i d e s on SDS-gels,  the  f o l l o w i n g m o l e c u l a r weight standards were used, bovine serum albumin (66.2K); ovalbumin (45  K), c a r b o n i c  inhibitor  lysozyme (14.4  (i)  (21.5  K)  Protein  Proteins  K),  soybean t r y p s i n  K) .  staining  i n SDS-PAGE were s t a i n e d  blue d i s s o l v e d r a t i o and  and  anhydrase (31  i n 1%  (wt/vol) Coomassie  i n g l a c i a l a c e t i c a c i d , methanol and  destained  i n s o l u t i o n w i t h the  brilliant  water at 1:4.5:4.5  same components (without the  dye)  at a 0.7:2:7.2 r a t i o .  (ii) LPS Frasch ethanol  (F)  LPS  g e l s were s t a i n e d by the  (1982).  P r o t e i n and  Pollack KDO  T h i s method was  p e r i o d a t e - s i l v e r method of T s a i  modified  to p r e f e r e n t i a l l y s t a i n LPS  Protein and  staining  isopropanol  instead  of  i n whole o u t e r membrane g e l s .  Keto-deoxyoctonate (KDO)  assays were done by  by u s i n g  and  assays  the m o d i f i e d  Lowry method of  Schacterle  (1973).  assays were performed by a m o d i f i c a t i o n  et a l . (1972) with a 15 min  hydrolysis  period  of the method by  i n H SO  .  LPS  Osborn  23  concentrations  were c a l c u l a t e d by  weight of KDO  (G)  (Darveau and  assuming t h a t  the LPS  contained  Enzymatic d i g e s t i o n of p r o t e i n F  enzymatically  aureus V8 pH  7.4  digested  protease  containing  with the  following  (Sigma Chemical Co., 35 mM  MgCl^.  The  ug enzyme per mg  (3) TPCK-Trypsin  at 0.1  mg  37°C f o r 60  enzyme per mg  (1)  protein.  EDTA 20 mM  Staphylococcus  i n 20 mM  The  Tris-HCl,  1:100.  T r i s - H C l , pH  (Sigma) i n 10 mM  cells  (2)  6.0  at  T r i s HCl,  50  pH  d i g e s t i o n s were performed at  min.  In assays u s i n g o p t i c a l density washed three  i n t a c t P.  at 600  nm  aeruginosa c e l l s ,  of 0.8  times i n 20 mM  enzymatic d i g e s t i o n s  were at 100  c u l t u r e s grown to  were c e n t r i f u g e d  Tris-HCl  resuspended i n t h i s b u f f e r to one  suspension.  whole  enzyme:protein r a t i o was  Sigma) i n 2 mM  proten.  enzymes:  S t . L o u i s , Mo.)  Papain (papainase Type 4,  8.0  by  Hancock, 1983).  P u r i f i e d p r o t e i n F, or p r o t e i n F i n o u t e r membranes and was  4.3%  (pH  7.4)  the  containing  t h i r d of the ug of the  and  cell pellets 5 mM  MgCl^  o r i g i n a l volume.  enzyme per ml  A l l r e a c t i o n s were stopped by h e a t i n g  an  of  and  All  cell  at 88°C f o r 10  min.  o and  (H)  the p r o t e o l y t i c p e p t i d e s kept f r o z e n  Chemical c l e a v a g e of p r o t e i n  Henning (1974).  p u r i f i e d p r o t e i n F was containing  dissolved  C.  F  Chemical c l e a v a g e of p r o t e i n F by method of Garten and  at -20  cyanogen bromide f o l l o w e d Briefly,  i n 0.8  1 M cyanogen bromide and  0.6  ml  60  of 98%  N HCl.  the  ug of l y o p h i l i z e d ( v o l / v o l ) formic  The  volume was  acid  made up  to  24  1 ml. vial  The sample was i n c u b a t e d at 37 C i n a f o i l - c o v e r e d  f o r 18 h r , and then d i l u t e d and l y o p h i l i z e d .  cyanogen bromide p e p t i d e s ) was r e d i s s o l v e d relyophilized.  tightly-capped  The r e s i d u e ( c o n t a i n i n g  i n d e i o n i z e d water and  The l y o p h i l i z e d p e p t i d e s were then r e d i s s o l v e d  i n the  o r i g i n a l volume of 10% (wt/vol) SDS, 10 mM T r i s - H C l , pH 7.4 b u f f e r . some cases, p r o t e i n F was denatured  i n 80% ( v o l / v o l ) a c e t i c  In  a c i d f o r 1 hr  p r i o r to cyanogen bromide treatment.  2.  Animals  (A)  Animals  used.  Mice used  in this  study were o f the BALB/c BYJ s t r a i n  o b t a i n e d from Jackson Labs (Bar-Harbour, the animal care u n i t  Maine).  and were  They were m a i n t a i n e d i n  i n the Department o f M i c r o b i o l o g y , U.B.C.  New  Zealand white female r a b b i t s o b t a i n e d from the animal c a r e u n i t at U.B.C. were a l s o  (B)  used.  Immunizing  Protocol  Mice, 6-8 weeks o l d , were g i v e n an i n i t i a l 20 yg o u t e r membrane o r 5 yg p u r i f i e d p r o t e i n volume o f Freund's saline.  subcutaneous  i n j e c t i o n of  i n a t o t a l o f 0.4 ml  incomplete adjuvant ( D i f c o Labs) d i l u t e d 1:1 w i t h  The animals were r e s t e d f o r 1 week and then  injected  i n t r a p e r i t o n e a l l y 2 t o 3 times a t one week i n t e r v a l s w i t h 10 yg o u t e r membranes or 5 yg p u r i f i e d p r o t e i n  i n phosphate  adjuvant.  i n j e c t i o n , mice were b l e d from the t a i l  One week a f t e r the l a s t  v e i n , and the s e r a t e s t e d f o r antibody response  b u f f e r e d s a l i n e without  t o the immunizing  antigen  25  by the ELISA assay d e s c r i b e d below.  Mice t o be used f o r p r o d u c t i o n of  monoclonal a n t i b o d i e s were r e s t e d f o r two weeks a f t e r the t h i r d i n j e c t i o n and then given  a final  antigen  antigen  injection either intraperitoneally  or i n t r a v e n o u s l y 3 days p r i o r to f u s i o n . Rabbits with  were i n j e c t e d subcutaneously f o u r times a t two week i n t e r v a l s  50 ug o f outer membranes  incomplete  adjuvant.  i n a t o t a l o f 0.8 ml o f Freund's  The r a b b i t s were then b l e d from the e a r v e i n and the  s e r a t e s t e d f o r antibody  response t o the immunizing a n t i g e n .  a n t i s e r a was used f o r the experiments i n Chapter 1. r a b b i t s were immunized once every membranes  two weeks w i t h  i n s a l i n e u n t i l 16 weeks and then b l e d .  This  T h e r e a f t e r the  25 ug o f o u t e r The a n t i s e r a were kept  o frozen  i n 0.5 ml volumes at -70 C u n t i l use.  from r a b b i t s immunized subcutaneously w i t h and  C o n t r o l s e r a were  Freund's incomplete  obtained adjuvant  s a l i n e f o r the same p e r i o d .  3.  Tissue  Culture  (A)  Myeloma c e l l  lines  Two myeloma c e l l  l i n e s o f BALB/c o r i g i n were used.  from P3-NS1/1, i s a c l o n e d c e l l myeloma  (Cowan e t al.. 1974).  i n t e r n a l l y degrades a MOPC-21  NS/1,  l i n e d e r i v e d from MOPC-21, a BALB/c  The NS/1 c e l l light  chain.  line  s y n t h e s i z e s and  On f u s i o n , t h e r e f o r e 25% o f  the s e c r e t e d immunoglobulins w i l l bear t h a t MOPC-21^ l i g h t w i l l have mixed l i g h t  chains.  abbreviated  The NS/1 c e l l  American Type C u l t u r e C o l l e c t i o n  l i n e was o b t a i n e d  ( R o c k v i l l e , Md).  SP2/0-Agl4 ( a b b r e v i a t e d SP2/0) a l s o o b t a i n e d  c h a i n and 50% from  The myeloma c e l l  from the American Type  line  26  C u l t u r e C o l l e c t i o n s was o r i g i n a l l y d e r i v e d by s e v e r a l r e c l o n i n g steps from a BALB/c myeloma l i n e SP2/HL-Ag, i t s e l f d e r i v e d by s e v e r a l steps from SP2/HLGK, the product o f a f u s i o n between the BALB/c myeloma X63-Ag8 (\G and K^) and a BALB/c mouse s p l e e n c e l l  (Schulman  e_t a l . , 1978).  SP2/0 i s a t o t a l h o n - s e c r e t o r of mouse immunoglobulin SP2/0 and NS/1 myeloma c e l l (Littlefield  l i n e s are r e s i s t a n t  1964) s i n c e they l a c k the enzyme  chains.  The Both the  t o 20 ug/ml 8 azaguanine  hypoxanthine-guanine  p h o s p h o r i b o s y l t r a n s f e r a s e , an enzyme of the s a l v a g e pathway o f n u c l e o t i d e synthesis.  (B)  Medium, maintenance Medium.  and f r e e z i n g o f c e l l  lines  Powdered Dulbecco's M o d i f i e d E a g l e s medium (DMEM; GIBCO  Labs, Grand I s l a n d , N.Y.), a b a l a n c e d s a l t , v i t a m i n , and amino a c i d mixture c o n t a i n i n g L-glutamine, D-glucose and phenol r e d , was d i s s o l v e d a t 13.7 g/1 d i s t i l l e d (41 mM) Mo.),  water.  The medium was supplemented  w i t h 3.5 g/1  sodium b i c a r b o n a t e , 0.11 g/1 sodium p y r u v a t e (Sigma, S t . L o u i s ,  1.2 g/1 (5 mM)  N-2-hydroxyethyl p i p e r a z i n e - N ' - 2 - e t h a n e sulphonate  (HEPES; Calbiochem-Behring, La J o l l a , Ca.) and 20 ug/ml  final  c o n c e n t r a t i o n o f g e n t a m i c i n s u l p h a t e (Garamycin, S c h e r i n g Corp, N . J . ) . The mixture was s t e r i l i z e d by f i l t r a t i o n  through a 0.2 urn f i l t e r  ( S c h l e i c h e r and S c h u e l l #1121, Keen, NH) and s t o r e d a t 4°C i n 500 y l volumes f o r a maximum o f 4 weeks.  B e f o r e use t h e medium was  w i t h 10% o r 20% ( v o l / v o l ) h e a t - i n a c t i v a t e d f e t a l  calf  supplemented  serum (FCS; GIBCO  and BOCKNECK, Rexdale, O n t a r i o ) ( p r e t e s t e d batches g i v i n g the b e s t growth  27  for  myeloma c e l l  l i n e s were s e l e c t e d ) .  The FCS was s t o r e d a t -20°C and  kept a t 4°C when i n use.  Maintenance.  Myeloma o r hybridoma c e l l  l i n e s were r o u t i n e l y grown i n  t i s s u e c u l t u r e f l a s k s ( L i n b r o Flow Labs, McLean, Va.) i n DMEM supplemented with  10% FCS a t 37°C i n a 10% C 0  obtained  a t 5 x 10^ c e l l s / m l .  2  incubator.  O p t i m a l growth was  C e l l numbers were kept below 1 x 10^  cells/ml.  Freezing. harvested  Cell lines  at 5 x 1 0  5  6  t o 1 x 1 0 c e l l s / m l were  by c e n t r i f u g a t i o n a t 1,200 rpm f o r 10 min.  resuspended g e n t l y  The p e l l e t was  i n i c e - c o l d DMEM (no HEPES) c o n t a i n i n g 80% ( v o l / v o l )  FCS, and the c e l l  count a d j u s t e d  medium.  volume o f i c e - c o l d FCS c o n t a i n i n g 18% ( v o l / v o l )  An equal  sulphoxide  t o 2 x 10^ c e l l s / m l i n t h e same  (DMSO, F i s h e r Co.) was added.  The c e l l  dimethyl  suspension i n 1.0 ml  o volumes was f r o z e n o v e r n i g h t  a t ^70 C u s i n g  a C r y o r a c k 10/16  ( S t r e c k Labs I n c . , Omaha, Ne.) p r e v i o u s l y kept a t -20°C. c u l t u r e s were t r a n s f e r r e d and s t o r e d  inliquid N  2  container  The f r o z e n  (-176°C).  When needed, the samples were q u i c k l y thawed a t 37°C, washed  twice  i n 10 ml volumes o f DMEM a t 1,200 rpm f o r 10 min. and resuspended i n 20 ml of DMEM c o n t a i n i n g 10% FCS f o r growth a t 37°C i n a 10% CO  incubator.  (C)  G e n e r a t i o n o f Monoclonal a n t i b o d i e s HAT S e l e c t i v e Medium Hypoxanthine  and thymidine  concentrated s o l u t i o n  (HT) were made up as a 100 times  (100 x HT) c o n t a i n i n g 0.1361 g hypoxanthine  (6-hydroxypurine, Sigma) and 0.07266 g thymidine [ l - ( 2 - D e o x y - B - D - r i b o f u r a n o s y l ) - 5 methyl u r a c i l , warmed up t o 70-80°C t o d i s s o l v e the s a l t s . by f i l t r a t i o n and kept f r o z e n Aminopterin  The s o l u t i o n was  sterilized  i n the dark a t -20°C i n 10 ml volumes.  ( 4 - a m i n o f o l i c a c i d , 4 amino p t e r o y l - g l u t a m i c a c i d , Sigma) was  made up as a 1000 f o l d  c o n c e n t r a t e d s t o c k s o l u t i o n by adding 22.6 mg o f  aminopterin to 80 ml d i s t i l l e d water, was  Sigma] per 100 ml o f water  added t o d i s s o l v e the a m i n o p t e r i n .  mis and the f i n a l s o l u t i o n s t e r i l i s e d  then enough 1.0 M sodium h y d r o x i d e The volume was then made up t o 100 by f i l t r a t i o n .  S e l e c t i o n o f myeloma/spleen c e l l h y b r i d s was accomplished by c u l t u r e of  the f u s i o n m i x t u r e  i n s e l e c t i v e medium c o n t a i n i n g hypoxanthine,  aminopterin and thymidine follows. of  Aminopterin  (HAT).  The mechanism o f HAT s e l e c t i o n  (an analogue  i s as  o f f o l i c a c i d ) b l o c k s de novo s y n t h e s i s  p u r i n e s and p y r i m i d i n e s , w h i l e the exogenous supply o f hypoxanthine and  thymidine a l l o w s f o r n u c l e o t i d e s y n t h e s i s v i a a salvage pathway. c e l l l i n e s l a c k the enzyme  hypoxanthine-guanine-phosphoribosyl  t r a n s f e r a s e , an enzyme o f the salvage pathway, and thus cannot HAT medium.  Myeloma  survive i n  S p l e e n / s p l e e n f u s i o n s are p a s s i v e l y s e l e c t e d o u t by t h e i r  l i m i t e d c a p a c i t y t o grow i n c u l t u r e  (Goding, 1980).  Thus o n l y c e l l s  with  the growth c a p a c i t y o f myelomas and the s a l v a g e pathway o f s p l e e n c e l l s can  survive.  29  The  s e l e c t i v e medium was made by the a d d i t i o n o f 1 ml o f the 100 x HT  and 0.1 ml o f the 1000 x aminopterin c o n t a i n i n g 20% v / v FCS.  The f i n a l  hypoxanthine, 7.3 ug/ml thymidine  (i) The Kohler  s t o c k s o l u t i o n s t o 99 mis o f DMEM  c o n c e n t r a t i o n s were 13.6 ug/ml and 0.22 ug/ml  aminopterin.  Cell fusion cell  f u s i o n procedure was a m o d i f i c a t i o n o f t h e t e c h n i q u e o f  and M i l s t e i n  (1975).  Spleen  c e l l s were o b t a i n e d  from  immunized BALB/c mice and were d i s p e r s e d i n t o s i n g l e c e l l  antigen  suspension  ( u s i n g a 22-gauge needle) i n DMEM c o n t a i n i n g 10% ( v o l / v o l ) FCS. The spleen c e l l s were washed once and counted i n a haemocytometer.  Myeloma  c e l l s grown t o 5 x 10^-1 x 10^ c e l l s / m l were h a r v e s t e d by c e n t r i f u g a t i o n a t 1200 rpm f o r 10 min, washed and resuspended i n DMEM-10% FCS and counted.  Spleen  and myeloma c e l l s were combined a t a r a t i o o f  10:1 and p e l l e t e d a t 1200 rpm f o r 10 min and the p e l l e t t a p p i n g the tube on bench. P r o t o c o l 1.  l o o s e n e d by  Two f u s i o n p r o t o c o l s were used:  Two ml o f 50% (wt/vol)  1500 p o l y e t h y l e n e  g l y c o l (PEG)  o (BDH) prewarmed t o 37 C were added over 2 min t o the l o o s e p e l l e t . p i p e t t e t i p was used t o g e n t l y break up the p e l l e t f u r t h e r 20 ml o f DMEM without  (The  as PEG was added) .  FCS was added over 3-5 min.  A  The c e l l s were  p e l l e t e d and resuspended, t o l e a v e s m a l l clumps, i n 20 ml o f DMEM c o n t a i n i n g 20% FCS, and incubated incubator.  f o r 3 h r a t 37°C i n a 10% C 0  2  A f t e r t h e i n c u b a t i o n p e r i o d , s e l e c t i v e HAT medium c o n t a i n i n g  thymocytes d e r i v e d from a 4-6 week o l d mouse was added t o t h e f u s e d 4 cells. The c e l l d e n s i t i e s were a d j u s t e d t o 1 x 10 myeloma c e l l s ,  30  1 x 10  spleen  of the c e l l  cells  and 1 x 10  thymocytes p e r ml.  Aliquots  suspension were then t r a n s f e r r e d t o each w e l l o f a 96 w e l l  t i s s u e c u l t u r e p l a t e ( L i n b r o , Flow Labs.) and incubated CO^  (0.2 ml)  a t 37°C i n a 10%  incubator. P r o t o c o l 2 - T h i s p r o t o c o l d i f f e r e d from the p r e v i o u s  protocol i n  t h a t two d i f f e r e n t PEG s o l u t i o n s were used, and the PEG was d i l u t e d out on cell  dilution.  41.6%  The procedure was:  cell  p e l l e t , 0.5 ml o f a  (wt/vol) PEG 1550 (Serva F e i n B i o c h i m i c a ) was added over 1 min.  m i x t u r e was rocked g e n t l y PEG  To t h e l o o s e  f o r 2 t o 3 min,  1550 (Serva) was added as b e f o r e .  then 0.5 ml o f a 25% (wt/vol)  Four ml o f DMEM c o n t a i n i n g 20%  ( v o l / v o l ) FCS was added over 3 t o 5 min w i t h g e n t l e m i x i n g . f u r t h e r 20 ml o f DMEM c o n t a i n i n g suspension incubated the  incubation,  the c e l l  suspension.  Then a  20% ( v o l / v o l ) FCS was added and the  f o r 3 h r a t 37°C i n a 10% CO^ i n c u b a t o r .  feeder  thymocytes i n DMEM c o n t a i n i n g The f i n a l c e l l  The  cell  After  20% FCS were added t o  count was a d j u s t e d  as d e s c r i b e d  for  p r o t o c o l 1, and 1 ml volumes were t r a n s f e r r e d t o each w e l l o f a 24 w e l l tissue culture plate Mass.).  ( L i n b r o Flow and C o s t a r  3524 C o s t e r ,  Cambridge,  A f t e r 24 h r , most o f the medium was removed from the w e l l and  r e p l a c e d w i t h 1 ml o f HAT s e l e c t i v e medium. w i t h HAT s e l e c t i v e medium c o n t a i n i n g  T h i s was r e p e a t e d on day 4  thymocytes.  For both p r o t o c o l s , the p l a t e s were checked f o r growth o f hybridoma clones.  The c u l t u r e medium was r e p l a c e d  s e l e c t i v e medium c o n t a i n i n g  thymocytes.  a f t e r 10 days w i t h f r e s h HAT Colonies  o f hybridoma c e l l s  be observed as e a r l y as 1 week o r as l a t e as 3 weeks a f t e r c e l l  could  fusion.  31  (ii)  Cloning.  Supernatants  from w e l l s t h a t c o n t a i n e d hybridoma  c l o n e s occupying one t h i r d o f the w e l l bottom, were removed and t e s t e d i n enzyme l i n k e d  immunosorbent assays  (ELISA)  as d e s c r i b e d below, f o r  s e c r e t i o n o f a n t i b o d i e s a g a i n s t the immunizing  antigen.  w e l l s t h a t showed p o s i t i v e ELISA r e s u l t s was performed  C e l l cloning as f o l l o w s :  from  The  hybridoma c o l o n i e s were d i s p e r s e d by mixing the w e l l c o n t e n t s w i t h a s t e r i l e Pasteur p i p e t t e .  Twenty y l o f the c e l l  suspension from 0.2 ml  w e l l s o r 50 y l from 1 ml w e l l s , was d i s p e n s e d i n t o the f i r s t  row o f a 96  w e l l p l a t e c o n t a i n i n g 0.1 ml o f HAT s e l e c t i v e medium w i t h thymocytes. w e l l c o n t e n t s were mixed 10 times and 20 y l t r a n s f e r r e d  The  i n t o 0.1 ml HAT  s e l e c t i v e medum w i t h thymocytes c o n t a i n e d i n the w e l l o f t h e next row. T h i s procedure was r e p e a t e d through  row 12.  After allowing s u f f i c i e n t  time f o r growth, w e l l s c o n t a i n i n g one c l o n e (seen from row 6 on) were t e s t e d f o r a n t i b o d y p r o d u c t i o n by ELISA.  Wells t h a t gave p o s i t i v e ELISA  r e s u l t s were r e c l o n e d and t e s t e d a g a i n , b e f o r e expansion t h a t hybridoma c e l l  (D)  and f r e e z i n g o f  line.  Ascites production Cloned hybridoma c e l l s  immunizing  secreting antibodies s p e c i f i c  6 a n t i g e n were grown t o 1 x 10 c e l l s / m l .  f o r the  The c e l l s were  h a r v e s t e d by c e n t r i f u g a t i o n a t 1200 rpm f o r 10 min and resuspended  i n DMEM  6 at 1 x 10  cells/ml.  BALB/c mice were i n j e c t e d  t o t a l o f 1 x 10"* c e l l s .  i n t r a p e r i t o n e a l l y with a  These mice had been primed  by i n t r a p e r i t o n e a l  i n j e c t i o n o f 1 ml o f P r i s t a n e (2, 6, 10, 1 4 - t e t r a m e t h y l pentadecane, Sigma) 3 weeks p r i o r t o i n j e c t i o n w i t h the hybridoma c e l l s  (Goding,  32  1980).  The mice were observed f o r abdominal d i s t e n s i o n , and a s c i t e s f l u i d  was removed from the p e r i t o n e a l c a v i t y with to 15 ml o f a s c i t i s  a sterile  18 gauge needle.  Up  c o u l d be c o l l e c t e d from one mouse i n 3 t o 5 such  procedures. The cells,  ascitic  f l u i d was c e n t r i f u g e d a t 1200 rpm f o r 10 min t o remove  t e s t e d f o r antibody  t i t r e against  the s p e c i f i c  antigen  by the ELISA  method and s t o r e d f r o z e n i n 0.3 mi volumes a t -70°C.  (E)  Antibody  Purification  Antibodies  were p u r i f i e d from a s c i t e s f l u i d o r immune s e r a by  ammonium sulphate The  p r e c i p i t a t i o n and f o l l o w e d by a f f i n i t y  immune s e r a or a s c i t i c  s a t u r a t e d ammonium sulphate hrs.  f l u i d was d i l u t e d 1:1 i n s a l i n e and 40% v o l / v o l added s l o w l y w i t h  stirring  a t 4°C f o r 4  The p r e c i p i t a t e was removed by c e n t r i f u g a t i o n at 1200 x g and  r e d i s s o l v e d a t the o r i g i n a l volume i n phosphate b u f f e r e d Section 4). 45%  chromatography.  s a l i n e (PBS see  The a n t i b o d i e s were then r e p r e c i p i t a t e d by the a d d i t i o n o f  (vol/vol) saturated  ammonium s u l p h a t e  as b e f o r e .  The p r e c i p i t a t e was  then d i s s o l v e d i n a minimal amount o f phosphate b u f f e r e d d i a l y s e d against  5 1 PBS o v e r n i g h t .  speed c e n t r i f u g a t i o n .  (Pharmacia) t o which the s p e c i f i c r a b b i t p o l y c l o n a l antibody to remove any anti-LPS  Any p r e c i p i t a t e was removed by low  When g r e a t e r p u r i t y o f antibody  a n t i b o d i e s were subsequently a f f i n i t y  s a l i n e and  was needed the  p u r i f i e d on a Sepharose CL-4B column  a n t i g e n was coupled.  F o r example, a  was chromatographed on an LPS a f f i n i t y  antibodies .  column  33  4.  Antibody C h a r a c t e r i z a t i o n  (A)  Enzyme L i n k e d Immunosorbent Assays - ELISA Phosphate b u f f e r e d s a l i n e , pH  NaCl; 0.2  g/1  (1.47  g/1  mM)  KC1;  (2.68  made i n 10 l i t r e  mM)  (polyoxyethylene  Kh^PO^ 2.9  and 0.2  batches  supplemented w i t h 1.02  7.4  (3.08  g/1  c o n t a i n e d 8.0  (20.43 mM)  Na HP0 ; 2  (136.89  0.2  4  was  at 5 f o l d c o n c e n t r a t i o n , d i l u t e d b e f o r e use  and  (5 mM)  mM)  g/1  PBS  g/1  g/1  (PBS)  sodium a z i d e NaN^.  MgCl .6H 0. 2  2  Tween 20  s o r b i t a n monolaurate, Sigma) was  The  (0.5  ml/1)  added to the PBS  where  specified.  Coating b u f f e r Carbonate/bicarbonate d i s t i l l e d Water 1.5 NaHC0 ; 1.02 3  b u f f e r was  g/1  b u f f e r (pH.  g (14.15 mM)  (5 mM)  9.6)  NaC0 , anhydrous; 2.93 3  M g C l . 6 H 0 and 0.2 2  a d j u s t e d to pH 9.6  c o n t a i n e d per l i t r e  2  with 0.1  g (3.08  M NaOH and  mM)  of  g (34.87 NaN^.  mM)  The  s t o r e d i n the dark at  4°C f o r a maximum of 4 weeks.  Diethanolamine  Buffer  Diethanolamine  b u f f e r (10% w t / v o l ) c o n t a i n e d 97 ml/1  (Baker), 0.2  g/1  The b u f f e r was 4°C,  (3.08  mM)  NaN^  and  a d j u s t e d to pH 9.6  100 mg/1  (0.49  mM)  diethanolamine  MgCl 6H 0. 2 >  2  w i t h 1 M HCl, s t o r e d i n the dark a t  and brought to room temperature b e f o r e  use.  mM)  34  ELISA The  ELISA procedure  Ruitenberg §_t a l (1974). ( F a l c o n 3912; 100  used was  Ninety s i x w e l l p o l y v i n y l c h l o r i d e p l a t e s  Becton-Dickinson  y l of a suspension  purified protein  and Co.,  were coated w i t h 50 or  of 20 yg/ml o u t e r membrane or 1 t o 5 yg/ml of buffer.  A f t e r a 16  2 hr at 37 C,  the p l a t e s were washed once i n  ( v o l / v o l ) Tween 20,  200 y l / w e l l of PBS  c o n t a i n i n g 1% FCS  b l o c k any f r e e p r o t e i n b i n d i n g s i t e s .  and 3 times  37°C or 16 hr at 4°C.  i n PBS  and  (PBS-FCS) f o r 30 min The  y l d i l u t i o n s of the t e s t antibody  PBS  i n c u b a t e d with  at 37°C, to  p l a t e s were then  incubated with  i n PBS-FCS f o r 1 t o 2 hr at  A f t e r washing w i t h PBS-Tween 20/PBS as above,  the p l a t e s were i n c u b a t e d i n the dark at 37°C f o r 1-2 y l of a goat  hr  o  i n c u b a t i o n at 4 C or  50 or 100  Oxnard, CA)  i n carbonate/bicarbonate  o  containing 0.01%  a m o d i f i c a t i o n of the method of  hr w i t h 50 or  anti-mouse F ( a b * ) ^ immunoglobulin fragments  antibody  coupled to a l k a l i n e phosphatase ( H e l i x B i o t e c h , Richmond, B.C.) been d i l u t e d 2000 f o l d  100  which  had  i n PBS-FCS.  A f u r t h e r PBS-Tween 20/PBS wash was  done, and  the p l a t e s  developed  w i t h the a l k a l i n e phosphatase enzyme s u b s t r a t e , disodium p - n i t r o p h e n y l phosphate (Sigma 104 t a b l e t per 5 mis development was  phosphatase s u b s t r a t e t a b l e t s ;  of 10% Diethanolamine  buffer.  The  5 mg/tablet)  at 1  yellow colour  read a f t e r 45 minutes u s i n g an ELISA T i t r e t e k M u l t i s c a n  photometer (Flow Labs., McLean, V i r g i n i a ) , s c r e e n i n g of a n t i b o d y  s e t a t 405  nm.  For  initial  s e c r e t i o n by hybridoma c l o n e s , the ELISA p l a t e s were  read a f t e r 2 - 4 hr c o l o u r development at room temperature.  Control wells  c o n t a i n e d e i t h e r no a n t i g e n or no t e s t a n t i b o d y , w h i l e p o s i t i v e  control  35  wells contained  as a n t i g e n ,  H103  outer  membranes and  the  serotype 5  s p e c i f i c monoclonal a n t i b o d y , MA1-8.  Whole C e l l  ELISA  ELISA assays u s i n g whole P. using e i t h e r f i x e d or n o n - f i x e d chloride plates  (PVC)  u l per w e l l of 0.5%  a e r u g i n o s a c e l l s were done by cells.  In the  (Flow) were i n c u b a t e d  at 37°C with  containing  1 mM  times by c e n t r i f u g a t i o n at 3000 rpm MgCl^ and  1.0%  50  (Sigma) d i l u t e d i n PBS.  aeruginosa c e l l s were grown to an o p t i c a l d e n s i t y at 600 and washed three  methods  f i r s t method, p o l y v i n y l  f o r 30 min  (vol/vol) glutaraldehyde  two  ( v o l / v o l ) FCS.  The  nm  P.  of 0.6-0.8,  f o r 10 min  in  PBS  c e l l s were 4  resuspended i n the u l of the c e l l  same b u f f e r t o g i v e 1 to 5 x 10  suspension was  aliquoted  cells/ml.  Fifty  i n t o each w e l l to g i v e a f i n a l  3 count per w e l l of a p p r o x i m a t e l y 1 x 10 glutaraldehyde.  The  PVC  cells  i n 0.25%  (vol/vol)  p l a t e s were c e n t r i f u g e d at 3,000 rpm  f o r 10  min  o and  incubated  p i of 0.1  f o r 30 min  at 37 C.  M glycine containing  inactivate free glutaraldehyde. r i n s e d by d i p p i n g  The  1% FCS  s u p e r n a t a n t was was  removed and  added to each w e l l  A f t e r a 10 min  into 3 separate containers  incubation  o f 500  ml PBS  100  to  the p l a t e s were containing  1  mM  MgCl . 2  The al.  second method was  (1982).  P.  a e r u g i n o s a c e l l s were not  c e n t r i f u g e d at 3,000 rpm procedure and described  a modification  during  f o r 10 min  of t h a t d e s c r i b e d fixed.  Instead,  between each step  the washing p r o c e d u r e s .  above f o r the ELISA assay, w i t h the  by Posner et the p l a t e s were  i n the ELISA assay  A l l other  procedures were as  exception  t h a t 2%  (vol/vol)  36  FCS  was used, the i n c u b a t i o n steps were a l l f o r 1 hr at 3 7 C.  A goat  anti-mouse antibody c o u p l e d t o h o r s e r a d i s h peroxidase enzyme (Kilkegaard-Perry Ltd.,  Flow Labs) was used as the second a n t i b o d y .  peroxidase enzyme s u b s t r a t e c o n t a i n e d 0 . 1  ml o f a 1 5 mg/ml diammonium  2,2'-Azino-di-(3-ethyl-benzthiazolin-sulphonate  ( 6 ) solution  Mannheim, W. Germany) i n 1 0 ml o f c i t r a t e b u f f e r buffer ml  contained 0 . 9 6  a c i d per 1 0 0  o f 3 N NaOH to a d j u s t the pH t o 4 . 0 .  (vol/vol) min  g citric  Before use,  0 . 3 3  ml o f 3 0 7 .  or 4 1 5  After 3 0  nm u s i n g a  photometer.  Western e l e c t r o p h o r e t i c The  The c i t r a t e  ml o f d i s t i l l e d water and 0 . 2  i n c u b a t i o n the green c o l o u r was r e c o r d e d a t 4 0 4  (B)  blot  transfers  method used was e s s e n t i a l l y t h a t  P r o t e i n s were e l e c t r o p h o r e t i c a l l y SDS-polyacrylamide g e l s and  (pH 4 . 0 ) .  (Boehringer-  hydrogen p e r o x i d e was added t o the s u b s t r a t e s o l u t i o n .  Titretek Multiscan  The  o f Towbin e t a l . ( 1 9 7 9 ) .  transferred  from one- o r two-dimensional  onto n i t r o c e l l u l o s e paper ( 0 . 4 5 um  Schuell) using a Biorad e l e c t r o b l o t  chamber a t 0 . 4  Schleicher  - 0 . 5 A  f o r 2 - 3 hr  o at 4 C or a t 1 0 mA f o r 1 8 h r a t room temperature.  In the f a s t  blot  method, the SDS-acrylamide g e l was l a i d on two sheets o f n i t r o c e l l u l o s e paper and prewashed i n PBS c o n t a i n i n g 0 . 1 7 o min  or 8 0 ° C  f o r 1 0 min.  (wt/vol) SDS a t 6 0 ° C  F o r 1 8 h r b l o t s , a sheet o f the n i t r o c e l l u l o s e  paper was l a i d on e i t h e r  side  of the g e l .  The g e l and n i t r o c e l l u l o s e  papers were e n c l o s e d between s t a c k s o f Whatman 3 M f i l t e r pads o f Scotch b r i t e . h o l d e r s and p l a c e d  for 3 0  papers and two  The sandwich was p l a c e d between two s l o t t e d p l a s t i c  i n the e l e c t r o b l o t  chamber o f a B i o r a d apparatus.  For  37  a f a s t b l o t , the n i t r o c e l l u l o s e was was  f i l l e d with 3 l i t r e s  p l a c e d on the anode s i d e .  of t r a n s f e r b u f f e r pH 8.3 43.2  Tris  from Schwarz/Mann) and 0.1%  u l t r a pure T r i s  glycine  [containing  ( v o l / v o l ) a b s o l u t e methanol, (25 mM,  g/1  The  (192 mM,  The Western b l o t s were developed as f o l l o w s :  chamber  20%  B i o r a d ) , 9.10  g/1  (wt/vol) SDS].  The b l o t s were  o incubated f o r 30 min albumin  at 37 C i n PBS  c o n t a i n i n g 3% (wt/vol) bovine serum  (BSA f r a c t i o n V, Sigma) to b l o c k a l l a v a i l a b l e p r o t e i n  s i t e s on the n i t r o c e l l u l o s e paper.  Subsequently, the b l o t s were i n c u b a t e d  w i t h 10 ml of the t e s t antibody d i l u t e d and then washed 3 times f o r 15 min steps were a 2-3  i n 3% BSA-PBS, f o r 16 hr at 4°C,  i n excess PBS  a t 37°C.  a l k a l i n e phosphatase, a l k a l i n e phosphatase  F(&b')^  substrate.  The  Double  Napthol-As-MX-phosphate  A f t e r the d e s i r e d c o l o u r  The  immunoperoxidase  staining 4.6.  Immunodiffusion  immunodiffusion  ( O u c h t e r l o n y , 1959)  was  performed  i n 2%  (wt/vol) agarose  (Standard low Mr  agarose, B i o r a d ) d i s s o l v e d  barbitone buffer  [12 g sodium  d i e t h y l b a r b i t u r a t e and 4.40  5'5  had  i n excess water, d r i e d between paper  done as d e s c r i b e d f o r c o l o n y immunoblots i n s e c t i o n  Double  10  20 mg/ml F a s t Red-TR S a l t  (Sigma) added i n t h a t o r d e r .  towels and s t o r e d out of d i r e c t l i g h t .  (C)  i n an  s u b s t r a t e s o l u t i o n c o n t a i n e d per  (pH 8.8)  developed, the b l o t s were r i n s e d  1000  a n t i b o d y c o n j u g a t e d to  ( 5 - c h l o r o - 2 - t o l u i d e n e diazonium, Sigma) and 10 mg  procedure was  next  3 more PBS washes, and c o l o u r development  Tris-HCl buffer  disodium s a l t  The  hr i n c u b a t i o n at 37°C i n 3% BSA-PBS c o n t a i n i n g a  f o l d d i l u t i o n of a goat anti-mouse  mis o f 50 mM  binding  in g  0.08% 5'5  38  diethylbarbituric with 1.0  a c i d per  M NaOH].  litre  T r i t o n X-100  of d i s t i l l e d water a d j u s t e d was  added to 1%  agarose s o l u t i o n poured on g l a s s s l i d e s . f i l l e d with 20 ug of the wells  The  s o l u b i l i z e d antigen  f i l l e d with u n d i l u t e d  to pH  ( v o l / v o l ) and  outer and  antigen  8 ml of  the c e n t r e  antiserum  f o l d d i l u t i o n s of a n t i s e r u m .  agarose p l a t e s were then incubated  f o r 16 hr at 37°C i n a humid  The  the  w e l l s were  or two  chamber.  8.2  The  w e l l s were f i l l e d w i t h s a l i n e b u f f e r , arid the p l a t e s kept at  o 4 C f o r a f u r t h e r 72 h r s .  The  s t a c k of Whatman 3M  papers, washed f i v e times i n PBS  37°C, d r i e d again,  filter  s t a i n e d i n 1%  d i s s o l v e d in absolute and  destained  (D)  i n the  Agglutination  same s o l u t i o n w i t h o u t the  preparations  at  blue  water at a r a t i o of  4:1:4  dye.  B a c t e r i a l a g g l u t i n a t i o n was  autoclaved  at 120°C f o r 1 hr,  resuspended i n s a l i n e , as d e s c r i b e d  (1979), or l i v e P.  f o r 45 min  Assays  aeruginosa c e l l s  c e n t r i f u g e d and  dry under a  (wt/vol> Coomassie b r i l l i a n t  methanol, a c e t i c a c i d and  Bacterial agglutination. e i t h e r P.  p l a t e s were then p r e s s e d  aeruginosa c e l l s .  were i n c u b a t e d  performed  using  then  by L a n y i  In both methods the  and  Bergan  cell  w i t h s e r i a l d i l u t i o n s of a n t i s e r u m f o r 1 hr  at  o 37 C.  The  granular  a g g l u t i n a t i o n of l i v e c e l l s was  b a c t e r i a l clumping, as opposed to the  h e a t - k i l l e d P.  a e r u g i n o s a w i t h homologous LPS  B a c t e r i a l a g g l u t i n a t i o n was to +4  c h a r a c t e r i z e d by  (strong r e a c t i o n ) .  scored  The  on  a coarse  f i n e r granular  clumping  O-antigen-specific  a s c a l e of +1  a g g l u t i n a t i o n was  (weak  of  serum.  agglutination)  a l s o done on g l a s s s l i d e s  39  using  a drop each o f antiserum and b a c t e r i a l p r e p a r a t i o n ,  and the r e s u l t s  observed a f t e r 3 t o 5 min.  Passive by Lanyi  agglutination.  Passive  and Bergan (1979) u s i n g  a g g l u t i n a t i o n was done as d e s c r i b e d  tanned sheep r e d blood  cells  aeruginosa LPS (heated f o r 1 hr a t 100°C p r i o r t o u s e ) . erythrocytes  were washed three  erythrocytes  ( v o l / v o l ) i n phosphate s a l i n e b u f f e r  M Na HP0 2  4  sheep  adjusted  t o 4%  (0.02 M KH^PO^, 0.06  and 0.12 M NaCl, pH 7.5) and 2.5 mg o f t a n n i c a c i d i n 50 ml  .  centrifuged  suspension.  After  o  a 15 min i n c u b a t i o n  at 37 C w i t h o c c a s i o n a l m i x i n g , the c e l l s were  (100 x g f o r 20 min) and washed w i t h 100 ml o f phosphate  saline buffer.  One h a l f o f the c e l l s were kept as a c o n t r o l i n b u f f e r  1 mM sodium a z i d e .  To the o t h e r  h a l f o f the c e l l s ,  of LPS o r p r o t e i n was added, and the m i x t u r e i n c u b a t e d with very  Briefly,  times i n s a l i n e (0.9% N a C l ) ,  phosphate s a l i n e b u f f e r was added t o 50 mis o f the c e l l  containing  and P.  gentle  were washed three  a g i t a t i o n at regular  intervals.  20 ug/ml  f o r 1 h r a t 37°C  The a n t i g e n - c o a t e d  cells  times i n s a l i n e and 1 mM NaN^ was added f o r storage a t  o 4 C.  The c e l l s were made up t o 1% ( v o l / v o l ) i n s a l i n e b e f o r e use. Passive  plates  a g g l u t i n a t i o n was done i n L i n b r o  ( L i n b r o , Flow Labs) u s i n g  50 u l o f a n t i s e r u m s e r i a l l y  s a l i n e and 50 u l o f 1% ( v o l / v o l ) a n t i g e n Control wells contained  96 w e l l c o n i c a l bottom  c o a t e d sheep  diluted in  erythrocytes.  the a n t i s e r u m and tanned, non-antigen-coated sheep  o erythrocytes.  The p l a t e s were i n c u b a t e d  t i t r e . was taken as the h i g h e s t  a t 37 C f o r 1 hr  serum d i l u t i o n  The serum  giving agglutination.  40  N o n - a g g l u t i n a t e d c e l l s gave a t i g h t b u t t o n o f c e l l s w e l l , while the a g g l u t i n a t e d c e l l s  (E)  Indirect  a t the bottom of the  formed a mat at the w e l l bottom.  immunofluorescence  Two methods were used.  In the f i r s t method, P. a e r u g i n o s a  cells,  grown to an o p t i c a l d e n s i t y a t 620 nm of 0.8, were washed t h r e e times i n PBS c o n t a i n i n g 2% FCS.  The b a c t e r i a were resuspended  volume and 0.8 ml d i s p e n s e d i n 1.5 ml p l a s t i c or  t o the o r i g i n a l  Eppendorf  tubes.  Monoclonal  p o l y c l o n a l serum a t an a p p r o p r i a t e d i l u t i o n was added and the c e l l  suspension v o r t e x e d a t s e t t i n g 3 of a V o r t e x Genie  (Fisher  Scientific).  o A f t e r a 45 min i n c u b a t i o n a t 37 C w i t h v i g o r o u s a g i t a t i o n , the c e l l suspension was washed t h r e e times  i n PBS/1% FCS/1 mM M g C l  c e n t r i f u g a t i o n f o r 1 min i n a Brinkmann M i c r o f u g e 5412).  The c e l l s were then  2  by  (Brinkmann c e n t r i f u g e  i n c u b a t e d a t 37°C f o r 45 min i n PBS/2% FCS  c o n t a i n i n g a 100 f o l d d i l u t i o n o f r a b b i t anti-mouse IgG (Sigma).  The  washing procedure was r e p e a t e d , then the c e l l s were i n c u b a t e d as b e f o r e , w i t h a goat a n t i - r a b b i t (FITC, Sigma).  antibody c o u p l e d t o F l u o r e s c e i n i s o t h i o c y a n a t e  A f t e r a f u r t h e r PBS wash, 10 y l o f the c e l l  suspension  was p l a c e d on a s l i d e under a c o v e r s l i p and examined w i t h a Z e i s s microscope  (Standard R.A., w i t h a condenser  f o r fluorescent  c o n t a i n i n g a halogen lamp and s u i t a b l e f i l t e r s f l u o r e s c e n c e e m i s s i o n of f l u o r e s c e i n experiments  microscopy)  f o r measuring  the  i s o t h i o c y a n a t e at 525 nm.  In  where p o l y c l o n a l r a b b i t a n t i s e r u m to P. a e r u g i n o s a o u t e r  membrane was used, the second F I T C - l a b e l l e d a n t i b o d y used  a n t i b o d y was o m i t t e d and a goat  (Sigma).  anti-rabbit  Negative c o n t r o l s contained  41  P.  aeruginosa c e l l s w i t h no f i r s t a n t i b o d y but a l l o t h e r a n t i b o d i e s , or  first  antibody and FITC antibody without  the second  antibody; other  c o n t r o l s were as d e s c r i b e d i n r e s u l t s . The (1979). was  second method used was The  e s s e n t i a l l y as d e s c r i b e d by H o f s t r a e t a l .  antibody steps were as above except  done on f i x e d P.  t h a t the whole  a e r u g i n o s a smears on g l a s s s l i d e s .  For  procedure  each o  i n c u b a t i o n s t e p , the s l i d e s were p l a c e d i n a humid chamber at 37 C f o r 10 minutes and were washed by d i p p i n g i n  (F)  Colony  PBS.  blotting  Colony b l o t t i n g was Henning e t a l (1979).  performed  by a m o d i f i c a t i o n o f the method of  B a c t e r i a l c o l o n i e s were t r a n s f e r r e d from  p l a t e s onto prewashed n i t r o c e l l u l o s e f i l t e r s of  the f i l t e r s  at 30°C f o r 30 min,  by c o n t a c t .  7.4;  2 mM  M g C l , 1 mM 2  NaCl  The  i n c u b a t i o n f o r 15 min  at 37°C  [10 mM  (TBS)] c o n t a i n i n g 37. (wt/vol)  then f o r 2 hr w i t h the t e s t antibody d i l u t e d (wt/vol) g e l a t i n .  After incubation  the c o l o n y b l o t s were s u c c e s s i v e l y  i n c u b a t e d with T r i s - b u f f e r e d s a l i n e f o r 45 min pH  i n TBS  c o n t a i n i n g 0.1%  Tris-HCl,  gelatin,  b u f f e r c o n t a i n i n g 1%  b l o t s were s u b s e q u e n t l y washed 3 times i n TBS  by  (wt/vol) g e l a t i n , and  i n c u b a t e d f o r 2 hr at 37°C w i t h a goat anti-mouse immunoglobulin to  horse-radish peroxidase  (Flow Labs, I n c . , McLean, V a . ) .  as above, the b l o t s were developed p e r o x i d a s e (5 mg  30%  (vol/vol)  of 4 - c h l o r o - l - n a p t h o l d i s s o l v e d  H 0 ). 2  2  then  coupled  A f t e r washing  using a histochemical substrate f o r  a b s o l u t e methanol and mixed at 23°C w i t h 10 mis of  agar  i n 1.67 o f TBS  mis o f  ice-cold  containing 5 u l  42  5.  Serum s e n s i t i v i t y Due  testing  to p r e v i o u s o b s e r v a t i o n s t h a t the apparent s e n s i t i v i t y of  P. a e r u g i n o s a to normal human serum was  dependent  on the technique used to  measure i t (De Matteo et a l . 1981), two d i f f e r e n t methods were used. Method 1.  In method 1, b a c t e r i a were p l a t e d on t r y p t i c a s e soy agar  p l a t e s and then scraped o f f and resuspended c o n t a i n i n g 1 mM  MgCl  o J  i n t r y p t i c a s e soy b r o t h ,  at an o p t i c a l d e n s i t y at 650 nm  i  0.1.  They were then grown to an On  o f 0.4  (0D, .) of 650 and c e n t r i f u g e d and C  6b0  resuspended  i n an e q u a l volume o f minimal e s s e n t i a l medium c o n t a i n i n g  [0.4% g l u c o s e ; 0.5 (NH )_S0.]•• 4 2 4 i  mM  MgS0 ; 10 uM FeSO^; 40 mM 4  T h i s c e l l suspension was  l^HPO^ and 7 mM  d i l u t e d 1 i n 1000  to 1 i n  10,000 i n t o minimal e s s e n t i a l medium and 0.1 ml of the d i l u t e d suspension mixed w i t h 0.1 ml of f r e s h normal human serum.  cell  A f t e r 1 hr at  o 37 C the b a c t e r i a were p l a t e d f o r v i a b l e counts on t r y p t i c a s e soy agar o plates.  Serum was  Method 2.  The  r o u t i n e l y s t o r e d a t -70 C p r i o r to use. second method was  method of Schneider and G r i f f i s s  based on the r e c e n t l y  published  (1982), and the well-known b r o t h d i l u t i o n  method f o r t e s t i n g o f a n t i m i c r o b i a l s u s c e p t i b i l i t y . of c e l l s were c e n t r i f u g e d and resuspended  Logarithmic cultures  i n p r o t e o s e peptone No. 4  (Gibco, O n t a r i o , Canada) at a f i n a l c o n c e n t r a t i o n of 5 x 10 ml.  Twenty u l of c e l l s were added  cells  2 broth  per  t o 80 u l o f f r e s h normal human  serum which had been s e r i a l l y d i l u t e d  i n p r o t e o s e peptone No.  2 b r o t h to  g i v e f i n a l serum c o n c e n t r a t i o n s ( a f t e r a d d i t i o n of c e l l s ) r a n g i n g from to 1.25% was  added  i n 7 s e r i a l two-fold d i l u t i o n s . to 20 u l o f c e l l s .  80%  As a c o n t r o l , 80 u l of medium  For convenience the assay was  performed i n  43  96 w e l l m i c r o t i t r e t r a y s ( L i n b r o t i s s u e c u l t u r e p l a t e s , Flow Labs, Va) a l l o w i n g comparison o f 12 d i f f e r e n t The p l a t e was  incubated  s t r a i n s on a s i n g l e m i c r o t i t r e t r a y .  f o r 18 hours at 37°C.  End p o i n t s were  as the h i g h e s t c o n c e n t r a t i o n of serum a l l o w i n g f u l l c o n t r o l without completely  serum.  prevented  U s u a l l y a two f o l d h i g h e r  growth.  recorded  growth compared  t o the  c o n c e n t r a t i o n o f serum  44  RESULTS CHAPTER I.  1.  OUTER MEMBRANE ANTIGENS OF P, AERUGINOSA  Outer membrane p a t t e r n s of s e r o t y p i n g i s o l a t e s The  outer membranes of P. a e r u g i n o s a PA01  at l e a s t 6 to 8 major p o l y p e p t i d e s . 4 can be present at 10  These major o u t e r membrane p r o t e i n s  5 to 10  c o p i e s / c e l l and  to the growth c o n d i t i o n s (Hancock and Carey, Kageyama, 1978a, 1979).  are expressed 1979,  1980;  according  Mizuno  and  U s i n g the method of Hancock and Carey  o u t e r membranes were i s o l a t e d from the 17 a e r u g i n o s a t h a t comprise and Farmer, 1979;  have been shown to c o n t a i n  serotype s t r a i n s of  (1979), P.  the I n t e r n a t i o n a l A n t i g e n T y p i n g Scheme (Brokopp  L a n y i and Bergan, 1979).  The  o u t e r membrane p r o t e i n  p a t t e r n s of these s t r a i n s were c h a r a c t e r i z e d on SDS-polyacrylamide The major p o l y p e p t i d e s were i d e n t i f i e d by c o - e l e c t r o p h o r e s i s w i t h aeruginosa PA01  o u t e r membrane p r o t e i n s under d i f f e r e n t  gels. P.  solubilization  conditions. Hancock and Carey SDS-polyacrylamide  (1979) showed t h a t the m o b i l i t i e s  on  g e l e l e c t r o p h o r e s i s of p r o t e i n s DI, HI and G v a r i e d  depending on the temperature  at which the o u t e r membranes were  solubilized  p r i o r to e l e c t r o p h o r e s i s . The m o b i l i t y of p r o t e i n F v a r i e d w i t h or absence of 2-mercaptoethanol  during s o l u b i l i z a t i o n  was  present  s t a i n e d o n l y when MgCl^ was Using the above c r i t e r i a ,  cystic  fibrosis  i s o l a t e s of P.  and l i p o p r o t e i n  i n the s o l u b i l i z a t i o n  I  buffer.  a l l the 17  serotype s t r a i n s  ( F i g . 1)  aeruginosa  ( F i g . 2) had p o l y p e p t i d e bands  t h a t c o - e l e c t r o p h o r e s e d w i t h the major o u t e r membrane p r o t e i n s of a e r u g i n o s a PA01.  presence  and  P.  However, the r e l a t i v e l e v e l s of these p r o t e i n s v a r i e d  45  F i g u r e 1.  SDS-polyacrylamide  of P. aeruginosa s t r a i n H103  g e l electrophoretograms and  of o u t e r membranes  serotyping strains.  The  17 l a n e s on  l e f t - h a n d s i d e were run a f t e r s o l u b i l i z a t i o n at 88°C f o r 10 min s o l u b i l i z a t i o n - r e d u c t i o n mix 2-mercaptoethanol, (pH 6.8)].  10%  [20 mM  EDTA, 2% (wt/vol) SDS,  ( v o l / v o l ) g l y c e r o l , and 62.5  mM  5%  the  in  (vol/vol)  Tris-HCl  Under these c o n d i t i o n s l i p o p r o t e i n I i s not v i s i b l e w h i l e the  other major p r o t e i n s m i g r a t e Carey, 1979).  The  to t h e i r heat-modified p o s i t i o n s  t h r e e l a n e s on the r i g h t hand-side  s o l u b i l i z a t i o n at 88°C f o r 10 min  i n the absence o f  were run  (Hancock and after  2-mercaptoethanol g  (causing p r o t e i n F to move w i t h a h i g h e r m o b i l i t y t o p o s i t i o n F ) and i n the presence o f 0.1  M MgCl  2  p r o t e i n s G and HI to s h i f t poorly:  Hancock and Carey,  and H2 are a l s o i n d i c a t e d .  ( c a u s i n g p r o t e i n I t o appear i n the g e l and t o t h e i r unmodified p o s i t i o n s , where they 1979). The  The  p o s i t i o n s o f the p r o t e i n s D2,  stain E  numbers below each l a n e r e f e r to the  s e r o t y p e s , w i t h the e x c e p t i o n o f our w i l d type H103  strain, labelled  W.  46  47  somewhat between the d i f f e r e n t strains,  serotype 1, 5 and  l i p o p r o t e i n H2,  D2 was  i n a l l s t r a i n s except  differences  serotype  7 s t r a i n s had low but d e t e c t a b l e l e v e l s  7 s t r a i n s had  m i s s i n g or a l t e r e d  observed  For example of the  whereas the s e r o t y p e 6 and  HI and serotypes 5 and observed  strains.  7 s t r a i n s had  reduced  l i p o p r o t e i n I.  serotype 7, 14 and  i n s e r o t y p e 5, 12,  reduced  protein  Protein G  16 s t r a i n s and  15 and  of  17 s t r a i n s .  was  protein These  i n the o u t e r membrane p r o t e i n p a t t e r n s c o u l d not  be  c o r r e l a t e d to s e r o t y p e - r e l a t e d d i f f e r e n c e s , as o n l y a s i n g l e r e p r e s e n t a t i v e from each s e r o t y p e was  2.  Cystic fibrosis The  isolates:  fibrosis  s i m i l a r to s t r a i n PA01. shown.  characteristics  outer membranes of 36 P.  p a t i e n t s with c y s t i c  and o u t e r membrane p a t t e r n s  aeruginosa s t r a i n s obtained  showed o u t e r membrane p r o t e i n  from  profiles  In F i g . 2 the p r o f i l e s of some of the s t r a i n s  A number of minor a l t e r a t i o n s  p r o f i l e s were observed  studied.  i n the o u t e r membrane p r o t e i n  but the g e n e r a l p a t t e r n s were s t r o n g l y conserved.  The most s u b s t a n t i a l a l t e r a t i o n s were observed s t r a i n s C46 mucoid and C46  non-mucoid.  i n s t r a i n CF 4349 and  S t r a i n CF4349 ( F i g . 2, l a n e  c o n t a i n e d a number of e x t r a p o l y p e p t i d e s as w e l l as an apparent i n p r o t e i n I.  are  S t r a i n s C46  mucoid and  had no d e t e c t a b l e p r o t e i n H2  deficiency  i t s spontaneous non-mucoid r e v e r t a n t  i n t h e i r o u t e r membranes.  Another  CF1278 a p p a r e n t l y l a c k e d l i p o p r o t e i n I ( F i g . 2, l a n e 11). F were, however, e x p r e s s e d  5)  i n a l l these P.  aeruginosa  strain  Proteins E  isolates.  and  48  F i g u r e 2.  SDS-polyacrylamide g e l e l e c t r o p h o r e t o g r a m s of o u t e r membrane  p r o t e i n s o f P. aeruginosa i s o l a t e s  from p a t i e n t s w i t h c y s t i c  fibrosis.  o Outer membranes were s o l u b i l i s e d of  2-mercaptoethanol. The e f f e c t  d e s c r i b e d i n F i g . 1.  i n r e d u c t i o n mix a t 88 C i n the absence of t h i s  solubilization  schedule i s  Lane 1, s t r a i n H103; Lane 2, s t r a i n CF3660-1;  Lane  3, CF9490; Lane 4, CF221, Lanes 5 CF 4349; Lane 6, CFC 6m; Lane 7, CF4522; Lane 8, CF832; Lane 9, CF3790; Lane 10, m o l e c u l a r weight standards  [Bovine  serum albumin (66.2 K), Ovalbumin (45 K ) , C a r b o n i c anhydrase (31 K ) , Soybean t r y p s i n K)];  inhibitor  (21.5 K) M y o g l o b u l i n  (16.7 K) and Lysozyme (14.4  Lane 11, CF1278; Lane 12, CF L; Lane 13, CF1452; Lane 14, CF6094;  Lane 15, CF2314; Lane 16, CFPlm; Lane 17, CFPlnm; Lane 18, CF C46m; Lane 19, CF C46nm.  49  i o  ~n mp  50  In c o n t r a s t to the g r e a t s i m i l a r i t y p r o f i l e s , these s t r a i n s d i f f e r e d of the f i f t e e n c y s t i c  fibrosis  i n the o u t e r membrane p r o t e i n  i n other c h a r a c t e r i s t i c s .  For example,  i s o l a t e s typed by the F i s h e r immunotyping  scheme (data of D.P. Speert and G.B. P i e r ; summarized i n T a b l e 1) o n l y t h r e e s t r a i n s were a g g l u t i n a t e d by a s i n g l e t y p i n g serum, whereas 8 s t r a i n s were a g g l u t i n a t e d with more than one serum and 4 were not a g g l u t i n a t e d by any serum.  N e i t h e r the p o l y a g g l u t i n a b l e (nor the  n o n - a g g l u t i n a b l e s t r a i n s ) were t y p a b l e by h e m a g g l u t i n a t i o n immunodiffusion  i n h i b i t i o n or  (Hancock e t a l . , 1981) , s u g g e s t i n g t h a t these  p o l y a g g l u t i n a b l e s t r a i n s d i d not express m u l t i p l e s e r o t y p e a n t i g e n s , but were i n s t e a d being a g g l u t i n a t e d by antibody t o non-serotype Four t y p a b l e i s o l a t e s H103) were r e s i s t a n t  ( i n c l u d i n g our l a b o r a t o r y w i l d type  to normal human serum (Table I ) . on LPS 0-antigen.  s t u d i e d were v e r y  sensitive  S e r o t y p i n g o f gram-negative b a c t e r i a i s  The LPS from these s t r a i n s was t h e r e f o r e  c h a r a c t e r i z e d by s t a i n i n g on SDS-polyacrylamide form  strain,  to pooled normal human serum, w h i l e the twelve  p o l y a g g l u t i n a b l e and n o n a g g l u t i n a b l e i s o l a t e s  based  determinants.  g e l s both  i n the p u r i f i e d  ( F i g . 2 of Hancock e t a l . , 1983) and i n the o u t e r membranes ( t o a v o i d  possible f r a c t i o n a t i o n during p u r i f i c a t i o n ) . of LPS i n the o u t e r membranes o f 16 c y s t i c a e r u g i n o s a and s t r a i n  F i g u r e 3 shows the s t a i n i n g  fibrosis  i s o l a t e s of P.  H103.  By the m o d i f i e d p e r i o d a t e s i l v e r  s t a i n i n g t e c h n i q u e , LPS s t a i n e d  orange w h i l e p r o t e i n s were brown or grey and were thus distinguishable.  clearly  As shown i n F i g . 3 ( l a n e s 5,6,13) o n l y s t r a i n s CF4349  and CF6094 (when overloaded)  c o n t a i n e d l e v e l s of smooth LPS s i m i l a r t o  Table I . Colony morphology, LPS phenotype, s e r o t y p a b i l i t y , and s e n s i t i v i t y to normal human serum of s e l e c t e d P. aeruginosa i s o l a t e s from p a t i e n t s with c y s t i c f i b r o s i s and rough L P S - a l t e r e d mutants Response to serum Strain  H103 CF2314 CF4349 CF6094 CF832 CF3790 CF4522 CF9490 CFPIM CFPINM CF96M CF96NM CF221 CF284 CF1278 CF1452 AK1160 AK1188 AK1012 AK1121 H223  Colony Morphology  NM NM NM PM NM  3  0 s i d e chains i n LPS'  ++ + ++ ++ +  +  NM NM M NM M NM NM NM M M NM NM NM NM NM  Typability  3  + +  ± + +  - • +  +  T T T T PA PA PA PA PA PA PA PA NT NT NT NT  0  % survival after exposure to 50% serum f o r 1 h r at 37°C >100% 89% >100% 86% 0% 6% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%  —  • —  % serum a l l o w i n g growth a f t e r 18 hr  >40% >40% >40% >40% 1.25% 1.25% 2.5% 1.25% 1.25% 1.25% 1.25% 1.25% 1.25% 1.25% 1.25% 1.25% 1.25% 1.25% 1.25% 1.25% 1.25%  a. NM - non -mucoid, smopth colony appearance, M - mucoid, PM - p a r t i a l l y mucoid. b. R e s u l t s are expressed as the minimum l e v e l of LPS which had to be loaded onto SDS p o l y a c r y l a m i d e g e l s i n order to see smooth type LPS ( c o n t a i n i n g 0 - s i d e c h a i n s ) upon s t a i n i n g by the method of T s a i and Frasch (1982) (data from Hancock e t . a l . , 1983). +++ 100 ng LPS; ++ 100-500 ng LPS; + 1-10 ug LPS; ± 10 Pg LPS; - no smooth LPS observed with 30 ug of LPS. c. (Data from Hancock e_t a_l. , 1983) T-typable by a g g l u t i n a t i o n ; PA - p o l y a g g l u t i n a b l e with more than one serum, non-typable by other means; NT - nontypable.  52  F i g u r e 3.  SDS-polyacrylamide  g e l o f o u t e r membranes o f P. a e r u g i n o s a  i s o l a t e s from p a t i e n t s w i t h c y s t i c  fibrosis.  The e l e c t r o p h o r e t o g r a m s  s t a i n e d f o r LPS by a m o d i f i c a t i o n o f the p e r i o d a t e - s i l v e r procedure  o f T s a i and F r a s c h (1982).  staining  The o u t e r membranes were  i n r e d u c t i o n mix c o n t a i n i n g 20 mM EDTA.  were  solubilized  Lane 1, s t r a i n H103  (underloaded); Lane 2, CF 3660-1; Lane 3, CF 9490; Lane 4, CF 221; Lanes 5 and 6, CF 4349; Lane 7, CF 4522; Lane 8, CF832; Lane 9, CF 3790; Lane 10, CF 1278; Lane 11, CF L; Lane 12, CF 1452; Lane 13, CF 6094; Lane 14, CF 2314; Lane 15, CF 284; Lane 16, Pirn; Lane 17, Plnm.  49  12  3 4 5 6 7 8 9  10 11 1213 14 15 16 17  54  s t r a i n H103  (on o v e r l o a d i n g ) .  O-side c h a i n s was  visible,  S t r a i n s CF284 had d e t e c t a b l e l e v e l s  in h e a v i l y overloaded  g e l s of p u r i f i e d LPS.  Rough type  i n a l l s t r a i n s t e s t e d , as a f a s t m i g r a t i n g band.  the O-side c h a i n - d e f i c i e n t  of LPS  However, i n  s t r a i n s , r e l a t i v e l y h i g h e r amounts of t h i s  core  LPS were p r e s e n t when compared to the O-side c h a i n - c o n t a i n i n g s t r a i n s . shown i n Table 1, these s t r a i n s a l s o d i f f e r e d  3.  I s o l a t i o n and  s t r a i n H103  antiserum was  aeruginosa  a g a i n s t the o u t e r membranes of P.  tested for a c t i v i t y  expressed  The  ELISA t i t r e  of the  as the d i l u t i o n of the antiserum which gave an  r e a d i n g at 405 The  aeruginosa  titre  nm  of 0.3  PA01  a g a i n s t the o u t e r membranes of  s e r o t y p i n g s t r a i n s by the ELISA method. was  i n c o l o n y morphology.  c h a r a c t e r i z a t i o n of a p o l y c l o n a l a n t i s e r a a g a i n s t the  o u t e r membranes of P. Rabbit  As  the  antiserum  absorbance  a f t e r 45 minutes of c o l o u r development at 37°C.  of the antiserum  a g a i n s t the homologous s t r a i n H103  outer  -8 membrane (10 all  ) was  one  t e s t e d heterologous  to t h r e e o r d e r s of magnitude h i g h e r than o u t e r membranes (Table I I ) .  against  This polyclonal  -4 a n t i s e r a had  a titre  ( T a b l e I I ) and  of o n l y 10  LPS  t h e r e f o r e most of the antibody must have been d i r e c t e d  a g a i n s t non-LPS a n t i g e n s because the s e r o t y p e 0-antigens,  a g a i n s t homologous s t r a i n H103  i n the o u t e r membrane.  s t r a i n s are h e t e r o l o g o u s  i t would seem t h a t at l e a s t  cross-reactivity  For t h i s reason,  w i t h r e s p e c t to t h e i r  LPS  some of the a n t i g e n i c  and d i f f e r e n c e s seen i n the ELISA t i t r e  s t r a i n s must be due  and  among these  to 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 p r o t e i n a n t i g e n s .  immunogenicity of o u t e r membrane p o r i n p r o t e i n s F, H2,  I , HI and D2  The was  Table I I . C r o s s - r e a c t i o n s o f a n t i s e r a to o u t e r membrane and p u r i f i e d outer membrane components with o u t e r membranes from v a r i o u s serotype s t r a i n s of P. a e r u g i n o s a .  Logi n ELISA Outer membrane antigen  PAOi serotype  CFPlm CFPlnm PAOI LPS  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17  t i t r e s using antisera to:  PAOI outer membranes  Protein D2  Protein F  8 6 6 5 7 7 5 6 6 6 6 6 6 6 6 6 7 6 5 5 4  3 3 3 2 2 2 2 3 3 3 3 3 3 3 3 2 3 3 2 2 2  3 2 2 1 1 3 4 2 1 3 3 2 3 1 2 1 3 3 2 2 1  Protein HI  3 3 3 2 3 2 4 3 3 4 4 4 4 3 4 3 2 4 4 4 1  Protein H2(+I)  4 4 3 1 3 3 4 4 3 4 4 4 4 3 4 3 3 4 3 2 < 1  Protein I .  3 2 2 2 2 2 2 2 2 3 3 2 3 2 2 2 2 3 2 2 1  NOTE: P. a e r u g i n o s a PAOI ( s t r a i n H103) was used as a s t a n d a r d s t r a i n f o r the i s o l a t i o n o f major o u t e r membrane a n t i g e n s .  56  demonstrated by the immunological c r o s s - r e a c t i v i t y o f p o l y c l o n a l a n t i s e r a against  these p r o t e i n s and o u t e r membranes from a l l o f the serotype  strains  (Table I I ) .  contained  The p o l y c l o n a l a n t i s e r a a g a i n s t these  some antibody  proteins  (ELISA t i t r e < 10) a g a i n s t homologous LPS,  i n d i c a t i n g t h a t LPS was a contaminant of the p u r i f i e d p r o t e i n but these anti-LPS  antibodies  c o u l d not account f o r the observed  extensive  immunological c r o s s - r e a c t i v i t y o f the a n t i s e r u m . P o l y c l o n a l a n t i s e r a a g a i n s t both o u t e r membranes and p r o t e i n F cross-reacted  i n ELISA assays w i t h  P. aeruginosa  s t r a i n obtained  spontaneous non-mucoid  4.  o u t e r membranes from CFPlm, a mucoid  from a c y s t i c  revertant.  I n t e r a c t i o n of p o l y c l o n a l sera with aeruginosa  f i b r o s i s p a t i e n t , and i t s  the o u t e r membranes o f P.  strains  To o b t a i n more s p e c i f i c  information  on the a n t i g e n i c r e l a t i o n s h i p o f  the major o u t e r membrane p r o t e i n s o f the serotype were t r a n s f e r r e d from SDS p o l y a c r y l a m i d e  s t r a i n s , the p r o t e i n s  g e l s onto n i t r o c e l l u l o s e  paper  125 and  reacted with  sera.  I - l a b e l l e d r a b b i t a n t i - o u t e r membrane p o l y c l o n a l  T h i s antiserum had been a f f i n i t y p u r i f i e d  (see methods).  t r a n s f e r o f p r o t e i n onto n i t r o c e l l u l o s e was very e f f i c i e n t HI,  H2 and I as judged by both the coommassie b r i l l i a n t  the SDS g e l s a f t e r e l e c t r o b l o t t i n g and amido b l a c k  The  f o r p r o t e i n s G,  blue  s t a i n i n g of  staining of b l o t s .  The  e f f i c i e n c y o f t r a n s f e r o f p r o t e i n s F and D2 was a p p r o x i m a t e l y 807. ( F i g . 4, right  panel).  57  Figure 4 .  L e f t , Autoradiogram o f an e l e c t r o p h o r e t i c b l o t o f o u t e r  membrane p r o t e i n s of P. a e r u g i n o s a  a f t e r treatment  a n t i b o d i e s to outer membrane p r o t e i n s . to the s e r o t y p e s ; W i s s t r a i n H 1 0 3 .  with r a d i o i o d i n a t e d  The numbers below the l a n e s  The i n d i c a t e d p o l y p e p t i d e s  refer  proteins  E, F, H 2 and I) were i d e n t i f i e d by amido b l a c k s t a i n i n g o f the electrophoretic blots.  The band l a b e l l e d F l a ( f l a g e l l i n ) was  by c o - e l e c t r o p h o r e s i s w i t h p u r i f i e d  flagellin.  R i g h t , SDS-polyacrylamide g e l e l e c t r o p h o r e t o g r a m electrophoretic  identified  after  t r a n s f e r o f o u t e r membrane p r o t e i n s t o n i t r o c e l l u l o s e .  Note o n l y p a r t i a l  (807o)  t r a n s f e r o f p r o t e i n F was  achieved.  59  The  autoradiograms of the Western immunoblots and the densitometer  scans of the autoradiograms r e v e a l e d with  the r a d i o l a b e l l e d a n t i b o d i e s  4 and 5) w h i l e strain  t h a t p r o t e i n s F, H2 and I , i n t e r a c t e d  i n a l l the t e s t e d o u t e r membranes ( F i g s .  p r o t e i n E was l a b e l l e d  (Fig. 5). This  i n a l l s t r a i n s except the serotype  4  s t r a i n appeared t o have a p r o t e i n E e q u i v a l e n t oh  SDS-polyacrylamide g e l s but i t i s p o s s i b l e t h a t p r o t e i n E was a n t i g e n i c a l l y a l t e r e d i n that s t r a i n . G were not l a b e l l e d  Outer membrane p r o t e i n s D2, HI and  i n any o u t e r membrane, and they would appear to be non  or weakly immunogenic by the d e s c r i b e d An  estimate  o f the amount o f a n t i b o d y  membrane p r o t e i n s was o b t a i n e d  schedule.  bound t o the major  from the d e n s i t o m e t e r scanning  autoradiogram ( F i g . 5 ) . As d e s c r i b e d labelled  immunizing  outer of the  above, p r o t e i n s F, H2 and I were  i n a l l s t r a i n s and t h e r e f o r e were a n t i g e n i c a l l y r e l a t e d i n a l l  s t r a i n s a t the p o l y c l o n a l l e v e l . show i d e n t i c a l a n t i b o d y  binding  However, the s e r o t y p e  t o these o u t e r membrane p r o t e i n as  demonstrated by d i f f e r e n c e s i n the peak h e i g h t s ( c o r r e l a t e d t o the d e n s i t y of the l a b e l ) . to the d i f f e r i n g  affinities  of the t e s t  membrane p r o t e i n s , o r by the d i f f e r i n g the v a r i o u s  o u t e r membranes.  s t r a i n s d i d not  o f the densitometer scans  This observation  antibodies  c o u l d be due  f o r the given  amounts o f t h e g i v e n p r o t e i n s i n  Thus s m a l l amounts o f a g i v e n  p r o t e i n i n an  o u t e r membrane would c o r r e l a t e t o the s m a l l amounts o f antibody the p r o t e i n i n t h a t  outer  bound to  strain.  P r o t e i n F was not q u a n t i t a t i v e l y t r a n s f e r r e d onto the Western b l o t s and  t h e r e f o r e q u a n t i t a t i v e statements on the a n t i g e n i c i t y o f t h i s  c o u l d not be made.  The s t r o n g l y l a b e l l e d p r o t e i n o f m o l e c u l a r  protein  weight  60  F i g u r e 5A and B.  Densitometer  t r a c i n g s o f autoradiograms  of separated  o u t e r membrane p r o t e i n s from Pseudomonas a e r u g i n o s a s t r a i n H103 s e r o t y p e 1-17 s t r a i n s , and i s o l a t e PI from a p a t i e n t w i t h c y s t i c  (W), fibrosis,  a f t e r treatment w i t h r a d i o i o d i n a t e d a n t i b o d i e s t o o u t e r membrane p r o t e i n . Exposed X-rays densitometer  f i l m s s i m i l a r t o those shown i n f i g u r e 4 were a n a l y z e d by  scanning.  I and f l a g e l l i n  The i n d i c a t e d p o l y p e p t i d e s ( p r o t e i n s E, F, H2, and  [ F l a ] ) were i d e n t i f i e d as d e s c r i b e d i n the F i g . 4 l e g e n d .  62  53,000 d a l t o n s i n the outer membranes of H103 flagellin gift  by c o e l e c t r o p h o r e s i s with p u r i f i e d  ( F i g . A ) was flagellin  identified  (flagellin  from R. Ansog, Hygiene I n s t i t u t e , U n i v e r s i t a t K r e u z b e r g i n ,  F e d e r a l R e p u b l i c of Germany). f l a g e l l i n must r e f l e c t  The  was  The  l a r g e amounts of antibody bound to  the h i g h immunogenicity  of t h i s p r o t e i n , s i n c e i t H103  outer membranes of s t r a i n s r e p r e s e n t i n g s e r o t y p e s 3  10, 11 and 12 showed l a r g e peaks of a n t i b o d y b i n d i n g to f l a g e l l i n s m a l l e r peaks were demonstrated by s e r o t y p e s 4, strains  ( F i g . 5).  5, 7, 16,  17 and  whereas PI  These r e s u l t s c o r r e l a t e w i t h the known a n t i g e n i c  v a r i a t i o n of f l a g e l l i n  5.  a  Gottingen  was,a v e r y minor component of the Coommassie b l u e - s t a i n e d s t r a i n outer membranes.  as  p r o t e i n among P.  aeruginosa s t r a i n s  (Pitt,  1980).  Summary In  P.  a e r u g i n o s a the c o n s e r v a t i o n of o u t e r membrane p o l y p e p t i d e s  among the 17  s e r o t y p e s t r a i n s and c y s t i c  fibrosis  isolates  was  demonstrated by the s t r o n g s i m i l a r i t y of o u t e r membrane p r o t e i n on SDS-polyacrylamide  gels.  The major o u t e r membrane p r o t e i n s D2,  H2 and I were g e n e r a l l y conserved, between  profiles  although t h e i r r e l a t i v e l e v e l s  E, F, varied  strains.  A n t i g e n i c c r o s s - r e a c t i v i t y was among a l l t e s t e d P.  demonstrated f o r p r o t e i n s F, H2  and  a e r u g i n o s a s t r a i n s at the p o l y c l o n a l l e v e l u s i n g an  antiserum to s t r a i n H103  outer membranes.  P r o t e i n E was  immunologically  c r o s s - r e a c t i v e i n 16 of the 17 s e r o t y p e s t r a i n s , but a n t i b o d i e s to p r o t e i n s D2, protocol.  HI and G were not d e t e c t e d u s i n g the d e s c r i b e d  immunization  I  64  A n t i g e n i c h e t e r o g e n e i t y a t the p o l y c l o n a l l e v e l was demonstrated f o r flagellin. Both s t r u c t u r a l and immunological h e t e r o g e n e i t y were demonstrated f o r the  LPS.  P. a e r u g i n o s a s t r a i n s  of the rough LPS phenotype  from c y s t i c f i b r o s i s p a t i e n t s were l a r g e l y  and were serum s e n s i t i v e .  65  Chapter  1.  II.  MONOCLONAL ANTIBODIES TO OUTER MEMBRANE ANTIGENS  I s o l a t i o n and c h a r a c t e r i z a t i o n of monoclonal a n t i b o d i e s A f t e r f u s i o n of NS1  from mice primed  or SP2/0-Agl4 myeloma c e l l s w i t h s p l e e n  e i t h e r w i t h P. a e r u g i n o s a PA01  cells  whole o u t e r membranes or  w i t h p a r t i a l l y p u r i f i e d o u t e r membrane p r o t e i n s , the o b t a i n e d hybridomas were c l o n e d out u n t i l a s i n g l e c e l l c l o n e per w e l l of a t i s s u e p l a t e was  evident.  By t h i s procedure  a number o f c l o n e s t h a t  culture produced  a n t i b o d i e s s p e c i f i c f o r o u t e r membrane a n t i g e n s were i s o l a t e d from s e v e r a l separate f u s i o n s .  These a n t i b o d i e s were judged  a n t i b o d i e s on the b a s i s of t h e i r s p e c i f i c i t y  to produce m o n o s p e c i f i c  towards a v a r i e t y of  a n t i g e n i c f r a c t i o n s from o u t e r membranes ( T a b l e I I I ) and of s i n g l e p r e c i p i t i n l i n e s  i n Ouchterlony  and r o c k e t Immunoelectrophoresis outer membranes s o l u b i l i z e d 8.0  and 10 mM  double  procedures  i n 1%  a n t i b o d i e s MA1-8, MA1-3 (Hancock e t a l . , 1982)], P a ) ] , were used  (A)  An LPS  in this  0-antigen  Monoclonal  and  5E4  [originally and 8A1  20 mM  PA01  Tris-HCl  In a d d i t i o n to the f o r rough LPS,  i s o l a t e d by A.A.  [from Centocor  Company  monoclonal Wieczoreck (Malvern,  study.  s p e c i f i c monoclonal  antibody MA1-8  pH  These a n t i b o d i e s were f u r t h e r  f o r p r o t e i n F and  and MA1-6  assays  u s i n g as a n t i g e n , s t r a i n  characterized for their antigenic s p e c i f i c i t i e s . monoclonal a n t i b o d i e s s p e c i f i c  t h e i r production  immunodiffusion  ( v o l / v o l ) T r i t o n X100,  EDTA ( R e s u l t s not shown).  crude  antibody  ( I g G l s u b c l a s s ) r e a c t e d s t r o n g l y i n ELISA  assays with p u r i f i e d o u t e r membranes from s t r a i n H103  and from  an  66  antibiotic  s u p e r s u s c e p t i b l e , L P S - a l t e r e d mutant Z61 o r i g i n a l l y d e r i v e d  from a c l i n i c a l  i s o l a t e o f P. a e r u g i n o s a  reacted strongly with p u r i f i e d partially purified  (Table IV).  also  s t r a i n H103 LPS and w i t h a v a r i e t y o f  o u t e r membrane p r o t e i n s from s t r a i n  shown by chemical means to c o n t a i n LPS ( T a b l e I I I ) . not i n t e r a c t  The antibody  H103 which were  MA1-8, however, d i d  with the o u t e r membranes from t h r e e i n d e p e n d e n t l y  isolated  LPS-0 a n t i g e n - d e f i c i e n t (rough) mutants d e r i v e d from s t r a i n H103 (Table IV).  The antibody p a s s i v e l y hemagglutinated  w i t h s t r a i n H103 LPS ( h e m a g g l u t i n a t i o n t i t r e  sheep r e d b l o o d c e l l s  coated  1400) but d i d not a g g l u t i n a t e  c e l l s coated w i t h the O - a n t i g e n - d e f i c i e n t (rough) LPS ( h e m a g g l u t i n a t i o n t i t r e < 2). O-antigen  (B)  These d a t a were c o n s i s t e n t w i t h MA1-8 b e i n g s p e c i f i c f o r the  o f s t r a i n H103 LPS ( F i g . 6, l a n e 1 ) .  LPS rough s p e c i f i c monoclonal a n t i b o d i e s Monoclonal  a n t i b o d i e s MA3-5 and MA3-6 r e a c t e d s t r o n g l y i n ELISA  assays w i t h outer membranes from P. a e r u g i n o s a s t r a i n s  H103, Z61 and  AK1012 (an O - a n t i g e n - d e f i c i e n t mutant o f PAOI) as w e l l as a l l p a r t i a l l y purified  outer membrane p r o t e i n s from s t r a i n H103 ( T a b l e s I I I , I V ) .  U n l i k e MA1-8, monoclonal a n t i b o d i e s MA3-5 and MA3-6 d i d not a g g l u t i n a t e any o f the above P. a e r u g i n o s a s t r a i n s .  In Western immunoblot  MA3-5 and MA3-6 i n t e r a c t e d w i t h a f a s t m i g r a t i n g band o f s i m i l a r weight from t h e LPS o f P. a e r u g i n o s a s t r a i n s lane 2 ) .  I t was t h e r e f o r e concluded  molecular  H103 and AK1012 ( F i g u r e 6,  t h a t these monoclonal a n t i b o d i e s were  s p e c i f i c f o r the rough core o f LPS. Monoclonal were o f lgM s u b c l a s s .  procedures,  a n t i b o d i e s MA3-5 and MA3-6  67  (C)  L i p i d A s p e c i f i c monoclonal a n t i b o d i e s Two  monoclonal a n t i b o d i e s 5E4  Corporation  ( P h i l a d e l p h i a , Pa)  c e l l s of E. c o l i  s t r a i n J5  and  after  8A1  produced  immunization  by  of mice w i t h heat  e_t aJL. , manuscript  (Bogard  et a l . , 1984), were t e s t e d f o r a c t i v i t y  Centocor  submitted;  killed  Mutharia  a g a i n s t P. a e r u g i n o s a s t r a i n s .  In  ELISA assays both of the a n t i b o d i e s i n t e r a c t e d with the o u t e r membranes, LPS  and L i p i d A from P. a e r u g i n o s a PA01  several d i f f e r e n t i a l observed.  (Table VI; see below).  However,  i n t e r a c t i o n s of the a n t i b o d i e s and a n t i g e n s were  For example antibody 8A1  r e a c t e d s t r o n g l y i n ELISA assays  with  s e v e r a l a n t i g e n s but very p o o r l y w i t h the same antigens i n Western b l o t assays.  These r e s u l t s may  reflect  Western immunoblot assays, 5E4 the o u t e r membrane or LPS  the mode of a n t i g e n p r e s e n t a t i o n .  In  i n t e r a c t e d with a f a s t m i g r a t i n g band i n  of s t r a i n H103  a n t i b o d i e s were of the IgGI s u b c l a s s by  ( F i g u r e 6, l a n e 5 ) .  These  i n t e r a c t i o n with subclass  specific  antisera.  (D)  A l i p o p r o t e i n H2  s p e c i f i c monoclonal  A monoclonal antibody MA1-6  antibody  r e a c t e d s t r o n g l y w i t h s t r a i n H103  membranes, and w i t h 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 s H2 contaminated  w i t h H2)  immunodiffusion  i n ELISA assays  I (which  In O u c h t e r l o n y  t e s t s , t h i s monoclonal antibody gave a s i n g l e  l i n e w i t h both l i p o p r o t e i n H2 precipitin  (Table I I I ) .  and  and  I preparations.  outer was double  precipitin  In a d d i t i o n , a s i n g l e  l i n e o c c u r r e d w i t h l i p o p r o t e i n H2 but not l i p o p r o t e i n I t h a t  had been cut out and T r i t o n X-100, 20 mM  e l u t e d from SDS-polyacrylamide Tris-HCl  (pH 8.0)  and 10 mM  g e l s w i t h 2%  EDTA.  The  (vol/vol)  antibody  was,  68  therefore s p e c i f i c  f o r l i p o p r o t e i n H2  as demonstrated by Western  blotting  ( F i g . 6, Lane 3 ) .  (E)  P r o t e i n F s p e c i f i c monoclonal a n t i b o d i e s F i v e monoclonal a n t i b o d i e s MA2-10, MA4-2, MA4-4, MA4-10 and MA5-8  were i s o l a t e d from d i f f e r e n t with spleen c e l l s s t r a i n H103  f u s i o n s of NS-1  or Sp2/0-Agl.4 myeloma c e l l s  from mice immunized w i t h e i t h e r p u r i f i e d p r o t e i n F or  o u t e r membranes.  These a n t i b o d i e s r e a c t e d i n ELISA  w i t h o u t e r membranes of s t r a i n H103  but not w i t h s t r a i n H103  assays  LPS.  When  t e s t e d w i t h a v a r i e t y of p a r t i a l l y p u r i f i e d o u t e r membrane p r o t e i n s , they reacted s p e c i f i c a l l y with p r o t e i n F (Table I I I ) . a n t i b o d i e s showed no  Furthermore,  i n t e r a c t i o n w i t h o u t e r membranes from P.  these aeruginosa  s t r a i n H283, a p r o t e i n F - d e f i c i e n t mutant d e r i v e d from s t r a i n H103 and Hancock, 1983).  These a n t i b o d i e s were s p e c i f i c  p o r i n p r o t e i n F ( F i g . 6, lane 4 ) .  Two  f o r P.  aeruginosa  H103  of these monoclonal a n t i b o d i e s  MA2-10 and MA4-10 were shown to be of the IgGI s u b c l a s s , by with s u b c l a s s s p e c i f i c  (Nicas  interaction  a n t i s e r a , w h i l e a n t i b o d i e s MA4-2, MA4-4 and MA5-8  were of the IgG3, IgG2a and IgG2b r e s p e c t i v e l y .  (F)  Monoclonal  antibody  MA1-3  A monoclonal antibody MA1-3 c e l l s and  the s p l e e n c e l l s  i s o l a t e d from the f u s i o n of NS-1  from a mouse immunized w i t h s t r a i n H103  membrane showed v e r y i n t e r e s t i n g r e s u l t s . f a i l e d to i n t e r a c t w i t h LPS D2,  F and HI but  myeloma  The monoclonal antibody  outer MA1-3  or p a r t i a l l y p u r i f i e d o u t e r membrane p r o t e i n s  i n t e r a c t e d w e l l with p r o t e i n I (Table I I I ) .  In an  69  Table I I I . I n t e r a c t i o n of monoclonal a n t i b o d i e s w i t h p a r t i a l l y major o u t e r membrane p r o t e i n s of P. a e r u g i n o s a PA01.  Log Antigen  H103 o u t e r membranes Protein D2 Protein F Protein H l P r o t e i n H2 Protein I LPS a  a  a  b  b  ELISA t i t r e s with monoclonal  MA1-8  MA1-6  5 3 2 2 < 1 < 1 5  3 < 1 1 < 1 4 4 0  MA1-3  < < ^ < <  purified  antiserum MA3-5  3 1 1 1 1 3 0  a. P a r t i a l l y p u r i f i e d p r o t e i n s D2, F, and HI were shown t o c o n t a i n s i g n i f i c a n t amounts of LPS [about 10% (wt/wt)]. b. P r o t e i n H2 and I were c r o s s - c o n t a m i n a t e d w i t h each o t h e r .  3 ND 2 2 2 ND 4  70  Table IV. I n t e r a c t i o n o f monoclonal a n t i b o d i e s w i t h the Outer membranes o f LPS - a l t e r e d s t r a i n s o f P. a e r u g i n o s a .  Outer Membrane A n t i g e n from s t r a i n  ' PAOI Z61 AK1160 AK1188 H223  ND - not done  L o g ^ ELISA t i t r e MA1-6 MA1-3 0  LPS d e f e c t  MA1-8  w i l d type L i p i d A (semi rough) rough rough rough  5 6 < 1 < 1 < 1  3 3 3 3 3  3 3 3 3 3  MA3-5  3 3 3 ND 3  71  F i g u r e 6. P.  Western e l e c t r o p h o r e t i c b l o t s o f o u t e r membranes o f  aeruginosa  s t r a i n H103 a f t e r treatment w i t h monoclonal a n t i b o d i e s MA1-8  ( l a n e 1 ) , MA3-5 (lane 2 ) , MA1-6 ( l a n e 3 ) , MA4-4 ( l a n e 4 ) , and 5E4 ( l a n e 5).  The immunostaining procedure was as d e s c r i b e d  Western b l o t s were from 5 separate the d e n s i t y o f the l a b e l .  i n methods.  (These  experiments, hence t h e d i f f e r e n c e s i n  The high backgrounds o b t a i n e d  with  t h e Western  b l o t u s i n g 5E4 c o n t r i b u t e d t o the f a i n t n e s s o f t h e l a b e l l e d band i n l a n e 5) .  72  1  2  3  4  5  73  Ouchterlony  double  d i f f u s i o n assay, MA1-3 gave a strong p r e c i p i t i n  when p r o t e i n I was used  as an a n t i g e n .  However, MA1-3  with p r o t e i n I e l u t e d from SDS-polyacrylamide of any P. a e r u g i n o s a  neither interacted  g e l s nor a g g l u t i n a t e d c e l l s  strains.  MA1-3 i n t e r a c t e d w i t h the o u t e r membranes from the s e r o t y p e of P. a e r u g i n o s a  (see below) i n ELISA a s s a y s .  i n t e r a c t w i t h Western b l o t s o f P. a e r u g i n o s a  2.  Use o f Monoclonal  line  strains  T h i s antibody d i d not s t r a i n H103 o u t e r membranes.  a n t i b o d i e s i n t h e study of a n t i g e n i c h e t e r o g e n e i t y  i n P. a e r u g i n o s a Monoclonal  a n t i b o d i e s p r o v i d e s e v e r a l advantages over c o n v e n t i o n a l  p o l y c l o n a l s e r a d e r i v e d from fractions.  immunization  o f animals with a n t i g e n i c  F o r example, they have d e f i n e d s p e c i f i c i t y f o r a s i n g l e  e p i t o p e ( i . e . a n t i g e n i c s i t e ) on one s p e c i e s o f macromolecule.  This  allows one t o a c c u r a t e l y q u a n t i f y and i d e n t i f y g i v e n macromolecules under a l l circumstances,  r e g a r d l e s s o f the presence  T h e r e f o r e , monoclonal a n t i b o d i e s were used  o f contaminating  antigens.  i n a study o f the a n t i g e n i c  r e l a t i o n s h i p s o f t h e major o u t e r membrane a n t i g e n s of P. a e r u g i n o s a .  (A)  S p e c i f i c i t y o f p r o t e i n F monoclonal a n t i b o d i e s The monoclonal a n t i b o d i e s s p e c i f i c  f o r p r o t e i n F were t e s t e d by ELISA  f o r b i n d i n g t o t h e o u t e r membranes o f t h e 17 serotype s t r a i n s o f P. a e r u g i n o s a and t o 16 c y s t i c f i b r o s i s p a t i e n t i s o l a t e s . MA4-4 and MA2-10 a r e p r e s e n t e d  i n T a b l e V.  Results  from  These monoclonal a n t i b o d i e s ,  as w e l l as MA4-2, MA4-10 and MA5-8, r e a c t e d w i t h a l l of the o u t e r  74  Table V:  I n t e r a c t i o n o f monoclonal a n t i b o d i e s MA 2-10 and MA 4-4 w i t h outer membranes o f P. a e r u g i n o s a serotype s t r a i n s and c y s t i c f i b r o s i s i s o l a t e s .  Source o f Outer Membrane Antigen  ELISA t i t r e o f monoclonal MA 4-4  PA01 (H103) Purified protein (from H103)  10 105  Serotype  2  F  (0.66) (1.16)  c  a , b  antibody MA 2-10  10 10  4  (1.42) (1.54)  5  strains  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 C F . isolates PI (mucoid) PI (non-mucoid) CF 3660-1 CF 9490 CF 221 CF 4349 CF 4522 CF 832 CF 3790 CF 1278 L CF 1452 CF 6094 CF 2314 CF 284  10 10 10 10 10 10 10 10 10 10 IO 10 10 10 10 10 10  10 10 10 IO 10 < 10 IO 10 10 10 10 10 10 10 10  4  3  3  4  4  5  4  4  4  4  4  4  4  4  4  4  4  3  4  4  5  5  2  5  3  4  2  5  5  2  4  4  4  (0.58) (0.24) (0.27) (0.40) (2.0) (1.54) (2.0) (0.89) (1.3) (1.4) (1.67) (0.72) (1.11) (0.13) (0.37) (0.71) (1.17)  10 10 10 10 10 10 10 10 10 10 10 10 10 10 IO IO 10  (0.2) (0.6) (0.24) (0.5) (0.58) (0.15) (0.69) (0.73) (0.48) (0.22) (0.7) (0.43) (0.15) (0.46) (0.53)  IO IO IO IO IO 10 10 IO IO IO IO 10 lp 10 IO  (0.62) (0.25) (0.24) (0.32) (2.0) (2.0) (1.79) (0.92) (1.0) (1.29) (2.0) (0.72) (1.2) (0.20) (0.42) (0.69) (1.09)  4  4  5  4  4  4  5  5  4  4  4  4  4  4  4  4  3  3  3  3  3  c  <  2  3  3  3  c  2  3  3  3  4  3  (0.54) (1.11) (0.29) (0.61) (0.62) (0.13) (0.72) (0.78) (0.55) (0.34) (0.71) (0.37) (0.18) (0.41) (0.48)  c  c  75  Table V,  a. ELISA readings  continued  are the average o f t h r e e  experiments.  b. Numbers i n parentheses r e p r e s e n t the average o p t i c a l d e n s i t y a t 405 nm a f t e r 1 hr o f i n c u b a t i o n a t 37°C w i t h p - n i t r o p h e n y l phosphate w i t h a d i l u t i o n o f antiserum g i v e n as the Log^g t i t r e . Controls lacking a n t i g e n o r u s i n g a s c i t e s f l u i d from NS1 myeloma c e l l s gave o p t i c a l d e n s i t i e s o f 0 - 0.04. c. W h i l e i n t e r a c t i o n o f a n t i b o d i e s with a n t i g e n s CF4349 and CF1248 was weak o r b a r e l y above background, a f t e r o v e r n i g h t i n c u b a t i o n c o l o u r appeared f o r a l l s t r a i n s . A l l o f the s t r a i n s i n t e r a c t e d w i t h these monoclonal a n t i b o d i e s on Western b l o t s , although s t r a i n CF 4349 showed weak i n t e r a c t i o n .  76  membranes o f P. a e r u g i n o s a s t r a i n s t e s t e d , a l t h o u g h the r e a c t i o n s v a r i e d from s t r a i n t o s t r a i n and with the d i f f e r e n t monoclonal a n t i b o d i e s .  Thus  the a n t i g e n i c s i t e s r e c o g n i z e d by these monoclonal a n t i b o d i e s on p r o t e i n F are a p p a r e n t l y common t o a l l these P. a e r u g i n o s a T h i s was confirmed by  strains.  f o r a l l p r o t e i n F - s p e c i f i c monoclonal a n t i b o d i e s  i n t e r a c t i o n w i t h e l e c t r o p h o r e t i c b l o t s o f s e p a r a t e d o u t e r membrane  p r o t e i n s from P. a e r u g i n o s a s e r o t y p i n g s t r a i n s  (see e.g. F i g . 7 ) .  G e n e r a l l y , o n l y one band, i d e n t i f i e d as p r o t e i n F by comparison w i t h i d e n t i c a l b l o t s s t a i n e d w i t h amido b l a c k , was e v i d e n t i n t h e immunolabelled fractions (Fig.  blots.  E x c e p t i o n s were observed  f o r purified protein F  ( F i g . 7, l a n e 9) o r o v e r l o a d e d o u t e r membrane p r o t e i n  samples  7, l a n e s 7 and 8 ) , f o r which h i g h e r m o l e c u l a r weight forms o f  p r o t e i n F were s t a i n e d , presumably due t o e i t h e r n a t i v e o l i g o m e r s o f p r o t e i n F (see c h a p t e r 3) o r p r o t e i n F-LPS, complexes both o f which have been i d e n t i f i e d 1983).  i n a chemical c r o s s - l i n k i n g  study  (Angus and Hancock,  With these e x c e p t i o n s , s i n g l e bands were o b t a i n e d w i t h a l l p r o t e i n  F - s p e c i f i c monoclonal a n t i b o d i e s and the i n t e n s i t y o f the l a b e l on the b l o t s c o r r e l a t e d w i t h the ELISA r e s u l t s f o r the d i f f e r e n t o u t e r membrane isolates.  As a c o n t r o l , no l a b e l l e d band was observed  on Western b l o t s  u s i n g o u t e r membranes from a p r o t e i n F - d e f i c i e n t mutant H283 (see below, F i g . 9, lane 2 ) . As d i s c u s s e d above, the outer membrane p r o t e i n p a t t e r n s o f the c y s t i c f i b r o s i s P. a e r u g i n o s a (Fig. 2).  i s o l a t e s were s i m i l a r t o those o f s t r a i n H103  P r o t e i n F from these i s o l a t e s  i n t e r a c t e d w e l l w i t h the p r o t e i n  F - s p e c i f i c monoclonal a n t i b o d i e s on ELISA ( T a b l e V) and Western b l o t s .  77  F i g u r e 7.  Western e l e c t r o p h o r e t i c  serotyping  s t r a i n s o f P. a e r u g i n o s a a f t e r treatment w i t h monoclonal  a n t i b o d y MA5-8.  b l o t s o f o u t e r membranes o f the  The b l o t was made by e l e c t r o p h o r e t i c  s e p a r a t e d o u t e r membrane p r o t e i n s n i t r o c e l l u l o s e paper.  t r a n s f e r of  from SDS-polyacrylamide g e l s  onto  Outer membrane samples are Lane 1-serotype 17; Lane  2-serotype 16; Lane 3-serotype 15; Lane 4-serotype 14; Lane - s e r o t y p e 13; Lane 6-serotype 12; Lane 7-serotype 11; Lane 8-serotype 10; Lane 9-purified protein  F; Lane 10-serotype 9; Lane 11-serotype 8; Lane  12-serotype 7; Lane 13-serotype 6; Lane 14-serotype 5; Lane 15-serotype 4; Lane 16-serotype 3; Lane 17-serotype 2; Lane 18-serotype 1; Lane 1 9 - w i l d type H103.  78  79  Figure  8.  I n t e r a c t i o n Of monoclonal a n t i b o d i e s  MA2-10 w i t h o u t e r  membranes from c y s t i c f i b r o s i s P. a e r u g i n o s a i s o l a t e s on Western b l o t . The outer membrane samples a r e : Lane 1-CF 3660-1; Lane 2-CF 9490;  Lane  3-CF 221; Lane 4-CF 283; Lane 5-CF 4522; Lane 6-CF 832; Lane 7-CF 3790; Lane 8-CF 1278; Lane 9 - S t r a i n  H103; Lane 10-CF L; Lane 11-CF 1452; Lane  12-CF 6094; Lane 13-CF 2314; Lane 14-CF 4349; Lane 15-CF 284; Lane Lane 1 7 - P u r i f i e d p r o t e i n F.  16-P1.  Only one band, i d e n t i f i e d as p r o t e i n F by  comparison w i t h s t a i n e d e l e c t r o p h o r e t i c b l o t s , was l a b e l l e d i n any o f the membranes.  Ho  81  F i g u r e 9.  Cross  r e a c t i v i t y of p r o t e i n F - s p e c i f i c monoclonal a n t i b o d y  MA4-4 w i t h outer membrane p r o t e i n s from other b a c t e r i a l s p e c i e s . e l e c t r o p h o r e t i c b l o t s of s e p a r a t e d  Western  o u t e r membrane p r o t e i n s and immune  s t a i n i n g were performed as d e s c r i b e d f o r F i g . 7.  Lanes 1 and  6-P.  s t r a i n H283, a p r o t e i n F  aeruginosa  s t r a i n H103; Lane 2-P. aeruginosa  d e f i c i e n t mutant; Lane 3 - E d w a r d s i e l l a  t a r d a 79054; Lane 4 - V i b r i o  a n g u i l l a r u m HT7602; Lane 5-Aeromonas h y d r o p h i l a ET-2; Lanes 7 and 14; p u r i f i e d p r o t e i n F; Lane 8-Pseudomonas a n g u i l l i s e p t i c a ET7601; Lanes 9 and 10; two P. f l u o r e s c e n s s t r a i n s ATCC 13525 and ATCC 949, r e s p e c t i v e l y ; l a n e 11-P. p u t i d a ATCC 12633; Lane 12-Azotobacter s y r i n g a e ATCC 19310.  v i n e l a n d i i OP; Lane  13-P.  82  83  S t r a i n CF 283 ( F i g . 8, lane 4) which f a i l e d t o i n t e r a c t was  subsequently  shown by f a t t y a c i d a n a l y s i s not t o be a P. a e r u g i n o s a s t r a i n personal  (L. Chan,  communication).  There was no i n t e r a c t i o n o f monoclonal antibody MA4-4 ( o r MA2-10, MA4-2 or MA4-10) w i t h o u t e r membrane p r o t e i n s o f E d w a r d s i e l l a t a r d a . V i b r i o a n g u i l l a r u m , P. a n g u i l l i s e p t i c a , Aeromonas h y d r o p h i l a , P. fluorescens, or Azotobacter v i n e l a n d i i .  ( F i g . 9 ) , although  they  interacted  s t r o n g l y w i t h a 39 kD p r o t e i n from P. p u t i d a and P. s y r i n g a e o u t e r membranes ( F i g . 9 ) . One monoclonal a n t i b o d y MA5-8, was h i g h l y s p e c i f i c f o r the p o r i n p r o t e i n F o f P. a e r u g i n o s a .  This antibody c r o s s - r e a c t e d  w i t h a l l t e s t e d s t r a i n s o f P. a e r u g i n o s a but no o t h e r gram-negative bacterial strains  (B)  (Table V I ) .  C r o s s - r e a c t i v i t y o f a l i p o p r o t e i n H 2 - s p e c i f i c monoclonal Monoclonal  antibody  antibody MA1-6 i n t e r a c t e d s p e c i f i c a l l y w i t h the major  outer membrane l i p o p r o t e i n H2 ( F i g . 6 ) . The a n t i g e n i c s i t e r e c o g n i z e d by t h i s antibody was p r e s e n t on l i p o p r o t e i n H2 from the 17 s e r o t y p e ( F i g . 10, T a b l e V I I ) and 28 o f the 30 c y s t i c  strains  f i b r o s i s P. a e r u g i n o s a  s t r a i n s examined (e.g. F i g . 1 1 ) . In a double  a n t i b o d y Western b l o t  ( F i g . 11) monoclonal a n t i b o d i e s  MA4-4, s p e c i f i c f o r p r o t e i n F, and MA1-6, s p e c i f i c used  to show the s p e c i f i c i t y o f the a n t i g e n - a n t i b o d y  Western b l o t , a band t h a t co-migrated lane 13) was l a b e l l e d isolates.  f o r p r o t e i n H2, were interaction.  In the  w i t h p u r i f i e d p r o t e i n F ( F i g . 11,  i n the o u t e r membranes o f a l l the c y s t i c  fibrosis  In c o n t r a s t , monoclonal a n t i b o d y MA1-6 showed no i n t e r a c t i o n  Table V I . C r o s s - r e a c t i o n of monoclonal a n t i b o d i e s s p e c i f i c for P_. aeruginosa outer membranes assessed by ELISA  a  •  Bacterial o u t e r membrane antigen  P. aeruginosa s t r a i n s serotype 5 s e r o t y p e s 7, 8, 10, 14, 1 6 Other s e r o t y p e s AK 1160 (LPS rough) CF 2218 P. p u t i d a P. syringae P. chlororaphis P. aureofaciens P. s t u t z e r i P. fluorescens P. a n g u i l l i s e p t i c a A. v i n e l a n d i i  ELISA readings A^(-^ using monoclonal a n t i b o d i e s s p e c i f i c f o r Outer Membrane P r o t e i n s Lipopolysaccharide H2 F F l(?) 0-antigen Rough c o r e (MA1-6) (MA4-4) (MA5-8) (MA1-3) (MA1-8) (MA3-5). 5  5  c  b  c  0  Lipid A (5E4) d  0.4  0.1  0.4  0.3  1.4  0.1  0.3  0.6-1.2 0.4-2.0 1.2 0.8-1.2 0.4 0.4 0.3 0.1 0.3 0.5 0.1 0.1  0.7-1.8 0.3-2.0 0.6 0.8-1.8 0.2 1.1  0.8-2 0.8-2 0.4-2 0.1-0.3  0.1-0.3 0.2-0.4 0.2 0.1-0.3 0.1 0.1 0.1 0.1 0.3 0.1  -  0.1-0.3  ND ND 0.1-0.3 0.2 0,2 ND ND ND ND 0.4 0.3 0.4  P. m a l t o p h i l i a P. P. P.  e  f  .—  -  ' .-  0.7  •-  -  •  '  -  -  . -  -  -  -  -  0.1  -  -  -  -  -  -  -  -  ND  acidovorans  -  -  -  -  ND  ND  solanacearum cepacia  -  -  -  -  -  -  ND +  CO  Table V I , continued  ELISA readings A405 u s i n g monoclonal a n t i b o d i e s s p e c i f i c for Outer Membrane P r o t e i n s Lipopolysaccharide c H2 F 0-antigen Rough c o r e K?)b (MA1-6) (MA4-4) (MA1-3) (MA5-8) (MA1-8) (MA3-5) 3  Bacterial o u t e r membrane antigen  E. S. E. Y.  coli typhimurium tarda pestis^  V. V. A. A.  cholera anguillarum salmonicida hydrophila  A.  tumefaciens PLT4 S1005.A6  b  c  c  F  _  -  -  -  -  -  -  ND  ND  ND  -  -  -  -  ND  ND  ND  ND  ND  ND  -  ND  -  (-)  (-)  -  -  -  -  —  -  -.  ND  ND  -  -  -  -  Lipid A (5E4) d  0.4 0.3 + +  ND  -  0  .  + 0.2 0.2 0.3  n  ND  ND  ND  ND  ND  ND  ND  ND  ND  ND  1.2 0.2  OO  86  VI Legend ND. a. b. c. d. e. f. g.  h.  not done; The ELISA data i s e x p r e s s e d as absorbance at 405 nm a f t e r 90 minute at 37°C. The monoclonal a n t i b o d y was used at 100 f o l d d i l u t i o n . The monoclonal antibody was used at 1,000 f o l d d i l u t i o n . 5E4 was used at 10 yg of lgG per ml. A l l other P. a e r u g i n o s a s e r o t y p e s i n t e r a c t e d w i t h the t e s t e d monoclonal a n t i b o d i e s except MA1-8 and MA3-5 (MA3-6). Three other L P S - ) - a n t i g e n d e f i c i e n t s t r a i n s H234, H223 and H1188 gave similar results. ' 3 6 d i f f e r e n t P. a e r u g i n o s a s t r a i n s from c y s t i c f i b r o s i s (CF) p a t i e n t s were t e s t e d . A l l s t r a i n s showed s i m i l a r r e s u l t s t o those i n T a b l e VI with some e x c e p t i o n s . S t r a i n CFC46 m and C46 nm d i d not i n t e r a c t with MA1-6, and o n l y 3 s t r a i n s i n t e r a c t e d w i t h MA3-5. LPS from Y. p e s t i s and A. tumefaciens was used as the a n t i g e n . (-) denote n e g a t i v e r e s u l t s by both Western b l o t and ELISA procedures. These d a t a were o b t a i n e d from G.W.K. C r o c k f o r d , U.B.C.  87  F i g u r e 10.  Western e l e c t r o p h o r e t i c b l o t s o f o u t e r membranes  o f the  s e r o t y p i n g s t r a i n s o f P. a e r u g i n o s a a f t e r treatment w i t h monoclonal antibody MA1-6.  Outer membrane samples were lane 1- w i l d type H103; l a n e  2 - serotype 1; lane 3 - s e r o t y p e 2; lane 4 - serotype 3; l a n e 5 serotype 4; lane 6 - s e r o t y p e 5; l a n e 7 - s e r o t y p e 6; l a n e 8 - s e r o t y p e 7; lane 9 - s e r o t y p e 8; l a n e 10 - s e r o t y p e 9; lane 11 - s e r o t y p e 10; l a n e 12 - serotype 11; lane 13 - s e r o t y p e 12; lane 14 - serotype 13; l a n e 15 serotype 14; lane 16 - s e r o t y p e 15; lane 17 - serotype 16; l a n e 18 serotype 17; (Lane S - SDS - e l e c t r o p h o r e t o g r a m Only one band was  labelled.  o f H103 o u t e r membrane).  88  89  F i g u r e 11.  Western e l e c t r o p h o r e t i c b l o t s o f o u t e r membranes o f 6 p a i r s  mucoid (m) and non-mucoid (nm) P. a e r u g i n o s a cystic  isolates  from p a t i e n t s w i t h  f i b r o s i s a f t e r i n t e r a c t i o n with monoclonal a n t i b o d i e s MA4-4 ( t o p  band), and MA1-6  (bottom band).  The o u t e r membrane samples were l a n e  1-wild type H103; lane 2 - Plm; l a n e 3 - CFClm; l a n d 4 - CFClnm; l a n e 5 CFC47m; lane 6 - CFC47nm; l a n e 7 - CFC46m; lane 8 - CFC46nm; l a n e 9 CFC4m; lane 10 - CFC4nm; l a n e 11 - CFC6m; lane 12 - CFC6nm; l a n e 13 p u r i f i e d p r o t e i n F.  1  2  3 A  5 6  7 8  9  10 11 12 13  91  F i g u r e 12.  Western e l e c t r o p h o r e t i c b l o t  antibody MA1-6 w i t h outer membranes from:  showing i n t e r a c t i o n  o f monoclonal  Lane 1, P. a n g u i l l i s e p t i c a ET2,  Lane 2, P. aeruginosa s t r a i n H103; Lane 3, P. f l u o r e s c e n s ATCC 949; Lane 4, P. s y r i n g a e ATCC 19310; Lane 5, P. a e r u g i n o s a CF46nm; Lane 6, P. putIda ATCC 12633; Lane 7, P. s t u t z e r i ATCC 17588; Lane 8, P. aeruginosa ATCC 19305; Lane 9, A, v i n e l a n d i i  OP; Lane 10, P. a u r e o f a c i e n s ATCC 13985; Lane  11, P. c e p a c i a ; Lane 12, P. c h l o r o r a p h i s ATCC 9446; Lane 13, P. pseudomallei ATCC 23343; Lane 14, P. a e r u g i n o s a  ATCC 9721.  92  1 2 3  4  5  6  7  8 9 10 11 12 13  K  93  with p u r i f i e d p r o t e i n F ( F i g . 11, l a n e 13) nor w i t h o u t e r membranes from s t r a i n CFC46 mucoid and CFC46 nonmucoid ( F i g . 11; lanes 8 and 9 ) .  As  d e s c r i b e d above ( F i g . 2 ) , these two s t r a i n s a l s o l a c k e d p r o t e i n H2 on SDS p o l y a c r y l a m i d e g e l s o f t h e i r outer membranes. Monoclonal a n t i b o d y MAl-6 a l s o r e a c t e d w i t h a p r o t e i n o f s i m i l a r molecular weight t o p r o t e i n H2 i n Western b l o t s o f P. a n g u i l l i s e p t i c a , P. f l u o r e s c e n s . P. p u t i d a and P. s y r i n g a e o u t e r membranes, w i t h a p r o t e i n o f higher m o l e c u l a r weight i n A z o t o b a c t e r v i n e l a n d i i , and s l i g h t l y  higher  m o l e c u l a r weight i n P. s t u t z e r i o u t e r membranes, and w i t h a p r o t e i n o f s l i g h t l y lower m o l e c u l a r weight i n P. a u r e o f a c i e n s and P. c h l o r o r a p h i s outer membranes ( F i g . 1 2 ) . There was no c r o s s - r e a c t i v i t y w i t h t h e o u t e r membranes o f P. c e p a c i a ( F i g . 12), E. c o l i , or  (C)  P. a c i d o v o r a n s . P. m a l t o p h i l i a  S. typhimurium ( T a b l e V I ) .  H e t e r o g e n e i t y o f t h e LPS rough c o r e i n P. a e r u g i n o s a Two rough c o r e - s p e c i f i c monoclonal a n t i b o d i e s were c h a r a c t e r i z e d i n  detail.  Antibody MA3-5 i n t e r a c t e d w i t h the LPS o f s e r o t y p e s 5 ( t h e  serotype o f s t r a i n H103), 7, 8, 10, 14 and 16 o f t h e I n t e r n a t i o n a l A n t i g e n Typing Scheme o f P. a e r u g i n o s a  (17 s e r o t y p e s )  ( F i g . 13), whereas MA3-6  i n t e r a c t e d o n l y w i t h s e r o t y p e s 5, 8, 10 and 16 ( d a t a not shown). 16 P. a e r u g i n o s a  i s o l a t e s from p a t i e n t s w i t h c y s t i c  fibrosis  t e s t e d , MA3-6  i n t e r a c t e d o n l y w i t h s t r a i n CF1452 whereas MA3-5 i n t e r a c t e d w i t h s t r a i n s , CF4522, CF221 and CF9490. evidence  T h i s p r o v i d e d the f i r s t  f o r LPS rough core h e t e r o g e n e i t y i n P. a e r u g i n o s a .  Of t h e  three  definitive  94  F i g u r e 13.  Western e l e c t r o p h o r e t i c  b l o t of o u t e r membranes of  P. aeruginosa serotype s t r a i n s a f t e r r e a c t i o n w i t h monoclonal MA3-5. antibody  The  numbers below the l a n e s are the s e r o t y p e s .  i n t e r a c t e d w i t h the rough core LPS  and 16 s t r a i n s , and s t r a i n H103  The  antibody  monoclonal  from s e r o t y p e 5, 7, 8, 10,  ( l a b e l l e d W).  14  95  W  1 2 3 4 5 6 7 8 9 10 111213141516 17  96  A v a r i e t y of other monoclonal a n t i b o d i e s showing specificities Although P.  f o r the P.  a e r u g i n o s a LPS  different  rough core were  isolated.  the r e s u l t s of the i n t e r a c t i o n of these monoclonal a n t i b o d i e s and  aeruginosa s t r a i n s  i s not p r e s e n t e d here,  some of the a n t i b o d i e s showed analogous  i t s h o u l d be mentioned t h a t  narrow c r o s s - r e a c t i v i t i e s  to  a n t i b o d i e s MA3-5 and MA3-6 w h i l e o t h e r s showed v e r y e x t e n s i v e c r o s s - r e a c t i v i t y among a l l P. a e r u g i n o s a s t r a i n s t e s t e d ( J . L i n , p e r s o n a l communication).  (D)  H e t e r o g e n e i t y of LPS  O-antigen  The monoclonal antibody MA1-8, s p e c i f i c  f o r the 0 a n t i g e n of  strain  PAOI, i n t e r a c t e d s t r o n g l y w i t h the o u t e r membrane of the serotype 5 s t r a i n (Table V I ) , i n agreement with a p r e v i o u s o b s e r v a t i o n t h a t s t r a i n PAOI i s type 5 i n the IATS t y p i n g scheme (Cox 1979).  Of the o t h e r 16  s t r a i n s examined, o n l y the o u t e r membranes of type 17 w i t h MA1-8  (Table V I I ) .  T h i s was  serotype  i n t e r a c t e d weakly  c o n s i s t e n t w i t h a s e r i e s of  experiments  t e s t i n g the a b i l i t y of our monoclonal a n t i b o d i e s t o a g g l u t i n a t e whole bacteria.  In these experiments,  of s t r a i n PAOI, s t r a i n Z61  MA1-8  showed a g g l u t i n a t i o n (4+)  and the IATS s e r o t y p e 5 s t r a i n but no  of c e l l s bacterial  a g g l u t i n a t i o n r e a c t i o n s were observed  u s i n g the other s e r o t y p i n g s t r a i n s .  In f u r t h e r t e s t s  shown to be s p e c i f i c f o r  P.  a e r u g i n o s a LPS  ( T a b l e VI) MA1-8 and  was  the antibody showed no c r o s s - r e a c t i v i t y w i t h o t h e r  gram-negative b a c t e r i a i n c l u d i n g o t h e r Pseudomonads.  97  Table V I I . I n t e r a c t i o n o f monoclonal a n t i b o d i e s MA1-3, MAl-6, and MA1-8 with the o u t e r membrane a n t i g e n s from the 17 s e r o t y p e s s t r a i n s o f P. a e r u g i n o s a .  Log^o ELISA t i t r e Outer membrane a n t i g e n from  MA1-8  w i t h monoclonal  antibody  MAl-6  MA1-3  4  Serotype  1 2 3 4 5 6 7 8 9 10 11 ' 12 13 14 15 16 17  PI mucoid PI r e v e r t a n t PA01  < < < < < < < < < < < < < < < < <  1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5  (0, .05) (0, .06) (0, .05) (0, .02) (2, .0) (0, .02) (0, .01) (0, .02) (0, .01) (0, .02) (0. .0) (0. .0) (0, .0) (0. .0) (0. .0) (0. .02) (0. 2) (0. .01) Co. 0) (1. ,99)  2 2 1 2 1 3 3 2 3 3 2 2 1 3 1 3 3 1 1 3  (0, .73) (0, .85) (0, .54) (0. .73) (0. .41) (1, .08) (1. .20) (0. .70) (1. .09) (1. .21) (0, .92) (1, .18) (0. 41) (1. 25) (0. ,40) (0. ,56) (> 2) (0. ,38) (0. ,20) (1. 15)  3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3  (0, .59) (0, .39) (0. .49) (0, .38) (0, .30) (0, .29) (0, .45) (0. .32) (0, .45) (0, .36) (0, .30) (0. .42) (0. 41) (0. ,39) (0. .37) (0. .50) (0. .58) (0. .44) (0. ,53) (0. ,58)  a. F o r t e s t i n g MA1-3, o u t e r membranes were s o l u b i l i z e d i n 2% T r i t o n X-100, 20 mM T r i s - H C l , pH 8.0, 10 mM EDTA, and then p r e c i p i t a t e d a t -20°C a f t e r a d d i t i o n o f 2 volumes o f e t h a n o l and 0.1 M NaCl p r i o r t o c o a t i n g of ELISA p l a t e s . Using u n s o l u b i l i z e d o u t e r membranes as a n t i g e n s gave i n c o n s i s t e n t r e s u l t s f o r MA1-3. F o r the o t h e r monoclonal a n t i b o d i e s u n s o l u b i l i z e d o u t e r membranes were used as the c o a t i n g a n t i g e n . The numbers i n parentheses r e p r e s e n t the o p t i c a l d e n s i t y a t 405 nm a f t e r 1 hr i n c u b a t i o n w i t h p - n i t r o - p h e n y l p h o s p h a t e w i t h the antibody d i l u t i o n given as the Log^Q t i t r e .  98  (E)  Conservation Chemical  of L i p i d A  c h a r a c t e r i z a t i o n of the L i p i d A p o r t i o n s o f the LPS of  d i v e r s e b a c t e r i a have i n d i c a t e d t h a t L i p i d A i s c h e m i c a l l y a s t r o n g l y conserved A bears  s t r u c t u r e ( R i e t s c h e l e t a l . , 1983).  the g e n e r a l name " e n d o t o x i n "  have s i m i l a r e f f e c t s on host c e l l s  Consistent with t h i s ,  Lipid  and L i p i d A's from d i f f e r e n t b a c t e r i a  ( B r a d l e y 1979).  Monoclonal a n t i b o d i e s  s p e c i f i c f o r L i p i d A were used to study the e x p r e s s i o n of s p e c i f i c e p i t o p e s on L i p i d A. For these s t u d i e s , outer membranes and l i p o p o l y s a c c h a r i d e s from a v a r i e t y of s p e c i e s and genera o f gram-negative b a c t e r i a were screened ELISA (and i n some i n s t a n c e s Western b l o t s ) f o r i n t e r a c t i o n w i t h  two  monoclonal a n t i b o d i e s which had been shown to be r e a c t i v e a g a i n s t L i p i d A from P. a e r u g i n o s a of  Bogard).  i n t e r a c t e d w i t h the gram p o s i t i v e c e l l s  walls  subtilis,  Staphylococcus  (W.C. Bogard, p e r s o n a l In the p r e s e n t  purified  and S. minnesota (Mutharia e_t a l . , 1984; data  G.W.K. C r o c k f o r d and W.C.  Bacillus  by  N e i t h e r of these a n t i b o d i e s (or c e l l walls) t e s t e d i . e .  aureus and S t r e p t o c o c c u s  faecalis  cell  communication).  study, monoclonal a n t i b o d i e s 5E4 and 8A1 were shown by  ELISA a n a l y s i s to e x h i b i t e x t e n s i v e c r o s s - r e a c t i v i t y w i t h the o u t e r membranes and LPS from a v a r i e t y o f P. a e r u g i n o s a bacterial strains  and gram n e g a t i v e  (Table V I ) .  To determine the s p e c i f i c component o f o u t e r membranes which was i n t e r a c t i n g w i t h the monoclonal a n t i b o d i e s 5E4 and 8A1 and t o c o n f i r m some of  the ELISA r e s u l t s  i n Table V I , Pseudomonas a e r u g i n o s a  membranes were s e p a r a t e d by SDS p o l y a c r y l a m i d e  s t r a i n PAOI o u t e r  g e l e l e c t r o p h o r e s i s and  99  t r a n s f e r r e d from the e l e c t r o p h o r e t o g r a m Western t e c h n i q u e .  The  separated o u t e r membranes were then  with monoclonal a n t i b o d i e s 5E4 antibodies migrated 0.8  amd  8A1.  interacted  Both of these monoclonal  compared t o the bromphenol b l u e dye  i n t e r a c t e d w i t h an LPS  Antibody 17 serotypes  band  since i t  from the rough mutant s t r a i n H146  and 14 c l i n i c a l  isolates  s e r i e s of c l o s e l y spaced bands of lower Although  ( F i g . 15)  of  r e l a t i v e m o b i l i t y was  t h i s o b s e r v a t i o n was  due  with a also  too i n c o n s i s t e n t t o a n a l y s e  to the low a f f i n i t y of the monoclonal a n t i b o d i e s f o r  h i g h e r m o l e c u l a r weight LPS), these bands may  have r e p r e s e n t e d smooth,  O - a n t i g e n - c o n t a i n i n g LPS which has been shown t o c o n s t i t u t e around of the LPS molecules Monoclonal membranes or LPS  and  s p e c i f i c monoclonal a n t i b o d y MA1-8.  O c c a s i o n a l l y , the i n t e r a c t i o n of a n t i b o d y 5E4  p r o p e r l y (perhaps  was  i n t e r a c t e d with a s i m i l a r f a s t - m i g r a t i n g band from a l l  ( F i g . 14)  P. aeruginosa.  The  rough c o r e - s p e c i f i c monoclonal a n t i b o d y MA3-5 but  O-antigen  5E4  front.  ( c o n t a i n i n g rough c o r e and l i p i d A)  comigrated w i t h a u t h e n t i c rough LPS  not with an LPS  had  p o l y a c r y l a m i d e g e l w i t h a r e l a t i v e m o b i l i t y of around  i d e n t i f i e d as rough LPS  observed.  the  i n t e r a c t e d with a s i n g l e major band (e.g. F i g . 14) which  i n the SDS  to 0.9  to n i t r o c e l l u l o s e paper by  i n some P. a e r u g i n o s a  a n t i b o d i e s 5E4  and 8A1  also  strains. i n t e r a c t e d w i t h the o u t e r  from o t h e r gram-negative b a c t e r i a .  These i n c l u d e d  s t r a i n s from the f a m i l i e s Pseudomonadaceae. R h i z o b i a c e a e , E n t e r o b a c t e r i a c e a e . and Vibronaceae  5-10%  (Table V I ) .  100  F i g u r e 14.  Western e l e c t r o p h o r e t i c b l o t o f s e p a r a t e d o u t e r membranes o f  the 17 Pseudomonas a e r u g i n o s a s e r o t y p e s t r a i n s a f t e r r e a c t i o n w i t h monoclonal antibody 5E4.  The o u t e r membranes  were l a n e 1-serotype 17;  2-serotype 16; 3-serotype 15; 4-serotype 14; 5-serotype 13, 6-serotype 12; 7-serotype 11; 8-serotype 10; 9-serotype 9; 10-serotype 8; 11-serotype 7; 12-serotype 6; 13-serotype 5; 14-serotype 4; 15-serotype 3; 16-serotype 2; 17-serotype 1; 18-P. a e r u g i n o s a PAOI.  C r e f e r s to a c o n t r o l lane  c o n t a i n i n g P. a e r u g i n o s a o u t e r membrane p r o t e i n F p u r i f i e d f r e e o f LPS.  101  1 2 3 4 5 6 7 8 C 9 10 11 12131415161718  102  F i g u r e 15.  R e a c t i o n o f monoclonal antibody 5E4 w i t h a Western  electrophoretic blot of P. a e r u g i n o s a .  o f the s e p a r a t e d o u t e r membranes o f c l i n i c a l  The o u t e r membranes were:  isolates  lane 1 - s t r a i n CF3660-1; 2  - s t r a i n CF9490; 3 - s t r a i n CF221; 4 - s t r a i n CF4349; 5 - s t r a i n CF284; 6 - s t r a i n CF4522; 7 - s t r a i n CF832; 8 - s t r a i n CF3780; 9 - s t r a i n  CF1278;  10 - s t r a i n L; 11 - s t r a i n CF1452; 12 - s t r a i n CF6094; 13 - s t r a i n 2314; 14 - s t r a i n CFP1M.  T h i s b l o t was made from an 11% a c r y l a m i d e , SDS  p o l y a c r y l a m i d e g e l ( u n l i k e F i g u r e 14 which was from a 14% a c r y l a m i d e gel).  T h e r e f o r e rough LPS m i g r a t e d w i t h the dye f r o n t  t i g h t band ( c . f . F i g u r e 1 4 ) .  and r e a c t e d as a  1 2 3 A 5 6 7 8  9 X) 11 12  13 IA  104  Summary A l l P. aeruginosa s t r a i n s conserved  s t u d i e d shared at l e a s t two s e p a r a t e  outer membrane a n t i g e n i c s i t e s on p r o t e i n F.  of two s t r a i n s  (CFC46 mucoid  and nonmucoid),  shared an a n t i g e n i c e p i t o p e on p r o t e i n H2. expressed  With the e x c e p t i o n  P. a e r u g i n o s a s t r a i n s  A l l t h r e e o f these s i t e s were  r e g a r d l e s s o f s o u r c e , s e r o t y p e , c o l o n y morphology  ( i . e . mucoid  o r nonmucoid;  rough o r smooth) o r serum s u s c e p t i b i l i t y o f the  P. aeruginosa  isolate.  A monoclonal a n t i b o d y MAl-6 s p e c i f i c p r o t e i n H2 of P. a e r u g i n o s a  also  f o r o u t e r membrane  lipoprotein  i n t e r a c t e d w i t h 28 P. a e r u g i n o s a s t r a i n s and  organisms r e p r e s e n t a t i v e o f 8 o t h e r Genera o f the Family o f Pseudomonadaceae (mostly members o f the f l u o r e s c e n t pseudomonads), as A z o t o b a c t e r v i n e l a n d i i .  T h i s a n t i b o d y d i d not i n t e r a c t  as w e l l  with  pseudomonads demonstrated t o be u n r e l a t e d to P. a e r u g i n o s a by rRNA homology s t u d i e s , n o r w i t h b a c t e r i a l bacterial  s t r a i n s from o t h e r gram-negative  Genera.  A group o f f o u r monoclonal a n t i b o d i e s a g a i n s t p o r i n p r o t e i n F o f Pseudomonas a e r u g i n o s a  i n t e r a c t e d w i t h a l l P. a e r u g i n o s a s t r a i n s t e s t e d as  w e l l as with P. p u t i d a and P. s y r i n g a e s t r a i n s but not w i t h pseudomonads.  other  Another p r o t e i n F - s p e c i f i c monoclonal a n t i b o d y MA5-8  i n t e r a c t e d o n l y w i t h the P. a e r u g i n o s a  strains.  The s p e c i f i c i t y o f a l l  f i v e o f these monoclonal a n t i b o d i e s f o r p r o t e i n F was demonstrated by the absence o f c r o s s - r e a c t i v i t y w i t h a p r o t e i n F - d e f i c i e n t mutant  s t r a i n H283  d e r i v e d from P. a e r u g i n o s a PA01 s t r a i n H103 (Nicas and Hancock,  1983), and  105  by t h e i r i n t e r a c t i o n w i t h p r o t e i n F on Western b l o t s of  SDS-polyacrylamide  g e l - s e p a r a t e d o u t e r membrane components. A monoclonal a n t i b o d y s p e c i f i c aeruginosa s t r a i n  f o r the O-antigen  of our w i l d type  i n t e r a c t e d w i t h s t r a i n s of e q u i v a l e n t s e r o t y p e  (0-5 i n  the I n t e r n a t i o n a l A n t i g e n Typing Scheme, IATS) and weakly w i t h s t r a i n s a r e l a t e d serotype Monoclonal c h a i n of one  antibodies specific  f o r the rough core or 0 - a n t i g e n i c s i d e  Pseudomonas a e r u g i n o s a s t r a i n demonstrated o n l y ( F i g . 13).  limited In c o n t r a s t ,  monoclonal a n t i b o d i e s s p e c i f i c f o r the l i p i d A p o r t i o n of LPS  14,15) and  of  (0-17) ( T a b l e V I I ) .  c r o s s - r e a c t i o n w i t h o t h e r P. a e r u g i n o s a s t r a i n s  with the LPS  P.  or o u t e r membrane of 36 P.  aeruginosa s t r a i n s  interacted  (Table VI; F i g .  22 o t h e r gram n e g a t i v e b a c t e r i a from 4 d i f f e r e n t F a m i l i e s and  16 separate Genera ( T a b l e V I ) . a n t i g e n i c a l l y conserved  T h i s demonstrated t h a t L i p i d A i s h i g h l y  i n many gram-negative  bacteria.  106  CHAPTER I I I .  CHARACTERIZATION OF OUTER MEMBRANE EPITOPES USING MONOCLONAL ANTIBODIES.  1.  C e l l s u r f a c e l o c a l i z a t i o n of the a n t i g e n i c s i t e s r e c o g n i z e d  by  monoclonal a n t i b o d i e s (A)  S u r f a c e l o c a l i z a t i o n s t u d i e s by The  immunofluorescence  a c c e s s i b i l i t y of an immunogenic p r o t e i n on the s u r f a c e of  bacterial cells  i s a c h a r a c t e r i s t i c of g r e a t  importance, e s p e c i a l l y when  such a p r o t e i n i s b e i n g c o n s i d e r e d f o r i t s p o t e n t i a l use identification,  i n e p i d e m i o l o g i c a l s t u d i e s and  a c t i v e and p a s s i v e v a c c i n e s . membrane p r o t e i n s has  has  in  clinical  i n the development of  S u r f a c e l o c a l i z a t i o n of P.  been i n d i c a t e d i n s t u d i e s u s i n g  r a d i o i o d i n a t i o n methods (Lambert and  intact  Booth, 1982).  aeruginosa  both outer  lactoperoxidase  However, t h i s method  been shown to l a b e l non-outer membrane p r o t e i n s as w e l l ( S u l l i v a n  Williams  1982), perhaps as a r e s u l t of d i s r u p t i o n o f o u t e r membrane  integrity.  Furthermore, i t does not r e v e a l whether the s u r f a c e a c c e s s i b l e  p o r t i o n s of the p r o t e i n are We  immunogenic.  were i n t e r e s t e d i n s t u d y i n g the topography of the major o u t e r  membrane p r o t e i n s on the c e l l  s u r f a c e of P.  monoclonal a n t i b o d i e s to p o r i n p r o t e i n F and  aeruginosa.  As  probes,  to l i p o p r o t e i n H2 were used.  Monoclonal antibody MA1-8, s p e c i f i c f o r the O-antigen of the LPS serotype  and  5 ( I n t e r n a t i o n a l A n t i g e n Typing  control. suspension  In these  s t u d i e s , i n t a c t P.  Scheme) was  aeruginosa  used as a p o s i t i v e  c e l l s were i n t e r a c t e d i n  with d i l u t i o n s of the monoclonal a n t i b o d y  f l u o r e s c e i n isothiocyanate-conjugated  of  and  then with  anti-mouse Ig a n t i b o d y .  The  a  107  l a b e l l e d c e l l s were then examined f o r f l u o r e s c e n c e w i t h a f l u o r e s c e n c e microscope c o n t a i n i n g a halogen lamp and The tested  r e s u l t s of these  (with one  patients,  appropriate  filters.  s t u d i e s showed t h a t a l l s t r a i n s of P.  exception), including isolates  from c y s t i c  aeruginosa  fibrosis  i n t e r a c t e d w i t h the t h r e e t e s t e d p o r i n p r o t e i n F - s p e c i f i c  monoclonal a n t i b o d i e s , MA2-10, MA4-4 and MA4-10 (Table V I I I : F i g . 16). None of these monoclonal a n t i b o d i e s i n t e r a c t e d w i t h  s t r a i n H283, a p r o t e i n  F - d e f i c i e n t mutant, as demonstrated by the l a c k of f l u o r e s c e n c e on H283 c e l l s The both  ( F i g . 16,  serotype  s t r a i n H103  panel E; Table V I I I ) .  5 L P S - s p e c i f i c monoclonal a n t i b o d y and  the  MA1-8  i n t e r a c t e d with  i t s p r o t e i n F - d e f i c i e n t d e r i v a t i v e H283 but not  with  s t r a i n ATCC33348 ( s e r o t y p e 1) or an 0 - a n t i g e n - d e f i c i e n t (rough) mutant, s t r a i n H233 ( a l s o d e r i v e d from s t r a i n H103) antibody MAl-6, s p e c i f i c f o r p r o t e i n H2,  (Table V I I I ) .  i n t e r a c t e d with  Monoclonal s t r a i n H223 the  rough mutant and w i t h CF221, CFC47 mucoid and CFC47 non-mucoid the rough c y s t i c f i b r o s i s 1 strains  (Table V I I I ) .  i s o l a t e s but not w i t h H103,  its  ( F i g . 11),  t h a t the l i p o p r o t e i n H2  a c c e s s i b i l i t y on the c e l l  wild-type  (smooth) P.  H283 or the  showed no f l u o r e s c e n c e .  i s e i t h e r not  s u r f a c e i s masked by LPS  aeruginosa  antibody MAl-6 w i t h rough P.  strains.  aeruginosa  The  0-side  chains i n  i n t e r a c t i o n of monoclonal  s t r a i n s may  be due  e i t h e r to on the LPS  the rough s t r a i n s o r to rearrangement of the macromolecules i n the strains.  These  s u r f a c e exposed o r  unmasking of t h i s p r o t e i n , because of the l a c k of 0-antigen  membranes of the rough  serotype  S t r a i n s CFC46 mucoid and CFC46 non-mucoid which  were rough but l a c k e d p r o t e i n H2 r e s u l t s suggested  strains,  of  outer  108  F i g u r e 16.  Indirect  immunofluorescent  tagged w i t h monoclonal  antibodies  l a b e l l i n g o f i n t a c t P. a e r u g i n o s a  t o o u t e r membrane components.  s t r a i n H103 i n t e r a c t e d w i t h p r o t e i n F - s p e c i f i c monoclonal  a n t i b o d y MA4-4.  Panel B - s t r a i n H223 i n t e r a c t e d w i t h p r o t e i n F - s p e c i f i c monoclonal MA2-10.  Panel A -  antibody  P a n e l C - s t r a i n H103 i n t e r a c t e d w i t h LPS O-antigen s p e c i f i c  monoclonal  a n t i b o d y MA1-8.  P a n e l D-rough, LPS d e f i c i e n t mutant s t r a i n  H223 i n t e r a c t e d w i t h l i p o p r o t e i n H2 s p e c i f i c monoclonal Panel E - s t r a i n H283 (as p r o t e i n F - s p e c i f i c monoclonal  a n t i b o d y MAl-6.  F- d e f i c i e n t ) i n t e r a c t e d w i t h  a n t i b o d y MA2-10.  (Note:  protein  The d i f f e r e n c e s  observed  i n the f l u o r e s c e n c e between p a n e l A and p a n e l B were due e n t i r e l y t o exposure  times  i n taking  the p i c t u r e , and not t o d i f f e r e n c e s  phenotype o f the two s t r a i n s u s e d ) .  i n the LPS  B.  C  D.  Table V I I I .  Bacterial Strain  a  Hl03 H283 H223 ATCC 33352 ATCC 33364 ATCC 33348 a  a  Demonstration of c e l l s u r f a c e l o c a l i z a t i o n o f o u t e r membrane components of P_. aeruginosa by immunofluorescence  Phenotype  MA2- •10  Wildtype serotype 5 Protein F deficient Rough, LPS d e f i c i e n t Serotype 5 Serotype 17 Serotype 1  C F . isolates rough CF 221° CF C46 mucoid non-mucoid r e v e r t a n t mucoid CFC47 non-mucoid r e v e r t a n t  a.  +  -  Protein F MA4-4 MA4-:LO  P r o t e i n H2 MA1-6  LPS 0- A n t i g e n MA1-8  Components Whole outer membrane rabbit antisera  +  +  —  +  +  -  -  -  +  +  +  +  + '  +  -  ND  + + +  + +  ND ND  -  + +  + +  +  ND  —  —  +  + + +  + +  ND  +  ND  -  + +• +  + +  + +  -  ND ND ND  + +  c  + +  s t r a i n s were i s o g e n i c . +, p o s i t i v e f l u o r e s c e n c e ; -, no f l u o r e s c e n c e ; 1 3 o t h e r P. a e r u g i n o s a c y s t i c f i b r o s i s i s o l a t e s and 9 other mucoid and non-mucoid r e v e r t a n t p a i r s gave r e s u l t s i d e n t i c a l to CF 221 and CF C47 r e s p e c t i v e l y . ND, not determined. d  Ill  In agreement w i t h the above d a t a , we observed b i n d i n g o f p r o t e i n Fs p e c i f i c monoclonal  a n t i b o d i e s to whole c e l l s o f s t r a i n H103 when these  c e l l s were used as the a n t i g e n i n ELISA assays on p o l y - L - l y s i n e - c o a t e d polyvinylchloride plates  (Table IX).  I n these experiments  the a n t i b o d y  t i t r e s o b t a i n e d were s i m i l a r t o those o b t a i n e d f o r s t r a i n H103 o u t e r membranes, although backgrounds tended t o be h i g h e r w i t h whole c e l l s p o s s i b l y due to n o n s p e c i f i c b i n d i n g o f the second antibody t o the c e l l s .  (B)  Surface l o c a l i z a t i o n A colony b l o t t i n g  s t u d i e s by c o l o n y  immunoblotting  immunoassay was developed t o f a c i l i t a t e  the r a p i d  s c r e e n i n g o f a v a r i e t y o f P. a e r u g i n o s a s t r a i n s f o r the e x p r e s s i o n o f the s p e c i f i c a n t i g e n i c determinants  r e c o g n i z e d by monoclonal  antibodies.  The  method used was a m o d i f i c a t i o n o f one developed by Henning e t a l . (1979) i n t h a t enzymatic a f t e r the b l o t t i n g  immunostaining step.  i n s t e a d o f radioimmunostaining was used  In these s t u d i e s , c o l o n i e s of the b a c t e r i a l  s t r a i n were r e p l i c a p l a t e d onto agar p l a t e s and then t r a n s f e r r e d t o nitrocellulose f i l t e r s  by d i r e c t c o n t a c t .  The f i l t e r s were then  immunostained u s i n g a procedure r e l a t e d t o t h a t used f o r Western b l o t s (see  Methods). Monoclonal  a n t i b o d i e s MA4-4, MA2-10, MA4-10 and MA5-8 were  interacted  by the colony b l o t t i n g procedure w i t h t h e type s t r a i n s from a l l 17 serotypes ( I n t e r n a t i o n a l A n t i g e n T y p i n g Scheme) o f P. a e r u g i n o s a , and w i t h a v a r i e t y o f l a b o r a t o r y s t r a i n s as w e l l as mucoid P. a e r u g i n o s a o b t a i n e d from p a t i e n t s w i t h c y s t i c non-mucoid r e v e r t a n t s .  fibrosis,  and t h e i r  isolates,  spontaneous  Although d i f f e r e n c e s were observed  i n the  112  Table IX.  I n t e r a c t i o n o f p r o t e i n F - s p e c i f i c monoclonal a n t i b o d i e s w i t h p u r i f i e d p r o t e i n F, o u t e r membranes and whole c e l l s o f s t r a i n H103, a s s e s s e d by ELISA  Log^o ELISA t i t r e s a g a i n s t :. a Purified Monoclonal antibody  MA2-10 MA4-4 MA4-10  S t r a i n H103 Protein F b  S t r a i n H103 whole c e l l s  0  S t r a i n H103 o u t e r membranes  4 2 3  a. ELISA t i t r e s e x p r e s s e d as Log^o v a l u e s were o b t a i n e d u s i n g a s c i t e s f l u i d as f i r s t a n t i b o d y . Whole c e l l ELISAs were performed as d e s c r i b e d i n Methods. b. P r o t e i n F was d e r i v e d from s t r a i n H103.  113  i n t e n s i t y of the blue c o l o u r developed b i n d i n g ) c o l o n i e s ( F i g . 17), a l l P.  with p o s i t i v e  aeruginosa  w i t h the monoclonal a n t i b o d i e s ( T a b l e X ) .  (antibody-  strains tested interacted  In c o n t r a s t , a P.  s t r a i n H283 t h a t l a c k s p r o t e i n F ( N i c a s and Hancock, 1983) non-P. aeruginosa gram-negative i s o l a t e  from a c y s t i c  aeruginosa  and CF283, a  fibrosis patient,  d i d not b i n d any of the p r o t e i n F - s p e c i f i c monoclonal a n t i b o d i e s . The  s p e c i f i c i t y of the c o l o n y b l o t t i n g procedure  u s i n g c o l o n i e s of the serotype s t r a i n s of P. fibrosis  was  demonstrated  a e r u g i n o s a and  the  cystic  i s o l a t e s , by showing t h a t monoclonal a n t i b o d y MA1-8, s p e c i f i c f o r  the serotype 5 LPS-O-antigen, showed p o s i t i v e b i n d i n g to s e r o t y p e s 5, and CFC1  mucoid and non-mucoid, but  o t h e r c o l o n i e s ( T a b l e X, F i g . 18).  showed no  V).  i n t e r a c t i o n w i t h any of the  Thus, the c o l o n y b l o t  r e f l e c t e d the s p e c i f i c i t y of a n t i b o d y MA1-8  results  as shown above by ELISA ( T a b l e  In a d d i t i o n , monoclonal a n t i b o d y MA1-6, which i s s p e c i f i c  membrane l i p o p r o t e i n H2, a l t e r e d mutants of P. F i g . 19). studies  i n t e r a c t e d o n l y w i t h c o l o n i e s of rough  a e r u g i n o s a and not w i t h  smooth s t r a i n s  T h i s confirmed the r e s u l t s of i n d i r e c t  (Table V I I I ) s u g g e s t i n g t h a t p r o t e i n H2  s u r f a c e of w i l d type  (smooth) P.  aeruginosa  Mucoid c o l o n i e s of P. a e r u g i n o s a and not shown any d i f f e r e n c e s c o l o n y b l o t procedure  in their  17  f o r outer LPS-  (Table X,  immunofluorescence  i s not e x p r e s s e d on  the  strains.  t h e i r non-mucoid r e v e r t a n t s d i d  i n t e r a c t i o n w i t h the a n t i b o d i e s by  the  ( F i g s . 17,18,19) d e s p i t e the f a c t t h a t the mucoid  material (alginate exopolysaccharide)  appeared  t o have been t r a n s f e r r e d t o  the n i t r o c e l l u l o s e b l o t t o g e t h e r w i t h the b a c t e r i a l c e l l s .  The  mucoid  m a t e r i a l d i d not a p p a r e n t l y mask the a c c e s s i b i l i t y o f the p r o t e i n to the  Table X. B i n d i n g o f 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 P.. aeruginosa outer membrane antigens to colony b l o t s • 3  Bacterial Strains  MA2-10  P. aeruginosa s t r a i n s H103 H283 ( p r o t e i n F d e f i c i e n t ) Serotype 5 Serotype l ^ ) Z61 LPS-rough s t r a i n s ^ )  + + + + +  Cystic Fibrosis isolates CF 221^ ^ ' CFC46 mucoid non-mucoid r e v e r t a n t  Protein F MA4-4  +  + + + + +  +  +. + +  + + +  + + +  + -  Other Pseudomonads Group l ^ ' Pseudomonads P. p u t i d a (2 s t r a i n s ) P. f l u o r e s c e n s (2 s t r a i n s ) P. syringae P. c h l o r o r a p h i s P_. a u r e o f a c i e n s P. s t u t z e r i P. a n g u i l l i s e p t i c a A. v i n e l a n d i i  + + -  + + -  -  + +  Other Pseudomonads (groups 2 , 3 , 4 )  -  -  -  a  c  d  + + +  P r o t e i n H2 MA1-6  MA5-8  +  LPS O-Antigen MA1-8  + + + + -  -  )  Other gram-negative (9 s t r a i n s )  -  +  -  + + + + +  -  -  ,  -  115  T a b l e X Legend  + p o s i t i v e c o l o u r development  on c o l o n y ,  a  A l l other methods.  s t r a i n s were as d e s c r i b e d  b  P. aeruginosa and H223.  c  The other P. a e r u g i n o s a i s o l a t e s from c y s t i c f i b r o s i s p a t i e n t s were (CF832, CF1452, CF2314, CF4522, CF3660-1, CF4349, CFL, CF6094, CF1278, CF284, CF3790, CF9490, and 9 mucoid s t r a i n s (CFC lm, CFC 21m, CFC 20m, CFC 6m, CFC 81m, CFC 47m, CFC 4m, CFC 91m, and CFC 96m) and t h e i r spontaneous non-mucoid r e v e r t a n t s o b t a i n e d from D.P. S p e e r t , C h i l d r e n ' s H o s p i t a l , Vancouver, and G.P. P i e r , Harvard. A s t r a i n , CF 283, d i d not i n t e r a c t w i t h any o f the monoclonal antibodies. T h i s s t r a i n was shown to not be a P. a e r u g i n o s a s t r a i n by f a t t y a c i d a n a l y s i s ( L . Chan). The rRNA homology grouping by DeVos and DeLey (1983) d i v i d e s Pseudomonads i n t o 4 groups w i t h A. v i n e l a n d i i i n group 1. P. p u t i d a s t r a i n s were ATCC 4359 and ATCC 12633; P. f l u o r e s c e n s s t r a i n s were ATCC 949 and 13525.  d  e  serotype  specific  LPS 0 a n t i g e n d e f i c i e n t  The o t h e r s t r a i n s were P. a c i d o v o r a n s , P. pseudomallei and P. m a l t o p h i l i a .  - no c o l o u r development  on  colony.  i n m a t e r i a l s and  s t r a i n s were AK1012, AK1282  P. solanacearum, P.  cepacia,  *• The gram-negative s t r a i n s were S a l m o n e l l a typhimurium LT2 SGSC205 and SGSC227; V i b r i o a n g u i l l a r u m HT 7602 Aeromonas h y d r o p h i l a ET2; Aeromonas s a l m o n i c i d a NCMB 2020; E s c h e r i c h i a c o l i CGSC 6041, CGSC 6044, PC 0479, and E d w a r d s i e l l a t a r d a ET 9054.  116  F i g u r e 17.  Colony  immunoblots showing i n t e r a c t i o n o f monoclonal  MA4-10, s p e c i f i c f o r p r o t e i n strains:  antibody  F o f P. a e r u g i n o s a w i t h the f o l l o w i n g  1 - P. a e r u g i n o s a s t r a i n PI non-mucoid; 2 - P. a e r u g i n o s a s t r a i n  PI mucoid; 3,4 - P. f l u o r e s c e n s ATCC 949 and 13525 (type respectively;  strain)  5 - P. a e r u g i n o s a PA01 s t r a i n H103; 6 - P. a e r u g i n o s a  strain  CFC1 mucoid; 7 - P. a e r u g i n o s a s t r a i n CFC1 non-mucoid; 8,9 and 10 Escherichia pseudomallei  coli  s t r a i n s CGSC6041, CGSC6044 and PC0479; 11 - P.  ATCC23343; 12 - P. solanacearum ATCC 11696; 13,14 - P. p u t i d a  ATCC 4359 and 12633 (type s t r a i n ) r e s p e c t i v e l y ;  15,16 - S a l m o n e l l a  typhimurium LT2 s t r a i n s SGSC206 and SGSC227 r e s p e c t i v e l y ;  17 - P.  a e r u g i n o s a ATCC 8689; 18 - P. a e r u g i n o s a PA01 s t r a i n H103; 19 - Aeromonas s a l m o n i c i d a NCMB 2020; 20 - Aeromonas h y d r o p h i l a ET2; 21 - P. c h l o r o r a p h i s ATCC 9446; 22 - P. a e r u g i n o s a  (type s t r a i n ) ATCC 19305; 23 - P.  a e r u g i n o s a s t r a i n Z61; 24 - P. a u r e o f a c i e n s ATCC 13985; 25 - P. s y r i n g a e ATCC 19310; 26 - P. a e r u g i n o s a CF4349; 27 - P. m a l t o p h i l i a  ATCC 13639; 28  - P. s t u t z e r i ATCC 17588; 29 and 30 - P. a e r u g i n o s a s t r a i n s AK1012 and AK1282.  117  118  F i g u r e 18. MA1-8  Colony immunoblot showing i n t e r a c t i o n  o f monoclonal a n t i b o d y  s p e c i f i c f o r serotype 5-0 a n t i g e n o f P. a e r u g i n o s a w i t h the  f o l l o w i n g serotype s t r a i n s . 2 - P. a e r u g i n o s a s t r a i n s  1-P. a e r u g i n o s a serotype 1 s t r a i n ;  H103; 3 - P. a e r u g i n o s a serotype 2 s t r a i n s ;  4 - s e r o t y p e 3; 5 - serotype 4; 6 - s e r o t y p e 5; 7 - s e r o t y p e 6; 8 - s e r o t y p e 7; 9 - serotype 8; 10 - s e r o t y p e 9; 11 - s e r o t y p e 10; 12 - s e r o t y p e 11; 13 - s e r o t y p e 12; 14 - s e r o t y p e 13; 15 - s e r o t y p e 15; 16 - s e r o t y p e 16; 17 - s e r o t y p e 17; 18 - P. a e r u g i n o s a s t r a i n H188; 19 P. a e r u g i n o s a CFPlnm; 20 - P. a e r u g i n o s a serotype 14; 21 - P. a e r u g i n o s a s t r a i n H223 (LPS rough); 22 - P. a e r u g i n o s a s t r a i n H235 (LPS rough); 23 - P. a e r u g i n o s a CFC91m; 24 - P. a e r u g i n o s a CFClm; 25 - P. a e r u g i n o s a CFClm; 26, 27 - P. a e r u g i n o s a CFC96m and CFC96nm.  120  F i g u r e 19.  Colony  immunoblots showing i n t e r a c t i o n o f monoclonal  MAl-6, s p e c i f i c f o r p r o t e i n strains:  1-P.  H2 o f P. a e r u g i n o s a w i t h the f o l l o w i n g  aeruginosa s t r a i n PI non-mucoid; 2 - P. a e r u g i n o s a  PI mucoid; 3,4 - P. f l u o r e s c e n s ATCC 949 and 13525 (type respectively; CFC1  antibody  strain  strain)  5 - P. a e r u g i n o s a PA01 s t r a i n H103; 6 - P. a e r u g i n o s a s t r a i n  mucoid; 7 - P. a e r u g i n o s a s t r a i n CFC1 non-mucoid; 8,9 and 10 -  Escherichia pseudomallei  coli  s t r a i n s CGSC6041, CGSC6044 and PC0479; 11 - P.  ATCC23343; 12 - P. solanacearum ATCC 11696; 13,14 - P. p u t i d a  ATCC 4359 and 12633 (type s t r a i n ) r e s p e c t i v e l y ;  15,16 - S a l m o n e l l a  typhimurium LT2 s t r a i n s SGSC206 and SGSC227 r e s p e c t i v e l y ;  17 - P.  a e r u g i n o s a ATCC 8689; 18 - P. a e r u g i n o s a PA01 s t r a i n H103; 19 - Aeromonas s a l m o n i c i d a NCMB 2020; 20 - Aeromonas h y d r o p h i l a ET2; 21 - P. c h l o r o r a p h i s ATCC 9446; 22 - P. a e r u g i n o s a  (type s t r a i n ) ATCC 19305; 23 - P.  a e r u g i n o s a s t r a i n H188; 24 - P. a u r e o f a c i e n s ATCC 13985; 25 - P. s y r i n g a e ATCC 19310; 26 - P. a e r u g i n o s a CF4349; 27 - P. m a l t b p h i l i a  ATCC 13639; 28  - P. s t u t z e r i ATCC 17588; 29 - P. a e r u g i n o s a s t r a i n H223; 30 and 31 P. a e r u g i n o s a s t r a i n s AK1012 and AK1282.  121  * 1 4 3 8  9  5 10  -» »-»  11 12  13  •  18 24 29  ft  15  14  20  21  26  27  30  I  16  » f/  22  23 /  */  122  antibody nor d i d i t adsorb the c o l o n y . was  The  a n t i b o d i e s n o n - s p e c i f i c a l l y to the s u r f a c e of  l a c k of n o n - s p e c i f i c antibody b i n d i n g to mucoid m a t e r i a l  demonstrated by the l a c k of i n t e r a c t i o n of monoclonal antibody MAl-6  ( s p e c i f i c f o r p r o t e i n H2)  w i t h c o l o n i e s of a mucoid d e r i v a t i v e of  CFC46, whose outer membranes were p r e v i o u s l y demonstrated t o be i n p r o t e i n H2 by both SDS-polyacrylamide Western immunoblot  ( F i g . 11)  analysis.  c o l o n i e s e x p r e s s i n g p r o t e i n H2 treatment  deficient  g e l e l e c t r o p h o r e s i s ( F i g . 2) Antibody  (wt/vol) SDS  and  MAl-6 i n t e r a c t e d with a l l  (as judged by Western b l o t s ) ,  of c o l o n i e s w i t h 0.1%  strain  or 0.1  after  M NaCl t o cause  membrane d i s r u p t i o n . The  a c c e s s i b i l i t y of LPS  was  a l s o found  mucoid e x o p o l y s a c c h a r i d e when the LPS antibody MA1-8  was  used  in this  d e r i v a t i v e s of s t r a i n CFC1, i n t e r a c t e d w i t h MA1-8  (C)  study.  the  O - a n t i g e n - s p e c i f i c monoclonal Both the mucoid and  which expressed  on c o l o n y b l o t s  t o be u n a f f e c t e d by  serotype 5  non-mucoid  0-antigen  ( F i g . 18, T a b l e X ) .  I n t e r a c t i o n of monoclonal a n t i b o d i e s w i t h o t h e r b a c t e r i a by colony immunoblot The  the  procedure  i n t e r a c t i o n of p r o t e i n F - s p e c i f i c monoclonal a n t i b o d i e s w i t h  other Pseudomonas s t r a i n s and v a r i o u s gram-negative b a c t e r i a was by the c o l o n y b l o t procedure Monoclonal  antibody MA5-8 was  studied  d e s c r i b e d above ( T a b l e X, F i g . 17,19). highly specific  f o r P.  aeruginosa  strains,  w h i l e MA4-4, MA2-10, and MA4-10 i n t e r a c t e d w i t h both s t r a i n s P. p u t i d a and the s i n g l e P.  syringae s t r a i n  t e s t e d ( F i g . 17; T a b l e X ) .  None of  these  monoclonal a n t i b o d i e s i n t e r a c t e d w i t h whole c e l l s of P. f l u o r e s c e n s ,  123  P. a n g u i l l i s e p t i c a , P. c h l o r o r a p h i s . P. a u r e o f a c i e n s . P. s t u t z e r i  o r A.  v i n e l a n d i i . a l l o f which had been p r e v i o u s l y demonstrated t o be r e l a t e d t o P. a e r u g i n o s a by rRNA homology experiments (DeVos and DeLey, 1983). c o n t r a s t , the l i p o p r o t e i n H 2 - s p e c i f i c monoclonal a n t i b o d y MAl-6  In  interacted  w i t h a l l group 1 Pseudomonads e x p r e s s i n g a p r o t e i n e q u i v a l e n t t o l i p o p r o t e i n H2 ( F i g . 1 9 ) . There was no i n t e r a c t i o n o f MAl-6 w i t h any o f the s t r a i n s from t h e E n t e r o b a c t e r i a c e a e , V i b r o n a c e a e , Aeromonas s t r a i n s or o t h e r Pseudomonads i n rRNA homology groups 2, 3 and 4 (DeVos and DeLey, 1981)  (Table X ) .  2.  S t u d i e s on p r o t e i n F e p i t o p e s r e c o g n i z e d by monoclonal  (A)  E f f e c t o f 2-mercaptoethanol on b i n d i n g o f p r o t e i n monoclonal  antibodies.  F-specific  antibodies  The high s p e c i f i c i t y o f monoclonal a n t i b o d i e s has been used t o study the s t r u c t u r e and a n t i g e n i c domains o f p r o t e i n s Virji  e_t a l . , 1983).  (Kenimer §_t a l . , 1983;  We used the p r o t e i n F - s p e c i f i c monoclonal  antibodies  MA5-8, MA4-4, MA2-10 and MA4-10, i n an attempt t o d e f i n e some o f t h e a n t i g e n i c domains ( e p i t o p e s ) o f t h i s  protein.  The apparent m o l e c u l a r weight, i . e t h e e l e c t r o p h o r e t i c m o b i l i t y o f p r o t e i n F o f P. a e r u g i n o s a  on SDS-polyacrylamide g e l s , i s markedly  i n f l u e n c e d by the s o l u b i l i z a t i o n c o n d i t i o n s also Figure 1).  When s o l u b i l i z e d  (Hancock and C a r e y , 1979; see  i n the presence o f 2-mercaptoethanol,  p r o t e i n F m i g r a t e s as a 41,000 d a l t o n p r o t e i n on a 14% p o l y a c r y l a m i d e gel.  The non-reduced p r o t e i n has an apparent m o l e c u l a r weight o f 37,000  daltons  (Hancock and Carey, 1979).  The reason f o r t h i s  i s almost  124  F i g u r e 20.  Western immunoblot o f p u r i f i e d p r o t e i n F - e f f e c t o f  2-mercaptoethanol.  P u r i f i e d p r o t e i n F was s e p a r a t e d on SDS 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 t o g r a m s a f t e r s o l u b i l i z a t i o n w i t h ( l a n e s A and D) o r without  ( l a n e s B, C, and E) 2-mercaptoethanol.  After  electrophoretic  t r a n s f e r t o n i t r o c e l l u l o s e , the b l o t s were i n t e r a c t e d w i t h MA4-4 ( l a n e s A and B) o r MA5-8 ( l a n e s C, D and E ) .  Lane E r e c e i v e d 5 times more p r o t e i n  F (5 yg) than Lanes A - D, i n o r d e r t o demonstrate not v i s i b l e  i n Lanes A - D.  the o l i g o m e r bands  125  126  certainly  the presence  of one or two  i n t r a c h a i n d i s u l p h i d e bonds. Thus,  when these d i s u l p h i d e bonds are not reduced,  the p r o t e i n runs  compact c o n f i g u r a t i o n w i t h a h i g h e r r e l a t i v e m o b i l i t y .  i n a more  Monoclonal  a n t i b o d i e s MA4-4, MA2-10 and MA4-10 i n t e r a c t e d o n l y w i t h the non-reduced form o f p r o t e i n F ( F i g . 20, l a n e B ) , w h i l e MA5-8 i n t e r a c t e d w i t h both  the  2-mercaptoethanol-reduced  D).  Monoclonal  and non-reduced forms ( F i g . 20,  a n t i b o d y MA5-8 was  h i g h e r m o l e c u l a r weight  a l s o unique  l a n e s C and  i n that i t i n t e r a c t e d with  or o l i g o m e r i c forms o f p r o t e i n F (as seen when  s u f f i c i e n t p r o t e i n F was  added t o the g e l ; F i g . 20, l a n e E ) .  These forms  of p r o t e i n F were not observed when o u t e r membranes o f the p r o t e i n F d e f i c i e n t mutant H283 were used  i n Western immunoblot s t u d i e s ( F i g . 9 ) ,  s u g g e s t i n g t h a t they d i d not r e p r e s e n t a r t e f a c t s due monoclonal  antibody MA5-8 w i t h o t h e r p r o t e i n s .  to c r o s s - r e a c t i o n o f  The o l i g o m e r i c  a s s o c i a t i o n s of p r o t e i n F were p r e s e n t i n v e r y low c o n c e n t r a t i o n s i n o u t e r membranes but were a p p a r e n t l y somewhat e n r i c h e d d u r i n g p u r i f i c a t i o n o f p r o t e i n F.  These d a t a p r o v i d e d evidence f o r the e x i s t e n c e o f  o l i g o m e r i c forms o f p r o t e i n  (B)  F.  I n t e r a c t i o n o f monoclonal of p r o t e i n  SDS-stable  a n t i b o d i e s w i t h cyanogen bromide  fragments  F.  P e p t i d e fragments cyanogen bromide.  o f p r o t e i n F were d e r i v e d by c h e m i c a l c l e a v a g e w i t h  Cyanogen bromide treatment  p r o t e i n F y i e l d e d s i x fragments, presence o f 2-mercaptoethanpl  two  o f n a t i v e or  o f which had  denatured  altered mobility  s u g g e s t i n g they c o n t a i n e d one  i n t r a c h a i n d i s u l p h i d e bonds o f p r o t e i n F ( F i g . 21).  The  i n the  or more o f the  t o t a l molecular  127  F i g u r e 21.  SDS-polyacrylamide  gel electrophoretogram  and Western  immunoblot o f p u r i f i e d p r o t e i n F b e f o r e and a f t e r d e g r a d a t i o n cyanogen bromide. B,D:  Lane A,C:  p u r i f i e d p r o t e i n F.  from an SDS-polyacrylamide  with  cyanogen bromide p e p t i d e s o f p r o t e i n F; Lane  P r o t e i n F and i t s p e p t i d e s were t r a n s f e r r e d g e l ( l a n e s A and B) t o n i t r o c e l l u l o s e  (lanes C  and D) and i n t e r a c t e d w i t h monoclonal antibody MA5-8 on the nitrocellulose. interact  The two cyanogen bromide p e p t i d e s o f p r o t e i n F which  w i t h antibody MA5-8 had m o l e c u l a r weights  o f 28,000 and 23,000.  In the cyanogen bromide c l e a v a g e p a t t e r n i n l a n e C, t h e n a t i v e p r o t e i n F formed a d o u b l e t due t o p a r t i a l heat m o d i f i c a t i o n o f t h e undegraded p r o t e i n F (Hancock and Carey, protein F preferentially visible  1979).  T h i s s m a l l r e s i d u e o f undegraded  bound the monoclonal a n t i b o d y , s i n c e i t was not  i n the g e l p a t t e r n i n lane A.  128  A B  C D  129  weight  o f the s i x p e p t i d e s , however, was approximately t h r e e times t h a t o f  monomeric p r o t e i n F.  The d i s c r e p a n c y i n m o l e c u l a r weight  to incomplete c l e a v a g e .  was p r o b a b l y due  T h i s has been shown t o occur e.g.  incomplete c l e a v a g e o f Met-Ser o r Met-Thr sequences,  due t o  which can form  homoserine and a r e t h e r e f o r e not c l e a v e d , l e a d i n g t o the p r o d u c t i o n o f o v e r l a p p i n g cyanogen bromide fragments than the n a t i v e p r o t e i n was  (Garten e t a l . , 1975).  a l s o observed w i t h denatured  digestion conditions. of the fragments.  o f t o t a l m o l e c u l a r weights  2-mercaptoethanol-modifiable  antibody MA5-8 i n t e r a c t e d w i t h any  i t i n t e r a c t e d w i t h the two  fragments  and non-reduced forms ( F i g . 21).  cleavage  p r o t e i n , and under more r i g o r o u s  Only monoclonal  Specifically,  T h i s incomplete  greater  o f p r o t e i n F i n both t h e reduced  The i n a b i l i t y  t o generate s m a l l e r  antibody r e a c t i v e p e p t i d e s under h a r s h e r c o n d i t i o n s ( p r e - t r e a t m e n t w i t h 85% g l a c i a l a c e t i c a c i d ) p r o b a b l y r e f l e c t e d a requirement f o r s t a b i l i z a t i o n o f t h e a n t i g e n i c e p i t o p e by maintenance o f t h e S D S - r e s i s t a n t , B-sheet  c o n f o r m a t i o n o f p r o t e i n F (Mizuno  and Kageyama,  1979), thus l i m i t i n g t h e i d e n t i f i c a t i o n o f t h e a n t i g e n i c domains.  (C)  I n t e r a c t i o n o f monoclonal  a n t i b o d i e s with p r o t e o l y t i c p e p t i d e s o f  protein F Enzymatic s h o r t times  d i g e s t i o n o f p r o t e i n F w i t h S. aureus V8 p r o t e a s e f o r v e r y  (< 1 min) y i e l d e d a s e r i e s o f p e p t i d e s o f m o l e c u l a r  weights  below 12,000 d a l t o n s , none o f which i n t e r a c t e d w i t h any o f t h e antibodies.  When p u r i f i e d p r o t e i n F was t r e a t e d w i t h p a p a i n , i t r a p i d l y  broke down t o two p e p t i d e fragments  w i t h m o l e c u l a r weights  o f 29,000 and  130  31,500 ( F i g . 22,  l a n e H).  Both of these p e p t i d e s were 2-mercaptoethanol  m o d i f i a b l e and r e a c t e d w i t h monoclonal a n t i b o d i e s MA2-10, MA4-4 and MA4-10 ( i n the unreduced form o n l y ) lane D). cells  ( F i g . 22,  l a n e H) but not w i t h MA5-8 ( F i g . 22  P r o t e i n F i n o u t e r membranes ( F i g . 22,  ( F i g . 22,  l a n e s N and 0) was  l a n e s I-M)  and  a l s o s u s c e p t i b l e to p a p a i n ,  intact although  f i v e - f o l d h i g h e r amounts of enzyme were r e q u i r e d and d i g e s t i o n d i d not proceed be due LPS.  as r e a d i l y to completion  ( e s p e c i a l l y w i t h whole c e l l s ) .  This  may  to p a r t i a l masking of the p a p a i n - s u s c e p t i b l e s i t e s of p r o t e i n F by In both o u t e r membranes and  intact c e l l s ,  p e p t i d e bands ( F i g . 22,  l a n e s L and N) of i n t e r m e d i a t e m o l e c u l a r weight between the undegraded p r o t e i n F (37 kD) were observed, due  and  the above two  p e p t i d e fragments (29 kD  i n c l u d i n g bands of 36,  35,  34,  33 and 32.5  and 31.5  kD.  However,  to the tendency of p r o t e i n F to run i n a v a r i e t y of p o s i t i o n s on  polyacrylamide  g e l s (Hancock and Carey, 1979)  c e r t a i n t y t h a t these bands r e p r e s e n t  i t is d i f f i c u l t  fragment of 31 kD  intermediates. When  used, a s i n g l e 2 - m e r c a p t o e t h a n o l - m o d i f i a b l e  formed.  SDS  to say w i t h  D i g e s t i o n of p r o t e i n F w i t h t r y p s i n gave s i m i l a r r e s u l t s . p u r i f i e d p r o t e i n F was  kD)  peptide  T h i s fragment i n t e r a c t e d w i t h monoclonal  a n t i b o d i e s MA2-10, MA4-4 and MA4-10 i n the unreduced form ( F i g . 22, l a n e F ) ] but not w i t h MA5-8 ( F i g . 22, membranes and whole c e l l s was times  l a n e K).  Again, p r o t e i n F i n outer  more r e s i s t a n t to t r y p s i n , r e q u i r i n g  as much enzyme, and r e s u l t i n g  m o l e c u l a r weight forms of 34.5,  33.5  i n the appearance of and  32.5  kD  intermediate  ( F i g . 22,  a l l of which i n t e r a c t e d w i t h MA2-10, MA4-4 and MA4-10. which appeared i n p u r i f i e d p r o t e i n F, o u t e r membrane and  ten  The  l a n e s M and 31 kD  intact  0)  fragment  cell  131  F i g u r e 22.  Western immunoblot o f n a t i v e and p r o t e o l y t i c a l l y t r e a t e d  p r o t e i n F from a p u r i f i e d p r o t e i n F sample ( l a n e s A - H), o u t e r membranes (lanes I - M) and whole c e l l s  l a n e s (N - 0 ) .  The p r o t e o l y t i c enzymes and  the monoclonal a n t i b o d i e s used t o r e v e a l the p o s i t i o n s o f the n a t i v e p r o t e i n F and i t s p r o t e o l y t i c  fragments were l a n e A - no  treatment,  antibody MA5-8; lane B - t r y p s i n , MA5-8; l a n e C - S. aureus V8 p r o t e a s e , MA5-8; lane D - papain, MA5-8; lane E - no treatment,  MA4-10, l a n e F -  t r y p s i n ; MA4-10; l a n e G - V8 p r o t e a s e , MA4-10, l a n e H - papain, lane I - no treatment,  MA4-10;  MA5-8; l a n e J - p a p a i n , MA5-8; lane K - t r y p s i n ,  MA5-8; l a n e L - papain, MA4-10; l a n e M - t r y p s i n , MA4-10; lane N T r y p s i n , MA4-10; l a n e 0 - papain, MA4-10.  132  133  F i g u r e 23.  SDS-polyacrylamide  from a time course p r o t e o l y s i s trypsin.  2-mercaptoethanol.  2, p u r i f i e d p r o t e i n  hr.,  of p u r i f i e d p r o t e i n  F w i t h the enzyme  The samples were s o l u b i l i z e d a t 88°C f o r 10 min i n r e d u c t i o n  mix w i t h o u t  protein  g e l electrophoretogram of peptides derived  Lane 1, m o l e c u l a r weight  s t a n d a r d s ; Lane  F ( c o n t r o l ) ; Lanes 3 and 9 show t r y p t i c p e p t i d e s o f  F a f t e r 30 min., 60 min., 90 min., 2 h r . , 2.5 h r . , 3 h r . , and 3.5  p r o t e o l y s i s with t r y p s i n  respectively.  134  135  Figure  24.  I n t e r a c t i o n o f p r o t e i n F - s p e c i f i c monoclonal a n t i b o d y MA2-10  with p r o t e o l y t i c a l l y t r e a t e d whole c e l l s o f P. a e r u g i n o s a CFC46nm and P. p u t i d a ATCC 12633.  The p r o t e o l y t i c enzymes and the s t r a i n  treated  were, Lane 1, P. a e r u g i n o s a CFC46nm, no treatment; Lane 2, CFC46nm, t r y p s i n ; Lane 3, CFC46nm, papain; Lane 4, P r o t e i n F c o n t r o l ; Lane 5, P. putida, papain.  no treatment; Lane 6, P. p u t i d a ,  t r y p s i n ; Lane 7, P. p u t i d a .  136  137  p r e p a r a t i o n s a f t e r treatment proteolysis  with t r y p s i n was r e s i s t a n t  ( F i g . 2 3 ) . Experiments  to f u r t h e r  with the p r o t e i n F e q u i v a l e n t i n P.  p u t i d a whole c e l l s demonstrated t h a t t h i s 39 kD p r o t e i n broke down t o a 31 kD,  2-mercaptoethanol-modifiable  tryptic  fragment which i n t e r a c t e d w i t h  monoclonal a n t i b o d y MA2-10 ( F i g . 24, lane 6 ) . T h i s P. p u t i d a p r o t e i n was a l s o s u s c e p t i b l e t o papain and a p e p t i d e o f a p p r o x i m a t e l y observed  29 kD was  ( l a n e 7 ) . D i g e s t i o n o f whole c e l l s o f P. a e r u g i n o s a CFC46nm w i t h  t r y p s i n and papain r e s u l t e d  i n the appearance o f a f a m i l y o f p e p t i d e s  ( F i g . 24, l a n e s 2 and 3) o f m o l e c u l a r weights  s i m i l a r t o those  observed  w i t h whole c e l l d i g e s t s o f P. a e r u g i n o s a s t r a i n H103 ( F i g . 22, lane N, 0).  These p e p t i d e s i n t e r a c t e d w i t h antibody MA2-10.  3.  Summary The use o f monoclonal a n t i b o d i e s i n the c o l o n y b l o t a n a l y s i s  enabled  r a p i d s c r e e n i n g o f P. a e r u g i n o s a s t r a i n s f o r the s u r f a c e exposure o f s i n g l e a n t i g e n s l i k e p o r i n p r o t e i n F.  The r e s u l t s o b t a i n e d  correlated  w e l l w i t h those o f the immunofluorescence s t a i n i n g a n a l y s i s . may w e l l prove  T h i s method  t o be a powerful t o o l f o r s c r e e n i n g f o r v a r i a n t s and  mutants o f o u t e r membrane components i n g e n e t i c s t u d i e s . S t u d i e s on the c r o s s r e a c t i v i t y o f monoclonal a n t i b o d i e s a g a i n s t outer membrane p r o t e i n F o f Pseudomonas a e r u g i n o s a demonstrated t h a t these monoclonal a n t i b o d i e s were o f two d i s t i n c t  specificities.  One antibody,  MA5-8, i n t e r a c t e d o n l y w i t h P. a e r u g i n o s a s t r a i n s whereas t h r e e o t h e r monoclonal a n t i b o d i e s , MA2-10, MA4-4 and MA4-10, c r o s s - r e a c t e d w i t h P. s y r i n g a e and P. p u t i d a s t r a i n s .  These two c l a s s e s o f a n t i b o d i e s were  d i s t i n g u i s h a b l e by r e a c t i v i t y w i t h 2 - m e r c a p t o e t h a n o l - t r e a t e d  protein F  138  ( F i g . 20), w i t h p r o t e i n F oligomers  ( F i g . 20), w i t h cyanogen bromide  fragments of p r o t e i n F ( F i g . 21), and w i t h p r o t e o l y t i c fragments of p r o t e i n F ( F i g . 22). e p i t o p e as judged F i g . 16) cells.  by  Both c l a s s e s of a n t i b o d i e s r e c o g n i z e d a s u r f a c e indirect  immunofluorescent  and c o l o n y immunoblotting The  labelling  ( T a b l e X) of i n t a c t P.  d a t a f a v o u r the e x i s t e n c e of two  (Table V I I I , aeruginosa  s e p a r a t e h i g h l y conserved  s u r f a c e e p i t o p e s on o u t e r membrane p r o t e i n F i n P. These s t u d i e s a l s o demonstrated the presence  aeruginosa. of a c o n s e r v e d a n t i g e n i c  e p i t o p e on l i p o p r o t e i n H2 of the rRNA homology group I Pseudomonadaceae (Table X, F i g . 12). immunoblotting  By both  indirect  immunofluorescence and  u s i n g antibody MA1-6, p r o t e i n H2 was  a c c e s s i b l e o n l y i n rough L P S - a l t e r e d  organisms.  colony  shown t o be s u r f a c e  139  DISCUSSION  The  gram-negative b a c t e r i a l c e l l  s u r f a c e (outer membrane) c o n t a i n s  the components, namely p r o t e i n s and LPS,  t h a t are i n v o l v e d i n the  i n t e r a c t i o n of these b a c t e r i a w i t h the host's immune system. of molecules  primary  Both c l a s s e s  were s t u d i e d i n t h i s t h e s i s and w i l l be d i s c u s s e d s e p a r a t e l y  below.  1.  C o n s e r v a t i o n and h e t e r o g e n e i t y o f LPS E x t e n s i v e s t u d i e s have c l e a r l y  LPS  i n the pathogenesis  epitopes  shown a v a r i e t y of p o t e n t i a l r o l e s f o r  of gram-negative b a c t e r i a  study monoclonal a n t i b o d i e s were used  ( B r a d l e y 1979).  In  this  t o probe the a n t i g e n i c c o n s e r v a t i o n  of d i f f e r e n t r e g i o n s of the LPS m o l e c u l e :  the immunodominant O - a n t i g e n i c  s i d e c h a i n s , the rough o l i g o s a c c h a r i d e core and  the l i p i d A p o r t i o n (see  T a b l e XI f o r a summary). A monoclonal a n t i b o d y MA1-8, which r e a c t e d w i t h the O - a n t i g e n i c r e g i o n of the LPS  of s e r o t y p e 5 s t r a i n s ,  showed weak c r o s s - r e a c t i o n w i t h  the outer membranes of a s e r o t y p e 17 s t r a i n , but no r e a c t i o n w i t h from 15 other s e r o t y p e s  ( T a b l e V) .  MA1-8  may  w e l l be d i r e c t e d a g a i n s t  a n t i g e n 2d which i s a p p a r e n t l y shared by serotype 5 and 17 Bergan, 1979).  The  h i g h s p e c i f i c i t y o f t h i s monoclonal a n t i b o d y  composition  The  s p e c i f i c i t y of MA1-8  agrees  i n the  i s responsible f o r serotyping differences  L a n y i and Bergan, 1979).  LPS  ( L a n y i and  w i t h the c o n c l u s i o n o f numerous o t h e r r e p o r t s t h a t v a r i a t i o n O-antigen  strains  (see  f o r the O-antigen  LPS e.g. was  Table X I .  R e a c t i o n of LPS s p e c i f i c monoclonal a n t i b o d i e s with outer membrane from Pseudombnas aeruginosa s t r a i n s i n ELISA assays  R e a c t i o n u s i n g monoclonal a n t i b o d i e s 0 Antigen type 5 Rough Core (MA1-8) (MA3-6) (MA3-5)  Outer Membranes  P. P. P. P. P. P. P. P.  a  aeruginosa aeruginosa aeruginosa aeruginosa aeruginosa aeruginosa aeruginosa  PAOI rough serotype 5 serotype 17 serotypes .7,8,10,14,16 serotype 11 serotypes 1 , 2 , 3 , 4 , 6 , 9 , 12,13,15 aeruginosa c l i n i c a l s t r a i n s  +  + +  + +  +  +  +  +  +  -  -  +  +  + +  +  (2/34)  b  reactions  out of the number of s t r a i n s  tested.  +  +  -  +/-(l/20)  weaker r e a c t i o n due to a shared determinant between serotype positive  Lipid A (5E4)  -  -  +/-  to LPS  b  +/-(3/20)  +  +(20/20)  b  17 and serotype  5.  b  141  shown by the l a c k o f i n t e r a c t i o n of the a n t i b o d y w i t h rough d e f i c i e n t ) d e r i v a t i v e s of a serotype 5 s t r a i n Monoclonal  antibodies s p e c i f i c  (O-antigen  (Table I V ) .  f o r rough LPS (MA3-5 and MA3-6)  r e a c t e d with a l i m i t e d subset o f the P. a e r u g i n o s a s e r o t y p e s t r a i n s and cystic  f i b r o s i s P. aeruginosa  demonstrated, f o r the f i r s t aeruginosa s t r a i n s . of  isolates  ( F i g . 13, T a b l e X I ) .  time, LPS rough c o r e h e t e r o g e n e i t y among P.  I n c o n t r a s t s t r u c t u r a l s t u d i e s on the hexose r e g i o n  the LPS-rough core o f 600 S a l m o n e l l a s p e c i e s suggested  c o r e (Jahnsson  These r e s u l t s  e t a l . , 1981).  However, more d e t a i l e d  s p e c i f i c monoclonal a n t i b o d i e s would be n e c e s s a r y  a s i n g l e type o f  studies using  t o c o n c l u s i v e l y show the  c o n s e r v a t i o n o f the s i n g l e a n t i g e n i c s t r u c t u r e o f t h i s r e g i o n o f the LPS in  Salmonella.  that the  D e s p i t e the observed  heterogeneity, the data  suggested  i n P. a e r u g i n o s a the rough core was a n t i g e n i c a l l y more conserved  than  O-antigen. The monoclonal a n t i b o d i e s t o l i p i d A i n t e r a c t e d w i t h a l l o f the  s t r a i n s o f P. a e r u g i n o s a t e s t e d , as w e l l as many o t h e r genera  o f many  gram-negative b a c t e r i a  These  ( F i g s . 14 and 15, T a b l e s VI and X I I ) .  r e s u l t s demonstrated t h a t the L P S - L i p i d A r e g i o n i s h i g h l y conserved gram-negative b a c t e r i a . A's  among  T h i s may e x p l a i n the s i m i l a r a c t i v i t i e s o f L i p i d  (endotoxin) from d i f f e r e n t b a c t e r i a  ( B r a d l e y , 1979).  Thus, i t would appear t h a t LPS h e t e r o g e n e i t y i n c r e a s e s f o r s u b s t i t u e n t s t h a t are f u r t h e r from the o u t e r membrane i . e . LPS components c l o s e s t t o the outer membrane s u r f a c e a r e g r e a t l y conserved The  (Table X I ) .  d i s t a l p o r t i o n s o f the LPS i n b a c t e r i a , e s p e c i a l l y the O-antigen  , are  142  usually 1980;  s t r a i n or s p e c i e s  s p e c i f i c (Table  XI,  see  a l s o Goldman and  B r a d l e y , 1979). The  extensive antigenic  c r o s s - r e a c t i v i t y o f the  l i p i d A moieties  many gram-negative b a c t e r i a would suggest a common e v o l u t i o n a r y the  l i p i d A moiety t h a t has  strong  conservation  been c o n s e r v e d over time.  i s u n c l e a r but,  of a gram-negative b a c t e r i a has be  t o d a t e , no  antigenic  cross-reactions  e t a l . . 1978; E. c o l i  J5  n a t u r e of E. c o l i  Pennington and  antiserum and  of l i p i d  LPS  of P.  J5 LPS  Menkes, 1981)  a n t i s e r u m to the  n o n - a g g l u t i n a b l i l i t y and  of two  explanation  for  may  the  or whole c e l l v a c c i n e s as w e l l  as the  (Braude  ability  of  l i p i d A-KDO (ReLPS) of Pseudomonas b a c t e r e m i a  patients fibrosis  phenotypes, p o l y a g g l u t i n a b i l i t y or  are thus e s s e n t i a l l y non-typable by  demonstrated t h a t  ( Z i e r d t and  the  W i l l i a m s 1975).  In t h i s  these n o n - t y p a b l e s t r a i n s were a l s o  serum s e n s i t i v e when compared to the  s e r o t y p a b l e s t r a i n s (Table  s t r a i n s were s e n s i t i v e to l e s s than 2%  human or mouse serum.  lipid A  a e r u g i n o s a s t r a i n s from c y s t i c  c o n v e n t i o n a l O-antigen based a n t i s e r a  most cases the  A - d e f i c i e n t mutant  aeruginosa i s o l a t e s from c y s t i c f i b r o s i s  demonstrate one  study i t was  reason f o r t h i s  Young e t a l . , 1975).  S i x t y p e r c e n t or more of P. patients  o r i g i n of  A from a v a r i e t y of gram-  S. minnesota to p a s s i v e l y p r o t e c t mice a g a i n s t (Braude et a l . , 1978;  of  cells.  negative b a c t e r i a also provides a p o s s i b l e cross-protective  lipid  The  been i s o l a t e d s u g g e s t i n g t h a t  an e s s e n t i a l component of these The  (A)  Leive,  highly  I).  In  ( v o l / v o l ) normal  These c h a r a c t e r i s t i c s i . e . n o n - t y p a b i l i t y  and  serum  143  s e n s i t i v i t y were c o r r e l a t e d t o d e f i c i e n c y  i n LPS O - a n t i g e n i c s i d e c h a i n s  (Hancock e t a l . 1983) but not t o presence  o r absence o f the mucoid  exopolysaccharide implications.  ( T a b l e 1, F i g . 3 ) .  F i r s t l y , they c a l l  O-antigen-based  Those o b s e r v a t i o n s have s e v e r a l  i n t o q u e s t i o n the broad  a n t i s e r a f o r s e r o t y p i n g P. a e r u g i n o s a , e s p e c i a l l y f o r  i s o l a t e s from the lungs of p a t i e n t s w i t h c y s t i c f i b r o s i s . tend t o suggest  Secondly,  they  t h a t immunotherapy u s i n g LPS-based v a c c i n e s w i l l not be  s u c c e s s f u l i n some d i s e a s e s i t u a t i o n s al.  application of  (Hanessian  e t a l . , 1971; M i l e r e t  1977; Woods e t a l . , 1983). The LPS-phenotypes of these s t r a i n s demonstrate a s i g n i f i c a n t  p r o p e r t y o f P. a e r u g i n o s a a s s o c i a t e d w i t h c y s t i c f i b r o s i s .  new  The p r o p e r t y  of serum s e n s i t i v i t y which seems t o be a s s o c i a t e d w i t h the LPS phenotype may w e l l e x p l a i n why p a t i e n t s w i t h c y s t i c P. a e r u g i n o s a  2.  fibrosis  rarely suffer  from  bacteremia.  A n t i g e n i c c o n s e r v a t i o n o f P. a e r u g i n o s a o u t e r membrane p r o t e i n s The  r o l e of o u t e r membrane p r o t e i n s i n host-pathogen  not been as e x t e n s i v e l y s t u d i e d as t h a t o f LPS.  i n t e r a c t i o n has  In the p r e s e n t study, the  c o n s e r v a t i o n and immunogenic p r o p e r t i e s o f s p e c i f i c o u t e r membrane p r o t e i n s o f P. a e r u g i n o s a were examined. T h i s study p r o v i d e d t h r e e l i n e s o f evidence p r o t e i n s are h i g h l y conserved  i n P. a e r u g i n o s a .  t h a t o u t e r membrane Amongst the type  strains  o f the 17 serotypes o f the I n t e r n a t i o n a l A n t i g e n T y p i n g Scheme and a v a r i e t y of c l i n i c a l patterns  i s o l a t e s , I observed  s i m i l a r o u t e r membrane p r o t e i n  ( F i g s . 1 and 2 ) , a n t i g e n i c c r o s s - r e a c t i o n u s i n g p o l y c l o n a l  144  a n t i s e r a t o the major o u t e r membrane p r o t e i n s ( F i g s . 4 and 5, Table I I ) , and  the c o n s e r v a t i o n o f s p e c i f i c a n t i g e n i c e p i t o p e s  on o u t e r membrane  p r o t e i n s F and H2 ( F i g s . 7, 8 and 10; T a b l e s V, VI and V I I ) .  (A)  Conservation  o f o u t e r membrane p r o t e i n p a t t e r n s  , Other gram-negative b a c t e r i a , u n l i k e P. a e r u g i n o s a , high s i m i l a r i t y  do not show such  i n t h e i r outer membrane p r o t e i n p r o f i l e s .  F o r example, a  v a r i e t y o f o u t e r membrane p r o t e i n p a t t e r n s have been observed f o r Haemophilus i n f l u e n z a e type b ( e i g h t t o n i n e  subtypes based on o u t e r  membrane p r o t e i n p a t t e r n s ; Barenkamp e t a l . , 1981), N. gonorrhoeae subtypes;  Buchanan and H i l d e b r a n d t , 1981), N. m e n i n g i t i d i s (15 subtypes;  T s a i e t a l . , 1981), E. c o l i V i b r i o cholerae subtypes;  (nine  (36 subtypes;  (two subtypes;  Overbeeke and Lugtenberg, 1980),  K a b i r , 1980) and V i b r i o a n g u i l l a r u m  Nakajima e t a l . , 1983).  (three  Indeed i n N. gonorrhoeae (Buchanan and  H i l d e b r a n d t , 1981) and i n H. i n f l u e n z a e type b (Barenkamp e t a l . 1981) o u t e r membrane p r o t e i n p a t t e r n s and a n t i s e r a t o o t h e r membrane p r o t e i n s 1  have been proposed as the b a s i s f o r s u b t y p i n g Therefore, studied  these  organisms.  i t i s h i g h l y s i g n i f i c a n t t h a t a l l P. a e r u g i n o s a  ( i n c l u d i n g 17 d i f f e r e n t s e r o t y p e  i s o l a t e s from c y s t i c  strains,  strains  and 34 d i f f e r e n t  f i b r o s i s p a t i e n t s , F i g . 1 and 2) have h i g h l y s i m i l a r  o u t e r membrane p r o t e i n p a t t e r n s . c o n s i s t e n t w i t h these r e s u l t s .  Two o b s e r v a t i o n s Sadoff  i n l i t e r a t u r e are  and A r t e n s t e i n (1974) showed  s i m i l a r p a t t e r n s i n the " n a t i v e complex" o f the seven F i s h e r immunotypes, whereas Mizuno and Kageyama (1978b) made s i m i l a r o b s e r v a t i o n s the o u t e r membrane p r o t e i n s o f f i v e d i f f e r e n t  P. a e r u g i n o s a  regarding  strains.  145  C o n s e r v a t i o n of o u t e r membrane p r o t e i n p a t t e r n s has the e e l pathogen P.  (B)  anguilliseptica  a l s o been r e p o r t e d f o r  (Nakaj ima e_t a l . , 1983).  Antigenic conservation revealed using p o l y c l o n a l  antiserum  A n t i g e n i c r e l a t i o n s h i p s between the o u t e r membrane p r o t e i n s of v a r i o u s P.  a e r u g i n o s a s t r a i n s were demonstrated by the c r o s s - r e a c t i v i t y i n  ELISA of antiserum  t o s t r a i n PAOI o u t e r membranes w i t h the o u t e r membranes  of s e r o t y p i n g s t r a i n s  (Table I I ) .  On Western b l o t s , the broad  reactivity  of the p o l y c l o n a l a n t i b o d i e s to i n d i v i d u a l p r o t e i n s from d i f f e r e n t suggested  t h a t each of p r o t e i n s F, H2  determinants E was  i n a l l P.  and  I p o s s e s s e d common a n t i g e n i c  a e r u g i n o s a o u t e r membranes t e s t e d ; and t h a t p r o t e i n  also closely related  i n 16 of the 17  serotype s t r a i n s  In c o n t r a s t , the a n t i g e n i c h e t e r o g e n e i t y o f f l a g e l l i n strains  ( P i t t , 1980)  flagellin  strains  was  confirmed by the d i f f e r e n t i a l  i n o n l y 7 (4 weakly) of the 17  serotype  ( F i g s . 4,  among P.  5).  aeruginosa  antibody b i n d i n g t o  strains.  D e s p i t e the s i m i l a r i t y of outer membrane p r o t e i n p a t t e r n s , not a l l serotype s t r a i n s proteins  showed i d e n t i c a l a n t i b o d y b i n d i n g t o the o u t e r membrane  ( F i g . 5).  observation.  There are at l e a s t  possible explanations for this  I n d i v i d u a l a n t i g e n i c s i t e s on g i v e n o u t e r membrane p r o t e i n s  c o u l d have d i f f e r i n g the g i v e n p r o t e i n observed  two  affinities  f o r the t e s t a n t i b o d y or the amounts of  i n the v a r i o u s o u t e r membranes c o u l d d i f f e r .  s m a l l amounts of p r o t e i n H2  Thus the  i n the s t r a i n s of s e r o t y p e s 5 and 7  ( F i g . 1) c o r r e l a t e d w i t h the s m a l l amount of a n t i b o d y bound to p r o t e i n of these s t r a i n s  ( F i g . 5).  S i m i l a r l y , the s e r o t y p e 7 s t r a i n had  amounts of l i p o p r o t e i n I and v e r y l i t t l e  a n t i b o d y was  H2  small  bound to l i p o p r o t e i n  146  I from t h i s  strain.  However, no s i m i l a r c o r r e l a t i o n c o u l d be made f o r  p r o t e i n E.  The d i f f e r e n t amounts of antibody to p r o t e i n E bound to these  s t r a i n s might be r e l a t e d t o a n t i b o d y - a n t i g e n There i s evidence  affinity.  i n the l i t e r a t u r e t h a t P. a e r u g i n o s a o u t e r membrane  p r o t e i n s are immunogenic i n v i v o d u r i n g the course of an Fernandes e t a l . (1981) demonstrated the presence with c y s t i c f i b r o s i s c e l l envelope  of i m m u n o p r e c i p i t a t i n g  p r o t e i n s of 37,000 d a l t o n s and  p r o t e i n F and f l a g e l l i n r e s p e c t i v e l y ) . al.  (1983), demonstrated the presence  o u t e r membrane p r o t e i n s F, H2 fibrosis  i n the s e r a of p a t i e n t s  a n t i b o d i e s t o P. 58,000 d a l t o n s e t a l . (1983),  aeruginosa (possibly  and Hancock e t  of a n t i b o d i e s a g a i n s t P.  aeruginosa  I i n the s e r a of 47 p a t i e n t s w i t h  and 4 p a t i e n t s w i t h acute P.  bacteremia The  and  Lam  infection.  a e r u g i n o s a pneumonia o r  as w e l l as i n r a t s c h r o n i c a l l y  i n f e c t e d w i t h P.  acute  aeruginosa.  simplest e x p l a n a t i o n f o r t h i s antibody c r o s s - r e a c t i o n data i s  t h a t the o u t e r membrane p r o t e i n a n t i g e n s are a n t i g e n i c a l l y conserved P.  cystic  aeruginosa s t r a i n s .  I t i s t h e r e f o r e p o s s i b l e t h a t the c r o s s p r o t e c t i o n  demonstrated by P. a e r u g i n o s a v a c c i n e s , such as r i b o s o m a l and o r i g i n a l e n d o t o x i c p r o t e i n  (Abe  e t a l . 1975)  c o n t a m i n a t i o n w i t h o u t e r membrane p r o t e i n s . o b s e r v a t i o n s ) observed  in  (Lieberman  v a c c i n e s i s due  1978)  to  Indeed Hancock ( u n p u b l i s h e d  t h a t the o r i g i n a l e n d o t o x i c p r o t e i n  contained  p r o t e i n s t h a t c o - e l e c t r o p h o r e s e d w i t h p u r i f i e d o u t e r membrane p r o t e i n s and r e a c t e d s t r o n g l y i n ELISA w i t h a n t i s e r a t o PA01  o u t e r membranes.  T h e r e f o r e , the major components of P. a e r u g i n o s a o u t e r membranes, p r o t e i n s and LPS,  are c a p a b l e of i n t e r a c t i n g w i t h the h o s t ' s  mechanisms and e l i c i t i n g  antibody responses.  immune  However the p r o t e i n s , u n l i k e  147  LPS,  are c r o s s - r e a c t i v e and  appear to be n o n - t o x i c to mice i n t h a t  i n j e c t i o n s of 5 ug/kg of p r o t e i n symptoms or l e t h a l i t y  i n t o mice caused  no obvious  clinical  (E.C.A. Mouat, p e r s o n a l communication).  e x p r e s s i o n i n P. a e r u g i n o s a of h i g h l y conserved outer membrane p r o t e i n s suggests  and  Thus the  apparently  t h a t common a n t i g e n s t h a t can  immunogenic provide  immunoprotection  a g a i n s t i n f e c t i o n s by the b a c t e r i u m s h o u l d e x i s t among  these p r o t e i n s .  Such p r o t e c t i o n has  been demonstrated i n N.  gonorrhoeae  (Buchanan e t al_. , 1977), i n e x p e r i m e n t a l  s a l m o n e l l o s i s (Kuusi e_t a l . ,  1979)  ( G i l l e l a n d , e t a l . , 1984;  and more r e c e n t l y i n P.  and Mouat, submitted  (C)  aeruginosa  Hancock  for publication).  C o n s e r v a t i o n of s p e c i f i c  e p i t o p e s on P.  a e r u g i n o s a o u t e r membrane  proteins The  c r o s s - r e a c t i v i t y of the monoclonal a n t i b o d i e s w i t h  p r o t e i n s i n the o u t e r membranes from d i f f e r e n t P.  specific  aeruginosa s t r a i n s  was  i n v e s t i g a t e d as an i n d i c a t o r of the c o n s e r v a t i o n of s p e c i f i c a n t i g e n i c sites  ( e p i t o p e s ) among these s t r a i n s .  Monoclonal  antibodies s p e c i f i c for  P. a e r u g i n o s a o u t e r membrane p o r i n p r o t e i n F and l i p o p r o t e i n s H2 were used t o study the d i s t r i b u t i o n of s p e c i f i c  and  antigenic epitopes  these p r o t e i n s i n the o u t e r membranes from a wide range of P.  I  on  aeruginosa  strains. A monoclonal a n t i b o d y MA1-3  was  d i s t i n g u i s h e d from MAl-6 on the b a s i s  of l a c k of i n t e r a c t i o n w i t h p a r t i a l l y p u r i f i e d P.  aeruginosa l i p o p r o t e i n  (Table I I I ) and w i t h the o u t e r membranes from o t h e r b a c t e r i a Antibody MA1-3  r e a c t e d i n ELISA w i t h o u t e r membranes from a l l  (Table V I ) .  I  148  P. a e r u g i n o s a s t r a i n s t e s t e d , i n c l u d i n g 17 s e r o t y p e s t r a i n s VII).  However i t d i d not r e a c t with denatured  SDS-PAGE g e l s or w i t h Western b l o t s of P.  and  a n t i g e n s e l u t e d from  a e r u g i n o s a o u t e r membranes.  T h e r e f o r e the a c t u a l nature of the a n t i g e n i c s i t e d i r e c t e d remains obscure.  (Tables VI  a g a i n s t which MAl-3 i s  However, i t i s p o s s i b l e t h a t MAl-3 i s d i r e c t e d  a g a i n s t a minor p r o t e i n i n the l i p o p r o t e i n I p r e p a r a t i o n , or a g a i n s t a complex of p r o t e i n s H2  and I , which may  p u r i f i e d p r o t e i n I p r e p a r a t i o n . The  be p r e s e n t  i n the  e x i s t e n c e of such  partially  a complex i n v i v o  has been p o s t u l a t e d from s e l e c t i v e s o l u b i l i z a t i o n experiments al.,  1981)  and has  experiments  a l s o been demonstrated by p r o t e i n - p r o t e i n c r o s s - l i n k i n g  (Angus and Hancock, 1983).  A second monoclonal antibody MAl-6, s p e c i f i c p r o t e i n H2 of P.  aeruginosa.  serotyping strains cystic strains  (Hancock e t  f o r an a n t i g e n i c s i t e  i n t e r a c t e d w i t h o u t e r membranes from  the  ( T a b l e V I I , F i g . 10), w i t h 34 of 36 t e s t e d s t r a i n s  f i b r o s i s p a t i e n t s ( T a b l e X, F i g . 11) ( T a b l e VI, F i g . 19).  Two  P.  and w i t h o t h e r P.  on  from  aeruginosa  a e r u g i n o s a s t r a i n s , CFC46 mucoid  and  i t s hon-mucoid d e r i v a t i v e , d i d not have p r o t e i n H2 when the o u t e r membranes of these s t r a i n s were examined by SDS-polyacrylamide e l e c t r o p h o r e s i s and Decreased envelopes  gel  f a i l e d t o i n t e r a c t w i t h MAl-6 ( F i g . 11).  amounts of p r o t e i n H2 had been observed  of P. a e r u g i n o s a s t r a i n H103  o b s e r v a t i o n s ) when the s t r a i n was  (K. P o o l e ,  grown i n serum.  p o s s i b l e to a t t r i b u t e the l o s s of p r o t e i n H2 and CFC46 non-mucoid t o serum e f f e c t s  i n the  cell  unpublished However, i t i s not  i n the s t r a i n s CFC46 mucoid  s i n c e the s t r a i n s  are  s e r u m - s e n s i t i v e l u n g i s o l a t e s which were not exposed t o serum i n v i v o .  In  149 a d d i t i o n a l l 34 o t h e r i s o l a t e s o f P. a e r u g i n o s a from c y s t i c f i b r o s i s patients  contained  Antigenic  p r o t e i n H2.  c r o s s - r e a c t i v i t y and c o n s e r v a t i o n  of s p e c i f i c  antigenic  s i t e s on p r o t e i n F was demonstrated among P. a e r u g i n o s a s t r a i n s u s i n g monoclonal a n t i b o d i e s f o r p r o t e i n F.  five  (MA5-8, MA4-4, MA4-10, MA2-10 and MA4-2) s p e c i f i c  The s p e c i f i c i t y o f these monoclonal a n t i b o d i e s  f o r protein  F was demonstrated by t h e absence o f c r o s s - r e a c t i v i t y with a p r o t e i n F - d e f i c i e n t mutant s t r a i n H283 d e r i v e d  from P. a e r u g i n o s a PAOI s t r a i n H103  ( F i g . 9 ) , and by t h e i r i n t e r a c t i o n w i t h p r o t e i n F on Western b l o t s o f o u t e r membrane p r o t e i n s  from s e r o t y p i n g  s t r a i n s and c y s t i c f i b r o s i s  i s o l a t e s o f P. a e r u g i n o s a ( F i g s . 7 and 8 ) . We observed d i f f e r e n c e s monoclonal a n t i b o d i e s (Fig. 7).  i n the i n t e r a c t i o n o f the p r o t e i n F - s p e c i f i c  w i t h p r o t e i n F from d i f f e r e n t P. a e r u g i n o s a s t r a i n s  S i n c e p r o t e i n F was not t r a n s f e r r e d q u a n t i t a t i v e l y from g e l s  onto Western b l o t s , i t was not p o s s i b l e observation.  t o determine the reason f o r t h i s  However, i t may be due t o s l i g h t  antigenic  s i t e recognized  differing  antigen-antibody  a l t e r a t i o n s o f the  by the monoclonal a n t i b o d y , g i v i n g r i s e t o affinities.  Thus, outer membranes o f P. a e r u g i n o s a c o n t a i n antigenic  s i t e s on p r o t e i n H2, F and p o s s i b l y I .  three  common conserved  These a n t i g e n i c  sites  are expressed i n a l l P. a e r u g i n o s a s t r a i n s t e s t e d w i t h the e x c e p t i o n two  (D)  cystic fibrosis  i s o l a t e s that lacked  p r o t e i n H2.  D i s t r i b u t i o n o f s p e c i f i c e p i t o p e s among o t h e r s p e c i e s The  of bacteria  immunological c r o s s - r e a c t i v i t y o f monoclonal a n t i b o d i e s  f o r P. a e r u g i n o s a o u t e r membrane p r o t e i n s  of  was t e s t e d  specific  i n ELISA assays and  150  Western b l o t s with o u t e r membranes from  a v a r i e t y o f s t r a i n s of the f a m i l y  Pseudomonadaceae and o t h e r organisms ( T a b l e s VI and X I I ) . Monoclonal  antibody MA1-3  i n t e r a c t e d weakly w i t h the two  P. f l u o r e s c e n s s t r a i n s , P. p u t i d a s t r a i n s , P. s y r i n g a e . P. c h l o r o r a p h i s . P. a u r e o f a c i e n s . P. s t u t z e r i  s t r a i n s t e s t e d and an A z o t o b a c t e r  strain', but not w i t h any other a n t i g e n s monoclonal antibody MA1-6  specific  (Table V I ) .  vinelandii  In c o n t r a s t  f o r P. a e r u g i n o s a l i p o p r o t e i n  H2  r e a c t e d s t r o n g l y w i t h the o u t e r membranes of two Pseudomonas f l u o r e s c e n s strains  ( i n c l u d i n g the type s t r a i n ATCC 13524), two P. p u t i d a s t r a i n s  ( i n c l u d i n g the type s t r a i n ATCC 12633) and one s t r a i n from each o f the f o l l o w i n g s p e c i e s , P. a n g u i l l i s e p t i c a . A. v i n e l a n d i i . P. c h l o r o r a p h i s . P. s y r i n g a e , P. s t u t z e r i and P. a u r e o f a c i e n s . w i t h o u t e r membranes of o t h e r Pseudomonas  There was no c r o s s - r e a c t i v i t y s t r a i n s from the f a m i l y  Pseudomonadaceae or r e p r e s e n t a t i v e s o f o t h e r F a m i l i e s o f b a c t e r i a V I , F i g . 19).  (Table  In agreement w i t h t h i s d a t a , Mizuno and Kageyama (1978b)  had demonstrated t h a t o u t e r membrane p r o t e i n H2 can be  immunoprecipitated  from t h r e e f l u o r e s c e n t psuedomonads, P. a e r u g i n o s a . P. f l u o r e s c e n s and P. p u t i d a , by crude a n t i s e r a a g a i n s t P. a e r u g i n o s a p r o t e i n H2.  The r e s u l t s  p r e s e n t e d here c o n f i r m taxonomic d a t a from rRNA homology, DNA homology and f a t t y a c i d a n a l y s i s s t u d i e s s u g g e s t i n g t h a t A. v i n e l a n d i i  i s taxonomically  r e l a t e d t o the f l u o r e s c e n t pseudomonads, w h i l e P. acidovorans m a l t o p h i l i a are t a x o n o m i c a l l y d i s t i n c t (Palleroni  from the f l u o r e s c e n t psuedomonads  e t a l . , 1973; DeVos and DeLey, 1983; Moss e t a l . , 1972; see  Table X I I ) . suggested  and P.  R e c e n t l y P a l l e r o n i e t a l . (1973) and DeVos and DeLey (1983)  t h a t the f a m i l y Pseudomonadaceae  c o n s i s t e d o f a number o f  Table X I I .  Potential  taxonomic v a l u e o f monoclonal  rRNA h o m o l o g y clusters'  Bacterial  Agrobacterium  A.  P.  P. s o l a n a c e a r u m P. c e p a c i a P_. p s e u d o m a l l e i  strain  5  r solanacearum  3 P.  2  acidovorans  P. f l u o r e s c e n s (A. v i n e l a n d i i )  I  P. P. P. - P. P. P. P. P. P. A.  antibodies  Cross-reactions MAl-6 MA4-4 o-H2 a-F  3  with monoclonal a n t i b o d i e s MA 1-8 MA5-8 5E4 a-Lipid A a-F a-OAg  tumefaciens (LPS)  acidovorans aeruginosa^) putida syringae chlororaphis aureofaciens stutzeri fluorescens anguilliseptica*' vinelandii  ND + + + + + + + + +  +  (+/-)  -  -  -  + + ND ND ND ND + + +  + + +  Xanthompnas  P. m a l t o p h i l i a  -  -  -  +  Enterobacteriaceae  E. c o l i S. t y p h i m u r i u m E. t a r d a  -  -  -  +  _  -  -  -  +  -  -  -  •+  Vibronaceae  V. V. A. A.  cholerae anguillarum salmonicida hydrophila  152 T a b l e XII  + denotes a p o s i t i v e  reaction;  —  Legend  a negative reaction;  ND  not  done.  DeVos  a  r e s u l t s from both ELISA and  D  c l a s s i f i c a t i o n by  c  1,2,3 and A denote rRNA homology groups of Pseudomonads by DeLey (1983).  Western  rRNA of DeVos and  blots. DeLey  ^ Tm(e)°C - Thermal e l u t i o n temperature of performed by DeVos and DeLey (1983). e  (1983).  the  DNA-rRNA  and  hybridizations  AO" d i f f e r e n t s t r a i n s of P. a e r u g i n o s a i n c l u d i n g the IATS s e r o t y p i n g s t r a i n s , c y s t i c f i b r o s i s i s o l a t e s and l a b o r a t o r y s t r a i n s as d e s c r i b e d i n m a t e r i a l s and methods. The p o s i t i v e (+) r e a c t i o n w i t h MA1-8 was o n l y seen with serotype 5 s t r a i n s .  153  d i s t a n t l y r e l a t e d taxonomic groups of b a c t e r i a . which MA1-6  reacted  were placed  A l l of the b a c t e r i a w i t h  i n the P. f l u o r e s c e n s  group 1 Pseudomonads), and the other Pseudomonas i n t e r a c t with a n t i b o d y MA1-6 Thus MA1-6  group ( a l s o c a l l e d  species  which f a i l e d t o  were found t o be t a x o n o m i c a l l y  distinct.  i n t e r a c t s s p e c i f i c a l l y w i t h o u t e r membranes of a s i n g l e  taxonomic s u b d i v i s i o n , the group 1 or " t r u e " pseudomonads,  indicating a  p o t e n t i a l r o l e i n taxonomy f o r t h i s monoclonal a n t i b o d y . Monoclonal a n t i b o d i e s according  to p o r i n p r o t e i n F f e l l  i n t o two groups  to t h e i r c r o s s - r e a c t i v i t y w i t h o t h e r s t r a i n s .  Antibodies  MA4-10, MA4-2, MA4-4, and MA2-10 i n t e r a c t e d w i t h a p r o t e i n i n the o u t e r membranes o f two P. p u t i d a  s t r a i n s and a P. s y r i n g a e  monoclonal a n t i b o d y MA5-8 was h i g h l y s p e c i f i c  s t r a i n , whereas  f o r P. a e r u g i n o s a  porin  p r o t e i n F ( F i g . 9; T a b l e X I I ) . The r e a c t i v i t y of these monoclonal a n t i b o d i e s  suggested t h a t  protein  F has at l e a s t two d i f f e r e n t conserved a n t i g e n i c  s i t e s defined  monoclonal a n t i b o d i e s .  c o n s e r v e d among o t h e r  Pseudomonadaceae s p e c i e s antigenic  drift  P r o t e i n F i s more p o o r l y  than p r o t e i n H2, s u g g e s t i n g t h a t  has o c c u r r e d  f o r p r o t e i n F.  f a r b e t t e r conserved than the p o r i n p r o t e i n s e t a l . , 1976) and H. i n f l u e n z a e antigenic v a r i a t i o n within  by these  greater  Nevertheless, protein F i s o f N. gonorrhoeae  (Johnston  (Barenkamp e t a l . , 1981) which show  a single  species.  154  3.  Surface The  accessibility  extensive  s t u d i e s u s i n g monoclonal  antibodies  c r o s s - r e a c t i v i t y o f monoclonal a n t i b o d i e s  specific for  p r o t e i n F and l i p o p r o t e i n H2 among P. a e r u g i n o s a s t r a i n s suggested these common antigens  would be l o g i c a l v a c c i n e  candidates.  that  One major  requirement f o r a good v a c c i n o g e n i s the a c c e s s i b i l i t y o f the a n t i g e n on the  s u r f a c e o f the i n t a c t organism.  outer membrane p r o t e i n s radioiodination  The s u r f a c e  l o c a l i z a t i o n of c e r t a i n  i n P. a e r u g i n o s a whole c e l l s has been i n d i c a t e d by  (Lambert and Booth, 1982).  shown t o l a b e l c e r t a i n p r o t e i n s ( S u l l i v a n and W i l l i a m s ,  t h a t are not i n the o u t e r  1982; S c h i n d l e r  i n other  phages (Braun and K r i e g e r - B r a u e r ,  membrane  and Teuber, 1979), s u g g e s t i n g  the procedure d i s r u p t s the o u t e r membrane. have a l s o been i d e n t i f i e d  However, t h i s method has been  Surface-localized proteins  b a c t e r i a by the use o f p r o t e i n 1977; D a t t a e t a l . , 1977).  these s t u d i e s do not r e v e a l i f the s u r f a c e - a c c e s s i b l e p o r t i o n s p r o t e i n are immunogenic.  f o r H. i n f l u e n z a e  specific  However, o f the  Thus, the p r e f e r r e d technique i s t o measure the  i n t e r a c t i o n o f tagged s p e c i f i c e.g.  that  a n t i b o d i e s w i t h whole organisms, as done  type b (Hansen e t a l . , 1981a), and E. c o l i  (Hofstra  et a l . , 1979) . In t h i s r e p o r t a study o f the s u r f a c e  antigenic  structures of  P. aeruginosa was performed u s i n g monoclonal a n t i b o d i e s membrane p r o t e i n s .  By i n d i r e c t  specific  immunofluorescence and c o l o n y  f o r outer  immunoblot  procedures, p r o t e i n F was demonstrated t o be s u r f a c e exposed and a c c e s s s i b l e t o monoclonal a n t i b o d i e s determinants on the p r o t e i n antibodies  d i d not b i n d  specific  f o r two s e p a r a t e  ( F i g s . 16 and 17; T a b l e s V I I I  antigenic  and X ) .  t o a p r o t e i n F - d e f i c i e n t P. a e r u g i n o s a  These  strain  (Fig. 9).  On c o l o n y b l o t s , the monoclonal a n t i b o d i e s i n t e r a c t e d w i t h a l l  P. aeruginosa  s t r a i n s t e s t e d i n c l u d i n g c o l o n i e s from smooth,  O-antigen-containing s e n s i t i v e and  and  resistant  rough O - a n t i g e n - d e f i c i e n t s t r a i n s , s t r a i n s , s t r a i n s of d i f f e r e n t  antigens.  One  and  fibrosis.  0-side c h a i n s nor the mucoid e x o p o l y s a c c h a r i d e  masked the surface-exposed  serum  serotypes,  mucoid and non-mucoid i s o l a t e s from p a t i e n t s w i t h c y s t i c N e i t h e r LPS  LPS  apparently  group of monoclonal a n t i b o d i e s ,  MA4-4, MA4-10 and MA2-10, a l s o i n t e r a c t e d w i t h c o l o n i e s of P. p u t i d a P.  P.  syringae  strains.  Monoclonal antibody MAl-6 d i d not b i n d to p r o t e i n H2  in intact  aeruginosa c e l l s but showed b i n d i n g to rough O-antigen  deficient  mutants (Tables V I I I and X ) . a n t i g e n i c determinants  T h i s may  be due  by l o s s of the 0-side c h a i n s of LPS  i n t e r a c t i o n of MAl-6 w i t h rough s t r a i n s aeruginosa  smooth  e i t h e r to the unmasking of or  re-arrangement of the o u t e r membrane components i n rough s t r a i n s .  P.  and  is significant  This  i n t h a t the rough  i s o l a t e s from c y s t i c f i b r o s i s p a t i e n t s ( T a b l e I) appear to  have p r o t e i n H2  as a s u r f a c e determinant  (Table X ) .  aeruginosa  s t r a i n s , however, l i p o p r o t e i n H2,  of E. c o l i  (Braun  e t a l . , 1976), i s not  In smooth  l i k e the Braun's  surface-exposed.  P. lipoprotein  Interestingly,  Braun's l i p o p r o t e i n does become exposed i n deep rough, L P S - a l t e r e d mutants. The  colony  immunoblot procedure  facilitated  rapid screening of a  v a r i e t y of s t r a i n s f o r e x p r e s s i o n of s p e c i f i c d e t e r m i n a n t s . s p e c i f i c i t y of the procedure  was  shown by the l a c k of b i n d i n g of MAl-6 to  CFC46 mucoid and CFC46 nonmucoid s t r a i n s and b i n d i n g (O-antigen  The  s p e c i f i c ) to serotype  the s p e c i f i c i t y of  5 strains.  The mucoid  MA1-8 capsule  t h e r e f o r e , d i d not appear to n o n - s p e c i f i c a l l y b i n d or adsorb a n t i b o d i e s .  156  4.  P a r t i a l c h a r a c t e r i z a t i o n o f two p r o t e i n F e p i t o p e s Studies  Table XIII.  on the a n t i g e n i c As d e s c r i b e d  protein F f e l l  s t r u c t u r e o f p r o t e i n F a r e summarized on  above, t h e monoclonal a n t i b o d i e s  i n t o two c l a s s e s by i n t e r a c t i o n w i t h Pseudomonas s t r a i n s .  Antibody MA5-8 r e a c t e d  s p e c i f i c a l l y w i t h P. a e r u g i n o s a s t r a i n s w h i l e  MA4-4, MA2-10, and MA4-10 c r o s s - r e a c t e d P. s y r i n g a e . 9).  w i t h a p r o t e i n , from P. p u t i d a and  o f s i m i l a r m o l e c u l a r weight t o P. a e r u g i n o s a p r o t e i n F. ( F i g .  The d i f f e r e n c e s  i n the a n t i g e n i c  classes of antibodies antibodies  specific for  domains r e c o g n i z e d  by these two  was f u r t h e r demonstrated by t h e b i n d i n g  o f these  t o p e p t i d e fragments o f p r o t e i n F and t o t h e n a t i v e  under d i f f e r e n t c o n d i t i o n s  (Table  XIII).  Monoclonal a n t i b o d y , MA5-8, u n l i k e t h e o t h e r a n t i b o d i e s , with oligomeric  protein  interacted  forms o f p r o t e i n F, as w e l l as t h e 2-mercaptoethanol-  reduced and non-reduced p r o t e i n  ( F i g . 20). This  antibody a l s o  interacted  w i t h two p a r t i a l cyanogen bromide fragments o f the p r o t e i n , a l t h o u g h the antigenic  s i t e recognized  by the a n t i b o d y was d e s t r o y e d by e i t h e r t r y p s i n  or papain ( F i g s . 21 and 22).  In c o n t r a s t , monoclonal a n t i b o d i e s  MA2-10,  MA4-4 and MA4-10 i n t e r a c t e d w i t h the non-reduced p o r i n , and w i t h t h e t r y p s i n and papain d e r i v e d ( F i g s . 20 and 22).  fragments o f p r o t e i n F i n the non-reduced form  The e p i t o p e o r e p i t o p e s r e c o g n i z e d  were d e s t r o y e d by  2-mercaptoethanol as w e l l as by c l e a v a g e o f t h e p r o t e i n w i t h cyanogen bromide ( F i g . 2 1 ) . These d a t a suggest t h a t p r o t e i n F has a t l e a s t two d i s t i n c t located antigenic  epitopes  (domains) r e c o g n i z e d  by d i f f e r e n t  surface  antibodies.  Both o f these domains a r e p r o b a b l y c o n f o r m a t i o n a l i n t h a t they  require  157  Table XIII. D i f f e r e n t i a t i o n o f two c l a s s e s o f monoclonal a n t i b o d i e s s p e c i f i c f o r P. a e r u g i n o s a p r o t e i n F.  MA5-8  R e a c t i v i t y with p u r i f i e d p r o t e i n  t a  +  +  Surface l a b e l l i n g of i n t a c t P. a e r u g i n o s a ^  +  +  R e a c t i v i t y with P. p u t i d a and P. s y r i n g a e p r o t e i n F ^ » ^  -  +  -  +  a  F  '  Monoclonal Antibody (MA2-10, MA4-4, MA4-10)  b  R e a c t i v i t y with a 31 kD t r y p s i n o r 29 kD papain p r o t e o l y t i c fragment of p r o t e i n F ^ ' c  R e a c t i v i t y w i t h cyanogen bromide fragments o f p r o t e i n F ^ ^  + (23,28 kD)  Antigenic r e a c t i v i t y stable 2-mercaptoethanol^ '  +  c  to  c  Binding to oligomers o f p r o t e i n on SDS-polyacrylamide g e l s ^ ^  F  +  c  a  b  c  determined by both t h e ELISA and Western b l o t p r o c e d u r e s . determined by t h e i n d i r e c t immunofluorescence and c o l o n y immunoblot procedures. determined by the Western b l o t p r o c e d u r e .  158  some t e r t i a r y  s t r u c t u r e which i s m a i n t a i n e d i n SDS.  Presumably  SDS-stable t e r t i a r y  s t r u c t u r e (demonstrated e x p e r i m e n t a l l y  Kageyama, 1979)  destroyed  degradation. extensive  The  by v i g o r o u s  complex 8 - s t r u c t u r e  studies with  reasonable  5.  was  this  by Mizuno  p r o t e o l y t i c or cyanogen bromide  of p o r i n p r o t e i n s w i l l  require  a l a r g e r l i b r a r y of monoclonal a n t i b o d i e s  s p e c u l a t i o n s on the  and  s t r u c t u r e of p r o t e i n F can  before  be made.  Prospectives The monoclonal a n t i b o d i e s  (epitopes)  on both LPS  and  r e p o r t e d here d e f i n e s i n g l e a n t i g e n i c  proteins.  The  antibodies f o r t h e i r p a r t i c u l a r epitope  high  has  s e r o l o g i c a l , e p i d e m i o l o g i c a l , taxonomic and A l i b r a r y of monoclonal a n t i b o d i e s used to f u r t h e r d e f i n e the aeruginosa  strains.  great  implications in  structural studies.  to the LPS  Combinations of rough core  o u t e r membrane a n t i g e n  s p e c i f i c i t y o f monoclonal  rough c o r e  s t r u c t u r a l heterogeneity  s p e c i f i c monoclonal a n t i b o d i e s  sites  could  of t h a t r e g i o n  specific  c o u l d be used t o g e t h e r  and  with  lipid  the  be i n P. A  specific  i n v a c c i n a t i o n or immunization s t u d i e s , thus  n e u t r a l i z i n g the e n d o t o x i c  a c t i o n of LPS.  Monoclonal a n t i b o d i e s  to  the  d i f f e r e n t O-antigen types of Pseudomonas would a l s o prove more s p e c i f i c serotyping The areas.  reagents than the p o l y c l o n a l a n t i s e r a p r e s e n t l y  p r o t e i n - s p e c i f i c monoclonal a n t i b o d i e s For example, MA1-6  (specific  taxonomic s t u d i e s of Pseudomonads. the group 1 Pseudomonads and Pseudomonadaceae.  can be used i n s e v e r a l  f o r p r o t e i n H2) The  antibody  used.  c o u l d be a p p l i e d to  showed i n t e r a c t i o n w i t h  thus can be used to d i f f e r e n t i a t e  S i m i l a r l y , antibody  the  MA5-8 c o u l d be used to d e f i n e  P.  as  159  aeruginosa  s t r a i n s s i n c e t h i s monoclonal antibody  P. aeruginosa  strains  showed s p e c i f i c i t y f o r  only.  Another area o f a p p l i c a t i o n i s t h a t o f d e f i n i n g a n t i g e n Monoclonal a n t i b o d i e s assist  in obtaining  described Since  to d i f f e r e n t peptide  the t h r e e d i m e n s i o n a l  s t r u c t u r e o f the p r o t e i n s  some o f the monoclonal a n t i b o d i e s  o p s o n i c - k i l l i n g assays to d e f i n e  passive  fragments c o u l d be used t o  here.  for surface-localized antigenic s i t e s ,  clearance  structure.  o f the b a c t e r i u m .  r e p o r t e d here a r e s p e c i f i c  they should  i f antibodies  These a n t i b o d i e s  be u s e f u l i n  t o the p r o t e i n  increase  c o u l d a l s o be used f o r  immunization.  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