Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1985_A1 M87.pdf [ 8.58MB ]
Metadata
JSON: 831-1.0096736.json
JSON-LD: 831-1.0096736-ld.json
RDF/XML (Pretty): 831-1.0096736-rdf.xml
RDF/JSON: 831-1.0096736-rdf.json
Turtle: 831-1.0096736-turtle.txt
N-Triples: 831-1.0096736-rdf-ntriples.txt
Original Record: 831-1.0096736-source.json
Full Text
831-1.0096736-fulltext.txt
Citation
831-1.0096736.ris

Full Text

IMMUNOCHEMISTRY OF PSEUDOMONAS AERUGINOSA OUTER MEMBRANE PROTEINS by Lucy Muthoni Mutharia B.Sc. (Hons), 1976, Univ e r s i t y of Nairobi, Kenya M.Sc, 1980, University of Nairobi, Kenya A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Faculty of Graduate Studies Department of Microbiology Uni v e r s i t y of B r i t i s h Columbia We accept t h i s thesis as conforming to the required standard / The University of B r i t i s h Columbia © Lucy Muthoni Mutharia,/Y&J. September, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of H y r . R - Q & I Q L-OQH ' The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 E-6 (3/81) ABSTRACT The immunochemistry and conservation of Pseudomonas aeruginosa outer membrane components was studied. Conservation of the major outer membrane proteins of P. aeruginosa was demonstrated by the strong s i m i l a r i t i e s in the SDS-polyacrylamide gel p r o f i l e s of outer membrane polypeptides from 47 d i f f e r e n t strains of P. aeruginosa. Immunological c r o s s - r e a c t i v i t y was demonstrated for proteins F, H2 and I among 17 serotype s t r a i n s of P. aeruginosa, using a rabbit polyclonal anti-outer membrane serum. In order to study the conservation of s p e c i f i c antigenic s i t e s among P. aeruginosa st r a i n s and other gram-negative bacteria, monoclonal antibodies s p e c i f i c f o r proteins F, H2 and lipopolysaccharide (LPS) were is o l a t e d . Monoclonal antibodies were used to study the antigenic conservation of the d i f f e r e n t regions of LPS. These antibodies demonstrated strong conservation of the l i p i d A region of LPS among a l l tested gram-negative bacteria, less conservation of the P. aeruginosa rough core and great heterogeneity of the LPS O-antigen. In contrast, protein H2 was shown to be highly conserved among the fluorescent Pseudomonads by the r e a c t i v i t y of the monoclonal antibody MA1-6. Monoclonal antibodies s p e c i f i c for protein F showed two d i s t i n c t s p e c i f i c i t i e s suggesting that there were at least two d i s t i n c t antigenic s i t e s on the protein. Antibodies MA4-4, MA4-2, MA2-10 and MA4-10 interacted with protein F in the outer membranes of a l l tested P. aeruginosa strains as well as P. syringae and P. putida s t r a i n s . These antibodies also interacted with defined p r o t e o l y t i c peptides of protein F. The epitope recognized by these antibodies was destroyed by 2-mercaptoethanol and cyanogen bromide treatment. In contrast, antibody MA5-8 interacted only with P. aeruginosa s t r a i n s , with two cyanogen bromide-derived peptides of the protein and with the 2-mercaptpethanol treated or untreated protein. None of these antibodies interacted with a protein F d e f i c i e n t P. aeruginosa s t r a i n . iv TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv L i s t of Tables x L i s t of Figures . . x i i ACKNOWLEDGEMENTS . . . . . . . . . . xv DEDICATION xvi INTRODUCTION . . . . . . . . . 1 1. The medical importance of Pseudomonas aeruginosa 1 2. The c e l l envelope of gram-negative b a c t e r i a 3 3. Components and structure of the outer membrane 5 (A) Lipopolysaccharide . 6 (B) Proteins • 7 4. C e l l envelope and the immune response 9 (A) Lipopolysaccharide 10 (B) Proteins 13 5. Aims of t h i s study 16 MATERIALS AND METHODS 1. Bacteria . . . . . . . 18 (A) Media and growth conditions 18 (B) B a c t e r i a l s t r a i n s 18 (C) C e l l envelope and outer membrane i s o l a t i o n 20 TABLE OF CONTENTS, continued Page (D) Protein and LPS p u r i f i c a t i o n . . . . . . . . 21 (E) Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis 21 ( i ) protein s t a i n i n g 22 ( i i ) LPS staini n g . 22 (F) Protein and Keto-deoxyoctonate (KDO) assays . . . . . 22 (G) Enzymatic digestion of protein F . . . . . . . . . . . 23 (H) Chemical cleavage of protein F . 23 2. Animals (A) Animals used 24 (B) Immunizing protocol . . . 24 3. Tissue Culture (A) Myeloma c e l l l i n e s 25 (B) Medium, maintenance and freezing of c e l l l i n e s . . . . 26 (C) Generation of monoclonal antibodies 28 ( i ) c e l l fusion 29 ( i i ) cloning . 31 (D) Ascites production 31 (E) Antibody p u r i f i c a t i o n 32 vi TABLE OF CONTENTS, continued Page 4. Antibody c h a r a c t e r i z a t i o n (A) Enzyme-linked immunosorbent assays-ELISA 33 (B) Western electrophoretic b l o t transfers . . . . . . . . 36 (C) Double immunodiffusion assay 37 (D) Agglutination assays . 38 (E) Indirect immunofluorescence 40 (F) Colony b l o t t i n g . . . . . . 41 5. Serum s e n s i t i v i t y t e s t i n g . . . . . . . . . . . . 42 RESULTS CHAPTER I. Outer membrane antigens of P. aeruginosa 1. Outer membrane patterns of serotyping s t r a i n s . 44 2. Cystic f i b r o s i s i s o l a t e s - ch a r a c t e r i z a t i o n and outer membrane patterns 47 3. I s o l a t i o n and cha r a c t e r i z a t i o n of a poly c l o n a l a n t i s e r a against outer membranes of P. aeruginosa 54 4. Interaction of polyclonal sera with the outer membranes of P. aeruginosa s t r a i n s 56 5. Summary . 63 CHAPTER I I . Monoclonal antibodies to outer membrane antigens 1. I s o l a t i o n and ch a r a c t e r i z a t i o n of monoclonal antibodies . . 65 (A) An LPS 0-Antigen s p e c i f i c monoclonal antibody . . . . 65 (B) Rough LPS s p e c i f i c monoclonal antibodies . 66 (C) L i p i d A s p e c i f i c monoclonal antibodies 67 v i i TABLE OF CONTENTS, continued Page (D) - A l i p o p r o t e i n H2 s p e c i f i c monoclonal antibody . . . . 67 (E) Protein F s p e c i f i c monoclonal antibodies . . 68 (F) Monoclonal antibody MA1-3 . . . 68 2. Use of monoclonal antibodies to study outer membrane antigen heterogeneity of P. aeruginosa . . . 73 (A) S p e c i f i c i t y of protein F s p e c i f i c monoclonal antibodies 73 (B) C r o s s - r e a c t i v i t y of a l i p o p r o t e i n H2 s p e c i f i c monoclonal antibody 83 (C) Heterogeneity of LPS-rough core in P. aeruginosa . . 93 (D) Heterogeneity of LPS O-antigen 96 (E) Conservation of L i p i d A 98 3. Summary 104 CHAPTER I I I . Characterization of outer membrane epitopes using monoclonal antibodies 1. C e l l surface l o c a l i z a t i o n of the antigenic s i t e s recognized by monoclonal antibodies . . ' . 106 (A) Surface l o c a l i z a t i o n studies by immunofluorescence . . 106 (B) Surface l o c a l i z a t i o n studies by colony immunoblotting . . . I l l (C) Interaction of monoclonal antibodies with other bacteria by colony immunoblotting 122 v i i i TABLE OF CONTENTS, continued Page 2. Studies on protein F epitopes recognized by monoclonal antibodies 123 (A) E f f e c t of 2-mercaptoethanol on binding of protein F s p e c i f i c monoclonal antibodies 123 (B) Interaction of monoclonal antibodies with cyanogen bromide fragments of protein F 126 (C) Interaction of monoclonal antibodies with p r o t e o l y t i c peptides of protein F 129 3. Summary 137 DISCUSSION 1. Conservation and heterogeneity of LPS epitopes 139 (A) LPS of P. aeruginosa i s o l a t e s from c y s t i c f i b r o s i s patients . . . . . . . . . . . . . 142 2. Antigenic conservation of P. aeruginosa outer membrane proteins . . . . . . . 143 (A) Conservation of outer membrane protein patterns . . . 144 (B) Antigenic conservation revealed using p o l y c l o n a l antiserum 145 (C) Conservation of s p e c i f i c epitopes on P. aeruginosa outer membrane proteins 147 (D) D i s t r i b u t i o n of s p e c i f i c epitopes among other species of b a c t e r i a . 149 ix TABLE OF CONTENTS, continued Page 3. Surface a c c e s s i b i l i t y studies using monoclonal antibodies . . . . . 154 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 protein F epitopes . . . . 156 5. Prospectives 158 LITERATURE CITED 160 X LIST OF TABLES Table Page 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 selected P. aeruginosa is o l a t e s from patients with c y s t i c f i b r o s i s and rough LPS-altered mutants. 51 II . Cross-reactions of antisera to outer membrane and p u r i f i e d outer membrane components with outer membranes from various serotype s t r a i n s of P. aeruginosa 55 I I I . Interaction of monoclonal antibodies with p a r t i a l l y p u r i f i e d major outer membrane proteins of P. aeruginosa PA01. . . . . . 69 IV. Interaction of monoclonal antibodies with the outer membranes of LPS alte r e d strains of P. aeruginosa. 70 V. Interaction of monoclonal antibodies MA2-10 and MA4-4 with outer membranes of P. aeruginosa serotype st 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 74 VI. Cross-reaction of monoclonal antibodies s p e c i f i c f o r P. aeruginosa outer membranes assessed by ELISA . 84 VII. Interaction of monoclonal antibodies MAi-3, MA1-6, and MA1-8 with the outer membrane antigens from 17 serotype s t r a i n s of P. aeruginosa 97 VIII. Demonstration of c e l l surface l o c a l i z a t i o n of outer membrane components of P. aeruginosa by immunofluorescence. 110 x i LIST OF TABLES, continued Table Page IX. Interaction of protein F s p e c i f i c monoclonal antibodies with p u r i f i e d protein- F, outer membranes and whole c e l l s of s t r a i n H103 assessed by ELISA. . 112 X. Binding of monoclonal antibodies d i r e c t e d against P. aeruginosa outer membrane antigens to colony blots 114 XI. Reaction of LPS s p e c i f i c monoclonal antibodies with outer membranes from P. aeruginosa st r a i n s in ELISA assays 140 XII. Pot e n t i a l taxonomic value of monoclonal antibodies 151 XIII. D i f f e r e n t i a t i o n of two classes of monoclonal antibodies s p e c i f i c for P. aeruginosa protein F . 157 xi i LIST OF FIGURES Figure Page 1. SDS-polyacrylamide gel electrophoretogram of outer membranes of P. aeruginosa s t r a i n H103 and serotyping strains 46 2. SDS-gel electrophoretogram of outer membrane proteins of P. aeruginosa i s o l a t e s from patients with c y s t i c f i b r o s i s . . . 49 3. SDS-gel electrophoretograms of outer membranes of P. aeruginosa isolates from patients with c y s t i c f i b r o s i s stained f o r LPS . . 53 4. Autoradiogram of ele c t r o p h o r e t i c b l o t of outer membrane proteins of P. aeruginosa a f t e r i n t e r a c t i o n with radioiodinated antibodies to outer membrane proteins 58 5. A. and B. Densitometer tracings of autoradiograms of separated outermembrane proteins from P. aeruginosa s t r a i n H103, serotype strains and c l i n i c a l i s o l a t e PI 61, 62 6. Western immunoblots of P. aeruginosa s t r a i n H103 a f t e r i n t e r a c t i o n with monoclonal antibodies MAl-8, MA3-5, MA1-6, MA4-4 and 5E4 . . . . . 72 7. Western immunoblots of outer membranes of the serotyping strains of P. aeruginosa a f t e r i n t e r a c t i o n with monoclonal antibody MA5-8 78 8. Interaction of monoclonal antibody MA2-10 with outer membranes from c y s t i c f i b r o s i s P. aeruginosa i s o l a t e s . 80 xi i i LIST OF FIGURES, continued Figure Page 9. C r o s s - r e a c t i v i t y of protein F s p e c i f i c monoclonal antibody MA4-4 with outer membranes from other b a c t e r i a l species . . . . 82 10. Western immunoblots of outer membranes of serotype s t r a i n s of P. aeruginosa a f t e r i n t e r a c t i o n with monoclonal antibody MA1-6 88 11. Western immunoblot of outer membranes of mucoid and non-mucoid P. aeruginosa i s o l a t e s interacted with monoclonal antibodies MA4-4 and MA1-6 90 12. Western immunoblots of outer membranes of d i f f e r e n t Pseudomonads interacted with monoclonal antibody MA1-6 92 13. Western immunoblot of outer membranes of P. aeruginosa serotype s t r a i n s a f t e r reaction with monoclonal antibody MA3-5. 95 14. Western immunoblot of outer membranes of P. aeruginosa serotype s t r a i n s a f t e r reaction with monoclonal antibody 5E4 . 101 15. Reaction of monoclonal antibody 5E4 with a Western b l o t of outer membranes of c l i n i c a l i s o l a t e s of P. aeruginosa 103 16. Indirect immunofluorescerit l a b e l l i n g of intact P. aeruginosa tagged with monoclonal antibodies to outer membrane components. 109 17. Colony immunoblot showing inte r a c t i o n of monoclonal antibody MA4-10 with 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, continued Figure Page 18. Colony immunoblot showing i n t e r a c t i o n of monoclonal antibody MAl-8 with serotype strains and c l i n i c a l i s o l a t e s of P. aeruginosa . . . . . . . . . . . . . 119 19. Colony immunoblots showing i n t e r a c t i o n of monoclonal antibody MA1-6 with d i f f e r e n t b a c t e r i a l strains 121 20. Western immunoblots of p u r i f i e d protein F: e f f e c t of 2-mercaptoethanol 125 21. SDS-gel electrophoretogram and Western immunoblots of p u r i f i e d protein F before and a f t e r degradation with cyanogen bromide 128 22. Western immunoblot of native and p r o t e o l y t i c a l l y treated protein F, from a p u r i f i e d protein F sample, outer membranes and whole c e l l s . 132 23. SDS-gel electrophoretograms of protein F peptides from a time course proteolysis of p u r i f i e d protein F with the enzyme tryps i n 134 24. Interaction of protein F s p e c i f i c monoclonal antibody MA2-10 with p r o t e o l y t i c a l l y treated whole c e l l s of P. aeruginosa CF C46nm and P. putida ATCC 12633 136 ACKNOWLEDGEMENTS I am si n c e r e l y indebted to my supervisor, Bob Hancock., for his constant advice, guidance, and friendship during the course of my studies. I am g r a t e f u l to the Department of Microbiology and the Canadian Commonwealth Scholarship Plan for f i n a n c i a l support. I wish to thank members of the Microbiology Department, and members of my supervisory committee for t h e i r help and advice. In p a r t i c u l a r , many thanks to the varied members of Bob's lab for t h e i r f r i e ndship and support. Thanks to Susan Heming for her patience in typing t h i s t h e s i s . xv i DEDICATION This thesis i s dedicated to my parents, E. Nj e r i and T. Mutharia, who have always supported our endeavours and aspirations. \ 1 INTRODUCTION The gram-negative bacterium Pseudomonas aeruginosa e x i s t s and p r o l i f e r a t e s in a wide range of natural environments due to i t s 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 a large v a r i e t y of carbon sources. During the l a s t 20-30 years P. aeruginosa has emerged as a major opportunistic pathogen associated with high m o r t a l i t y in h o s p i t a l acquired i n f e c t i o n s . 1. The medical importance of P. aeruginosa An impressive increase in the incidence of gram-negative b a c t e r i a l infections occurred with the introduction of p e n i c i l l i n a n t i b i o t i c s and the subsequent elimination of Streptococci and Staphylococci as the major causes of nosocomial i n f e c t i o n s . Since then, the intensive a p p l i c a t i o n of broad spectrum a n t i b i o t i c s has resulted in b a c t e r i a l s u b s t i t u t i o n , with more a n t i b i o t i c r e s i s t a n t s t r a i n s replacing highly a n t i b i o t i c susceptible ones. In p a r t i c u l a r , P. aeruginosa which i s well known for i t s high i n t r i n s i c resistance to a n t i b i o t i c s , detergents and a n t i s e p t i c s (Bryan,, 1979; Hancock, 1981) has been associated with every aspect of the h o s p i t a l environment. Although only 4-10% of the normal human adult population are f e c a l or pharyngeal c a r r i e r s of P. aeruginosa (Bodey §_t a l . , 1983), the c a r r i e r rate i s reported to r i s e to 43% among patients h o s p i t a l i z e d for more than 15 days (Bodey et a l . , 1983). Young et a l . (1982) reported that although P. aeruginosa caused only 20% of the t o t a l gram-negative i n f e c t i o n s , i t 2 was associated with 84% mortality. The high a n t i b i o t i c resistance of P. aeruginosa as well as the large repertoire of virulence factors elaborated by this organism presumably contribute to i t s prevalence and virulence (Young, 1980; L i u , 1974). Among the gram-negative bacteria causing nosocomial i n f e c t i o n s , P. aeruginosa i s exceptional in i t s a b i l i t y to i n f e c t s p e c i f i c sub-populations of patients due eit h e r to the underlying disease condition or as a r e s u l t of therapeutic measures. Among those most susceptible are immunocompromised patients, e s p e c i a l l y those with acute leukemia or neoplastic diseases which r e s u l t in neutropenia (Young, 1979; Young and Pollack, 1980) as well as patients receiving immunosuppressive therapy, such as cytotoxic and c o r t i c o s t e r o i d therapy f o r cancer, ti s s u e or organ transplantations (Schimpff ejt a l . , 1970). Among these patients, f a t a l i t y due to P. aeruginosa bacteremia has been reported to be as high as 50-807. (Rodriquez and Bodey, 1979). P. aeruginosa i s also the predominant gram-negative bacterium of burn wound in f e c t i o n s . P r u i t t and Lindenberg (1979) reported that 50% of burn victims were colonised by the bacterium within 48 hours of h o s p i t a l i z a t i o n . The frequency of P. aeruginosa i n f e c t i o n s is also high among patients requiring invasive s u r g i c a l procedures or in-dwelling urinary or vascular catheters, and among diabetics and intravenous drug addicts, where the bacterium gains access to s t e r i l e t i s s u e . P. aeruginosa i s well known for chronic i n f e c t i o n s in c y s t i c f i b r o s i s patients where i t i s often associated with terminal pulmonary i l l n e s s . In these patients, the bacterium elaborates massive amounts of a mucoid 3 exopolysaccharide. The f a i l u r e of pulmonary clearance of P. aeruginosa may be due to the copious amounts of mucoid material which is p r e c i p i t a t e d by the high amounts of calcium present in the lung f l u i d s of these patients, as well as the presence in serum of a postulated " c y s t i c f i b r o s i s f a c t o r " which prevents normal c i l i a r y movement (Bowman et a l . , 1975). Thomassen e_t a l . (1979) reported the presence of a fac t o r in the serum of these patients that impaired normal phagocytic function of rabbit and human alveolar macrophages. In addition, P. aeruginosa i s a common cause of hospital-acquired pneumonias, urinary t r a c t infections and endocarditis and sometimes causes f a t a l meningitis. 2. The c e l l envelope of gram-negative b a c t e r i a The c e l l envelope of P. aeruginosa, l i k e that of other gram-negative bacteria, consists of three layers, the cytoplasmic (inner) membrane, a peptidoglycan (murein) layer, and the outer membrane. The cytoplasmic membrane is a phospholipid b i l a y e r containing 40-60% by weight of proteins. Localized in t h i s membrane are s p e c i f i c energy-coupled transport systems and components of the c e l l u l a r energy-generating systems, as well as enzymes f o r the synthesis and export of outer membrane and peptidoglycan components. The periplasm, located between the outer and inner membranes, i s r i c h in oligosaccharides, cata b o l i c enzymes, binding proteins and enzymes involved in degradation of toxic substances (Benveniste and Davies, 1973) . 4 The c e l l shape and osmotic s t a b i l i t y is maintained by the r i g i d structure of the peptidoglycan layer. The chemical composition and structure of the peptidoglycan is s i m i l a r in a l l gram-negative ba c t e r i a (Meadow e_t a l . , 1978). In P. aeruginosa PA01, unlike E. c o l i . there i s no convincing evidence of covalent-linkage of t h i s layer to the outer membrane components, although strong non-covalent linkage has been demonstrated (Hancock e_t a l . , 1981) . The c e l l surface (outer) membrane of gram-negative bacteria forms the interface between the c e l l and i t s environment. Among i t s functional properties, the outer membrane prevents leakage of periplasmic proteins and i n h i b i t s the entry of toxic substances l i k e s a l t s , detergents 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 nutrients. Thus, i t constitutes a major physical and f u n c t i o n a l b a r r i e r for c e l l s . In P. aeruginosa, the outer membrane has been shown to be the main b a r r i e r responsible f o r the high i n t r i n s i c resistance of t h i s bacterium to a n t i b i o t i c s (Nicas and Hancock, 1983; Angus et a l . , 1982). Of greater importance to the present study, the outer membrane represents the c e l l u l a r component involved in the primary i n t e r a c t i o n with the hosts* immune system and therefore contributes to the a b i l i t y of the organism to invade, p e r s i s t , evade and r e s i s t the host's defence mechanisms and therefore manifest the disease syndrome. The outer membrane components involved in each of the above f u n c t i o n a l , p h y s i c a l and protective functions have not a l l been defined, but a b r i e f overview of the structure and components of the outer membrane as r e l a t e d to function i s presented below. 5 In addition to these components of the c e l l envelopes, some but not a l l gram-negative b a c t e r i a contain a d d i t i o n a l c e l l envelope associated structures. In many ba c t e r i a , the c e l l surface is covered by eit h e r an amorphous or a highly ordered capsular layer that can be of protein (Kay et a l . , 1981, Winter §_t a l . , 1978) or polysaccharide material (Moorhouse et a l . , 1977). P. aeruginosa strains do not have a true capsule but those strains i s o l a t e d from c y s t i c f i b r o s i s patients are often surrounded by a mucoid exopolysaccharide resembling seaweed alginate and composed of mannuronic and guluronic acid residues. Anchored in the c e l l envelope are c e l l u l a r appendages l i k e f l a g e l l a , p i l i and fimbriae. F l a g e l l a are associated with a l l three layers of the c e l l envelope and are responsible for m o t i l i t y (Craven and Montie, 1981), while p i l i ( e s s e n t i a l f o r conjugation) and the much smaller, r i g i d fimbriae have been implicated in adhesion (Woods et a l . , 1980a, b). Another component of the Pseudomonas aeruginosa c e l l envelope i s a common antigen composed mainly of protein. This common antigen exhibits extensive antigenic 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 of gram-negative bacteria (Sompolinsky et a l . , 1980a). 3. Components and structure of the outer membrane The structure and functions of the outer membrane of ent e r i c b a c t e r i a have been extensively studied (Osborn and Wu, 1980; Lugtenberg and Van Alphen, 1983). The outer membrane, as determined by electron microscopy, is a b i l a y e r composed of phospholipids, proteins and lipopolysaccharide (LPS). The outer membrane of gram-negative bacteria i s a very unusual 6 b i o l o g i c a l membrane in that i t has a highly asymmetrical d i s t r i b u t i o n of i t s components. A l l the LPS i s present in the outer monolayer where i t i s the major l i p i d i c molecule, while v i r t u a l l y a l l the phospholipid is in the inner l e a f l e t (with the possible exception of some LPS mutants of E. c o l i and Salmonella typhimurium); proteins are present in both layers and in some-cases are membrane-spanning. (A) Lipopolysaccharide (LPS) LPS i s an amphiphilic molecule and has been shown to be s t r u c t u r a l l y very s i m i l a r in a l l gram-negative ba c t e r i a studied (Orskov et a l . , 1977 ; Rietschel et a l . , 1983). St r u c t u r a l studies indicate a t r i p a r t i t e structure (Nikaido and Nakae, 1979; Wilkinson, 1983) c o n s i s t i n g of a hydrophobic region, the L i p i d A (also known as endotoxin), covalently attached to a rough core region which is often, but not always, capped with a hydrophilic 0 - polysaccharide chain extending from the c e l l surface. More d e t a i l e d studies on LPS have shown the l i p i d A to be a g l y c o l i p i d usually containing a diglucosamine residue that i s substituted with phosphate and/or pyrophosphate residues and 5 to 6 f a t t y acids, two of which are amide linked to the diglucosamine. The f a t t y acids are almost always saturated, and include B-OH f a t t y acids that are amide linked to the diglucosamine. These hydroxy f a t t y acids 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 contains a highly unusual octosaccharide, Z-keto-3-deoxyoctonate (KDO) (Nikaido and Nakae, 1979) at i t s proximal end; t h i s octose i s also highly s p e c i f i c to LPS. In addition, the rough core contains a v a r i e t y of hexoses and heptoses which 7 may be substituted with phosphate and ethanolamine phosphate. The rough core region of LPS is s t r u c t u r a l l y highly conserved among the i n d i v i d u a l Enterobacterial species (Lugtenberg and Van Alphen, 1983). The L i p i d A-rough core region contains a high density of negatively-charged residues and has been shown to be the divalent cation binding s i t e of the outer membrane (Schindler and Osborn, 1979). The hydrophilic oligosaccharide portion of LPS consists of a highly variable number of repeated t r i - to pentasaccharide u n i t s . The substantial v a r i a b i l i t y in composition of th i s region, which occurs even within a single species (or genus) allows f o r tremendous v a r i a t i o n in the molecular make-up of the c e l l surface. This v a r i a t i o n of the polysaccharide chain has been used as a fine s e r o l o g i c a l typing t o o l , r e s u l t i n g in the name "0" antigen for t h i s region (Brokopp and Farmer, 1979; Lanyi and Bergan, 1979). (B) Proteins SDS-polyacrylamide gel electrophoresis techniques have contributed greatly 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 of b a c t e r i a l outer membrane proteins. SDS-gel electrophoretograms of outer membranes of P. aeruginosa (Hancock and Carey, 1979), l i k e those of other gram-negative bac t e r i a , show the presence of a few, very prominent "major" polypeptide bands and numerous other "minor" polypeptides (For reviews see Nikaido and Nakae, 1979; Osborn and Wu, 1980; Lugtenberg and Van Alphen, 1983). The reason for the presence of a r e s t r i c t e d number of major proteins 4 5 in high copy number (2 x 10 to 3 x 10 copies per c e l l ) i s uncertain 8 although in some cases important s t r u c t u r a l and functional roles have been demonstrated. For example in P. aeruginosa l i p o p r o t e i n I, analogous to the E. c o l i peptidoglycan bound l i p o p r o t e i n (Braun, 1975), l i p o p r o t e i n H2 and protein F a l l have s t r u c t u r a l r o l e s , being noncovalently associated with the peptidoglycan (Mizuno, 1979; Hancock, et a l . , 1981). A major function of the outer membrane i s as a size dependent permeability b a r r i e r . This b a r r i e r function i s l a r g e l y determined by a family of transmembrane proteins named porins. These porin proteins are present in a l l gram-negative b a c t e r i a examined to date, and t h e i r function has been extensively studied (Reviews by Lugtenberg and Val Alphen 1983; Nikaido and Nakae, 1979). They have been shown to form quite non-specific, w a t e r - f i l l e d hydrophilic channels across the outer membrane. 4 5 Porin proteins are present in high copy numbers (10 to 10 copies per c e l l ) , have apparent molecular weights of 32 - 42,000 daltons, are non-covalently associated with the peptidoglycan (DiRienzo e_t a l . , 1978) and are organized as trimers in the outer membrane (Angus and Hancock, 1983; Yu et a l . , 1979; I s h i i and Nakae, 1980). They form channels with a f a i r l y constant diameter and thus determine the molecular size of compounds entering the c e l l . For example, the exclusion l i m i t of the E. c o l i and S. typhimurium porins i s about 500 - 650 daltons (Nakae, 1975; 1976b). In P. aeruginosa the outer membrane is permeable to molecules of up to 6,000 daltons (Hancock, §_t a l . , 1979), yet P. aeruginosa exhibits lower permeability to a n t i b i o t i c s than E. c o l i (Nicas and Hancock, 1983). This property has been a t t r i b u t e d to P. aeruginosa having less than 1% of i t s porin protein molecules forming 9 functional open pores (Benz and Hancock, 1981; Nicas and Hancock, 1983). In P. aeruginosa. protein F i s the main non-specific hydrophilic pore-forming protein contributing to the general permeability of the outer membrane (Nicas and Hancock, 1983). The a l t e r n a t i v e porin proteins DI and P are induced by growth in glucose and under phosphate l i m i t i n g conditions respectively (Hancock and Carey, 1980; Hancock et a l . , 1982). 4. C e l l envelope and the Immune Response B a c t e r i a l c e l l surface components are involved in the primary inte r a c t i o n of the bacterium with the host's immune system and, therefore, contribute in part to the a b i l i t y of the b a c t e r i a to i n f e c t the susceptible host. In th i s respect, the c e l l surface components can be considered as virulence f a c t o r s . One i n d i c a t i o n of these interactions i s the generation of s p e c i f i c antibodies i n the infec t e d host, against the foreign ( b a c t e r i a l ) antigens. The e a r l i e s t studies on the a n t i g e n i c i t y and role in virulence of b a c t e r i a l surfaces were involved with surface appendages. Most of the c l i n i c a l i s o l a t e s possessed appendages l i k e p i l i , fimbriae, f l a g e l l a or capsules, which were often lacking in a v i r u l e n t s t r a i n s . Each of these components has therefore been ascribed a r o l e in b a c t e r i a l pathogenesis. P i l i and fimbriae are involved in b a c t e r i a l adhesion to s p e c i f i c e p i t h e l i a l c e l l s (Woods, 1980a, b). F l a g e l l a (the basis of H-antigen schemes) have been correlated to invasiveness of P. aeruginosa in burn tissue (Holder e_t aJL. , 1981) . Capsules increase the virulence of Aeromonas salmonicida. N e i s s e r i a meningitidis and Haemophilus influenzae 10 type b (Frasch and Robbins, 1978; Munn et a l . , 1982) and may be involved in persistence and resistance to phagocytosis of E. c o l i c e l l s (Blackwood and Pennington, 1981; Costerton e_t a l . , 1983). However, where studied, these components can vary s u b s t a n t i a l l y from s t r a i n to s t r a i n . The antigenic v a r i a t i o n of these components ( P i t t , 1980; Brinton, 1982; V i r j i et a l . , 1983) and hence t h e i r i n a b i l i t y to provide active immunity against heterologous organisms of the same species, has focussed attention on other b a c t e r i a l c e l l surface components. The outer membrane contains two immunologically important molecules, the LPS and proteins. Most of the studies on the immune response to outer membrane components have been performed on LPS. Antibodies to LPS have been shown to agglutinate, and promote opsonic phagocytosis of homologous bacteria (Ka'ss and Wolff, 1973). In contrast, outer membrane proteins have been subjected to intensive research only in recent years, with the search for non-toxic, LPS-free vaccines. (A) LPS Ba c t e r i a l LPS has been the subject of extensive s t r u c t u r a l and immunological studies (Kass and Wolff, 1973, Lanyi and Bergan, 1979; Wilkinson, 1983). The b i o l o g i c a l or immunological a c t i v i t i e s of i s o l a t e d LPS depend on i t s source and to some extent on the procedures used to pur i f y i t . A major b i o l o g i c a l property of P. aeruginosa LPS, l i k e that of other gram-negative b a c t e r i a , i s endotoxicity which i s a major virulence factor in inf e c t i o n s (Owen, 1981). In a series of d e t a i l e d studies, Homma and colleagues (Cho e t a l . , 1979; Tanamoto and Homma 1982) i s o l a t e d and 11 fractionated LPS from E. c o l i , P. aeruginosa. S. typhimurium and V. cholerae and showed that the intact L i p i d A region was the endotoxin component of LPS and was also e s s e n t i a l for i t s pyrogenicity in rabbits and for i t s mitogenicity towards mouse B - c e l l lymphocytes. In addition to these important b i o l o g i c a l properties, LPS demonstrates a plethora of e f f e c t s on infected hosts (Bradley, 1979; Tanamoto and Homma, 1982; Tanamoto et a l . , 1979). The strong immunogenic a c t i v i t y of LPS i s a function of the polysaccharide O-antigenic side chain when lin k e d to i t s own natural adjuvant ( 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 in the chemical composition and consequently the a n t i g e n i c i t y of the LPS O-antigen of d i f f e r e n t strains of b a c t e r i a within a species i s the basis of the f i n e typing schemes of gram negative b a c t e r i a (Orskov et_ a l . , 1977; Brokopp and Farmer, 1979) * In P. aeruginosa a va r i e t y of serotyping schemes have been described (Brokopp and Farmer, 1979; Lanyi and Bergan, 1979) . One of these, the International Antigen Typing Scheme comprises 17 d i s t i n c t serotype st r a i n s and contains, as subsets, the serotypes of a l l other schemes. One major problem of these serotyping schemes i s the high frequency in P. aeruginosa disease i s o l a t e s of non-typable or polyagglutinable s t r a i n s , a property now shown to c o r r e l a t e to deficiences in the O-antigen (Hancock et a l . , 1983). This observation brings into question the broad a p p l i c a t i o n of these O-antigen s p e c i f i c antisera in serotyping and in epidemiological studies of P. aeruginosa. The strong a n t i g e n i c i t y of the O-side chain of LPS has made i t a popular candidate for vaccines. However, the antigenic 12 v a r i a t i o n and endotoxicity noted above as we'll as the lack of a memory (IgG) response with the '0' antigen, are factors which tend to argue against LPS as a good vaccine. Structural s i m i l a r i t i e s in the inner core region of both smooth and rough form LPS among i n d i v i d u a l species of gram-negative bacteria have been shown (Ziegler et a l . , 1975; Braude et a l . , 1983; Galanos et a l . , 1984) using antisera s p e c i f i c for that region. Antiserum to the rough core region of the E. c o l i J5 (LPS mutant) LPS has been shown to protect animals against gram-negative bacteremia and endotoxemia including P. aeruginosa bacteremia (Braude et a l . , 1977, 1978; Chedid et a l . , 1968; Pollack and Young, 1979). However, p r i o r to the present study, no s p e c i f i c information was available on the s p e c i f i c epitopes of t h i s s t r u c t u r a l l y complex region of the LPS. Other important roles of b a c t e r i a l LPS are in resistance to k i l l i n g by serum factors and in resistance to phagocytosis (Taylor 1983), as well as in resistance to detergents and a n t i b i o t i c s (Kropinski e t a l . , 1978). The property of serum b a c t e r i c i d a l resistance (Rowley, 1968) was correlated to the length of the 0-antigenic side chains (smooth strains with long 0-side chains being serum r e s i s t a n t and rough s t r a i n s with no 0-side chains being serum s e n s i t i v e ) . The long side chains were proposed to block the binding of activated serum complement complexes to the b a c t e r i a l surface (reviewed by Taylor, 1983). The composition of the LPS 0-antigen was also correlated to resistance to phagocytosis by mouse macrophage c e l l s i n Salmonella typhimurium (Liang-Takasaki et a l . , 1982). The mechanism of resistance was proposed to be of bacterial-phagocyte 13 a f f i n i t y , because the rate of k i l l i n g of phagocytosed b a c t e r i a was the same for a l l LPS O-antigen variants. The importance of the '0' antigen in pathogenesis can be summed up by the observation that generally only smooth rather than rough (0 antigen-deficient) gram-negative b a c t e r i a are is o l a t e d from bacteremic i n f e c t i o n s , where resistance to serum b a c t e r i c i d a l a c t i v i t y and phagocytosis would be major factors in b a c t e r i a l persistence. (B) Proteins Studies on the a n t i g e n i c i t y of b a c t e r i a l outer membrane proteins have progressed r a p i d l y in recent years, due to the need to develop non-toxic, LPS-free vaccines providing heterologous protection against a l l serotypes of a given bacterium. Surface a c c e s s i b i l i t y of outer membrane proteins has been shown by bacteriophage binding to outer membrane protein receptors (Braun and Krieger-Brauer 1977; Datta et a l . , 1977), surface 125 l a b e l l i n g with "non-penetrating" r a d i o l a b e l l e d dextrans or I (Kamio and Nikaido, 1977), and i n d i r e c t immunofluorescence using p o l y c l o n a l sera (Hofstra et al'.', 1979). The mitogenicity of outer membrane proteins f o r B c e l l lymphocytes has been shown for P. aeruginosa protein F, and li p o p r o t e i n s H2 and I (Chen e_t a l . , 1980), for N. meningitidis surface antigens (Melancon et a l . , 1983) and for the E. c o l i OmpF, OmpA and Lpo proteins (Bessler and Henning, 1979). In p r i n c i p l e , the a n t i g e n i c i t y of outer membrane proteins was shown by the a b i l i t y of active immunization with outer membrane proteins, to lead to protection of mice against experimental salmonellosis 14 in suckling mice (Kuusi et a l , , 1979) and against k e r a t o c o n j u c t i v i t i s sh i g e l l o s a (Adamus §_t a l . ; 1980). Outer membrane proteins have also been used in t r i a l vaccine studies in H. influenzae (Lam et a l . , 1980b; Hansen et a l . , 1982), and Ne i s s e r i a sp. ( Z o l l i n g e r et. a l . , 1978, Buchanan, 1977) . In P. aeruginosa strong evidence for the immunogenicity of outer membrane proteins has been demonstrated in c y s t i c f i b r o s i s patients colonized with t h i s bacterium (Hancock et a l . , 1984) and in convalescent patients recovering from Pseudomonas bacteremia (Lam et a l . , 1983, Fernandes et a l . , 1981) as well as in rats with induced chronic P. aeruginosa lung i n f e c t i o n s (Lam et a l . , 1983). In a l l these cases, antibodies that i n t e r a c t with P. aeruginosa outer membrane proteins were i d e n t i f i e d . Hofstra and colleagues (Hofstra and Dankert, 1979; Hofstra e_t a l . , 1980) demonstrated that polyclonal antisera r a i s e d against the E. c o l i K12 porin proteins OmpF and OmpC showed extensive immunological c r o s s - r e a c t i v i t y with proteins of 32-42,000 daltons from the outer membranes of other Enterobacteriaceae. Similar c r o s s - r e a c t i v i t y was demonstrated for the 33,000 dalton OmpA protein of E. c o l i with s i m i l a r proteins from other Enterobacteriaceae s t r a i n s (Hofstra e_t a l . , 1980) . In simi l a r studies, antigenic cross-reactions were shown by Braun et a l . , (1976) for the peptidoglycan-bound l i p o p r o t e i n of E. c o l i and other enterobacterial s t r a i n s . In i n d i r e c t immunofluorescent studies, Braun et a l . , (1976) also showed that t h i s l i p o p r o t e i n was not surface l o c a l i s e d or accessible in int a c t organisms. In contrast to the extensive 15 immunological c r o s s - r e a c t i v i t y among the major porins of Enterobacteriaceae, antisera to the E. c o l i LamB protein did not react with any other protein. Monoclonal antibodies s p e c i f i c for outer membrane proteins have been used to show the surface a c c e s s i b i l i t y of those proteins in i n t a c t organisms of 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). In addition, surface exposed antigens have been te n t a t i v e l y i d e n t i f i e d in N. gonorrhoeae (Swanson et a l . , 1982) and H. influenzae type b (Robertson et a l . , 1982; Gulig et a l . , 1982) by 125 inte r a c t i o n of monoclonal antibodies with i n t a c t I - l a b e l l e d b a c t e r i a , followed by detergent l y s i s and p r e c i p i t a t i o n of the antigen-antibody complexes. However, these studies are not r e a l l y d e f i n i t i v e since r a d i o l a b e l l i n g has been shown to perturb outer membrane i n t e g r i t y (Sullivan and Williams, 1982). In recent years, great i n t e r e s t has developed in the a p p l i c a t i o n of outer membrane proteins in c l a s s i f i c a t i o n of some b a c t e r i a l species, as an alternative to serogrouping based on LPS O-antigen. Substantial v a r i a t i o n in the outer membrane protein patterns of i n d i v i d u a l s t r a i n s have been demonstrated for E. c o l i (Overbeeke and Lugtenberg, 1980), V. cholerae (Kabir and Mann, 1980), N. men i g i t i d i s (Tsai et a l . , 1981), N. gonorrhoeae (Buchanan and Hildebrandt, 1981), H. influenzae (Loeb and Smith, 1982; Barenkamp, 1981), and V. anguillarum (Nakajima et a l . , 1983). For many of these species, a biotyping scheme based on grouping according to the s p e c i f i c outer membrane protein patterns of i n d i v i d u a l s t r a i n s has been proposed. The heterogeneity of protein patterns is often 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 antisera to s p e c i f i c outer membrane proteins has 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 and Hildebrandt, 1981; Knapp et a l . , 1984) H. influenzae (McDade and Johnson, 1980), and Legionella pneumophila (Joly, et a l . , 1983). In contrast to LPS, the d i r e c t contribution of outer membrane proteins to virulence by enhancing the s u r v i v a l of a bacterium iri vivo has only been shown for N. gonorrhoeae (Lambden et a l . , 1979), A. salmonicida (Kay e_t a l . , 1981), and E. c o l i [where resistance to serum b a c t e r i c i d a l a c t i v i t y was due to the presence of an outer membrane plasmid-encoded (the TraT) protein (Moll 1980)]. 5. Aims of t h i s study The objective of t h i s i n v e s t i g a t i o n was to look at the a n t i g e n i c i t y and surface immunochemietry of major outer membrane proteins of P. aeruginosa. Studies using polyclonal antiserum to s t r a i n H103 outer membranes demonstrated antigenic c r o s s - r e a c t i v i t y at the polyclonal l e v e l of the major outer membrane proteins. However, the expression of s p e c i f i c antigenic determinants on these proteins was studied in order to demonstrate the antigenic r e l a t i o n s h i p s of these antigens. To a s s i s t in t h i s aim, monoclonal antibodies s p e c i f i c for porin protein F and l i p o p r o t e i n H2 were developed. These monoclonal antibodies were used to study the antigenic d i s t r i b u t i o n of these proteins in both laboratory s t r a i n s and c l i n i c a l i s o l a t e s of P. aeruginosa. The l o c a l i z a t i o n of the proteins on i n t a c t b a c t e r i a was studied using the monoclonal antibodies in i n d i r e c t immunofluorescent assays and in d i r e c t antiserum absorption with 17 in t a c t organisms. The r e s u l t s from these studies were compared to those obtained with a monoclonal antibody s p e c i f i c f o r the O-antigen of one serotype of P. aeruginosa and 5 other monoclonal antibodies recognizing ei t h e r the rough core or L i p i d A regions of LPS. The protein and L i p i d A - s p e c i f i c monoclonal antibodies were used in a taxonomic study of antigenic conservation in the outer membrane of gram-negative bacteria from d i f f e r e n t taxonomic groups. In addition, the i n t e r a c t i o n of the protein F - s p e c i f i c monoclonal antibodies with protein F peptides obtained by cyanogen bromide cleavage and enzyme pr o t e o l y s i s of p u r i f i e d protein F, protein F in outer membranes and whole c e l l s were studied. 18 MATERIALS AND METHODS I Bacteria (A) Media and growth conditions One % (wt/vol) Proteose-peptone no. 2 (PP2) (Difco Labs, Det r o i t , Mi.) was used as a r i c h medium for the growth of a l l b a c t e r i a l s t r a i n s , except in s p e c i f i e d cases where 1% (wt/vol) Trypticase Soy broth (BBL, Cockseyville, Md) supplemented with 1 mM MgCl 2.6H 20 was used. L i q u i d cultures were grown with vigorous aeration at 37°C or 30°C. (B) B a c t e r i a l s t r a i n s Pseudomonas aeruginosa PA01 s t r a i n H103 was used as the wild type and reference s t r a i n throughout the study. Sources and properties of the b a c t e r i a l s t r a i n s used in t h i s study are l i s t e d below. Stra i n H283 was a mutant of H103, lacking the outer membrane porin protein F. The s t r a i n was i s o l a t e d by random heavy mutagenesis of s t r a i n H103 as described by Nicas and Hancock (1983). Strains AK1160, AK1188, AK1012 and AK1121 were LPS-altered mutants of P. aeruginosa PA01 obtained from A. Kropinski (Queen's University, Kingston, Ontario). S t r a i n H223 was obtained as a mutant of H103 r e s i s t a n t to smooth LPS-specific bacteriophage 44 (Hancock et a l . , 1982) . A set of 17 serotype-specific s t r a i n s representative of the International Antigen Typing Scheme (IATS, commercially marketed by Difco Ltd., D e t r o i t , Mi.) was a kind g i f t from P. L i u (University of L o u i s v i l l e , L o u i s v i l l e , Kentucky). These str a i n s were named as follows: 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 set of 12 P. aeruginosa c l i n i c a l i s o l a t e s from patients with c y s t i c f i b r o s i s were obtained from G.B. Pier (Harvard Medical School, Mass.). These strains 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; s t r a i n L, also obtained from G.B. Pier, was a blood i s o l a t e . In addition, 12 pairs of mucoid P. aeruginosa c l i n i c a l i s o l a t e s from c y s t i c f i b r o s i s patients, and t h e i r spontaneous non-mucoid revertants, were obtained from D.P. Speert (Children's Hospital, Vancouver, B.C.). These s t r a i n s were named Pirn and Plnm; Clm and nm; C2lm and nm; C20m and nm; C6m and nm; C81m and nm; C47m and nm; C46m and nm; C4m and nm; C91m and nm; C81m and nm; and C96m and nm. Other Pseudomonadaceae s t r a i n s used in t h i s study were, T two strains of P. putida ATCC 4359 and ATCC 12633 ; two P. fluorescens strains ATCC 949 and ATCC 13525 T; P. aeruginosa ATCC 9721, ATCC 8689 and ATCC 19305 T; P. syringae ATCC 19310 T; P. s t u t z e r i ATCC 17588 T; P. solanacearum ATCC 11696 T; P. m a l t o p h i l i a ATCC 13639 T; P. T T chlororaphis ATCC 9446 ; P. cepacia ATCC 25416 ; P. aureofacxens ATCC T T 13985 ; P. pseudomallei ATCC 23343 ; P. a n g u i l l i s e p t i c a ET2 and ET7601; and P. acidovorans ATCC 9353 (superscript T denoted Type s t r a i n ) . C e l l envelopes or lipopolysaccharide (LPS) preparations were also obtained in our laboratory from Escherichia c o l i CGSC 6044 and PC0479, and Salmonella typhimurium LT2 SGSC205 and SGSC206. 20 Outer membranes from Edwardsiella tarda, E79054, V i b r i o cholerae PS 7910, V i b r i o anguillarum ET208 and HT 7602, Aeromonas salmonicida NCMB2020, Aeromonas hydrophila ET2, 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 in our laboratory, and LPS from Agrobacterium tumefaciens PLT4 and PLT S-1005 and Y e r s i n i a pestis EV76 was i s o l a t e d by R.P. Darveau in our laboratory. Azotobacter  v i n e l a n d i i OP outer membranes were a kind g i f t from R. Moore, University of B r i t i s h Columbia. (C) C e l l envelope and outer membrane i s o l a t i o n For both preparations, c e l l s from logarithmic phase cultures were co l l e c t e d and resuspended in 10 mM Tr i s - H C l (pH 7.4) containing 10 ug/ml pancreatic deoxyribonuclease 1 (Sigma Chemical Co., St. Louis, Mo.) and broken in a French Press at 15,000 p s i . Whole c e l l s were removed by centrifugation at 3,000 rpm for 10 min. For c e l l envelopes, the above supernatant was d i l u t e d i n d i s t i l l e d water and centrifuged at 160,000 x g for 2 hr and the p e l l e t resuspended in water. Outer membranes were i s o l a t e d by the one step sucrose gradient method of Hancock and Carey (1979). In t h i s method, the French Press lysate in 10 mM Tris-HCl (pH 7.4) and 20% (wt/vol) sucrose was layered onto a two-step sucrose gradient co n s i s t i n g of 70% (wt/vol) sucrose in the bottom layer and 52% (wt/vol) sucrose in the top layer, and centrifuged at 21,000 rpm in a Beckman SW27 rotor f or 16 hrs. This rapid method gave two membrane bands. The higher density band was the p u r i f i e d outer membrane f r a c t i o n . 21 (D) Protein and LPS p u r i f i c a t i o n Outer membrane protein F (porin), and l i p o p r o t e i n H2 were p u r i f i e d from P. aeruginosa s t r a i n H103 by L. Chan (Microbiology, U.B.C.) using the method of Hancock e_t a l . (1979). LPS was i s o l a t e d in our laboratory by L. Chan and R. Darveau using the method of Darveau and Hancock (1983). This method has been shown to re s u l t i n high y i e l d s of protein-free LPS from both rough and smooth s t r a i n s . (E) Sodium dodecyl sulphate (SDS) - polyacrylamide gel electrophoresis The method used was as described by Hancock and Carey (1979). The acrylamide concentration in the lower separating gel was 14% (wt/vol) acrylamide and 0.12% ((wt/vol) N,N' methylene bis acrylamide ( i n 12.5 ml of gel mix also containing 3.125 ml of 1.5 M Tris - H C l pH 8.8 and 0.25 ml of 10% (wt/vol) SDS). The gel was polymerized by the addition of 20 y l of TEMED (N, N, N, N'-tetramethylethylenediamine - Biorad) and 0.3 ml of 1% (wt/vol) ammonium persulphate (Biorad). The separating gel also contained 0.07 M NaCl for better r e s o l u t i o n of the lower molecular weight proteins (Hancock and Carey, 1979). The upper stacking gel contained 3% (wt/vol) acrylamide and 0.12% (wt/vol) bis acrylamide (in 5 ml of gel mix also containing 50 y l of 10% (wt/vol) SDS and 1.25 ml of 0.5 M Tris-HCl pH 6.8) and was polymerized by the addition of 10 y l TEMED and 0.12 ml of 1% (wt/vol) ammonium persulphate. The electrode buffer contained 25.6 mM T r i s base, 191.82 mM glycine and 0.1% (wt/vol) SDS (pH 8.4). 22 Samples were s o l u b i l i z e d by the addition of an equal volume of s o l u b i l i z i n g mixture containing 4% (wt/vol) SDS, 20% (vol/vol) g l y c e r o l in 0.25 M Tris-HCl (pH 6.8) and where s p e c i f i e d 10% (vol/vol) 2-Mercaptoethanol (Biorad). The samples were heated at 88°C for 10 minutes. For estimation of molecular weights of peptides on SDS-gels, the following molecular weight standards were used, bovine serum albumin (66.2K); ovalbumin (45 K), carbonic anhydrase (31 K), soybean t r y p s i n i n h i b i t o r (21.5 K) and lysozyme (14.4 K) . ( i ) Protein staining Proteins in SDS-PAGE were stained in 1% (wt/vol) Coomassie b r i l l i a n t blue dissolved in g l a c i a l acetic acid, methanol and water at 1:4.5:4.5 r a t i o and destained in solution with the same components (without the dye) at a 0.7:2:7.2 r a t i o . ( i i ) LPS s t a i n i n g LPS gels were stained by the p e r i o d a t e - s i l v e r method of Tsai and Frasch (1982). This method was modified by using isopropanol instead of ethanol to p r e f e r e n t i a l l y s t a i n LPS in whole outer membrane gels. (F) Protein and Keto-deoxyoctonate (KDO) assays Protein assays were done by the modified Lowry method of Schacterle and Pollack (1973). KDO assays were performed by a modification of the method by Osborn et a l . (1972) with a 15 min hydrolysis period in H SO . LPS 23 concentrations were calculated by assuming that the LPS contained 4.3% by weight of KDO (Darveau and Hancock, 1983). (G) Enzymatic digestion of protein F P u r i f i e d protein F, or protein F in outer membranes and whole c e l l s was enzymatically digested with the following enzymes: (1) Staphylococcus  aureus V8 protease (Sigma Chemical Co., St. Louis, Mo.) in 20 mM T r i s - H C l , pH 7.4 containing 35 mM MgCl^. The enzyme:protein r a t i o was 1:100. (2) Papain (papainase Type 4, Sigma) in 2 mM EDTA 20 mM T r i s - H C l , pH 6.0 at 50 ug enzyme per mg proten. (3) TPCK-Trypsin (Sigma) in 10 mM T r i s HCl, pH 8.0 at 0.1 mg enzyme per mg protein. The digestions were performed at 37°C for 60 min. In assays using intact P. aeruginosa c e l l s , cultures grown to an o p t i c a l density at 600 nm of 0.8 were centrifuged and the c e l l p e l l e t s washed three times in 20 mM Tris-HCl (pH 7.4) containing 5 mM MgCl^ and resuspended in t h i s buffer to one t h i r d of the o r i g i n a l volume. A l l enzymatic digestions were at 100 ug of the enzyme per ml of c e l l suspension. A l l reactions were stopped by heating at 88°C for 10 min. o and the p r o t e o l y t i c peptides kept frozen at -20 C. (H) Chemical cleavage of protein F Chemical cleavage of protein F by cyanogen bromide followed the method of Garten and Henning (1974). B r i e f l y , 60 ug of l y o p h i l i z e d p u r i f i e d protein F was dissolved in 0.8 ml of 98% (vol/vol) formic a c i d containing 1 M cyanogen bromide and 0.6 N HCl. The volume was made up to 24 1 ml. The sample was incubated at 37 C in a f o i l - c o v e r e d tightly-capped v i a l for 18 hr, and then d i l u t e d and l y o p h i l i z e d . The residue (containing cyanogen bromide peptides) was redissolved in deionized water and re l y o p h i l i z e d . The l y o p h i l i z e d peptides were then r e d i s s o l v e d in 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 buffer. In some cases, protein F was denatured in 80% (vol/vol) a c e t i c acid f o r 1 hr pri o r to cyanogen bromide treatment. 2. Animals (A) Animals used. Mice used in t h i s study were of the BALB/c BYJ s t r a i n and were obtained from Jackson Labs (Bar-Harbour, Maine). They were maintained in the animal care unit i n the Department of Microbiology, U.B.C. New Zealand white female rabbits obtained from the animal care unit at U.B.C. were also used. (B) Immunizing Protocol Mice, 6-8 weeks old, were given an i n i t i a l subcutaneous i n j e c t i o n of 20 yg outer membrane or 5 yg p u r i f i e d protein in a t o t a l of 0.4 ml volume of Freund's incomplete adjuvant (Difco Labs) d i l u t e d 1:1 with sa l i n e . The animals were rested f o r 1 week and then i n j e c t e d i n t r a p e r i t o n e a l l y 2 to 3 times at one week i n t e r v a l s with 10 yg outer membranes or 5 yg p u r i f i e d protein i n phosphate buffered s a l i n e without adjuvant. One week a f t e r the l a s t i n j e c t i o n , mice were bled from the t a i l vein, and the sera tested for antibody response to the immunizing antigen 25 by the ELISA assay described below. Mice to be used for production of monoclonal antibodies were rested f o r two weeks a f t e r the t h i r d antigen i n j e c t i o n and then given a f i n a l antigen i n j e c t i o n e i t h e r i n t r a p e r i t o n e a l l y or intravenously 3 days p r i o r to fusion. Rabbits were injected subcutaneously four times at two week intervals with 50 ug of outer membranes in a t o t a l of 0.8 ml of Freund's incomplete adjuvant. The rabbits were then bled from the ear vein and the sera tested for antibody response to the immunizing antigen. This antisera was used f or the experiments i n Chapter 1. Thereafter the rabbits were immunized once every two weeks with 25 ug of outer membranes i n saline u n t i l 16 weeks and then bled. The an t i s e r a were kept o frozen in 0.5 ml volumes at -70 C u n t i l use. Control sera were obtained from rabbits immunized subcutaneously with Freund's incomplete adjuvant and saline for the same period. 3. Tissue Culture (A) Myeloma c e l l l i n e s Two myeloma c e l l l i n e s of BALB/c o r i g i n were used. NS/1, abbreviated from P3-NS1/1, i s a cloned c e l l l i n e derived from MOPC-21, a BALB/c myeloma (Cowan et al.. 1974). The NS/1 c e l l l i n e synthesizes and i n t e r n a l l y degrades a MOPC-21 l i g h t chain. On fusion, therefore 25% of the secreted immunoglobulins w i l l bear that MOPC-21^ l i g h t chain and 50% w i l l have mixed l i g h t chains. The NS/1 c e l l l i n e was obtained from American Type Culture C o l l e c t i o n ( R o c k v i l l e , Md). The myeloma c e l l l i n e SP2/0-Agl4 (abbreviated SP2/0) also obtained from the American Type 26 Culture C o l l e c t i o n s was o r i g i n a l l y derived by several recloning steps from a BALB/c myeloma l i n e SP2/HL-Ag, i t s e l f derived by several steps from SP2/HLGK, the product of a fusion between the BALB/c myeloma X63-Ag8 (\G and K^) and a BALB/c mouse spleen c e l l (Schulman e_t a l . , 1978). The SP2/0 is a t o t a l hon-secretor of mouse immunoglobulin chains. Both the SP2/0 and NS/1 myeloma c e l l l i n e s are r e s i s t a n t to 20 ug/ml 8 azaguanine ( L i t t l e f i e l d 1964) since they lack the enzyme hypoxanthine-guanine phosphoribosyl transferase, an enzyme of the salvage pathway of nucleotide synthesis. (B) Medium, maintenance and freezing of c e l l l i n e s Medium. Powdered Dulbecco's Modified Eagles medium (DMEM; GIBCO Labs, Grand Island, N.Y.), a balanced s a l t , vitamin, and amino acid mixture containing L-glutamine, D-glucose and phenol red, was dissolved at 13.7 g/1 d i s t i l l e d water. The medium was supplemented with 3.5 g/1 (41 mM) sodium bicarbonate, 0.11 g/1 sodium pyruvate (Sigma, St. Louis, Mo.), 1.2 g/1 (5 mM) N-2-hydroxyethyl piperazine-N'-2-ethane sulphonate (HEPES; Calbiochem-Behring, La J o l l a , Ca.) and 20 ug/ml f i n a l concentration of gentamicin sulphate (Garamycin, Schering 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 (Schleicher and Schuell #1121, Keen, NH) and stored at 4°C in 500 y l volumes for a maximum of 4 weeks. Before use the medium was supplemented with 10% or 20% (vol/vol) heat-inactivated f e t a l c a l f serum (FCS; GIBCO and BOCKNECK, Rexdale, Ontario) (pretested batches giving the best growth 27 for myeloma c e l l l i n e s were selected). The FCS was stored at -20°C and kept at 4°C when in use. Maintenance. Myeloma or hybridoma c e l l l i n e s were r o u t i n e l y grown in tissue culture f l a s k s (Linbro Flow Labs, McLean, Va.) in DMEM supplemented with 10% FCS at 37°C in a 10% C0 2 incubator. Optimal growth was obtained at 5 x 10^ c e l l s / m l . C e l l numbers were kept below 1 x 10^ ce l l s / m l . Freezing. C e l l l i n e s at 5 x 10 5 to 1 x 10 6 c e l l s / m l were harvested by centrifugation at 1,200 rpm for 10 min. The p e l l e t was resuspended gently i n ic e - c o l d DMEM (no HEPES) containing 80% (vol/vol) FCS, and the c e l l count adjusted to 2 x 10^ c e l l s / m l in the same medium. An equal volume of i c e - c o l d FCS containing 18% (vol/vol) dimethyl sulphoxide (DMSO, Fisher Co.) was added. The c e l l suspension in 1.0 ml o volumes was frozen overnight at ^ 70 C using a Cryorack 10/16 container (Streck Labs Inc., Omaha, Ne.) previously kept at -20°C. The frozen cultures were transferred and stored in l i q u i d N 2 (-176°C). When needed, the samples were quickly thawed at 37°C, washed twice in 10 ml volumes of DMEM at 1,200 rpm for 10 min. and resuspended in 20 ml of DMEM containing 10% FCS for growth at 37°C in a 10% CO incubator. (C) Generation of Monoclonal antibodies  HAT Selective Medium Hypoxanthine and thymidine (HT) were made up as a 100 times concentrated so l u t i o n (100 x HT) containing 0.1361 g hypoxanthine (6-hydroxypurine, Sigma) and 0.07266 g thymidine [l-(2-Deoxy-B-D-ribofuranosyl)-5 methyl u r a c i l , Sigma] per 100 ml of water warmed up to 70-80°C to dissolve the s a l t s . The solu t i o n was s t e r i l i z e d by f i l t r a t i o n and kept frozen in the dark at -20°C in 10 ml volumes. Aminopterin (4-aminofolic acid, 4 amino pteroyl-glutamic acid, Sigma) was made up as a 1000 f o l d concentrated stock solution by adding 22.6 mg of aminopterin to 80 ml d i s t i l l e d water, then enough 1.0 M sodium hydroxide was added to di s s o l v e the aminopterin. The volume was then made up to 100 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 by f i l t r a t i o n . Selection of myeloma/spleen c e l l hybrids was accomplished by culture of the fusion mixture in s e l e c t i v e medium containing hypoxanthine, aminopterin and thymidine (HAT). The mechanism of HAT s e l e c t i o n i s as follows. Aminopterin (an analogue of f o l i c acid) blocks de novo synthesis of purines and pyrimidines, while the exogenous supply of hypoxanthine and thymidine allows f o r nucleotide synthesis v i a a salvage pathway. Myeloma c e l l l i n e s lack the enzyme hypoxanthine-guanine-phosphoribosyl transferase, an enzyme of the salvage pathway, and thus cannot survive i n HAT medium. Spleen/spleen fusions are passively selected out by t h e i r l i m i t e d capacity to grow in culture (Goding, 1980). Thus only c e l l s with the growth capacity of myelomas and the salvage pathway of spleen c e l l s can survive. 29 The s e l e c t i v e medium was made by the addition of 1 ml of the 100 x HT and 0.1 ml of the 1000 x aminopterin stock solutions to 99 mis of DMEM containing 20% v/v FCS. The f i n a l concentrations were 13.6 ug/ml hypoxanthine, 7.3 ug/ml thymidine and 0.22 ug/ml aminopterin. ( i ) C e l l fusion The c e l l fusion procedure was a modification of the technique of Kohler and M i l s t e i n (1975). Spleen c e l l s were obtained from antigen immunized BALB/c mice and were dispersed into single c e l l suspension (using a 22-gauge needle) in DMEM containing 10% (vol/vol) FCS. The spleen c e l l s were washed once and counted in a haemocytometer. Myeloma c e l l s grown to 5 x 10^-1 x 10^ c e l l s / m l were harvested by centrifugation at 1200 rpm for 10 min, washed and resuspended in DMEM-10% FCS and counted. Spleen and myeloma c e l l s were combined at a r a t i o of 10:1 and pe l l e t e d at 1200 rpm for 10 min and the p e l l e t loosened by tapping the tube on bench. Two fusion protocols were used: Protocol 1. Two ml of 50% (wt/vol) 1500 polyethylene g l y c o l (PEG) o (BDH) prewarmed to 37 C were added over 2 min to the loose p e l l e t . (The pipette t i p was used to gently break up the p e l l e t as PEG was added) . A further 20 ml of DMEM without FCS was added over 3-5 min. The c e l l s were pe l l e t e d and resuspended, to leave small clumps, i n 20 ml of DMEM containing 20% FCS, and incubated for 3 hr at 37°C in a 10% C0 2 incubator. A f t e r the incubation period, s e l e c t i v e HAT medium containing thymocytes derived from a 4-6 week old mouse was added to the fused 4 c e l l s . The c e l l d e n s i t i e s were adjusted to 1 x 10 myeloma c e l l s , 30 1 x 10 spleen c e l l s and 1 x 10 thymocytes per ml. Aliquots (0.2 ml) of the c e l l suspension were then transferred to each well of a 96 well tissue culture plate (Linbro, Flow Labs.) and incubated at 37°C in a 10% CO^ incubator. Protocol 2 - This protocol d i f f e r e d from the previous protocol in that two d i f f e r e n t PEG solutions were used, and the PEG was d i l u t e d out on c e l l d i l u t i o n . The procedure was: To the loose c e l l p e l l e t , 0.5 ml of a 41.6% (wt/vol) PEG 1550 (Serva Fein Biochimica) was added over 1 min. The mixture was rocked gently for 2 to 3 min, then 0.5 ml of a 25% (wt/vol) PEG 1550 (Serva) was added as before. Four ml of DMEM containing 20% (vol/vol) FCS was added over 3 to 5 min with gentle mixing. Then a further 20 ml of DMEM containing 20% (vol/vol) FCS was added and the c e l l suspension incubated for 3 hr at 37°C i n a 10% CO^ incubator. After the incubation, feeder thymocytes in DMEM containing 20% FCS were added to the c e l l suspension. The f i n a l c e l l count was adjusted as described for protocol 1, and 1 ml volumes were tra n s f e r r e d to each well of a 24 well tissue culture plate (Linbro Flow and Costar 3524 Coster, Cambridge, Mass.). Af t e r 24 hr, most of the medium was removed from the well and replaced with 1 ml of HAT s e l e c t i v e medium. This was repeated on day 4 with HAT se l e c t i v e medium containing thymocytes. For both protocols, the plates were checked for growth of hybridoma clones. The culture medium was replaced a f t e r 10 days with fresh HAT se l e c t i v e medium containing thymocytes. Colonies of hybridoma c e l l s could be observed as early as 1 week or as l a t e as 3 weeks a f t e r c e l l fusion. 31 ( i i ) Cloning. Supernatants from wells that contained hybridoma clones occupying one t h i r d of the well bottom, were removed and tested i n enzyme linked immunosorbent assays (ELISA) as described below, for secretion of antibodies against the immunizing antigen. C e l l cloning from wells that showed p o s i t i v e ELISA r e s u l t s was performed as follows: The hybridoma colonies were dispersed by mixing the well contents with a s t e r i l e Pasteur pipette. Twenty y l of the c e l l suspension from 0.2 ml wells or 50 y l from 1 ml wells, was dispensed into the f i r s t row of a 96 well plate containing 0.1 ml of HAT se l e c t i v e medium with thymocytes. The well contents were mixed 10 times and 20 y l transferred into 0.1 ml HAT se l e c t i v e medum with thymocytes contained in the well of the next row. This procedure was repeated through row 12. A f t e r allowing s u f f i c i e n t time for growth, wells containing one clone (seen from row 6 on) were tested f or antibody production by ELISA. Wells that gave p o s i t i v e ELISA r e s u l t s were recloned and tested again, before expansion and freezing of that hybridoma c e l l l i n e . (D) Ascites production Cloned hybridoma c e l l s secreting antibodies s p e c i f i c for the 6 immunizing antigen were grown to 1 x 10 c e l l s / m l . The c e l l s were harvested by centrifugation at 1200 rpm for 10 min and resuspended in DMEM 6 at 1 x 10 c e l l s / m l . BALB/c mice were injected i n t r a p e r i t o n e a l l y with a t o t a l of 1 x 10"* c e l l s . These mice had been primed by in t r a p e r i t o n e a l i n j e c t i o n of 1 ml of Pristane (2, 6, 10, 14-tetramethyl pentadecane, Sigma) 3 weeks p r i o r to i n j e c t i o n with the hybridoma c e l l s (Goding, 32 1980). The mice were observed for abdominal diste n s i o n , and ascite s f l u i d was removed from the peritoneal cavity with a s t e r i l e 18 gauge needle. Up to 15 ml of a s c i t i s could be c o l l e c t e d from one mouse in 3 to 5 such procedures. The a s c i t i c f l u i d was centrifuged at 1200 rpm for 10 min to remove c e l l s , tested for antibody t i t r e against the s p e c i f i c antigen by the ELISA method and stored frozen in 0.3 mi volumes at -70°C. (E) Antibody P u r i f i c a t i o n Antibodies were p u r i f i e d from ascites f l u i d or immune sera by ammonium sulphate p r e c i p i t a t i o n and followed by a f f i n i t y chromatography. The immune sera or a s c i t i c f l u i d was d i l u t e d 1:1 in sa l i n e and 40% v o l / v o l saturated ammonium sulphate added slowly with s t i r r i n g at 4°C for 4 hrs. 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 redissolved at the o r i g i n a l volume in phosphate buffered s a l i n e (PBS see Section 4). The antibodies were then r e p r e c i p i t a t e d by the addition of 45% (vol/vol) saturated ammonium sulphate as before. The p r e c i p i t a t e was then dissolved in a minimal amount of phosphate buffered s a l i n e and dialysed against 5 1 PBS overnight. Any p r e c i p i t a t e was removed by low speed c e n t r i f u g a t i o n . When greater p u r i t y of antibody was needed the antibodies were subsequently a f f i n i t y p u r i f i e d on a Sepharose CL-4B column (Pharmacia) to which the s p e c i f i c antigen was coupled. For example, a rabbit polyclonal antibody was chromatographed on an LPS a f f i n i t y column to remove any anti-LPS antibodies . 33 4. Antibody Characterization (A) Enzyme Linked Immunosorbent Assays - ELISA Phosphate buffered s a l i n e , pH 7.4 (PBS) contained 8.0 g/1 (136.89 mM) NaCl; 0.2 g/1 (1.47 mM) Kh^PO^ 2.9 g/1 (20.43 mM) Na 2HP0 4; 0.2 g/1 (2.68 mM) KC1; and 0.2 g/1 (3.08 mM) sodium azide NaN^. The PBS was made in 10 l i t r e batches at 5 f o l d concentration, d i l u t e d before use and supplemented with 1.02 g/1 (5 mM) MgCl 2.6H 20. Tween 20 (0.5 ml/1) (polyoxyethylene sorbitan monolaurate, Sigma) was added to the PBS where sp e c i f i e d . Coating buffer Carbonate/bicarbonate buffer (pH. 9.6) contained per l i t r e of d i s t i l l e d Water 1.5 g (14.15 mM) NaC0 3, anhydrous; 2.93 g (34.87 mM) NaHC03; 1.02 g/1 (5 mM) MgCl 2.6H 20 and 0.2 g (3.08 mM) NaN^. The buffer was adjusted to pH 9.6 with 0.1 M NaOH and stored in the dark at 4°C for a maximum of 4 weeks. Diethanolamine Buffer Diethanolamine buffer (10% wt/vol) contained 97 ml/1 diethanolamine (Baker), 0.2 g/1 (3.08 mM) NaN^ and 100 mg/1 (0.49 mM) MgCl 2 >6H 20. The buffer was adjusted to pH 9.6 with 1 M HCl, stored in the dark at 4°C, and brought to room temperature before use. 34 ELISA The ELISA procedure used was a modification of the method of Ruitenberg §_t a l (1974). Ninety six well p o l y v i n y l chloride plates (Falcon 3912; Becton-Dickinson and Co., Oxnard, CA) were coated with 50 or 100 y l of a suspension of 20 yg/ml outer membrane or 1 to 5 yg/ml of p u r i f i e d protein in carbonate/bicarbonate buffer. A f t e r a 16 hr o o incubation at 4 C or 2 hr at 37 C, the plates were washed once in PBS containing 0.01% (vol/vol) Tween 20, and 3 times in PBS and incubated with 200 y l / w e l l of PBS containing 1% FCS (PBS-FCS) for 30 min at 37°C, to block any free protein binding s i t e s . The plates were then incubated with 50 or 100 y l d i l u t i o n s of the test antibody in PBS-FCS f o r 1 to 2 hr at 37°C or 16 hr at 4°C. After washing with PBS-Tween 20/PBS as above, the plates were incubated in the dark at 37°C f o r 1-2 hr with 50 or 100 y l of a goat anti-mouse F(ab*)^ immunoglobulin fragments antibody coupled to a l k a l i n e phosphatase (Helix Biotech, Richmond, B.C.) which had been d i l u t e d 2000 f o l d in PBS-FCS. A further PBS-Tween 20/PBS wash was done, and the plates developed with the a l k a l i n e phosphatase enzyme substrate, disodium p-nitrophenyl phosphate (Sigma 104 phosphatase substrate t a b l e t s ; 5 mg/tablet) at 1 tablet per 5 mis of 10% Diethanolamine buffer. The yellow colour development was read a f t e r 45 minutes using an ELISA T i t r e t e k Multiscan photometer (Flow Labs., McLean, V i r g i n i a ) , set at 405 nm. For i n i t i a l screening of antibody secretion by hybridoma clones, the ELISA plates were read a f t e r 2 - 4 hr colour development at room temperature. Control wells contained e i t h e r no antigen or no t e s t antibody, while p o s i t i v e control 35 wells contained as antigen, H103 outer membranes and the serotype 5 s p e c i f i c monoclonal antibody, MA1-8. Whole C e l l ELISA ELISA assays using whole P. aeruginosa c e l l s were done by two methods using either f i x e d or non-fixed c e l l s . In the f i r s t method, p o l y v i n y l chloride plates (PVC) (Flow) were incubated f o r 30 min at 37°C with 50 u l per well of 0.5% (vol/vol) glutaraldehyde (Sigma) d i l u t e d in PBS. P. aeruginosa c e l l s were grown to an o p t i c a l density at 600 nm of 0.6-0.8, and washed three times by c e n t r i f u g a t i o n at 3000 rpm for 10 min in PBS containing 1 mM MgCl^ and 1.0% (vol/vol) FCS. The c e l l s were 4 resuspended in the same buffer to give 1 to 5 x 10 c e l l s / m l . F i f t y u l of the c e l l suspension was aliquoted into each well to give a f i n a l 3 count per well of approximately 1 x 10 c e l l s in 0.25% (vol/vol) glutaraldehyde. The PVC plates were centrifuged at 3,000 rpm for 10 min o and incubated for 30 min at 37 C. The supernatant was removed and 100 p i of 0.1 M glycine containing 1% FCS was added to each well to inactivate free glutaraldehyde. A f t e r a 10 min incubation the plates were rinsed by dipping into 3 separate containers of 500 ml PBS containing 1 mM MgCl 2. The second method was a modification of that described by Posner et a l . (1982). P. aeruginosa c e l l s were not f i x e d . Instead, the plates were centrifuged at 3,000 rpm for 10 min between each step in the ELISA assay procedure and during the washing procedures. A l l other procedures were as described above f o r the ELISA assay, with the exception that 2% (vol/vol) 3 6 FCS was used, the incubation steps were a l l for 1 hr at 3 7 C. A goat anti-mouse antibody coupled to horseradish peroxidase enzyme (Kilkegaard-Perry Ltd., Flow Labs) was used as the second antibody. The peroxidase enzyme substrate contained 0 . 1 ml of a 1 5 mg/ml diammonium 2 , 2 ' - A z i n o - d i - ( 3 - e t h y l - b e n z t h i a z o l i n - s u l p h o n a t e ( 6 ) s olution (Boehringer-Mannheim, W. Germany) in 1 0 ml of c i t r a t e buffer (pH 4 . 0 ) . The c i t r a t e buffer contained 0 . 9 6 g c i t r i c acid per 1 0 0 ml of d i s t i l l e d water and 0 . 2 ml of 3 N NaOH to adjust the pH to 4 . 0 . Before use, 0 . 3 3 ml of 3 0 7 . ( vol/vol) hydrogen peroxide was added to the substrate s o l u t i o n . A f t e r 3 0 min incubation the green colour was recorded at 4 0 4 or 4 1 5 nm using a T i t r e t e k Multiscan photometer. (B) Western electrophoretic b l o t transfers The method used was e s s e n t i a l l y that of Towbin et a l . ( 1 9 7 9 ) . Proteins were e l e c t r o p h o r e t i c a l l y transferred from one- or two-dimensional SDS-polyacrylamide gels onto n i t r o c e l l u l o s e paper ( 0 . 4 5 um Schleicher and Schuell) using a Biorad e l e c t r o b l o t chamber at 0 . 4 - 0 . 5 A f o r 2 - 3 hr o at 4 C or at 1 0 mA for 1 8 hr at room temperature. In the f a s t blot method, the SDS-acrylamide gel was l a i d on two sheets of n i t r o c e l l u l o s e paper and prewashed in PBS containing 0 . 1 7 o (wt/vol) SDS at 6 0 ° C f o r 3 0 min or 8 0 ° C f o r 1 0 min. For 1 8 hr b l o t s , a sheet of the n i t r o c e l l u l o s e paper was l a i d on ei t h e r side of the ge l . The gel and n i t r o c e l l u l o s e papers were enclosed between stacks of Whatman 3 M f i l t e r papers and two pads of Scotch b r i t e . The sandwich was placed between two s l o t t e d p l a s t i c holders and placed in the e l e c t r o b l o t chamber of a Biorad apparatus. For 37 a fast b l o t , the n i t r o c e l l u l o s e was placed on the anode side. The chamber was f i l l e d with 3 l i t r e s of transfer buffer pH 8.3 [containing 20% (vol/vol) absolute methanol, 43.2 g/1 glycine (192 mM, Biorad), 9.10 g/1 T r i s (25 mM, u l t r a pure T r i s from Schwarz/Mann) and 0.1% (wt/vol) SDS]. The Western blots were developed as follows: The blots were o incubated for 30 min at 37 C in PBS containing 3% (wt/vol) bovine serum albumin (BSA f r a c t i o n V, Sigma) to block a l l a v a i l a b l e protein binding s i t e s on the n i t r o c e l l u l o s e paper. Subsequently, the blots were incubated with 10 ml of the t e s t antibody d i l u t e d in 3% BSA-PBS, for 16 hr at 4°C, and then washed 3 times for 15 min in excess PBS at 37°C. The next steps were a 2-3 hr incubation at 37°C in 3% BSA-PBS containing a 1000 f o l d d i l u t i o n of a goat anti-mouse F(&b')^ antibody conjugated to alk a l i n e phosphatase, 3 more PBS washes, and colour development in an alk a l i n e phosphatase substrate. The substrate so l u t i o n contained per 10 mis of 50 mM Tris-HCl buffer (pH 8.8) 20 mg/ml Fast Red-TR S a l t (5-chloro-2-toluidene diazonium, Sigma) and 10 mg Napthol-As-MX-phosphate disodium s a l t (Sigma) added in that order. A f t e r the desired colour had developed, the blots were rinsed in excess water, dri e d between paper towels and stored out of d i r e c t l i g h t . The immunoperoxidase st a i n i n g procedure was done as described f or colony immunoblots in section 4.6. (C) Double Immunodiffusion Double immunodiffusion (Ouchterlony, 1959) was performed in 2% (wt/vol) agarose (Standard low Mr agarose, Biorad) dissolved in 0.08% barbitone buffer [12 g sodium 5'5 d i e t h y l barbiturate and 4.40 g 5'5 38 d i e t h y l b a r b i t u r i c acid per l i t r e of d i s t i l l e d water adjusted to pH 8.2 with 1.0 M NaOH]. T r i t o n X-100 was added to 1% (vol/vol) and 8 ml of the agarose solution poured on glass s l i d e s . The outer antigen wells were f i l l e d with 20 ug of the s o l u b i l i z e d antigen and the centre antiserum wells f i l l e d with undiluted or two f o l d d i l u t i o n s of antiserum. The agarose plates were then incubated for 16 hr at 37°C in a humid chamber. The wells were f i l l e d with s a l i n e buffer, arid the plates kept at o 4 C for a further 72 hrs. The plates were then pressed dry under a stack of Whatman 3M f i l t e r papers, washed f i v e times in PBS for 45 min at 37°C, dried again, stained in 1% (wt/vol> Coomassie b r i l l i a n t blue dissolved in absolute methanol, acetic acid and water at a r a t i o of 4:1:4 and destained in the same solution without the dye. (D) Agglutination Assays B a c t e r i a l agglutination. B a c t e r i a l agglutination was performed using either P. aeruginosa c e l l s autoclaved at 120°C f o r 1 hr, then centrifuged and resuspended in s a l i n e , as described by Lanyi and Bergan (1979), or l i v e P. aeruginosa c e l l s . In both methods the c e l l preparations were incubated with s e r i a l d i l u t i o n s of antiserum for 1 hr at o 37 C. The agglutination of l i v e c e l l s was characterized by a coarse granular b a c t e r i a l clumping, as opposed to the f i n e r granular clumping of h e a t - k i l l e d P. aeruginosa with homologous LPS O-antigen-specific serum. B a c t e r i a l agglutination was scored on a scale of +1 (weak agglutination) to +4 (strong reaction). The agglutination was also done on glass s l i d e s 39 using a drop each of antiserum and b a c t e r i a l preparation, and the re s u l t s observed after 3 to 5 min. Passive agglutination. Passive agglutination was done as described by Lanyi and Bergan (1979) using tanned sheep red blood c e l l s and P. aeruginosa LPS (heated for 1 hr at 100°C p r i o r to use). B r i e f l y , sheep erythrocytes were washed three times in sa l i n e (0.9% NaCl), adjusted to 4% erythrocytes (vol/vol) in phosphate s a l i n e buffer (0.02 M KH^PO^, 0.06 M Na 2HP0 4 and 0.12 M NaCl, pH 7.5) and 2.5 mg of tannic a c i d i n 50 ml phosphate saline buffer was added to 50 mis of the c e l l suspension. A f t e r . o a 15 min incubation at 37 C with occasional mixing, the c e l l s were centrifuged (100 x g for 20 min) and washed with 100 ml of phosphate saline buffer. One hal f of the c e l l s were kept as a co n t r o l in buffer containing 1 mM sodium azide. To the other h a l f of the c e l l s , 20 ug/ml of LPS or protein was added, and the mixture incubated f o r 1 hr at 37°C with very gentle a g i t a t i o n at regular i n t e r v a l s . The antigen-coated c e l l s were washed three times in saline and 1 mM NaN^ was added f o r storage at o 4 C. The c e l l s were made up to 1% (vol/vol) in saline before use. Passive agglutination was done i n Linbro 96 well c o n i c a l bottom plates (Linbro, Flow Labs) using 50 u l of antiserum s e r i a l l y d i l u t e d i n sali n e and 50 u l of 1% (vol/vol) antigen coated sheep erythrocytes. Control wells contained the antiserum and tanned, non-antigen-coated sheep o erythrocytes. The plates were incubated at 37 C for 1 hr The serum t i t r e . was taken as the highest serum d i l u t i o n giving agglutination. 40 Non-agglutinated c e l l s gave a ti g h t button of c e l l s at the bottom of the well, while the agglutinated c e l l s formed a mat at the well bottom. (E) Indirect immunofluorescence Two methods were used. In the f i r s t method, P. aeruginosa c e l l s , grown to an o p t i c a l density at 620 nm of 0.8, were washed three times in PBS containing 2% FCS. The bacteria were resuspended to the o r i g i n a l volume and 0.8 ml dispensed in 1.5 ml p l a s t i c Eppendorf tubes. Monoclonal or polyclonal serum at an appropriate d i l u t i o n was added and the c e l l suspension vortexed at se t t i n g 3 of a Vortex Genie (Fisher S c i e n t i f i c ) . o Aft e r a 45 min incubation at 37 C with vigorous a g i t a t i o n , the c e l l suspension was washed three times in PBS/1% FCS/1 mM MgCl 2 by centrifugation f o r 1 min in a Brinkmann Microfuge (Brinkmann centrifuge 5412). The c e l l s were then incubated at 37°C f o r 45 min in PBS/2% FCS containing a 100 f o l d d i l u t i o n of rabb i t anti-mouse IgG (Sigma). The washing procedure was repeated, then the c e l l s were incubated as before, with a goat a n t i - r a b b i t antibody coupled to Fluorescein isothiocyanate (FITC, Sigma). Af t e r a further PBS wash, 10 y l of the c e l l suspension was placed on a s l i d e under a c o v e r s l i p and examined with a Zeiss microscope (Standard R.A., with a condenser f o r fluorescent microscopy) containing a halogen lamp and suitable f i l t e r s f o r measuring the fluorescence emission of f l u o r e s c e i n isothiocyanate at 525 nm. In experiments where polyclonal rabbit antiserum to P. aeruginosa outer membrane was used, the second antibody was omitted and a goat a n t i - r a b b i t FITC-labelled antibody used (Sigma). Negative controls contained 41 P. aeruginosa c e l l s with no f i r s t antibody but a l l other antibodies, or f i r s t antibody and FITC antibody without the second antibody; other controls were as described in r e s u l t s . The second method used was e s s e n t i a l l y as described by Hofstra et a l . (1979). The antibody steps were as above except that the whole procedure was done on f i x e d P. aeruginosa smears on glass s l i d e s . For each o incubation step, the s l i d e s were placed in a humid chamber at 37 C for 10 minutes and were washed by dipping in PBS. (F) Colony b l o t t i n g Colony b l o t t i n g was performed by a modification of the method of Henning et a l (1979). B a c t e r i a l colonies were transferred from agar plates onto prewashed n i t r o c e l l u l o s e f i l t e r s by contact. A f t e r incubation of the f i l t e r s at 30°C for 30 min, the colony blots were successively incubated with T r i s - b u f f e r e d saline f o r 45 min at 37°C [10 mM T r i s - H C l , pH 7.4; 2 mM MgCl 2, 1 mM NaCl (TBS)] containing 37. (wt/vol) g e l a t i n , then for 2 hr with the test antibody d i l u t e d in TBS buffer containing 1% (wt/vol) g e l a t i n . The blots were subsequently washed 3 times by incubation for 15 min in TBS containing 0.1% (wt/vol) g e l a t i n , and then incubated for 2 hr at 37°C with a goat anti-mouse immunoglobulin coupled to horse-radish peroxidase (Flow Labs, Inc., McLean, Va.). A f t e r washing as above, the blots were developed using a histochemical substrate for peroxidase (5 mg of 4-chloro-l-napthol dissolved in 1.67 mis of i c e - c o l d absolute methanol and mixed at 23°C with 10 mis of TBS containing 5 u l of 30% (vol/vol) H 2 0 2 ) . 42 5. Serum s e n s i t i v i t y testing Due to previous observations that the apparent s e n s i t i v i t y of P. aeruginosa 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, ba c t e r i a were plated on try p t i c a s e soy agar plates and then scraped o f f and resuspended in trypticase soy broth, containing 1 mM MgCl o J at an o p t i c a l density at 650 nm (0D, C.) of i 650 0.1. They were then grown to an On of 0.4 and centrifuged and 6 b 0 resuspended in an equal volume of minimal e s s e n t i a l medium containing [0.4% glucose; 0.5 mM MgS04; 10 uM FeSO^; 40 mM l^HPO^ and 7 mM (NH i)_S0.]•• This c e l l suspension was d i l u t e d 1 in 1000 to 1 in 4 2 4 10,000 into minimal e s s e n t i a l medium and 0.1 ml of the d i l u t e d c e l l suspension mixed with 0.1 ml of fresh normal human serum. After 1 hr at o 37 C the bacteria were plated f or v i a b l e counts on t r y p t i c a s e soy agar o plates. Serum was routinely stored at -70 C p r i o r to use. Method 2. The second method was based on the recently published method of Schneider and G r i f f i s s (1982), and the well-known broth d i l u t i o n method for t e s t i n g of antimicrobial s u s c e p t i b i l i t y . Logarithmic cultures of c e l l s were centrifuged and resuspended in proteose peptone No. 2 broth 4 (Gibco, Ontario, Canada) at a f i n a l concentration of 5 x 10 c e l l s per ml. Twenty u l of c e l l s were added to 80 u l of fresh normal human serum which had been s e r i a l l y d i l u t e d in proteose peptone No. 2 broth to give f i n a l serum concentrations ( a f t e r addition of c e l l s ) ranging from 80% to 1.25% in 7 s e r i a l two-fold d i l u t i o n s . As a control, 80 u l of medium was added to 20 u l of c e l l s . For convenience the assay was performed in 43 96 well m i c r o t i t r e trays (Linbro tissue culture plates, Flow Labs, Va) allowing comparison of 12 d i f f e r e n t s t r a i n s on a single m i c r o t i t r e tray. The plate was incubated for 18 hours at 37°C. End points were recorded as the highest concentration of serum allowing f u l l growth compared to the cont r o l without serum. Usually a two f o l d higher concentration of serum completely prevented growth. 44 RESULTS CHAPTER I. OUTER MEMBRANE ANTIGENS OF P, AERUGINOSA 1. Outer membrane patterns of serotyping i s o l a t e s The outer membranes of P. aeruginosa PA01 have been shown to contain at least 6 to 8 major polypeptides. These major outer membrane proteins 4 5 can be present at 10 to 10 c o p i e s / c e l l and are expressed according to the growth conditions (Hancock and Carey, 1979, 1980; Mizuno and Kageyama, 1978a, 1979). Using the method of Hancock and Carey (1979), outer membranes were i s o l a t e d from the 17 serotype s t r a i n s of P. aeruginosa that comprise the International Antigen Typing Scheme (Brokopp and Farmer, 1979; Lanyi and Bergan, 1979). The outer membrane protein patterns of these s t r a i n s were characterized on SDS-polyacrylamide gels. The major polypeptides were i d e n t i f i e d by co-electrophoresis with P. aeruginosa PA01 outer membrane proteins under d i f f e r e n t s o l u b i l i z a t i o n conditions. Hancock and Carey (1979) showed that the m o b i l i t i e s on SDS-polyacrylamide gel electrophoresis of proteins DI, HI and G varied depending on the temperature at which the outer membranes were s o l u b i l i z e d p r i o r to electrophoresis. The mobility of protein F varied with presence or absence of 2-mercaptoethanol during s o l u b i l i z a t i o n and l i p o p r o t e i n I was stained only when MgCl^ was present in the s o l u b i l i z a t i o n buffer. Using the above c r i t e r i a , a l l the 17 serotype s t r a i n s ( F i g . 1) and c y s t i c f i b r o s i s i s o l a t e s of P. aeruginosa ( F i g . 2) had polypeptide bands that co-electrophoresed with the major outer membrane proteins of P. aeruginosa PA01. However, the r e l a t i v e l e v e l s of these proteins varied 45 Figure 1. SDS-polyacrylamide gel electrophoretograms of outer membranes of P. aeruginosa s t r a i n H103 and serotyping s t r a i n s . The 17 lanes on the left-hand side were run a f t e r s o l u b i l i z a t i o n at 88°C for 10 min in s o l u b i l i z a t i o n - r e d u c t i o n mix [20 mM EDTA, 2% (wt/vol) SDS, 5% (vol/vol) 2-mercaptoethanol, 10% (vol/vol) g l y c e r o l , and 62.5 mM Tr i s - H C l (pH 6.8)]. Under these conditions l i p o p r o t e i n I i s not v i s i b l e while the other major proteins migrate to t h e i r heat-modified positions (Hancock and Carey, 1979). The three lanes on the r i g h t hand-side 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 in the absence of 2-mercaptoethanol g (causing protein F to move with a higher mobility to p o s i t i o n F ) and in the presence of 0.1 M MgCl 2 (causing protein I to appear in the gel and proteins G and HI to s h i f t to t h e i r unmodified p o s i t i o n s , where they s t a i n poorly: Hancock and Carey, 1979). The positions of the proteins D2, E and H2 are also indicated. The numbers below each lane r e f e r to the serotypes, with the exception of our wild type H103 s t r a i n , l a b e l l e d W. 46 47 somewhat between the d i f f e r e n t s t r a i n s . For example of the serotype s t r a i n s , serotype 1, 5 and 7 strains had low but detectable l e v e l s of l i p o p r o t e i n H2, whereas the serotype 6 and 7 st r a i n s had reduced protein HI and serotypes 5 and 7 st r a i n s had reduced l i p o p r o t e i n I. Protein G was observed in a l l s t r a i n s except serotype 7, 14 and 16 s t r a i n s and protein D2 was missing or a l t e r e d in serotype 5, 12, 15 and 17 s t r a i n s . These observed differences in the outer membrane protein patterns could not be correlated to serotype-related di f f e r e n c e s , as only a si n g l e representative from each serotype was studied. 2. Cystic f i b r o s i s i s o l a t e s : c h a r a c t e r i s t i c s and outer membrane patterns The outer membranes of 36 P. aeruginosa st r a i n s obtained from patients with c y s t i c f i b r o s i s showed outer membrane protein p r o f i l e s s i m i l a r to s t r a i n PA01. In F i g . 2 the p r o f i l e s of some of the st r a i n s are shown. A number of minor a l t e r a t i o n s in the outer membrane protein p r o f i l e s were observed but the general patterns were strongly conserved. The most substantial a l t e r a t i o n s were observed in s t r a i n CF 4349 and str a i n s C46 mucoid and C46 non-mucoid. Strain CF4349 ( F i g . 2, lane 5) contained a number of extra polypeptides as well as an apparent deficiency in protein I. Strains C46 mucoid and i t s spontaneous non-mucoid revertant had no detectable protein H2 in t h e i r outer membranes. Another s t r a i n CF1278 apparently lacked l i p o p r o t e i n I (Fig. 2, lane 11). Proteins E and F were, however, expressed in a l l these P. aeruginosa i s o l a t e s . 48 Figure 2. SDS-polyacrylamide gel electrophoretograms of outer membrane proteins of P. aeruginosa i s o l a t e s from patients with c y s t i c f i b r o s i s . o Outer membranes were s o l u b i l i s e d in reduction mix at 88 C in the absence of 2-mercaptoethanol. The e f f e c t of t h i s s o l u b i l i z a t i o n schedule i s described in F i g . 1. 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, molecular weight standards [Bovine serum albumin (66.2 K), Ovalbumin (45 K), Carbonic anhydrase (31 K), Soybean try p s i n i n h i b i t o r (21.5 K) Myoglobulin (16.7 K) and Lysozyme (14.4 K)]; 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. 4 9 i o ~n mp 50 In contrast to the great s i m i l a r i t y in the outer membrane protein p r o f i l e s , these s t r a i n s d i f f e r e d in other c h a r a c t e r i s t i c s . For example, of the f i f t e e n c y s t i c f i b r o s i s i s o l a t e s typed by the Fisher immunotyping scheme (data of D.P. Speert and G.B. Pie r ; summarized in Table 1) only three strains were agglutinated by a single typing serum, whereas 8 stra i n s were agglutinated with more than one serum and 4 were not agglutinated by any serum. Neither the polyagglutinable (nor the non-agglutinable st r a i n s ) were typable by hemagglutination i n h i b i t i o n or immunodiffusion (Hancock et a l . , 1981) , suggesting that these polyagglutinable s t r a i n s did not express multiple serotype antigens, but were instead being agglutinated by antibody to non-serotype determinants. Four typable i s o l a t e s (including our laboratory wild type s t r a i n , H103) were r e s i s t a n t to pooled normal human serum, while the twelve polyagglutinable and nonagglutinable i s o l a t e s studied were very s e n s i t i v e to normal human serum (Table I ) . Serotyping of gram-negative b a c t e r i a i s based on LPS 0-antigen. The LPS from these s t r a i n s was therefore characterized by st a i n i n g on SDS-polyacrylamide gels both in the p u r i f i e d form ( F i g . 2 of Hancock et a l . , 1983) and in the outer membranes (to avoid 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 ) . Figure 3 shows the st a i n i n g of LPS in the outer membranes of 16 c y s t i c f i b r o s i s i s o l a t e s of P. aeruginosa and s t r a i n H103. By the modified periodate s i l v e r s t a i n i n g technique, LPS stained orange while proteins were brown or grey and were thus c l e a r l y distinguishable. As shown in F i g . 3 (lanes 5,6,13) only s t r a i n s CF4349 and CF6094 (when overloaded) contained l e v e l s of smooth LPS s i m i l a r to 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 selected P. aeruginosa isolates from patients with c y s t i c f i b r o s i s and rough LPS-altered mutants Response to serum Stra i n Colony 0 side chains T y p a b i l i t y 0 % su r v i v a l a f t e r % serum allowing Morphology 3 i n LPS'3 exposure to 50% serum growth a f t e r 18 hr for 1 hr at 37°C H103 NM ++ T >100% >40% CF2314 NM + T 89% >40% CF4349 NM ++ T >100% >40% CF6094 PM ++ T 86% >40% CF832 NM + PA 0% 1.25% CF3790 + PA 6% 1.25% CF4522 NM - PA 0% 2.5% CF9490 NM + PA 0% 1.25% CFPIM M + PA 0% 1.25% CFPINM NM ± PA 0% 1.25% CF96M M + PA 0% 1.25% CF96NM NM + PA 0% 1.25% CF221 NM - • NT 0% 1.25% CF284 NM + NT 0% 1.25% CF1278 M - NT 0% 1.25% CF1452 M + NT 0% 1.25% AK1160 NM - 1.25% AK1188 NM - - 1.25% AK1012 NM 1.25% AK1121 NM - • - 1.25% H223 NM — — 1.25% a. NM - non -mucoid, smopth colony appearance, M - mucoid, PM - p a r t i a l l y mucoid. b. Results are expressed as the minimum le v e l of LPS which had to be loaded onto SDS polyacrylamide gels i n order to see smooth type LPS (containing 0-side chains) upon stainin g by the method of Tsai 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 agglutination; PA - polyagglutinable with more than one serum, non-typable by other means; NT - nontypable. 52 Figure 3. SDS-polyacrylamide gel of outer membranes of P. aeruginosa is o l a t e s from patients with c y s t i c f i b r o s i s . The electrophoretograms were stained for LPS by a modification of the p e r i o d a t e - s i l v e r s t a i n i n g procedure of Tsai and Frasch (1982). The outer membranes were s o l u b i l i z e d in reduction mix containing 20 mM EDTA. 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 1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 54 s t r a i n H103 (on overloading). Strains CF284 had detectable l e v e l s of O-side chains in heavily overloaded gels of p u r i f i e d LPS. Rough type LPS was v i s i b l e , in a l l strains tested, as a fast migrating band. However, in the O-side cha i n - d e f i c i e n t s t r a i n s , r e l a t i v e l y higher amounts of t h i s core LPS were present when compared to the O-side chain-containing s t r a i n s . As shown in Table 1, these st r a i n s also d i f f e r e d in colony morphology. 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 of a polyclonal a n t i s e r a against the  outer membranes of P. aeruginosa Rabbit antiserum against the outer membranes of P. aeruginosa PA01 s t r a i n H103 was tested for a c t i v i t y against the outer membranes of the serotyping st r a i n s by the ELISA method. The ELISA t i t r e of the antiserum was expressed as the d i l u t i o n of the antiserum which gave an absorbance reading at 405 nm of 0.3 a f t e r 45 minutes of colour development at 37°C. The t i t r e of the antiserum against the homologous s t r a i n H103 outer -8 membrane (10 ) was one to three orders of magnitude higher than against a l l tested heterologous outer membranes (Table I I ) . This p o l y c l o n a l -4 antisera had a t i t r e of only 10 against homologous s t r a i n H103 LPS (Table II) and therefore most of the antibody must have been dire c t e d against non-LPS antigens in the outer membrane. For t h i s reason, and because the serotype st r a i n s are heterologous with respect to t h e i r LPS 0-antigens, i t would seem that at l e a s t some of the antigenic c r o s s - r e a c t i v i t y and differences seen in the ELISA t i t r e among these s t r a i n s must be due to antibodies d i r e c t e d against protein antigens. The immunogenicity of outer membrane porin proteins F, H2, I, HI and D2 was Table I I . Cross-reactions of antisera to outer membrane and p u r i f i e d outer membrane components with outer membranes from various serotype s t r a i n s of P. aeruginosa. Logi n ELISA t i t r e s using antisera to: Outer PAOI Protein Protein Protein Protein Protein membrane outer D2 F HI H2(+I) I . antigen membranes PAOi 8 3 3 3 4 3 serotype 1 6 3 2 3 4 2 2 6 3 2 3 3 2 3 5 2 1 2 1 2 4 7 2 1 3 3 2 5 7 2 3 2 3 2 6 5 2 4 4 4 2 7 6 3 2 3 4 2 8 6 3 1 3 3 2 9 6 3 3 4 4 3 10 6 3 3 4 4 3 11 6 3 2 4 4 2 12 6 3 3 4 4 3 13 6 3 1 3 3 2 14 6 3 2 4 4 2 15 6 2 1 3 3 2 16 7 3 3 2 3 2 17 6 3 3 4 4 3 CFPlm 5 2 2 4 3 2 CFPlnm 5 2 2 4 2 2 PAOI LPS 4 2 1 1 < 1 1 NOTE: P. aeruginosa PAOI ( s t r a i n H103) was used as a standard s t r a i n f o r the i s o l a t i o n of major outer membrane antigens. 56 demonstrated by the immunological c r o s s - r e a c t i v i t y of pol y c l o n a l antisera against these proteins and outer membranes from a l l of the serotype strains (Table I I ) . The polyclonal antisera against these proteins contained some antibody (ELISA t i t r e < 10) against homologous LPS, indicati n g that LPS was a contaminant of the p u r i f i e d protein but these anti-LPS antibodies could not account f o r the observed extensive immunological c r o s s - r e a c t i v i t y of the antiserum. Polyclonal a ntisera against both outer membranes and protein F cross-reacted in ELISA assays with outer membranes from CFPlm, a mucoid P. aeruginosa s t r a i n obtained from a c y s t i c f i b r o s i s patient, and i t s spontaneous non-mucoid revertant. 4. Interaction of polyclonal sera with the outer membranes of P.  aeruginosa s t r a i n s To obtain more s p e c i f i c information on the antigenic r e l a t i o n s h i p of the major outer membrane proteins of the serotype s t r a i n s , the proteins were transferred from SDS polyacrylamide gels onto n i t r o c e l l u l o s e paper 125 and reacted with I - l a b e l l e d rabbit anti-outer membrane poly c l o n a l sera. This antiserum had been a f f i n i t y p u r i f i e d (see methods). The transfer of protein 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 f o r proteins G, HI, H2 and I as judged by both the coommassie b r i l l i a n t blue s t a i n i n g of the SDS gels a f t e r e l e c t r o b l o t t i n g and amido black s t a i n i n g of b l o t s . The e f f i c i e n c y of transf e r of proteins F and D2 was approximately 807. (F i g . 4, rig h t panel). 57 Figure 4 . L e f t , Autoradiogram of an electrophoretic bl o t of outer membrane proteins of P. aeruginosa a f t e r treatment with radioiodinated antibodies to outer membrane proteins. The numbers below the lanes r e f e r to the serotypes; W i s s t r a i n H 1 0 3 . The indicated polypeptides proteins E, F, H 2 and I) were i d e n t i f i e d by amido black s t a i n i n g of the electrophoretic b l o t s . The band l a b e l l e d F l a ( f l a g e l l i n ) was i d e n t i f i e d by co-electrophoresis with p u r i f i e d f l a g e l l i n . Right, SDS-polyacrylamide gel electrophoretogram a f t e r electrophoretic transfer of outer membrane proteins to n i t r o c e l l u l o s e . Note only p a r t i a l ( 8 0 7 o ) t r a n s f e r of protein F was achieved. 59 The autoradiograms of the Western immunoblots and the densitometer scans of the autoradiograms revealed that proteins F, H2 and I, interacted with the r a d i o l a b e l l e d antibodies in a l l the tested outer membranes (Figs. 4 and 5) while protein E was l a b e l l e d i n a l l s t r a i n s except the serotype 4 s t r a i n ( F i g . 5). This s t r a i n appeared to have a protein E equivalent oh SDS-polyacrylamide gels but i t i s possible that protein 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 . Outer membrane proteins D2, HI and G were not l a b e l l e d i n any outer membrane, and they would appear to be non or weakly immunogenic by the described immunizing schedule. An estimate of the amount of antibody bound to the major outer membrane proteins was obtained from the densitometer scanning of the autoradiogram (Fig. 5). As described above, proteins F, H2 and I were l a b e l l e d in a l l s t r a i n s and therefore were a n t i g e n i c a l l y r e l a t e d in a l l st r a i n s at the po l y c l o n a l l e v e l . However, the serotype s t r a i n s did not show i d e n t i c a l antibody binding to these outer membrane protein as demonstrated by differences i n the peak heights of the densitometer scans (correlated to the density of the l a b e l ) . This observation could be due to the d i f f e r i n g a f f i n i t i e s of the t e s t antibodies f o r the given outer membrane proteins, or by the d i f f e r i n g amounts of the given proteins i n the various outer membranes. Thus small amounts of a given protein in an outer membrane would c o r r e l a t e to the small amounts of antibody bound to the protein in that s t r a i n . Protein 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 blots and therefore quantitative statements on the a n t i g e n i c i t y of t h i s protein could not be made. The strongly l a b e l l e d protein of molecular weight 60 Figure 5A and B. Densitometer tracings of autoradiograms of separated outer membrane proteins from Pseudomonas aeruginosa s t r a i n H103 (W), serotype 1-17 s t r a i n s , and i s o l a t e PI from a patient with c y s t i c f i b r o s i s , a f t e r treatment with radioiodinated antibodies to outer membrane protein. Exposed X-rays films s i m i l a r to those shown in figure 4 were analyzed by densitometer scanning. The indicated polypeptides (proteins E, F, H2, and I and f l a g e l l i n [Fla]) were i d e n t i f i e d as described in the F i g . 4 legend. 62 53,000 daltons in the outer membranes of H103 ( F i g . A ) was i d e n t i f i e d as f l a g e l l i n by coelectrophoresis with p u r i f i e d f l a g e l l i n ( f l a g e l l i n was a g i f t from R. Ansog, Hygiene I n s t i t u t e , U n i v e r s i t a t Kreuzbergin, Gottingen Federal Republic of Germany). The large amounts of antibody bound to f l a g e l l i n must r e f l e c t the high immunogenicity of t h i s protein, since i t was,a very minor component of the Coommassie blue-stained s t r a i n H103 outer membranes. The outer membranes of s t r a i n s representing serotypes 3 10, 11 and 12 showed large peaks of antibody binding to f l a g e l l i n whereas smaller peaks were demonstrated by serotypes 4, 5, 7, 16, 17 and PI str a i n s ( F i g . 5). These r e s u l t s c o r r e l a t e with the known antigenic v a r i a t i o n of f l a g e l l i n protein among P. aeruginosa s t r a i n s ( P i t t , 1980). 5. Summary In P. aeruginosa the conservation of outer membrane polypeptides among the 17 serotype st 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 was demonstrated by the strong s i m i l a r i t y of outer membrane protein p r o f i l e s on SDS-polyacrylamide gels. The major outer membrane proteins D2, E, F, H2 and I were generally conserved, although t h e i r r e l a t i v e l e v e l s varied between s t r a i n s . Antigenic c r o s s - r e a c t i v i t y was demonstrated f o r proteins F, H2 and I among a l l tested P. aeruginosa s t r a i n s at the po l y c l o n a l l e v e l using an antiserum to s t r a i n H103 outer membranes. Protein E was immunologically cross-reactive in 16 of the 17 serotype s t r a i n s , but antibodies to proteins D2, HI and G were not detected using the described immunization protocol. 64 Antigenic heterogeneity at the polyclonal l e v e l was demonstrated f or f l a g e l l i n . Both s t r u c t u r a l and immunological heterogeneity were demonstrated f or the LPS. P. aeruginosa s t r a i n s from c y s t i c f i b r o s i s patients were l a r g e l y of the rough LPS phenotype and were serum s e n s i t i v e . 65 Chapter I I . MONOCLONAL ANTIBODIES TO OUTER MEMBRANE ANTIGENS 1. I s o l a t i o n and ch a r a c t e r i z a t i o n of monoclonal antibodies After fusion of NS1 or SP2/0-Agl4 myeloma c e l l s with spleen c e l l s from mice primed e i t h e r with P. aeruginosa PA01 whole outer membranes or with p a r t i a l l y p u r i f i e d outer membrane proteins, the obtained hybridomas were cloned out u n t i l a single c e l l clone per well of a ti s s u e culture plate was evident. By t h i s procedure a number of clones that produced antibodies s p e c i f i c f o r outer membrane antigens were i s o l a t e d from several separate fusions. These antibodies were judged to produce monospecific antibodies on the basis of t h e i r s p e c i f i c i t y towards a v a r i e t y of crude antigenic frac t i o n s from outer membranes (Table III) and t h e i r production of single p r e c i p i t i n l i n e s in Ouchterlony double immunodiffusion assays and rocket Immunoelectrophoresis procedures using as antigen, s t r a i n PA01 outer membranes s o l u b i l i z e d in 1% (vol/vol) T r i t o n X100, 20 mM Tr i s - H C l pH 8.0 and 10 mM EDTA (Results not shown). These antibodies were further characterized for t h e i r antigenic s p e c i f i c i t i e s . In addition to the monoclonal antibodies s p e c i f i c f o r protein F and for rough LPS, monoclonal antibodies MA1-8, MA1-3 and MA1-6 [ o r i g i n a l l y i s o l a t e d by A.A. Wieczoreck (Hancock et a l . , 1982)], and 5E4 and 8A1 [from Centocor Company (Malvern, Pa)], were used in t h i s study. (A) An LPS 0-antigen s p e c i f i c monoclonal antibody Monoclonal antibody MA1-8 (IgGl subclass) reacted strongly in ELISA assays with p u r i f i e d outer membranes from s t r a i n H103 and from an 66 a n t i b i o t i c supersusceptible, LPS-altered mutant Z61 o r i g i n a l l y derived from a c l i n i c a l i s o l a t e of P. aeruginosa (Table IV). The antibody also reacted strongly with p u r i f i e d s t r a i n H103 LPS and with a vari e t y of p a r t i a l l y p u r i f i e d outer membrane proteins from s t r a i n H103 which were shown by chemical means to contain LPS (Table I I I ) . MA1-8, however, d i d not interact with the outer membranes from three independently i s o l a t e d LPS-0 antigen-deficient (rough) mutants derived from s t r a i n H103 (Table IV). The antibody passively hemagglutinated sheep red blood c e l l s coated with s t r a i n H103 LPS (hemagglutination t i t r e 1400) but did not agglutinate c e l l s coated with the O-antigen-deficient (rough) LPS (hemagglutination t i t r e < 2). These data were consistent with MA1-8 being s p e c i f i c f o r the O-antigen of s t r a i n H103 LPS (F i g . 6, lane 1). (B) LPS rough s p e c i f i c monoclonal antibodies Monoclonal antibodies MA3-5 and MA3-6 reacted strongly i n ELISA assays with outer membranes from P. aeruginosa s t r a i n s H103, Z61 and AK1012 (an O-antigen-deficient mutant of PAOI) as well as a l l p a r t i a l l y p u r i f i e d outer membrane proteins from s t r a i n H103 (Tables I I I , IV). Unlike MA1-8, monoclonal antibodies MA3-5 and MA3-6 did not agglutinate any of the above P. aeruginosa s t r a i n s . In Western immunoblot procedures, MA3-5 and MA3-6 interacted with a fa s t migrating band of s i m i l a r molecular weight from the LPS of P. aeruginosa s t r a i n s H103 and AK1012 (Figure 6, lane 2). I t was therefore concluded that these monoclonal antibodies were s p e c i f i c for the rough core of LPS. Monoclonal antibodies MA3-5 and MA3-6 were of lgM subclass. 67 (C) L i p i d A s p e c i f i c monoclonal antibodies Two monoclonal antibodies 5E4 and 8A1 produced by Centocor Corporation (Philadelphia, Pa) a f t e r immunization of mice with heat k i l l e d c e l l s of E. c o l i s t r a i n J5 (Bogard e_t aJL. , manuscript submitted; Mutharia et a l . , 1984), were tested for a c t i v i t y against P. aeruginosa s t r a i n s . In ELISA assays both of the antibodies interacted with the outer membranes, LPS and L i p i d A from P. aeruginosa PA01 (Table VI; see below). However, several d i f f e r e n t i a l interactions of the antibodies and antigens were observed. For example antibody 8A1 reacted strongly in ELISA assays with several antigens but very poorly with the same antigens in Western blo t assays. These r e s u l t s may r e f l e c t the mode of antigen presentation. In Western immunoblot assays, 5E4 interacted with a f a s t migrating band in the outer membrane or LPS of s t r a i n H103 (Figure 6, lane 5). These antibodies were of the IgGI subclass by int e r a c t i o n with subclass s p e c i f i c antisera. (D) A l i p o p r o t e i n H2 s p e c i f i c monoclonal antibody A monoclonal antibody MA1-6 reacted strongly with s t r a i n H103 outer membranes, and with p a r t i a l l y p u r i f i e d l ipoproteins H2 and I (which was contaminated with H2) in ELISA assays (Table I I I ) . In Ouchterlony double immunodiffusion t e s t s , t h i s monoclonal antibody gave a single p r e c i p i t i n l i n e with both l i p o p r o t e i n H2 and I preparations. In addition, a single p r e c i p i t i n l i n e occurred with 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 that had been cut out and eluted from SDS-polyacrylamide gels with 2% (vol/vol) T r i t o n X-100, 20 mM Tris-HCl (pH 8.0) and 10 mM EDTA. The antibody was, 68 therefore s p e c i f i c for l i p o p r o t e i n H2 as demonstrated by Western b l o t t i n g (Fig. 6, Lane 3). (E) Protein F s p e c i f i c monoclonal antibodies Five monoclonal antibodies 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 fusions of NS-1 or Sp2/0-Agl.4 myeloma c e l l s with spleen c e l l s from mice immunized with e i t h e r p u r i f i e d protein F or s t r a i n H103 outer membranes. These antibodies reacted in ELISA assays with outer membranes of s t r a i n H103 but not with s t r a i n H103 LPS. When tested with 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 outer membrane proteins, they reacted s p e c i f i c a l l y with protein F (Table I I I ) . Furthermore, these antibodies showed no i n t e r a c t i o n with outer membranes from P. aeruginosa s t r a i n H283, a protein F - d e f i c i e n t mutant derived from s t r a i n H103 (Nicas and Hancock, 1983). These antibodies were s p e c i f i c f o r P. aeruginosa H103 porin protein F ( F i g . 6, lane 4). Two of these monoclonal antibodies MA2-10 and MA4-10 were shown to be of the IgGI subclass, by i n t e r a c t i o n with subclass s p e c i f i c antisera, while antibodies 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 i s o l a t e d from the fusion of NS-1 myeloma c e l l s and the spleen c e l l s from a mouse immunized with s t r a i n H103 outer membrane showed very i n t e r e s t i n g r e s u l t s . The monoclonal antibody MA1-3 f a i l e d to i n t e r a c t with LPS or p a r t i a l l y p u r i f i e d outer membrane proteins D2, F and HI but interacted well with protein I (Table I I I ) . In an 69 Table I I I . Interaction of monoclonal antibodies with p a r t i a l l y p u r i f i e d major outer membrane proteins of P. aeruginosa PA01. Log ELISA t i t r e s with monoclonal antiserum Antigen MA1-8 MA1-6 MA1-3 MA3-5 H103 outer membranes 5 3 3 3 Protein D2 a 3 < 1 < 1 ND Protein F a 2 1 < 1 2 Protein H l a 2 < 1 ^ < 1 2 Protein H2 b < 1 4 < 1 2 Protein I b < 1 4 3 ND LPS 5 0 0 4 a. P a r t i a l l y p u r i f i e d proteins D2, F, and HI were shown to contain s i g n i f i c a n t amounts of LPS [about 10% (wt/wt)]. b. Protein H2 and I were cross-contaminated with each other. 70 Table IV. Interaction of monoclonal antibodies with the Outer membranes of LPS - altered strains of P. aeruginosa. Outer Membrane Antigen Log^ 0 ELISA t i t r e from s t r a i n LPS defect MA1-8 MA1-6 MA1-3 MA3-5 ' PAOI wild type 5 3 3 3 Z61 L i p i d A (semi rough) 6 3 3 3 AK1160 rough < 1 3 3 3 AK1188 rough < 1 3 3 ND H223 rough < 1 3 3 3 ND - not done 71 Figure 6. Western electrophoretic blots of outer membranes of P. aeruginosa s t r a i n H103 af t e r treatment with monoclonal antibodies MA1-8 (lane 1), MA3-5 (lane 2), MA1-6 (lane 3), MA4-4 (lane 4), and 5E4 (lane 5). The immunostaining procedure was as described i n methods. (These Western blots were from 5 separate experiments, hence the differences in the density of the l a b e l . The high backgrounds obtained with the Western blot using 5E4 contributed to the faintness of the l a b e l l e d band i n lane 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 l i n e when protein I was used as an antigen. However, MA1-3 neither interacted with protein I eluted from SDS-polyacrylamide gels nor agglutinated c e l l s of any P. aeruginosa s t r a i n s . MA1-3 interacted with the outer membranes from the serotype st r a i n s of P. aeruginosa (see below) in ELISA assays. This antibody d i d not interact with Western blots of P. aeruginosa s t r a i n H103 outer membranes. 2. Use of Monoclonal antibodies i n the study of antigenic heterogeneity  in P. aeruginosa Monoclonal antibodies provide several advantages over conventional polyclonal sera derived from immunization of animals with antigenic f r a c t i o n s . For example, they have defined s p e c i f i c i t y for a single epitope ( i . e . antigenic s i t e ) on one species of macromolecule. This allows one to accurately quantify and i d e n t i f y given macromolecules under a l l circumstances, regardless of the presence of contaminating antigens. Therefore, monoclonal antibodies were used in a study of the antigenic re l a t i o n s h i p s of the major outer membrane antigens of P. aeruginosa. (A) S p e c i f i c i t y of protein F monoclonal antibodies The monoclonal antibodies s p e c i f i c f o r protein F were tested by ELISA for binding to the outer membranes of the 17 serotype strains of P. aeruginosa and to 16 c y s t i c f i b r o s i s patient i s o l a t e s . Results from MA4-4 and MA2-10 are presented in Table V. These monoclonal antibodies, as well as MA4-2, MA4-10 and MA5-8, reacted with a l l of the outer 74 Table V: Interaction of monoclonal antibodies MA 2-10 and MA 4-4 with outer membranes of P. aeruginosa 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 of Outer ELISA t i t r e of monoclonal a n t i b o d y a , b Membrane Antigen MA 4-4 MA 2-10 PA01 (H103) 10 2 P u r i f i e d protein F 105 (from H103) Serotype str a i n s 1 10 4 2 10 3 3 10 3 4 10 4 5 10 4 6 10 5 7 10 4 8 10 4 9 10 4 10 10 4 11 IO 4 12 10 4 13 10 4 14 10 4 15 10 4 16 10 4 17 10 4 C F . is o l a t e s PI (mucoid) 10 3 PI (non-mucoid) 10 4 CF 3660-1 10 4 CF 9490 IO 5 CF 221 10 5 CF 4349 < 10 2 CF 4522 IO 5 CF 832 10 3 CF 3790 10 4 CF 1278 10 2 L 10 5 CF 1452 10 5 CF 6094 10 2 CF 2314 10 4 CF 284 10 4 (0.66) c 10 4 (1.42) (1.16) 10 5 (1.54) (0.58) 10 4 (0.62) (0.24) 10 4 (0.25) (0.27) 10 4 (0.24) (0.40) 10 5 (0.32) (2.0) 10 4 (2.0) (1.54) 10 4 (2.0) (2.0) 10 4 (1.79) (0.89) 10 5 (0.92) (1.3) 10 5 (1.0) (1.4) 10 4 (1.29) (1.67) 10 4 (2.0) (0.72) 10 4 (0.72) (1.11) 10 4 (1.2) (0.13) 10 4 (0.20) (0.37) IO 4 (0.42) (0.71) IO 4 (0.69) (1.17) 10 4 (1.09) (0.2) IO 3 (0.54) (0.6) IO 3 (1.11) (0.24) IO 3 (0.29) (0.5) IO 3 (0.61) (0.58) IO 3 (0.62) (0.15) c < 10 2 (0.13) c (0.69) 10 3 (0.72) (0.73) IO 3 (0.78) (0.48) IO 3 (0.55) (0.22) c IO 2 (0.34) c (0.7) IO 3 (0.71) (0.43) 10 3 (0.37) (0.15) l p 3 (0.18) (0.46) 10 3 (0.41) (0.53) IO 4 (0.48) 75 Table V, continued a. ELISA readings are the average of three experiments. b. Numbers in parentheses represent the average o p t i c a l density at 405 nm aft e r 1 hr of incubation at 37°C with p-nitrophenyl phosphate with a d i l u t i o n of antiserum given as the Log^g t i t r e . Controls lacking antigen or using ascites f l u i d from NS1 myeloma c e l l s gave o p t i c a l densities of 0 - 0.04. c. While i n t e r a c t i o n of antibodies with antigens CF4349 and CF1248 was weak or barely above background, a f t e r overnight incubation colour appeared for a l l s t r a i n s . A l l of the st r a i n s interacted with these monoclonal antibodies 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 of P. aeruginosa strains tested, although the reactions varied from s t r a i n to s t r a i n and with the d i f f e r e n t monoclonal antibodies. Thus the antigenic s i t e s recognized by these monoclonal antibodies on protein F are apparently common to a l l these P. aeruginosa s t r a i n s . This was confirmed for a l l protein F - s p e c i f i c monoclonal antibodies by i n t e r a c t i o n with electrophoretic blots of separated outer membrane proteins from P. aeruginosa serotyping s t r a i n s (see e.g. F i g . 7). Generally, only one band, i d e n t i f i e d as protein F by comparison with i d e n t i c a l b l o t s stained with amido black, was evident i n the immunolabelled b l o t s . Exceptions were observed f o r p u r i f i e d protein F fr a c t i o n s ( F i g . 7, lane 9) or overloaded outer membrane protein samples (Fig. 7, lanes 7 and 8), for which higher molecular weight forms of protein F were stained, presumably due to e i t h e r native oligomers of protein F (see chapter 3) or protein F-LPS, complexes both of which have been i d e n t i f i e d in a chemical c r o s s - l i n k i n g study (Angus and Hancock, 1983). With these exceptions, single bands were obtained with a l l protein F - s p e c i f i c monoclonal antibodies and the i n t e n s i t y of the l a b e l on the blots c o r r e l a t e d with the ELISA r e s u l t s f o r the d i f f e r e n t outer membrane i s o l a t e s . As a c o n t r o l , no l a b e l l e d band was observed on Western blots using outer membranes from a protein F - d e f i c i e n t mutant H283 (see below, F i g . 9, lane 2). As discussed above, the outer membrane protein patterns of the c y s t i c f i b r o s i s P. aeruginosa i s o l a t e s were s i m i l a r to those of s t r a i n H103 (Fi g . 2). Protein F from these i s o l a t e s interacted well with the protein F - s p e c i f i c monoclonal antibodies on ELISA (Table V) and Western b l o t s . 77 Figure 7. Western electrophoretic blots of outer membranes of the serotyping s t r a i n s of P. aeruginosa a f t e r treatment with monoclonal antibody MA5-8. The blot was made by electrophoretic transfer of separated outer membrane proteins from SDS-polyacrylamide gels onto n i t r o c e l l u l o s e paper. Outer membrane samples are Lane 1-serotype 17; Lane 2-serotype 16; Lane 3-serotype 15; Lane 4-serotype 14; Lane -serotype 13; Lane 6-serotype 12; Lane 7-serotype 11; Lane 8-serotype 10; Lane 9-pu r i f i e d 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 19-wild type H103. 78 79 Figure 8. Interaction Of monoclonal antibodies MA2-10 with outer membranes from c y s t i c f i b r o s i s P. aeruginosa i s o l a t e s on Western b l o t . The outer membrane samples are: 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-Strain 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 16-P1. Lane 17-Purified protein F. Only one band, i d e n t i f i e d as protein F by comparison with stained electrophoretic b l o t s , was l a b e l l e d in any of the membranes. Ho 81 Figure 9. Cross r e a c t i v i t y of protein F - s p e c i f i c monoclonal antibody MA4-4 with outer membrane proteins from other b a c t e r i a l species. Western electrophoretic blots of separated outer membrane proteins and immune staining were performed as described for F i g . 7. Lanes 1 and 6-P. aeruginosa s t r a i n H103; Lane 2-P. aeruginosa s t r a i n H283, a protein F d e f i c i e n t mutant; Lane 3-Edwardsiella tarda 79054; Lane 4-Vibrio  anguillarum HT7602; Lane 5-Aeromonas hydrophila ET-2; Lanes 7 and 14; p u r i f i e d protein 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. fluorescens s t r a i n s ATCC 13525 and ATCC 949, r e s p e c t i v e l y ; lane 11-P. putida ATCC 12633; Lane 12-Azotobacter v i n e l a n d i i OP; Lane 13-P. syringae ATCC 19310. 82 83 Strain CF 283 (Fig. 8, lane 4) which f a i l e d to int e r a c t was subsequently shown by f a t t y acid analysis not to be a P. aeruginosa s t r a i n (L. Chan, personal communication). There was no i n t e r a c t i o n of monoclonal antibody MA4-4 (or MA2-10, MA4-2 or MA4-10) with outer membrane proteins of Edwardsiella tarda. V i b r i o anguillarum, P. a n g u i l l i s e p t i c a , Aeromonas hydrophila, P. fluorescens, or Azotobacter v i n e l a n d i i . ( F i g . 9), although they interacted strongly with a 39 kD protein from P. putida and P. syringae outer membranes (Fig. 9). One monoclonal antibody MA5-8, was highly s p e c i f i c for the porin protein F of P. aeruginosa. This antibody cross-reacted with a l l tested s t r a i n s of P. aeruginosa but no other gram-negative b a c t e r i a l strains (Table VI). (B) C r o s s - r e a c t i v i t y of a l i p o p r o t e i n H2-specific monoclonal antibody Monoclonal antibody MA1-6 interacted s p e c i f i c a l l y with the major outer membrane l i p o p r o t e i n H2 ( F i g . 6). The antigenic s i t e recognized by thi s antibody was present on l i p o p r o t e i n H2 from the 17 serotype st r a i n s (Fig. 10, Table VII) and 28 of the 30 c y s t i c f i b r o s i s P. aeruginosa strains examined (e.g. F i g . 11). In a double antibody Western blo t ( Fig. 11) monoclonal antibodies MA4-4, s p e c i f i c f o r protein F, and MA1-6, s p e c i f i c f o r protein H2, were used to show the s p e c i f i c i t y of the antigen-antibody i n t e r a c t i o n . In the Western b l o t , a band that co-migrated with p u r i f i e d protein F (Fig. 11, lane 13) was l a b e l l e d in the outer membranes of a l l the c y s t i c f i b r o s i s i s o l a t e s . In contrast, monoclonal antibody MA1-6 showed no int e r a c t i o n Table V I . Cross -react ion of monoclonal ant ibodies s p e c i f i c for P_. aeruginosa outer membranes assessed by ELISA • ELISA readings A^(-^ ausing monoclonal ant ibodies s p e c i f i c for B a c t e r i a l Outer Membrane Proteins Lipopolysaccharide outer membrane H25 F 5 F c l ( ? ) b 0 -ant igen c Rough c o r e 0 L i p i d A antigen (MA1-6) (MA4-4) (MA5-8) (MA1-3) (MA1-8) (MA3-5). (5E4) d P. aeruginosa s t ra ins serotype 5 0.4 0.1 0.4 0.3 1.4 0.1 0.3 serotypes 7, 8, 10, 14, 16 e 0.6-1.2 0.7-1.8 0.8-2 0.1-0.3 - 0.1-0.3 ND Other serotypes 0.4-2.0 0.3-2.0 0.8-2 0.2-0.4 - - ND AK 1160 f (LPS rough) 1.2 0.6 0.4-2 0.2 0.7 0.1-0.3 CF 2218 0.8-1.2 0.8-1.8 0.1-0.3 0.1-0.3 - 0.2 P. put ida 0.4 0.2 ' - 0.1 - - 0,2 P. syringae 0.4 1.1 0.1 - - ND P. ch lororaphis 0.3 . — - 0.1 - ND P. aureofaciens 0.1 - . - 0.1 - - ND P. s t u t z e r i 0.3 - 0.3 • - • ' - ND P. f luorescens 0.5 - - 0.1 - - 0.4 P. a n g u i l l i s e p t i c a 0.1 - . - - - - 0.3 A . v i n e l a n d i i 0.1 - - 0.1 - - 0.4 P. m a l t o p h i l i a - - - - - - ND P. acidovorans - - - - ND ND P. solanacearum - - - - ND P. cepacia - - - - - + CO Table V I , continued ELISA readings A4053 using monoclonal ant ibodies s p e c i f i c for B a c t e r i a l Outer Membrane Proteins Lipopolysacchar ide outer membrane H2 b F c F c K ? ) b 0-ant igen c Rough c o r e 0 L i p i d A antigen (MA1-6) (MA4-4) (MA5-8) (MA1-3) (MA1-8) (MA3-5) (5E4) d E . c o l i _ 0.4 S. typhimurium - - - - - 0.3 E . tarda - - - - - ND + Y . pest is^ ND ND ND ND ND ND + V . cholera ND ND - ND ND ND + V . angui l larum - (-) (-) - ND 0.2 A. salmonicida - - - - - - . 0.2 A . hydrophi la — - -. - - - 0.3 A. tumefaciens n PLT4 ND ND ND ND ND 1.2 S1005.A6 ND ND ND ND ND 0.2 OO 86 VI Legend ND. not done; a. The ELISA data i s expressed as absorbance at 405 nm a f t e r 90 minute at 37°C. b. The monoclonal antibody was used at 100 f o l d d i l u t i o n . c. The monoclonal antibody was used at 1,000 f o l d d i l u t i o n . d. 5E4 was used at 10 yg of lgG per ml. e. A l l other P. aeruginosa serotypes interacted with the tested monoclonal antibodies except MA1-8 and MA3-5 (MA3-6). f. Three other LPS-)-antigen d e f i c i e n t s t r a i n s H234, H223 and H1188 gave sim i l a r r e s u l t s . g. '36 d i f f e r e n t P. aeruginosa s t r a i n s from c y s t i c f i b r o s i s (CF) patients were tested. 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 to those in Table VI with some exceptions. 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 only 3 s t r a i n s interacted with MA3-5. h. LPS from Y. pestis and A. tumefaciens was used as the antigen. (-) denote negative r e s u l t s by both Western blo t and ELISA procedures. These data were obtained from G.W.K. Crockford, U.B.C. 87 Figure 10. Western electrophoretic blots of outer membranes of the serotyping strains of P. aeruginosa a f t e r treatment with monoclonal antibody MA1-6. Outer membrane samples were lane 1- wild type H103; lane 2 - serotype 1; lane 3 - serotype 2; lane 4 - serotype 3; lane 5 -serotype 4; lane 6 - serotype 5; lane 7 - serotype 6; lane 8 - serotype 7; lane 9 - serotype 8; lane 10 - serotype 9; lane 11 - serotype 10; lane 12 - serotype 11; lane 13 - serotype 12; lane 14 - serotype 13; lane 15 -serotype 14; lane 16 - serotype 15; lane 17 - serotype 16; lane 18 -serotype 17; (Lane S - SDS - electrophoretogram of H103 outer membrane). Only one band was l a b e l l e d . 88 89 Figure 11. Western electrophoretic blots of outer membranes of 6 pairs mucoid (m) and non-mucoid (nm) P. aeruginosa i s o l a t e s from patients with c y s t i c 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 antibodies MA4-4 (top band), and MA1-6 (bottom band). The outer membrane samples were lane 1-wild type H103; lane 2 - Plm; lane 3 - CFClm; land 4 - CFClnm; lane 5 CFC47m; lane 6 - CFC47nm; lane 7 - CFC46m; lane 8 - CFC46nm; lane 9 -CFC4m; lane 10 - CFC4nm; lane 11 - CFC6m; lane 12 - CFC6nm; lane 13 -p u r i f i e d protein F. 1 2 3 A 5 6 7 8 9 10 11 12 13 91 Figure 12. Western electrophoretic b l o t showing inte r a c t i o n of monoclonal antibody MA1-6 with outer membranes from: 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. fluorescens ATCC 949; Lane 4, P. syringae ATCC 19310; Lane 5, P. aeruginosa 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. aureofaciens ATCC 13985; Lane 11, P. cepacia; Lane 12, P. chlororaphis ATCC 9446; Lane 13, P. pseudomallei ATCC 23343; Lane 14, P. aeruginosa 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 protein F (Fig. 11, lane 13) nor with outer membranes from s t r a i n CFC46 mucoid and CFC46 nonmucoid ( F i g . 11; lanes 8 and 9). As described above ( F i g . 2), these two st r a i n s also lacked protein H2 on SDS polyacrylamide gels of t h e i r outer membranes. Monoclonal antibody MAl-6 also reacted with a protein of s i m i l a r molecular weight to protein H2 in Western blots of P. a n g u i l l i s e p t i c a , P. fluorescens. P. putida and P. syringae outer membranes, with a protein of higher molecular weight in Azotobacter v i n e l a n d i i , and s l i g h t l y higher molecular weight in P. s t u t z e r i outer membranes, and with a protein of s l i g h t l y lower molecular weight i n P. aureofaciens and P. chlororaphis outer membranes ( F i g . 12). There was no c r o s s - r e a c t i v i t y with the outer membranes of P. cepacia (Fig. 12), E. c o l i , P. acidovorans. P. m a l t o p h i l i a or S. typhimurium (Table VI). (C) Heterogeneity of the LPS rough core in P. aeruginosa Two rough c o r e - s p e c i f i c monoclonal antibodies were characterized i n d e t a i l . Antibody MA3-5 interacted with the LPS of serotypes 5 (the serotype of s t r a i n H103), 7, 8, 10, 14 and 16 of the International Antigen Typing Scheme of P. aeruginosa (17 serotypes) ( F i g . 13), whereas MA3-6 interacted only with serotypes 5, 8, 10 and 16 (data not shown). Of the 16 P. aeruginosa i s o l a t e s from patients with c y s t i c f i b r o s i s tested, MA3-6 interacted only with s t r a i n CF1452 whereas MA3-5 interacted with three s t r a i n s , CF4522, CF221 and CF9490. This provided the f i r s t d e f i n i t i v e evidence for LPS rough core heterogeneity in P. aeruginosa. 94 Figure 13. Western electrophoretic blot of outer membranes of P. aeruginosa serotype s t r a i n s a f t e r reaction with monoclonal antibody MA3-5. The numbers below the lanes are the serotypes. The monoclonal antibody interacted with the rough core LPS from serotype 5, 7, 8, 10, 14 and 16 s t r a i n s , and s t r a i n H103 ( l a b e l l e d W). 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 antibodies showing d i f f e r e n t s p e c i f i c i t i e s for the P. aeruginosa LPS rough core were is o l a t e d . Although the r e s u l t s of the i n t e r a c t i o n of these monoclonal antibodies and P. aeruginosa strains is not presented here, i t should be mentioned that some of the antibodies showed analogous narrow c r o s s - r e a c t i v i t i e s to antibodies MA3-5 and MA3-6 while others showed very extensive c r o s s - r e a c t i v i t y among a l l P. aeruginosa s t r a i n s tested ( J . L i n , personal communication). (D) Heterogeneity of LPS O-antigen The monoclonal antibody MA1-8, s p e c i f i c f o r the 0 antigen of s t r a i n PAOI, interacted strongly with the outer membrane of the serotype 5 s t r a i n (Table VI), in agreement with a previous observation that s t r a i n PAOI i s type 5 in the IATS typing scheme (Cox 1979). Of the other 16 serotype s t r a i n s examined, only the outer membranes of type 17 interacted weakly with MA1-8 (Table V I I ) . This was consistent with a series of experiments t e s t i n g the a b i l i t y of our monoclonal antibodies to agglutinate whole bact e r i a . In these experiments, MA1-8 showed agglutination (4+) of c e l l s of s t r a i n PAOI, s t r a i n Z61 and the IATS serotype 5 s t r a i n but no b a c t e r i a l agglutination reactions were observed using the other serotyping s t r a i n s . In further tests (Table VI) MA1-8 was shown to be s p e c i f i c f o r P. aeruginosa LPS and the antibody showed no c r o s s - r e a c t i v i t y with other gram-negative ba c t e r i a including other Pseudomonads. 97 Table VII. Interaction of monoclonal antibodies MA1-3, MAl-6, and MA1-8 with the outer membrane antigens from the 17 serotypes s t r a i n s of P. aeruginosa. Log^o ELISA t i t r e with monoclonal antibody Outer membrane MA1-8 MAl-6 MA1-3 ant i g e n 4 from Serotype 1 < 1 (0, .05) 2 (0, .73) 3 (0, .59) 2 < 1 (0, .06) 2 (0, .85) 3 (0, .39) 3 < 1 (0, .05) 1 (0, .54) 3 (0. .49) 4 < 1 (0, .02) 2 (0. .73) 3 (0, .38) 5 3 (2, .0) 1 (0. .41) 3 (0, .30) 6 < 1 (0, .02) 3 (1, .08) 3 (0, .29) 7 < 1 (0, .01) 3 (1. .20) 3 (0, .45) 8 < 1 (0, .02) 2 (0. .70) 3 (0. .32) 9 < 1 (0, .01) 3 (1. .09) 3 (0, .45) 10 < 1 (0, .02) 3 (1. .21) 3 (0, .36) 11 ' < 1 (0. .0) 2 (0, .92) 3 (0, .30) 12 < 1 (0. .0) 2 (1, .18) 3 (0. .42) 13 < 1 (0, .0) 1 (0. 41) 3 (0. 41) 14 < 1 (0. .0) 3 (1. 25) 3 (0. ,39) 15 < 1 (0. .0) 1 (0. ,40) 3 (0. .37) 16 < 1 (0. .02) 3 (0. ,56) 3 (0. .50) 17 1 (0. 2) 3 (> 2) 3 (0. .58) PI mucoid < 1 (0. .01) 1 (0. ,38) 3 (0. .44) PI revertant < 1 Co. 0) 1 (0. ,20) 3 (0. ,53) PA01 5 (1. ,99) 3 (1. 15) 3 (0. ,58) a. For testing MA1-3, outer membranes were s o l u b i l i z e d in 2% T r i t o n X-100, 20 mM Tris-HCl, pH 8.0, 10 mM EDTA, and then p r e c i p i t a t e d at -20°C aft e r addition of 2 volumes of ethanol and 0.1 M NaCl p r i o r to coating of ELISA plates. Using u n s o l u b i l i z e d outer membranes as antigens gave inconsistent r e s u l t s f o r MA1-3. For the other monoclonal antibodies unsolubilized outer membranes were used as the coating antigen. The numbers in parentheses represent the o p t i c a l density at 405 nm aft e r 1 hr incubation with p-nitro-phenylphosphate with the antibody d i l u t i o n given as the Log^Q t i t r e . 98 (E) Conservation of L i p i d A Chemical c h a r a c t e r i z a t i o n of the L i p i d A portions of the LPS of diverse bacteria have indicated that L i p i d A i s chemically a strongly conserved structure (Rietschel et a l . , 1983). Consistent with t h i s , L i p i d A bears the general name "endotoxin" 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 have s i m i l a r e f f e c t s on host c e l l s (Bradley 1979). Monoclonal antibodies s p e c i f i c for L i p i d A were used to study the expression of s p e c i f i c epitopes on L i p i d A. For these studies, outer membranes and lipopolysaccharides from a va r i e t y of species and genera of gram-negative bacteria were screened by ELISA (and in some instances Western blots) f o r i n t e r a c t i o n with two monoclonal antibodies which had been shown to be reactive against p u r i f i e d L i p i d A from P. aeruginosa and S. minnesota (Mutharia e_t a l . , 1984; data of G.W.K. Crockford and W.C. Bogard). Neither of these antibodies interacted with the gram p o s i t i v e c e l l s (or c e l l walls) tested i . e . B a c i l l u s s u b t i l i s , Staphylococcus aureus and Streptococcus f a e c a l i s c e l l walls (W.C. Bogard, personal communication). In the present study, monoclonal antibodies 5E4 and 8A1 were shown by ELISA analysis to ex h i b i t extensive c r o s s - r e a c t i v i t y with the outer membranes and LPS from a v a r i e t y of P. aeruginosa and gram negative b a c t e r i a l s t r a i n s (Table VI). To determine the s p e c i f i c component of outer membranes which was int e r a c t i n g with the monoclonal antibodies 5E4 and 8A1 and to confirm some of the ELISA r e s u l t s in Table VI, Pseudomonas aeruginosa s t r a i n PAOI outer membranes were separated by SDS polyacrylamide gel electrophoresis and 99 transferred from the electrophoretogram to n i t r o c e l l u l o s e paper by the Western technique. The separated outer membranes were then interacted with monoclonal antibodies 5E4 amd 8A1. Both of these monoclonal antibodies interacted with a single major band (e.g. F i g . 14) which had migrated in the SDS polyacrylamide gel with a r e l a t i v e m o b i l i t y of around 0.8 to 0.9 compared to the bromphenol blue dye f r o n t . The band was i d e n t i f i e d as rough LPS (containing rough core and l i p i d A) since i t comigrated with authentic rough LPS from the rough mutant s t r a i n H146 and interacted with an LPS rough c o r e - s p e c i f i c monoclonal antibody MA3-5 but not with an LPS O-antigen s p e c i f i c monoclonal antibody MA1-8. Antibody 5E4 interacted with a s i m i l a r fast-migrating band from a l l 17 serotypes ( F i g . 14) and 14 c l i n i c a l i s o l a t e s ( F i g . 15) of P. aeruginosa. Occasionally, the i n t e r a c t i o n of antibody 5E4 with a series of c l o s e l y spaced bands of lower r e l a t i v e m o b i l i t y was also observed. Although t h i s observation was too inconsistent to analyse properly (perhaps due to the low a f f i n i t y of the monoclonal antibodies for higher molecular weight LPS), these bands may have represented smooth, O-antigen-containing LPS which has been shown to c o n s t i t u t e around 5-10% of the LPS molecules in some P. aeruginosa s t r a i n s . Monoclonal antibodies 5E4 and 8A1 also interacted with the outer membranes or LPS from other gram-negative b a c t e r i a . These included strains from the f a m i l i e s Pseudomonadaceae. Rhizobiaceae, Enterobacteriaceae. and Vibronaceae (Table VI). 100 Figure 14. Western electrophoretic b l o t of separated outer membranes of the 17 Pseudomonas aeruginosa serotype s t r a i n s a f t e r reaction with monoclonal antibody 5E4. The outer membranes were lane 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. aeruginosa PAOI. C r e f e r s to a c o n t r o l lane containing P. aeruginosa outer membrane protein F p u r i f i e d free of LPS. 101 1 2 3 4 5 67 8 C 9 10 11 12131415161718 102 Figure 15. Reaction of monoclonal antibody 5E4 with a Western electrophoretic blot of the separated outer membranes of c l i n i c a l i s o l a t e s of P. aeruginosa. The outer membranes were: 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. This b l o t was made from an 11% acrylamide, SDS polyacrylamide gel (unlike Figure 14 which was from a 14% acrylamide g e l ) . Therefore rough LPS migrated with the dye front and reacted as a tig h t band ( c . f . Figure 14). 1 2 3 A 5 6 7 8 9 X) 11 12 13 IA 104 Summary A l l P. aeruginosa st r a i n s studied shared at le a s t two separate conserved outer membrane antigenic s i t e s on protein F. With the exception of two strains (CFC46 mucoid and nonmucoid), P. aeruginosa s t r a i n s also shared an antigenic epitope on protein H2. A l l three of these s i t e s were expressed regardless of source, serotype, colony morphology ( i . e . mucoid or nonmucoid; rough or smooth) or serum s u s c e p t i b i l i t y of the P. aeruginosa i s o l a t e . A monoclonal antibody MAl-6 s p e c i f i c for outer membrane l i p o p r o t e i n protein H2 of P. aeruginosa interacted with 28 P. aeruginosa s t r a i n s and organisms representative of 8 other Genera of the Family of Pseudomonadaceae (mostly members of the fluorescent pseudomonads), as well as Azotobacter v i n e l a n d i i . This antibody did not in t e r a c t with pseudomonads demonstrated to be unrelated to P. aeruginosa by rRNA homology studies, nor with b a c t e r i a l s t r a i n s from other gram-negative b a c t e r i a l Genera. A group of four monoclonal antibodies against porin protein F of Pseudomonas aeruginosa interacted with a l l P. aeruginosa s t r a i n s tested as well as with P. putida and P. syringae st r a i n s but not with other pseudomonads. Another protein F - s p e c i f i c monoclonal antibody MA5-8 interacted only with the P. aeruginosa s t r a i n s . The s p e c i f i c i t y of a l l f i v e of these monoclonal antibodies f o r protein F was demonstrated by the absence of c r o s s - r e a c t i v i t y with a protein F - d e f i c i e n t mutant s t r a i n H283 derived from P. aeruginosa 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 with protein F on Western blots of SDS-polyacrylamide gel-separated outer membrane components. A monoclonal antibody s p e c i f i c for the O-antigen of our wild type P. aeruginosa s t r a i n interacted with s t r a i n s of equivalent serotype (0-5 in the International Antigen Typing Scheme, IATS) and weakly with s t r a i n s of a re l a t e d serotype (0-17) (Table V I I ) . Monoclonal antibodies s p e c i f i c f o r the rough core or 0-antigenic side chain of one Pseudomonas aeruginosa s t r a i n demonstrated only l i m i t e d cross-reaction with other P. aeruginosa s t r a i n s ( F i g . 13). In contrast, monoclonal antibodies s p e c i f i c f o r the l i p i d A portion of LPS interacted with the LPS or outer membrane of 36 P. aeruginosa strains (Table VI; F i g . 14,15) and 22 other gram negative b a c t e r i a from 4 d i f f e r e n t Families and 16 separate Genera (Table VI). This demonstrated that L i p i d A i s highly a n t i g e n i c a l l y conserved in many gram-negative bac t e r i a . 106 CHAPTER I I I . CHARACTERIZATION OF OUTER MEMBRANE EPITOPES USING MONOCLONAL  ANTIBODIES. 1. C e l l surface l o c a l i z a t i o n of the antigenic s i t e s recognized by monoclonal antibodies (A) Surface l o c a l i z a t i o n studies by immunofluorescence The a c c e s s i b i l i t y of an immunogenic protein on the surface of i n t a c t b a c t e r i a l c e l l s i s a c h a r a c t e r i s t i c of great importance, e s p e c i a l l y when such a protein i s being considered for i t s p o t e n t i a l use i n c l i n i c a l i d e n t i f i c a t i o n , i n epidemiological studies and in the development of both active and passive vaccines. Surface l o c a l i z a t i o n of P. aeruginosa outer membrane proteins has been indicated in studies using lactoperoxidase radio i o d i n a t i o n methods (Lambert and Booth, 1982). However, t h i s method has been shown to l a b e l non-outer membrane proteins as well ( S u l l i v a n and Williams 1982), perhaps as a r e s u l t of d i s r u p t i o n of outer membrane i n t e g r i t y . Furthermore, i t does not reveal whether the surface accessible portions of the protein are immunogenic. We were interested in studying the topography of the major outer membrane proteins on the c e l l surface of P. aeruginosa. As probes, monoclonal antibodies to porin protein F and 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 of serotype 5 (International Antigen Typing Scheme) was used as a p o s i t i v e c o n t r o l . In these studies, intact P. aeruginosa c e l l s were interacted in suspension with d i l u t i o n s of the monoclonal antibody and then with a f l u o r e s c e i n isothiocyanate-conjugated anti-mouse Ig antibody. The 107 l a b e l l e d c e l l s were then examined for fluorescence with a fluorescence microscope containing a halogen lamp and appropriate f i l t e r s . The results of these studies showed that a l l s t r a i n s of P. aeruginosa tested (with one exception), including i s o l a t e s from c y s t i c f i b r o s i s patients, interacted with the three tested porin protein F - s p e c i f i c monoclonal antibodies, MA2-10, MA4-4 and MA4-10 (Table VIII: F i g . 16). None of these monoclonal antibodies interacted with s t r a i n H283, a protein F - d e f i c i e n t mutant, as demonstrated by the lack of fluorescence on the H283 c e l l s (Fig. 16, panel E; Table V I I I ) . The serotype 5 LPS-specific monoclonal antibody MA1-8 interacted with both s t r a i n H103 and i t s protein 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 (serotype 1) or an 0-antigen-deficient (rough) mutant, s t r a i n H233 (also derived from s t r a i n H103) (Table V I I I ) . Monoclonal antibody MAl-6, s p e c i f i c f o r protein H2, interacted with s t r a i n H223 the rough mutant and with CF221, CFC47 mucoid and CFC47 non-mucoid s t r a i n s , the rough c y s t i c f i b r o s i s i s o l a t e s but not with H103, H283 or the serotype 1 st r a i n s (Table V I I I ) . Strains CFC46 mucoid and CFC46 non-mucoid which were rough but lacked protein H2 ( F i g . 11), showed no fluorescence. These r e s u l t s suggested that the l i p o p r o t e i n H2 i s e i t h e r not surface exposed or i t s a c c e s s i b i l i t y on the c e l l surface i s masked by LPS 0-side chains in wild-type (smooth) P. aeruginosa s t r a i n s . The i n t e r a c t i o n of monoclonal antibody MAl-6 with rough P. aeruginosa s t r a i n s may be due e i t h e r to unmasking of t h i s protein, because of the lack of 0-antigen on the LPS of the rough strains or to rearrangement of the macromolecules in the outer membranes of the rough s t r a i n s . 108 Figure 16. Indirect immunofluorescent l a b e l l i n g of in t a c t P. aeruginosa tagged with monoclonal antibodies to outer membrane components. Panel A -s t r a i n H103 interacted with protein F - s p e c i f i c monoclonal antibody MA4-4. Panel B-strain H223 interacted with protein F - s p e c i f i c monoclonal antibody MA2-10. Panel C- s t r a i n H103 interacted with LPS O-antigen s p e c i f i c monoclonal antibody MA1-8. Panel D-rough, LPS d e f i c i e n t mutant s t r a i n H223 interacted with l i p o p r o t e i n H2 s p e c i f i c monoclonal antibody MAl-6. Panel E - s t r a i n H283 (as protein F- d e f i c i e n t ) interacted with protein F - s p e c i f i c monoclonal antibody MA2-10. (Note: The differences observed in the fluorescence between panel A and panel B were due e n t i r e l y to exposure times in taking the p i c t u r e , and not to differences in the LPS phenotype of the two st r a i n s used). B. C D. Table VIII. Demonstration of c e l l surface l o c a l i z a t i o n of outer membrane components of P_. aeruginosa by immunofluorescence Protein F Protein H2 LPS Components B a c t e r i a l Phenotype MA2-•10 MA4-4 MA4-: LO MA1-6 0 -Antigen Whole outer membrane Str a i n MA1-8 rabbit antisera Hl03 a Wildtype serotype 5 + + + — + + H283 a Protein F d e f i c i e n t - - - - + + H223 a Rough, LPS d e f i c i e n t + + + ' + - ND ATCC 33352 Serotype 5 + + ND - + + ATCC 33364 Serotype 17 + + ND - + + ATCC 33348 Serotype 1 + + ND — — + C F . i s o l a t e s CF 221° rough + + ND + - + CF C46 mucoid + + ND - - +• non-mucoid revertant + ND - - + CFC47 mucoid + + ND + - + non-mucoid revertant + + ND + + a. st r a i n s were isogenic. +, po s i t i v e fluorescence; -, no fluorescence; c13 other P. aeruginosa c y s t i c f i b r o s i s i s o l a t e s and d9 other mucoid and non-mucoid revertant pairs gave r e s u l t s i d e n t i c a l to CF 221 and CF C47 respectively. ND, not determined. I l l In agreement with the above data, we observed binding of protein F-s p e c i f i c monoclonal antibodies to whole c e l l s of s t r a i n H103 when these c e l l s were used as the antigen in ELISA assays on poly-L-lysine-coated polyvinylchloride plates (Table IX). In these experiments the antibody t i t r e s obtained were s i m i l a r to those obtained f o r s t r a i n H103 outer membranes, although backgrounds tended to be higher with whole c e l l s possibly due to nonspecific binding of the second antibody to the c e l l s . (B) Surface l o c a l i z a t i o n studies by colony immunoblotting A colony b l o t t i n g immunoassay was developed to f a c i l i t a t e the rapid screening of a v a r i e t y of P. aeruginosa s t r a i n s f o r the expression of the s p e c i f i c antigenic determinants recognized by monoclonal antibodies. The method used was a modification of one developed by Henning et a l . (1979) in that enzymatic immunostaining instead of radioimmunostaining was used af t e r the b l o t t i n g step. In these studies, colonies 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 plated onto agar plates and then transferred to n i t r o c e l l u l o s e f i l t e r s by d i r e c t contact. The f i l t e r s were then immunostained using a procedure r e l a t e d to that used for Western blots (see Methods). Monoclonal antibodies MA4-4, MA2-10, MA4-10 and MA5-8 were interacted by the colony b l o t t i n g procedure with the type st r a i n s from a l l 17 serotypes (International Antigen Typing Scheme) of P. aeruginosa, and with a va r i e t y of laboratory s t r a i n s as well as mucoid P. aeruginosa i s o l a t e s , obtained from patients with c y s t i c f i b r o s i s , and t h e i r spontaneous non-mucoid revertants. Although di f f e r e n c e s were observed in the 112 Table IX. Interaction of protein F - s p e c i f i c monoclonal antibodies with p u r i f i e d protein F, outer membranes and whole c e l l s of s t r a i n H103, assessed by ELISA Log^o ELISA t i t r e s against: . a P u r i f i e d Monoclonal antibody Str 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 outer membranes MA2-10 MA4-4 MA4-10 4 2 3 a. ELISA t i t r e s expressed as Log^o values were obtained using a s c i t e s f l u i d as f i r s t antibody. Whole c e l l ELISAs were performed as described in Methods. b. Protein F was derived from s t r a i n H103. 113 i n t e n s i t y of the blue colour developed with p o s i t i v e (antibody-binding) colonies ( F i g . 17), a l l P. aeruginosa s t r a i n s tested interacted with the monoclonal antibodies (Table X). In contrast, a P. aeruginosa s t r a i n H283 that lacks protein F (Nicas and Hancock, 1983) and CF283, a non-P. aeruginosa gram-negative i s o l a t e from a c y s t i c f i b r o s i s patient, did not bind any of the protein F - s p e c i f i c monoclonal antibodies. The s p e c i f i c i t y of the colony b l o t t i n g procedure was demonstrated using colonies of the serotype s t r a i n s of P. aeruginosa and the c y s t i c f i b r o s i s i s o l a t e s , by showing that monoclonal antibody 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 binding to serotypes 5, 17 and CFC1 mucoid and non-mucoid, but showed no i n t e r a c t i o n with any of the other colonies (Table X, F i g . 18). Thus, the colony blot r e s u l t s r e f l e c t e d the s p e c i f i c i t y of antibody MA1-8 as shown above by ELISA (Table V). In addition, monoclonal antibody MA1-6, which i s s p e c i f i c f o r outer membrane l i p o p r o t e i n H2, interacted only with colonies of rough LPS-alt e r e d mutants of P. aeruginosa and not with smooth s t r a i n s (Table X, F i g . 19). This confirmed the r e s u l t s of i n d i r e c t immunofluorescence studies (Table VIII) suggesting that protein H2 i s not expressed on the surface of wild type (smooth) P. aeruginosa s t r a i n s . Mucoid colonies of P. aeruginosa and t h e i r non-mucoid revertants did not shown any differences in t h e i r i n t e r a c t i o n with the antibodies by the colony blot procedure (Figs. 17,18,19) despite the f a c t that the mucoid material (alginate exopolysaccharide) appeared to have been transferred to the n i t r o c e l l u l o s e blot together with the b a c t e r i a l c e l l s . The mucoid material did not apparently mask the a c c e s s i b i l i t y of the protein to the Table X. Binding of monoclonal antibodies directed against P.. aeruginosa outer membrane antigens to colony b l o t s 3 • B a c t e r i a l Protein F Protein H2 LPS O-Antigen Strains MA2-10 MA4-4 MA5-8 MA1-6 MA1-8 P. aeruginosa strains H103 + + + - + H283 (protein F d e f i c i e n t ) - - - - + Serotype 5 + + + - + Serotype l ^ a ) + + + -Z61 + + + - + LPS-rough s t r a i n s ^ ) + + + + -Cystic F i b r o s i s isolates CF 221^ c^ ' +. + + + CFC46 mucoid + + + -non-mucoid revertant + + + - -Other Pseudomonads Group l ^ d ' ) Pseudomonads P. putida (2 strains) + + - + -P. fluorescens (2 strains) - - - + P. syringae + + - + -P. chlororaphis - - - + -P_. aureofaciens - - - + -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 ) - - - - , -Other gram-negative (9 s t r a i n s ) 115 Table X Legend + p o s i t i v e colour development on colony, - no colour development on colony. a A l l other serotype s p e c i f i c s t r a i n s were as described in materials and methods. b P. aeruginosa LPS 0 antigen d e f i c i e n t strains were AK1012, AK1282 and H223. c The other P. aeruginosa i s o l a t e s from c y s t i c f i b r o s i s patients were (CF832, CF1452, CF2314, CF4522, CF3660-1, CF4349, CFL, CF6094, CF1278, CF284, CF3790, CF9490, and 9 mucoid str 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 revertants obtained from D.P. Speert, Children's Hospital, 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 with any of the monoclonal antibodies. This s t r a i n was shown to not be a P. aeruginosa s t r a i n by f a t t y acid analysis (L. Chan). d The rRNA homology grouping by DeVos and DeLey (1983) divides Pseudomonads into 4 groups with A. v i n e l a n d i i in group 1. P. putida s t r a i n s were ATCC 4359 and ATCC 12633; P. fluorescens s t r a i n s were ATCC 949 and 13525. e The other strains were P. acidovorans, P. solanacearum, P. cepacia, P. pseudomallei and P. m a l t o p h i l i a . *• The gram-negative s t r a i n s were Salmonella typhimurium LT2 SGSC205 and SGSC227; V i b r i o anguillarum HT 7602 Aeromonas hydrophila ET2; Aeromonas  salmonicida NCMB 2020; Escherichia c o l i CGSC 6041, CGSC 6044, PC 0479, and Edwardsiella tarda ET 9054. 116 Figure 17. Colony immunoblots showing i n t e r a c t i o n of monoclonal antibody MA4-10, s p e c i f i c for protein F of P. aeruginosa with the following s t r a i n s : 1 - P. aeruginosa s t r a i n PI non-mucoid; 2 - P. aeruginosa s t r a i n PI mucoid; 3,4 - P. fluorescens ATCC 949 and 13525 (type st r a i n ) respectively; 5 - P. aeruginosa PA01 s t r a i n H103; 6 - P. aeruginosa s t r a i n CFC1 mucoid; 7 - P. aeruginosa s t r a i n CFC1 non-mucoid; 8,9 and 10 -Escherichia c o l i strains CGSC6041, CGSC6044 and PC0479; 11 - P. pseudomallei ATCC23343; 12 - P. solanacearum ATCC 11696; 13,14 - P. putida 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 - Salmonella  typhimurium LT2 strains SGSC206 and SGSC227 r e s p e c t i v e l y ; 17 - P. aeruginosa ATCC 8689; 18 - P. aeruginosa PA01 s t r a i n H103; 19 - Aeromonas  salmonicida NCMB 2020; 20 - Aeromonas hydrophila ET2; 21 - P. chlororaphis ATCC 9446; 22 - P. aeruginosa (type strain) ATCC 19305; 23 - P. aeruginosa s t r a i n Z61; 24 - P. aureofaciens ATCC 13985; 25 - P. syringae ATCC 19310; 26 - P. aeruginosa 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. aeruginosa s t r a i n s AK1012 and AK1282. 117 118 Figure 18. Colony immunoblot showing i n t e r a c t i o n of monoclonal antibody MA1-8 s p e c i f i c for serotype 5-0 antigen of P. aeruginosa with the following serotype s t r a i n s . 1-P. aeruginosa serotype 1 s t r a i n ; 2 - P. aeruginosa strains H103; 3 - P. aeruginosa serotype 2 s t r a i n s ; 4 - serotype 3; 5 - serotype 4; 6 - serotype 5; 7 - serotype 6; 8 - serotype 7; 9 - serotype 8; 10 - serotype 9; 11 - serotype 10; 12 - serotype 11; 13 - serotype 12; 14 - serotype 13; 15 - serotype 15; 16 - serotype 16; 17 - serotype 17; 18 - P. aeruginosa s t r a i n H188; 19 -P. aeruginosa CFPlnm; 20 - P. aeruginosa serotype 14; 21 - P. aeruginosa s t r a i n H223 (LPS rough); 22 - P. aeruginosa s t r a i n H235 (LPS rough); 23 - P. aeruginosa CFC91m; 24 - P. aeruginosa CFClm; 25 - P. aeruginosa CFClm; 26, 27 - P. aeruginosa CFC96m and CFC96nm. 120 Figure 19. Colony immunoblots showing i n t e r a c t i o n of monoclonal antibody MAl-6, s p e c i f i c for protein H2 of P. aeruginosa with the following s t r a i n s : 1 - P . aeruginosa s t r a i n PI non-mucoid; 2 - P. aeruginosa s t r a i n PI mucoid; 3,4 - P. fluorescens ATCC 949 and 13525 (type st r a i n ) respectively; 5 - P. aeruginosa PA01 s t r a i n H103; 6 - P. aeruginosa s t r a i n CFC1 mucoid; 7 - P. aeruginosa s t r a i n CFC1 non-mucoid; 8,9 and 10 -Escherichia c o l i strains CGSC6041, CGSC6044 and PC0479; 11 - P. pseudomallei ATCC23343; 12 - P. solanacearum ATCC 11696; 13,14 - P. putida ATCC 4359 and 12633 (type st r a i n ) r e s p e c t i v e l y ; 15,16 - Salmonella  typhimurium LT2 strains SGSC206 and SGSC227 r e s p e c t i v e l y ; 17 - P. aeruginosa ATCC 8689; 18 - P. aeruginosa PA01 s t r a i n H103; 19 - Aeromonas  salmonicida NCMB 2020; 20 - Aeromonas hydrophila ET2; 21 - P. chlororaphis ATCC 9446; 22 - P. aeruginosa (type st r a i n ) ATCC 19305; 23 - P. aeruginosa s t r a i n H188; 24 - P. aureofaciens ATCC 13985; 25 - P. syringae ATCC 19310; 26 - P. aeruginosa 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. aeruginosa s t r a i n H223; 30 and 31 -P. aeruginosa strains AK1012 and AK1282. 121 11 12 13 18 24 * 1 3 4 5 8 9 10 -» f t I »-» 16 14 15 » f / 20 21 22 23 / • 26 27 * / 29 30 122 antibody nor did i t adsorb antibodies n o n - s p e c i f i c a l l y to the surface of the colony. The lack of non-specific antibody binding to mucoid material was demonstrated by the lack 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 protein H2) with colonies of a mucoid d e r i v a t i v e of s t r a i n CFC46, whose outer membranes were previously demonstrated to be d e f i c i e n t in protein H2 by both SDS-polyacrylamide gel electrophoresis ( F i g . 2) and Western immunoblot ( F i g . 11) a n a l y s i s . Antibody MAl-6 interacted with a l l colonies expressing protein H2 (as judged by Western b l o t s ) , a f t e r treatment of colonies with 0.1% (wt/vol) SDS or 0.1 M NaCl to cause membrane disr u p t i o n . The a c c e s s i b i l i t y of LPS was also found to be unaffected by the mucoid exopolysaccharide when the LPS O-antigen-specific monoclonal antibody MA1-8 was used in t h i s study. Both the mucoid and non-mucoid derivatives of s t r a i n CFC1, which expressed serotype 5 0-antigen interacted with MA1-8 on colony blots ( Fig. 18, Table X). (C) Interaction of monoclonal antibodies with other b a c t e r i a by the  colony immunoblot procedure The i n t e r a c t i o n of protein F - s p e c i f i c monoclonal antibodies with other Pseudomonas st r a i n s and various gram-negative b a c t e r i a was studied by the colony blot procedure described above (Table X, F i g . 17,19). Monoclonal antibody MA5-8 was highly s p e c i f i c f o r P. aeruginosa s t r a i n s , while MA4-4, MA2-10, and MA4-10 interacted with both s t r a i n s P. putida and the single P. syringae s t r a i n tested ( F i g . 17; Table X). None of these monoclonal antibodies interacted with whole c e l l s of P. fluorescens, 123 P. a n g u i l l i s e p t i c a , P. chlororaphis. P. aureofaciens. P. s t u t z e r i or A. v i n e l a n d i i . a l l of which had been previously demonstrated to be re l a t e d to P. aeruginosa by rRNA homology experiments (DeVos and DeLey, 1983). In contrast, the l i p o p r o t e i n H2-specific monoclonal antibody MAl-6 interacted with a l l group 1 Pseudomonads expressing a protein equivalent to li p o p r o t e i n H2 ( F i g . 19). There was no i n t e r a c t i o n of MAl-6 with any of the strains from the Enterobacteriaceae, Vibronaceae, Aeromonas st r a i n s or other Pseudomonads in rRNA homology groups 2, 3 and 4 (DeVos and DeLey, 1981) (Table X). 2. Studies on protein F epitopes recognized by monoclonal antibodies. (A) E f f e c t of 2-mercaptoethanol on binding of protein F - s p e c i f i c  monoclonal antibodies The high s p e c i f i c i t y of monoclonal antibodies has been used to study the structure and antigenic domains of proteins (Kenimer §_t a l . , 1983; V i r j i e_t a l . , 1983). We used the protein F-specif i c monoclonal antibodies MA5-8, MA4-4, MA2-10 and MA4-10, in an attempt to define some of the antigenic domains (epitopes) of t h i s protein. The apparent molecular weight, i.e the elec t r o p h o r e t i c m o b i l i t y of protein F of P. aeruginosa on SDS-polyacrylamide gels, i s markedly influenced by the s o l u b i l i z a t i o n conditions (Hancock and Carey, 1979; see also Figure 1). When s o l u b i l i z e d i n the presence of 2-mercaptoethanol, protein F migrates as a 41,000 dalton protein on a 14% polyacrylamide ge l . The non-reduced protein has an apparent molecular weight of 37,000 daltons (Hancock and Carey, 1979). The reason f o r t h i s i s almost 124 Figure 20. Western immunoblot of p u r i f i e d protein F - e f f e c t of 2-mercaptoethanol. P u r i f i e d protein F was separated on SDS polyacrylamide gel electrophoretograms a f t e r s o l u b i l i z a t i o n with (lanes A and D) or without (lanes B, C, and E) 2-mercaptoethanol. A f t e r e l e c t r o p h o r e t i c transfer to n i t r o c e l l u l o s e , the blots were interacted with MA4-4 (lanes A and B) or MA5-8 (lanes C, D and E). Lane E received 5 times more protein F (5 yg) than Lanes A - D, in order to demonstrate the oligomer bands not v i s i b l e in Lanes A - D. 125 126 c e r t a i n l y the presence of one or two intrachain disulphide bonds. Thus, when these disulphide bonds are not reduced, the protein runs in a more compact configuration with a higher r e l a t i v e m o b i l i t y . Monoclonal antibodies MA4-4, MA2-10 and MA4-10 interacted only with the non-reduced form of protein F ( F i g . 20, lane B), while MA5-8 interacted with both the 2-mercaptoethanol-reduced and non-reduced forms (Fig. 20, lanes C and D). Monoclonal antibody MA5-8 was also unique in that i t interacted with higher molecular weight or oligomeric forms of protein F (as seen when s u f f i c i e n t protein F was added to the g e l ; F i g . 20, lane E). These forms of protein F were not observed when outer membranes of the protein F d e f i c i e n t mutant H283 were used in Western immunoblot studies ( F i g . 9), suggesting that they d i d not represent a r t e f a c t s due to cross-reaction of monoclonal antibody MA5-8 with other proteins. The oligomeric associations of protein F were present in very low concentrations in outer membranes but were apparently somewhat enriched during p u r i f i c a t i o n of protein F. These data provided evidence for the existence of SDS-stable oligomeric forms of protein F. (B) Interaction of monoclonal antibodies with cyanogen bromide fragments  of protein F. Peptide fragments of protein F were derived by chemical cleavage with cyanogen bromide. Cyanogen bromide treatment of native or denatured protein F yielded six fragments, two of which had altered m o b i l i t y in the presence of 2-mercaptoethanpl suggesting they contained one or more of the intrachain disulphide bonds of protein F ( F i g . 21). The t o t a l molecular 127 Figure 21. SDS-polyacrylamide gel electrophoretogram and Western immunoblot of p u r i f i e d protein F before and a f t e r degradation with cyanogen bromide. Lane A,C: cyanogen bromide peptides of protein F; Lane B,D: p u r i f i e d protein F. Protein F and i t s peptides were tra n s f e r r e d from an SDS-polyacrylamide gel (lanes A and B) to n i t r o c e l l u l o s e (lanes C and D) and interacted with monoclonal antibody MA5-8 on the n i t r o c e l l u l o s e . The two cyanogen bromide peptides of protein F which inte r a c t with antibody MA5-8 had molecular weights of 28,000 and 23,000. In the cyanogen bromide cleavage pattern in lane C, the native protein F formed a doublet due to p a r t i a l heat modification of the undegraded protein F (Hancock and Carey, 1979). This small residue of undegraded protein F p r e f e r e n t i a l l y bound the monoclonal antibody, since i t was not v i s i b l e in the gel pattern i n lane A. 128 A B C D 129 weight of the six peptides, however, was approximately three times that of monomeric protein F. The discrepancy in molecular weight was probably due to incomplete cleavage. This has been shown to occur e.g. due to incomplete cleavage of Met-Ser or Met-Thr sequences, which can form homoserine and are therefore not cleaved, leading to the production of overlapping cyanogen bromide fragments of t o t a l molecular weights greater than the native protein (Garten et a l . , 1975). This incomplete cleavage was also observed with denatured protein, and under more rigorous digestion conditions. Only monoclonal antibody MA5-8 interacted with any of the fragments. S p e c i f i c a l l y , i t interacted with the two 2-mercaptoethanol-modifiable fragments of protein F in both the reduced and non-reduced forms ( F i g . 21). The i n a b i l i t y to generate smaller antibody reactive peptides under harsher conditions (pre-treatment with 85% g l a c i a l a c e tic acid) probably 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 of the antigenic epitope by maintenance of the SDS-resistant, B-sheet conformation of protein F (Mizuno and Kageyama, 1979), thus l i m i t i n g the i d e n t i f i c a t i o n of the antigenic domains. (C) Interaction of monoclonal antibodies with p r o t e o l y t i c peptides of  protein F Enzymatic d i g e s t i o n of protein F with S. aureus V8 protease f o r very short times (< 1 min) yi e l d e d a series of peptides of molecular weights below 12,000 daltons, none of which interacted with any of the antibodies. When p u r i f i e d protein F was treated with papain, i t r a p i d l y broke down to two peptide fragments with molecular weights of 29,000 and 130 31,500 (Fig. 22, lane H). Both of these peptides were 2-mercaptoethanol modifiable and reacted with monoclonal antibodies MA2-10, MA4-4 and MA4-10 (i n the unreduced form only) (Fig. 22, lane H) but not with MA5-8 (Fig. 22 lane D). Protein F in outer membranes ( F i g . 22, lanes I-M) and intact c e l l s (Fig. 22, lanes N and 0) was also susceptible to papain, although f i v e - f o l d higher amounts of enzyme were required and d i g e s t i o n did not proceed as r e a d i l y to completion ( e s p e c i a l l y with whole c e l l s ) . This may be due to p a r t i a l masking of the papain-susceptible s i t e s of protein F by LPS. In both outer membranes and i n t a c t c e l l s , peptide bands ( F i g . 22, lanes L and N) of intermediate molecular weight between the undegraded protein F (37 kD) and the above two peptide fragments (29 kD and 31.5 kD) were observed, including bands of 36, 35, 34, 33 and 32.5 kD. However, due to the tendency of protein F to run in a v a r i e t y of positions on SDS polyacrylamide gels (Hancock and Carey, 1979) i t is d i f f i c u l t to say with c e r t a i n t y that these bands represent intermediates. Digestion of protein F with t r y p s i n gave s i m i l a r r e s u l t s . When p u r i f i e d protein F was used, a single 2-mercaptoethanol-modifiable peptide fragment of 31 kD formed. This fragment interacted with monoclonal antibodies MA2-10, MA4-4 and MA4-10 in the unreduced form ( F i g . 22, lane F)] but not with MA5-8 (F i g . 22, lane K). Again, protein F in outer membranes and whole c e l l s was 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 ten times as much enzyme, and r e s u l t i n g in the appearance of intermediate molecular weight forms of 34.5, 33.5 and 32.5 kD (Fig. 22, lanes M and 0) a l l of which interacted with MA2-10, MA4-4 and MA4-10. The 31 kD fragment which appeared in p u r i f i e d protein F, outer membrane and i n t a c t c e l l 131 Figure 22. Western immunoblot of native and p r o t e o l y t i c a l l y treated protein F from a p u r i f i e d protein F sample (lanes A - H), outer membranes (lanes I - M) and whole c e l l s lanes (N - 0). The p r o t e o l y t i c enzymes and the monoclonal antibodies used to reveal the positions of the native protein F and i t s p r o t e o l y t i c fragments were lane A - no treatment, antibody MA5-8; lane B - t r y p s i n , MA5-8; lane C - S. aureus V8 protease, MA5-8; lane D - papain, MA5-8; lane E - no treatment, MA4-10, lane F -try p s i n ; MA4-10; lane G - V8 protease, MA4-10, lane H - papain, MA4-10; lane I - no treatment, MA5-8; lane J - papain, MA5-8; lane K - t r y p s i n , MA5-8; lane L - papain, MA4-10; lane M - t r y p s i n , MA4-10; lane N -Trypsin, MA4-10; lane 0 - papain, MA4-10. 132 133 Figure 23. SDS-polyacrylamide gel electrophoretogram of peptides derived from a time course prot e o l y s i s of p u r i f i e d protein F with the enzyme tr y p s i n . The samples were s o l u b i l i z e d at 88°C f or 10 min in reduction mix without 2-mercaptoethanol. Lane 1, molecular weight standards; Lane 2, p u r i f i e d protein F ( c o n t r o l ) ; Lanes 3 and 9 show t r y p t i c peptides of protein F a f t e r 30 min., 60 min., 90 min., 2 hr., 2.5 hr., 3 hr., and 3.5 hr., p r o t e o l y s i s with t r y p s i n r e s p e c t i v e l y . 134 135 Figure 24. Interaction of protein F - s p e c i f i c monoclonal antibody MA2-10 with p r o t e o l y t i c a l l y treated whole c e l l s of P. aeruginosa CFC46nm and P. putida 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. aeruginosa CFC46nm, no treatment; Lane 2, CFC46nm, try p s i n ; Lane 3, CFC46nm, papain; Lane 4, Protein F c o n t r o l ; Lane 5, P. putida, no treatment; Lane 6, P. putida, t r y p s i n ; Lane 7, P. putida. papain. 136 137 preparations a f t e r treatment with tr y p s i n was r e s i s t a n t to further proteolysis ( F i g . 23). Experiments with the protein F equivalent in P. putida whole c e l l s demonstrated that t h i s 39 kD protein broke down to a 31 kD, 2-mercaptoethanol-modifiable t r y p t i c fragment which interacted with monoclonal antibody MA2-10 (Fig. 24, lane 6). This P. putida protein was also susceptible to papain and a peptide of approximately 29 kD was observed (lane 7). Digestion of whole c e l l s of P. aeruginosa CFC46nm with try p s i n and papain re s u l t e d in the appearance of a family of peptides (Fig. 24, lanes 2 and 3) of molecular weights s i m i l a r to those observed with whole c e l l digests of P. aeruginosa s t r a i n H103 ( F i g . 22, lane N, 0). These peptides interacted with antibody MA2-10. 3. Summary The use of monoclonal antibodies in the colony b l o t analysis enabled rapid screening of P. aeruginosa s t r a i n s f o r the surface exposure of single antigens l i k e porin protein F. The r e s u l t s obtained co r r e l a t e d well with those of the immunofluorescence s t a i n i n g a n a l y s i s . This method may well prove to be a powerful t o o l for screening f o r variants and mutants of outer membrane components in genetic studies. Studies on the cross r e a c t i v i t y of monoclonal antibodies against outer membrane protein F of Pseudomonas aeruginosa demonstrated that these monoclonal antibodies were of two d i s t i n c t s p e c i f i c i t i e s . One antibody, MA5-8, interacted only with P. aeruginosa s t r a i n s whereas three other monoclonal antibodies, MA2-10, MA4-4 and MA4-10, cross-reacted with P. syringae and P. putida s t r a i n s . These two classes of antibodies were distinguishable by r e a c t i v i t y with 2-mercaptoethanol-treated protein F 138 (Fig. 20), with protein F oligomers ( F i g . 20), with cyanogen bromide fragments of protein F (Fig. 21), and with p r o t e o l y t i c fragments of protein F ( F i g . 22). Both classes of antibodies recognized a surface epitope as judged by i n d i r e c t immunofluorescent l a b e l l i n g (Table VIII, F i g . 16) and colony immunoblotting (Table X) of i n t a c t P. aeruginosa c e l l s . The data favour the existence of two separate highly conserved surface epitopes on outer membrane protein F in P. aeruginosa. These studies also demonstrated the presence of a conserved antigenic epitope 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). By both i n d i r e c t immunofluorescence and colony immunoblotting using antibody MA1-6, protein H2 was shown to be surface accessible only in rough LPS-altered organisms. 139 DISCUSSION The gram-negative b a c t e r i a l c e l l surface (outer membrane) contains the components, namely proteins and LPS, that are involved in the primary inte r a c t i o n of these b a c t e r i a with the host's immune system. Both classes of molecules were studied in t h i s thesis and w i l l be discussed separately below. 1. Conservation and heterogeneity of LPS epitopes Extensive studies have c l e a r l y shown a v a r i e t y of p o t e n t i a l roles f or LPS in the pathogenesis of gram-negative ba c t e r i a (Bradley 1979). In t h i s study monoclonal antibodies were used to probe the antigenic conservation of d i f f e r e n t regions of the LPS molecule: the immunodominant O-antigenic side chains, the rough oligosaccharide core and the l i p i d A portion (see Table XI for a summary). A monoclonal antibody MA1-8, which reacted with the O-antigenic region of the LPS of serotype 5 s t r a i n s , showed weak cross-reaction with the outer membranes of a serotype 17 s t r a i n , but no reaction with s t r a i n s from 15 other serotypes (Table V) . MA1-8 may well be d i r e c t e d against LPS antigen 2d which i s apparently shared by serotype 5 and 17 (Lanyi and Bergan, 1979). The high s p e c i f i c i t y of t h i s monoclonal antibody agrees with the conclusion of numerous other reports that v a r i a t i o n in the LPS O-antigen composition i s responsible f o r serotyping differences (see e.g. Lanyi and Bergan, 1979). The s p e c i f i c i t y of MA1-8 for the O-antigen was Table X I . Reaction of LPS s p e c i f i c monoclonal antibodies from Pseudombnas aeruginosa s tra ins in ELISA with outer assays membrane Reaction using monoclonal ant ibodies to LPS 0 Antigen Outer Membranes type 5 Rough Core L i p i d A (MA1-8) (MA3-5) (MA3-6) (5E4) P. aeruginosa PAOI + + + + P. aeruginosa rough - + + + P. aeruginosa serotype 5 + + + + P. aeruginosa serotype 17 - - + P. aeruginosa serotypes .7,8,10,14,16 - + + + P. aeruginosa serotype 11 - + + P. aeruginosa serotypes 1 ,2 ,3 ,4 ,6 ,9 , 12,13,15 - - + P. aeruginosa c l i n i c a l s t ra ins +/- (2/34) b + / - ( l / 2 0 ) b +/ - (3 /20) b +(20/20) b aweaker reac t ion due to a shared determinant between serotype 17 and serotype 5. p o s i t i v e react ions out of the number of s t ra ins tested. 141 shown by the lack of inter a c t i o n of the antibody with rough (O-antigen d e f i c i e n t ) derivatives of a serotype 5 s t r a i n (Table IV). Monoclonal antibodies s p e c i f i c f o r rough LPS (MA3-5 and MA3-6) reacted with a l i m i t e d subset of the P. aeruginosa serotype strains and c y s t i c f i b r o s i s P. aeruginosa i s o l a t e s ( F i g . 13, Table XI). These re s u l t s demonstrated, f o r the f i r s t time, LPS rough core heterogeneity among P. aeruginosa s t r a i n s . In contrast s t r u c t u r a l studies on the hexose region of the LPS-rough core of 600 Salmonella species suggested a single type of core (Jahnsson et a l . , 1981). However, more d e t a i l e d studies using s p e c i f i c monoclonal antibodies would be necessary to conclusively show the conservation of the single antigenic structure of t h i s region of the LPS in Salmonella. Despite the observed heterogeneity, the data suggested that in P. aeruginosa the rough core was a n t i g e n i c a l l y more conserved than the O-antigen. The monoclonal antibodies to l i p i d A interacted with a l l of the str a i n s of P. aeruginosa tested, as well as many other genera of many gram-negative bacteria (Figs. 14 and 15, Tables VI and XII). These r e s u l t s demonstrated that the LPS-Lipid A region i s highly conserved among gram-negative bac t e r i a . This may explain the s i m i l a r a c t i v i t i e s of L i p i d A's (endotoxin) from d i f f e r e n t b a c t e r i a (Bradley, 1979). Thus, i t would appear that LPS heterogeneity increases f o r substituents that are further from the outer membrane i . e . LPS components clo s e s t to the outer membrane surface are gr e a t l y conserved (Table XI). The d i s t a l portions of the LPS in b a c t e r i a , e s p e c i a l l y the O-antigen , are 142 usually s t r a i n or species s p e c i f i c (Table XI, see also Goldman and Leive, 1980; Bradley, 1979). The extensive antigenic c r o s s - r e a c t i v i t y of the l i p i d A moieties of many gram-negative bacteria would suggest a common evolutionary o r i g i n of the l i p i d A moiety that has been conserved over time. The reason for t h i s strong conservation i s unclear but, to date, no l i p i d A - d e f i c i e n t mutant of a gram-negative bacteria has been i s o l a t e d suggesting that l i p i d A may be an e s s e n t i a l component of these c e l l s . The antigenic cross-reactions of l i p i d A from a v a r i e t y of gram-negative b a c t e r i a also provides a possible explanation for the cross-protective nature of E. c o l i J5 LPS or whole c e l l vaccines (Braude et a l . . 1978; Pennington and Menkes, 1981) as well as the a b i l i t y of E. c o l i J5 antiserum and antiserum to the l i p i d A-KDO (ReLPS) of S. minnesota to passively protect mice against Pseudomonas bacteremia (Braude et a l . , 1978; Young et a l . , 1975). (A) LPS of P. aeruginosa i s o l a t e s from c y s t i c f i b r o s i s patients Sixty percent or more of P. aeruginosa s t r a i n s from c y s t i c f i b r o s i s patients demonstrate one of two phenotypes, p o l y a g g l u t i n a b i l i t y or n o n - a g g l u t i n a b l i l i t y and are thus e s s e n t i a l l y non-typable by the conventional O-antigen based antisera ( Z i e r d t and Williams 1975). In t h i s study i t was demonstrated that these non-typable st r a i n s were also highly serum s e n s i t i v e when compared to the serotypable st r a i n s (Table I ) . In most cases the strains were s e n s i t i v e to l e s s than 2% (vol/vol) normal human or mouse serum. These c h a r a c t e r i s t i c s i . e . non-typability and serum 143 s e n s i t i v i t y were correlated to def i c i e n c y in LPS O-antigenic side chains (Hancock et a l . 1983) but not to presence or absence of the mucoid exopolysaccharide (Table 1, F i g . 3). Those observations have several implications. F i r s t l y , they c a l l into question the broad a p p l i c a t i o n of O-antigen-based antisera for serotyping P. aeruginosa, e s p e c i a l l y f o r is o l a t e s from the lungs of patients with c y s t i c f i b r o s i s . Secondly, they tend to suggest that immunotherapy using LPS-based vaccines w i l l not be successful in some disease sit u a t i o n s (Hanessian et a l . , 1971; Mi l e r et a l . 1977; Woods et 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 new property of P. aeruginosa associated with c y s t i c f i b r o s i s . The property of serum s e n s i t i v i t y which seems to be associated with the LPS phenotype may well explain why patients with c y s t i c f i b r o s i s r a r e l y s u f f e r from P. aeruginosa bacteremia. 2. Antigenic conservation of P. aeruginosa outer membrane proteins The r o l e of outer membrane proteins i n host-pathogen i n t e r a c t i o n has not been as extensively studied as that of LPS. In the present study, the conservation and immunogenic properties of s p e c i f i c outer membrane proteins of P. aeruginosa were examined. This study provided three l i n e s of evidence that outer membrane proteins are highly conserved in P. aeruginosa. Amongst the type st r a i n s of the 17 serotypes of the International Antigen Typing Scheme and a var i e t y of c l i n i c a l i s o l a t e s , I observed s i m i l a r outer membrane protein patterns (Figs. 1 and 2), antigenic cross-reaction using po l y c l o n a l 144 antisera to the major outer membrane proteins (Figs. 4 and 5, Table I I ) , and the conservation of s p e c i f i c antigenic epitopes on outer membrane proteins F and H2 (Figs. 7, 8 and 10; Tables V, VI and VI I ) . (A) Conservation of outer membrane protein patterns , Other gram-negative bacteria, unlike P. aeruginosa, do not show such high s i m i l a r i t y i n t h e i r outer membrane protein p r o f i l e s . For example, a va r i e t y of outer membrane protein patterns have been observed f o r Haemophilus influenzae type b (eight to nine subtypes based on outer membrane protein patterns; Barenkamp et a l . , 1981), N. gonorrhoeae (nine subtypes; Buchanan and Hildebrandt, 1981), N. meningitidis (15 subtypes; Tsai et a l . , 1981), E. c o l i (36 subtypes; Overbeeke and Lugtenberg, 1980), V i b r i o cholerae (two subtypes; Kabir, 1980) and V i b r i o anguillarum (three subtypes; Nakajima et a l . , 1983). Indeed i n N. gonorrhoeae (Buchanan and Hildebrandt, 1981) and in H. influenzae type b (Barenkamp et a l . 1981) outer membrane protein patterns and a n t i s e r a to other membrane proteins 1 have been proposed as the basis for subtyping these organisms. Therefore, i t i s highly s i g n i f i c a n t that a l l P. aeruginosa st r a i n s studied (including 17 d i f f e r e n t serotype s t r a i n s , and 34 d i f f e r e n t i s o l a t e s from c y s t i c f i b r o s i s patients, F i g . 1 and 2) have highly s i m i l a r outer membrane protein patterns. Two observations i n l i t e r a t u r e are consistent with these r e s u l t s . Sadoff and Artenstein (1974) showed s i m i l a r patterns in the "native complex" of the seven Fisher immunotypes, whereas Mizuno and Kageyama (1978b) made s i m i l a r observations regarding the outer membrane proteins of f i v e d i f f e r e n t P. aeruginosa s t r a i n s . 145 Conservation of outer membrane protein patterns has also been reported for the e e l pathogen P. a n g u i l l i s e p t i c a (Nakaj ima e_t a l . , 1983). (B) Antigenic conservation revealed using p o l y c l o n a l antiserum Antigenic r e l a t i o n s h i p s between the outer membrane proteins of various P. aeruginosa strains were demonstrated by the c r o s s - r e a c t i v i t y in ELISA of antiserum to s t r a i n PAOI outer membranes with the outer membranes of serotyping s t r a i n s (Table I I ) . On Western b l o t s , the broad r e a c t i v i t y of the polyclonal antibodies to i n d i v i d u a l proteins from d i f f e r e n t strains suggested that each of proteins F, H2 and I possessed common antigenic determinants in a l l P. aeruginosa outer membranes tested; and that protein E was also c l o s e l y r e l a t e d in 16 of the 17 serotype s t r a i n s (Figs. 4, 5). In contrast, the antigenic heterogeneity of f l a g e l l i n among P. aeruginosa str a i n s ( P i t t , 1980) was confirmed by the d i f f e r e n t i a l antibody binding to f l a g e l l i n in only 7 (4 weakly) of the 17 serotype s t r a i n s . Despite the s i m i l a r i t y of outer membrane protein patterns, not a l l serotype s t r a i n s showed i d e n t i c a l antibody binding to the outer membrane proteins ( F i g . 5). There are at l e a s t two possible explanations for t h i s observation. I n d i v i d u a l antigenic s i t e s on given outer membrane proteins could have d i f f e r i n g a f f i n i t i e s for the t e s t antibody or the amounts of the given protein in the various outer membranes could d i f f e r . Thus the observed small amounts of protein H2 in the s t r a i n s of serotypes 5 and 7 (F i g . 1) c o r r e l a t e d with the small amount of antibody bound to protein H2 of these s t r a i n s ( F i g . 5). S i m i l a r l y , the serotype 7 s t r a i n had small amounts of l i p o p r o t e i n I and very l i t t l e antibody was bound to l i p o p r o t e i n 146 I from t h i s s t r a i n . However, no s i m i l a r c o r r e l a t i o n could be made for protein E. The d i f f e r e n t amounts of antibody to protein E bound to these str a i n s might be r e l a t e d to antibody-antigen a f f i n i t y . There i s evidence in the l i t e r a t u r e that P. aeruginosa outer membrane proteins are immunogenic in vivo during the course of an i n f e c t i o n . Fernandes et a l . (1981) demonstrated the presence in the sera of patients with c y s t i c f i b r o s i s of immunoprecipitating antibodies to P. aeruginosa c e l l envelope proteins of 37,000 daltons and 58,000 daltons (possibly protein F and f l a g e l l i n r e s p e c t i v e l y ) . Lam et a l . (1983), and Hancock et a l . (1983), demonstrated the presence of antibodies against P. aeruginosa outer membrane proteins F, H2 and I in the sera of 47 patients with c y s t i c f i b r o s i s and 4 patients with acute P. aeruginosa pneumonia or acute bacteremia as well as in rats c h r o n i c a l l y i n f e c t e d with P. aeruginosa. The simplest explanation for t h i s antibody cross-reaction data i s that the outer membrane protein antigens are a n t i g e n i c a l l y conserved in P. aeruginosa s t r a i n s . I t i s therefore possible that the cross protection demonstrated by P. aeruginosa vaccines, such as ribosomal (Lieberman 1978) and o r i g i n a l endotoxic protein (Abe et a l . 1975) vaccines i s due to contamination with outer membrane proteins. Indeed Hancock (unpublished observations) observed that the o r i g i n a l endotoxic protein contained proteins that co-electrophoresed with p u r i f i e d outer membrane proteins and reacted strongly in ELISA with a n t i s e r a to PA01 outer membranes. Therefore, the major components of P. aeruginosa outer membranes, proteins and LPS, are capable of i n t e r a c t i n g with the host's immune mechanisms and e l i c i t i n g antibody responses. However the proteins, unlike 147 LPS, are cross-reactive and appear to be non-toxic to mice in that injections of 5 ug/kg of protein into mice caused no obvious c l i n i c a l symptoms or l e t h a l i t y (E.C.A. Mouat, personal communication). Thus the expression in P. aeruginosa of highly conserved and apparently immunogenic outer membrane proteins suggests that common antigens that can provide immunoprotection against i n f e c t i o n s by the bacterium should e x i s t among these proteins. Such protection has been demonstrated in N. gonorrhoeae (Buchanan et al_. , 1977), i n experimental salmonellosis (Kuusi e_t a l . , 1979) and more recently in P. aeruginosa ( G i l l e l a n d , et a l . , 1984; Hancock and Mouat, submitted for p u b l i c a t i o n ) . (C) Conservation of s p e c i f i c epitopes on P. aeruginosa outer membrane  proteins The c r o s s - r e a c t i v i t y of the monoclonal antibodies with s p e c i f i c proteins in the outer membranes from d i f f e r e n t P. aeruginosa s t r a i n s was investigated as an i n d i c a t o r of the conservation of s p e c i f i c antigenic s i t e s (epitopes) among these s t r a i n s . Monoclonal antibodies s p e c i f i c f o r P. aeruginosa outer membrane porin protein F and l i p o p r o t e i n s H2 and I were used to study the d i s t r i b u t i o n of s p e c i f i c antigenic epitopes on these proteins in the outer membranes from a wide range of P. aeruginosa s t r a i n s . A monoclonal antibody MA1-3 was d i s t i n g u i s h e d from MAl-6 on the basis of lack of i n t e r a c t i o n with 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 I (Table III) and with the outer membranes from other bacteria (Table VI). Antibody MA1-3 reacted i n ELISA with outer membranes from a l l 148 P. aeruginosa st r a i n s tested, including 17 serotype s t r a i n s (Tables VI and VII). However i t d i d not react with denatured antigens eluted from SDS-PAGE gels or with Western blots of P. aeruginosa outer membranes. Therefore the actual nature of the antigenic s i t e against which MAl-3 is directed remains obscure. However, i t i s possible that MAl-3 i s directed against a minor protein in the l i p o p r o t e i n I preparation, or against a complex of proteins H2 and I, which may be present in the p a r t i a l l y p u r i f i e d protein I preparation. The existence of such a complex in vivo has been postulated 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 (Hancock et a l . , 1981) and has also been demonstrated by protein-protein c r o s s - l i n k i n g experiments (Angus and Hancock, 1983). A second monoclonal antibody MAl-6, s p e c i f i c f o r an antigenic s i t e on protein H2 of P. aeruginosa. interacted with outer membranes from the serotyping st r a i n s (Table VII, F i g . 10), with 34 of 36 tested strains from c y s t i c f i b r o s i s patients (Table X, F i g . 11) and with other P. aeruginosa str a i n s (Table VI, F i g . 19). Two P. aeruginosa 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 , did not have protein H2 when the outer membranes of these s t r a i n s were examined by SDS-polyacrylamide gel electrophoresis and f a i l e d to i n t e r a c t with MAl-6 ( F i g . 11). Decreased amounts of protein H2 had been observed in the c e l l envelopes of P. aeruginosa s t r a i n H103 (K. Poole, unpublished observations) when the s t r a i n was grown in serum. However, i t i s not possible to a t t r i b u t e the loss of protein H2 in the s t r a i n s CFC46 mucoid and CFC46 non-mucoid to serum e f f e c t s since the s t r a i n s are serum-sensitive lung i s o l a t e s which were not exposed to serum in vivo. In 149 addition a l l 34 other i s o l a t e s of P. aeruginosa from c y s t i c f i b r o s i s patients contained protein H2. Antigenic c r o s s - r e a c t i v i t y and conservation of s p e c i f i c antigenic s i t e s on protein F was demonstrated among P. aeruginosa s t r a i n s using f i v e monoclonal antibodies (MA5-8, MA4-4, MA4-10, MA2-10 and MA4-2) s p e c i f i c f o r protein F. The s p e c i f i c i t y of these monoclonal antibodies for protein F was demonstrated by the absence of c r o s s - r e a c t i v i t y with a protein F - d e f i c i e n t mutant s t r a i n H283 derived from P. aeruginosa PAOI s t r a i n H103 (Fig. 9), and by t h e i r i n t e r a c t i o n with protein F on Western blots of outer membrane proteins from serotyping 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 of P. aeruginosa (Figs. 7 and 8). We observed differences i n the i n t e r a c t i o n of the protein F - s p e c i f i c monoclonal antibodies with protein F from d i f f e r e n t P. aeruginosa s t r a i n s (Fig. 7). Since protein F was not tra n s f e r r e d q u a n t i t a t i v e l y from gels onto Western b l o t s , i t was not possible to determine the reason for t h i s observation. However, i t may be due to s l i g h t a l t e r a t i o n s of the antigenic s i t e recognized by the monoclonal antibody, giving r i s e to d i f f e r i n g antigen-antibody a f f i n i t i e s . Thus, outer membranes of P. aeruginosa contain three common conserved antigenic s i t e s on protein H2, F and possibly I. These antigenic s i t e s are expressed in a l l P. aeruginosa s t r a i n s tested with the exception of two c y s t i c f i b r o s i s i s o l a t e s that lacked protein H2. (D) D i s t r i b u t i o n of s p e c i f i c epitopes among other species of b a c t e r i a The immunological c r o s s - r e a c t i v i t y of monoclonal antibodies s p e c i f i c f o r P. aeruginosa outer membrane proteins was tested i n ELISA assays and 150 Western blots with outer membranes from a v a r i e t y of strains of the family Pseudomonadaceae and other organisms (Tables VI and XII). Monoclonal antibody MA1-3 interacted weakly with the two P. fluorescens s t r a i n s , P. putida s t r a i n s , P. syringae. P. chlororaphis. P. aureofaciens. P. s t u t z e r i s t r a i n s tested and an Azotobacter v i n e l a n d i i strain', but not with any other antigens (Table V I ) . In contrast monoclonal antibody MA1-6 s p e c i f i c f o r P. aeruginosa l i p o p r o t e i n H2 reacted strongly with the outer membranes of two Pseudomonas fluorescens strains (including the type s t r a i n ATCC 13524), two P. putida st r a i n s (including the type s t r a i n ATCC 12633) and one s t r a i n from each of the following species, 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. chlororaphis. P. syringae, P. s t u t z e r i and P. aureofaciens. There was no c r o s s - r e a c t i v i t y with outer membranes of other Pseudomonas s t r a i n s from the family Pseudomonadaceae or representatives of other Families of ba c t e r i a (Table VI, F i g . 19). In agreement with t h i s data, Mizuno and Kageyama (1978b) had demonstrated that outer membrane pro t e i n H2 can be immunoprecipitated from three fluorescent psuedomonads, P. aeruginosa. P. fluorescens and P. putida, by crude antisera against P. aeruginosa protein H2. The r e s u l t s presented here confirm taxonomic data from rRNA homology, DNA homology and f a t t y acid analysis studies suggesting that A. v i n e l a n d i i i s taxonomically r e l a t e d to the fluorescent pseudomonads, while P. acidovorans and P. ma l t o p h i l i a are taxonomically d i s t i n c t from the fluorescent psuedomonads (Pa l l e r o n i et a l . , 1973; DeVos and DeLey, 1983; Moss et a l . , 1972; see Table XII). Recently P a l l e r o n i et a l . (1973) and DeVos and DeLey (1983) suggested that the family Pseudomonadaceae consisted of a number of T a b l e X I I . P o t e n t i a l taxonomic v a l u e o f mono c l o n a l a n t i b o d i e s rRNA homology c l u s t e r s ' 5 C r o s s - r e a c t i o n s w i t h m o n o c l o n a l a n t i b o d i e s 3 B a c t e r i a l s t r a i n MAl-6 o-H2 MA4-4 a-F MA5-8 a-F MA 1-8 a-OAg 5E4 a - L i p i d A r 3 2 A g r o b a c t e r i u m P. solanacearum P. a c i d o v o r a n s I A. t u m e f a c i e n s (LPS) 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 P. a c i d o v o r a n s ND P. a e r u g i n o s a ^ ) + + + (+/-) + P. p u t i d a + + - - + - P. s y r i n g a e + + - - ND P. f l u o r e s c e n s P. c h l o r o r a p h i s + - - - ND (A. v i n e l a n d i i ) P. a u r e o f a c i e n s + - - - ND P. s t u t z e r i + - - - ND P. f l u o r e s c e n 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 + - - - + Xanthompnas P. m a l t o p h i l i a - - - + E. c o l i E n t e r o b a c t e r i a c e a e S. t y p h i m u r i u m - - - + E. t a r d a V. c h o l e r a e _ - - - + V i b r o n a c e a e V. a n g u i l l a r u m A. s a l m o n i c i d a A. h y d r o p h i l a - - - • + Table XII Legend 152 + denotes a p o s i t i v e reaction; — a negative re a c t i o n ; ND not done. a r e s u l t s from both ELISA and Western b l o t s . D c l a s s i f i c a t i o n by rRNA of DeVos and DeLey (1983). c 1,2,3 and A denote rRNA homology groups of Pseudomonads by DeVos and DeLey (1983). ^ Tm(e)°C - Thermal e l u t i o n temperature of the DNA-rRNA hyb r i d i z a t i o n s performed by DeVos and DeLey (1983). e AO" d i f f e r e n t s t r a i n s of P. aeruginosa including the IATS serotyping 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 laboratory s t r a i n s as described in materials and methods. The p o s i t i v e (+) r e a c t i o n with MA1-8 was only seen with serotype 5 s t r a i n s . 153 d i s t a n t l y related taxonomic groups of b a c t e r i a . A l l of the ba c t e r i a with which MA1-6 reacted were placed in the P. fluorescens group (also c a l l e d group 1 Pseudomonads), and the other Pseudomonas species which f a i l e d to inte r a c t with antibody MA1-6 were found to be taxonomically d i s t i n c t . Thus MA1-6 int e r a c t s s p e c i f i c a l l y with outer membranes of a single taxonomic subdivision, the group 1 or "true" pseudomonads, i n d i c a t i n g a po t e n t i a l role i n taxonomy for t h i s monoclonal antibody. Monoclonal antibodies to porin protein F f e l l into two groups according to t h e i r c r o s s - r e a c t i v i t y with other s t r a i n s . Antibodies MA4-10, MA4-2, MA4-4, and MA2-10 interacted with a protein i n the outer membranes of two P. putida strains and a P. syringae s t r a i n , whereas monoclonal antibody MA5-8 was highly s p e c i f i c f o r P. aeruginosa porin protein F (Fig. 9; Table XII). The r e a c t i v i t y of these monoclonal antibodies suggested that protein F has at l e a s t two d i f f e r e n t conserved antigenic s i t e s defined by these monoclonal antibodies. Protein F i s more poorly conserved among other Pseudomonadaceae species than protein H2, suggesting that greater antigenic d r i f t has occurred for protein F. Nevertheless, protein F i s fa r better conserved than the porin proteins of N. gonorrhoeae (Johnston et a l . , 1976) and H. influenzae (Barenkamp et a l . , 1981) which show antigenic v a r i a t i o n within a single species. 154 3. Surface a c c e s s i b i l i t y studies using monoclonal antibodies The extensive c r o s s - r e a c t i v i t y of monoclonal antibodies s p e c i f i c f o r protein F and l i p o p r o t e i n H2 among P. aeruginosa st r a i n s suggested that these common antigens would be l o g i c a l vaccine candidates. One major requirement for a good vaccinogen i s the a c c e s s i b i l i t y of the antigen on the surface of the i n t a c t organism. The surface l o c a l i z a t i o n of c e r t a i n outer membrane proteins in P. aeruginosa whole c e l l s has been indicated by radioio d i n a t i o n (Lambert and Booth, 1982). However, t h i s method has been shown to l a b e l c e r t a i n proteins that are not in the outer membrane (Su l l i v a n and Williams, 1982; Schindler and Teuber, 1979), suggesting that the procedure disrupts the outer membrane. Su r f a c e - l o c a l i z e d proteins have also been i d e n t i f i e d in other b a c t e r i a by the use of protein s p e c i f i c phages (Braun and Krieger-Brauer, 1977; Datta et a l . , 1977). However, these studies do not reveal i f the surface-accessible portions of the protein are immunogenic. Thus, the preferred technique i s to measure the int e r a c t i o n of tagged s p e c i f i c antibodies with whole organisms, as done e.g. for H. influenzae type b (Hansen et a l . , 1981a), and E. c o l i (Hofstra et a l . , 1979) . In t h i s report a study of the surface antigenic structures of P. aeruginosa was performed using monoclonal antibodies s p e c i f i c f o r outer membrane proteins. By i n d i r e c t immunofluorescence and colony immunoblot procedures, protein F was demonstrated to be surface exposed and accesssible to monoclonal antibodies s p e c i f i c f o r two separate antigenic determinants on the protein (Figs. 16 and 17; Tables VIII and X). These antibodies did not bind to a protein F - d e f i c i e n t P. aeruginosa s t r a i n (Fig. 9). On colony b l o t s , the monoclonal antibodies interacted with a l l P. aeruginosa st r a i n s tested including colonies from smooth, LPS O-antigen-containing and rough O-antigen-deficient s t r a i n s , serum sensitive and r e s i s t a n t s t r a i n s , s t r a i n s of d i f f e r e n t serotypes, and mucoid and non-mucoid i s o l a t e s from patients with c y s t i c f i b r o s i s . Neither LPS 0-side chains nor the mucoid exopolysaccharide apparently masked the surface-exposed antigens. One group of monoclonal antibodies, MA4-4, MA4-10 and MA2-10, also interacted with colonies of P. putida and P. syringae s t r a i n s . Monoclonal antibody MAl-6 d i d not bind to protein H2 in i n t a c t smooth P. aeruginosa c e l l s but showed binding to rough O-antigen d e f i c i e n t mutants (Tables VIII and X). This may be due e i t h e r to the unmasking of antigenic determinants by los s of the 0-side chains of LPS or re-arrangement of the outer membrane components in rough s t r a i n s . This in t e r a c t i o n of MAl-6 with rough s t r a i n s i s s i g n i f i c a n t in that the rough P. aeruginosa i s o l a t e s from c y s t i c f i b r o s i s patients (Table I) appear to have protein H2 as a surface determinant (Table X). In smooth P. aeruginosa s t r a i n s , however, l i p o p r o t e i n H2, l i k e the Braun's l i p o p r o t e i n of E. c o l i (Braun et a l . , 1976), i s not surface-exposed. I n t e r e s t i n g l y , Braun's l i p o p r o t e i n does become exposed in deep rough, LPS-altered mutants. The colony immunoblot procedure f a c i l i t a t e d rapid screening of a v a r i e t y of st r a i n s for expression of s p e c i f i c determinants. The s p e c i f i c i t y of the procedure was shown by the lack of binding of MAl-6 to CFC46 mucoid and CFC46 nonmucoid s t r a i n s and the s p e c i f i c i t y of MA1-8 binding (O-antigen s p e c i f i c ) to serotype 5 s t r a i n s . The mucoid capsule therefore, did not appear to n o n - s p e c i f i c a l l y bind or adsorb antibodies. 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 of two protein F epitopes Studies on the antigenic structure of protein F are summarized on Table XIII. As described above, the monoclonal antibodies s p e c i f i c f o r protein F f e l l into two classes by i n t e r a c t i o n with Pseudomonas s t r a i n s . Antibody MA5-8 reacted s p e c i f i c a l l y with P. aeruginosa s t r a i n s while MA4-4, MA2-10, and MA4-10 cross-reacted with a protein, from P. putida and P. syringae. of s i m i l a r molecular weight to P. aeruginosa protein F. (F i g . 9). The differences in the antigenic domains recognized by these two classes of antibodies was further demonstrated by the binding of these antibodies to peptide fragments of protein F and to the native protein under d i f f e r e n t conditions (Table X I I I ) . Monoclonal antibody, MA5-8, unlike the other antibodies, interacted with oligomeric forms of protein F, as well as the 2-mercaptoethanol-reduced and non-reduced protein ( F i g . 20). This antibody also interacted with two p a r t i a l cyanogen bromide fragments of the protein, although the antigenic s i t e recognized by the antibody was destroyed by e i t h e r t r y p s i n or papain (Figs. 21 and 22). In contrast, monoclonal antibodies MA2-10, MA4-4 and MA4-10 interacted with the non-reduced porin, and with the try p s i n and papain derived fragments of protein F in the non-reduced form (Figs. 20 and 22). The epitope or epitopes recognized were destroyed by 2-mercaptoethanol as well as by cleavage of the protein with cyanogen bromide (Fig. 21). These data suggest that protein F has at l e a s t two d i s t i n c t surface located antigenic epitopes (domains) recognized by d i f f e r e n t antibodies. Both of these domains are probably conformational in that they require 157 Table XIII. D i f f e r e n t i a t i o n of two classes of monoclonal antibodies s p e c i f i c for P. aeruginosa protein F. Monoclonal Antibody  MA5-8 (MA2-10, MA4-4, MA4-10) Reac t i v i t y with p u r i f i e d protein F t a ' + + Surface l a b e l l i n g of int a c t + + P. a e r u g i n o s a ^ Rea c t i v i t y with P. putida and - + P. syringae protein F^ a» b^ React i v i t y with a 31 kD t r y p s i n or 29 kD papain p r o t e o l y t i c fragment - + of protein F^ c' React i v i t y with cyanogen bromide + (23,28 kD) fragments of protein F^ c^ Antigenic r e a c t i v i t y stable to + 2-mercaptoethanol^ c' Binding to oligomers of protein F + on SDS-polyacrylamide g e l s ^ c ^ a determined by both the ELISA and Western blo t procedures. b determined by the i n d i r e c t immunofluorescence and colony immunoblot procedures. c determined by the Western blo t procedure. 158 some t e r t i a r y structure which is maintained in SDS. Presumably t h i s SDS-stable t e r t i a r y structure (demonstrated experimentally by Mizuno and Kageyama, 1979) was destroyed by vigorous p r o t e o l y t i c or cyanogen bromide degradation. The complex 8-structure of porin proteins w i l l require extensive studies with a l a r g e r l i b r a r y of monoclonal antibodies before reasonable speculations on the structure of protein F can be made. 5. Prospectives The monoclonal antibodies reported here define s i n g l e antigenic s i t e s (epitopes) on both LPS and proteins. The high s p e c i f i c i t y of monoclonal antibodies for t h e i r p a r t i c u l a r epitope has great implications in s e r o l o g i c a l , epidemiological, taxonomic and s t r u c t u r a l studies. A l i b r a r y of monoclonal antibodies to the LPS rough core could be used to further define the s t r u c t u r a l heterogeneity of that region in P. aeruginosa s t r a i n s . Combinations of rough core s p e c i f i c and l i p i d A s p e c i f i c monoclonal antibodies could be used together with the s p e c i f i c outer membrane antigen in vaccination or immunization studies, thus n e u t r a l i z i n g the endotoxic action of LPS. Monoclonal antibodies to the d i f f e r e n t O-antigen types of Pseudomonas would also prove more s p e c i f i c as serotyping reagents than the polyclonal antisera presently used. The p r o t e i n - s p e c i f i c monoclonal antibodies can be used in several areas. For example, MA1-6 ( s p e c i f i c f or protein H2) could be applied to taxonomic studies of Pseudomonads. The antibody showed i n t e r a c t i o n with the group 1 Pseudomonads and thus can be used to d i f f e r e n t i a t e the Pseudomonadaceae. S i m i l a r l y , antibody MA5-8 could be used to define P. 159 aeruginosa s t r a i n s since t h i s monoclonal antibody showed s p e c i f i c i t y f o r P. aeruginosa s t r a i n s only. Another area of app l i c a t i o n i s that of defining antigen structure. Monoclonal antibodies to d i f f e r e n t peptide fragments could be used to as s i s t in obtaining the three dimensional structure of the proteins described here. Since some of the monoclonal antibodies reported here are s p e c i f i c for s u r f a c e - l o c a l i z e d antigenic s i t e s , they should be useful i n o p s o n i c - k i l l i n g assays to define i f antibodies to the protein increase clearance of the bacterium. These antibodies could also be used f o r passive immunization. F i n a l l y , t h e i r s p e c i f i c i t y and a v a i l a b i l i t y in large amounts makes monoclonal antibodies good candidates in studies where there i s a need to define antigenemia i n patients serum, as well as in antigen p u r i f i c a t i o n . 160 REFERENCES Abe, C., H. Shionoya, Y. Hirao, K. Okada, and J.Y. Homma. 1975. Common protective antigen (OEP) of Pseudomonas aeruginosa. Jpn. J. Exp. Med. 45:355-359. Adamus, G., M. Mulczyk, D. Witkowska, and E. Romanowska. 1980. Protection against k e r a t o c o n j u c t i v i t i s s h i g e l l o s a induced by immunization with outer membrane proteins of S h i g e l l a spp. Infect. Immun. 30:321-324. Angus, B.L., Carey, A.M., Caron, D.A., Kropinski, A.M.B., and Hancock, R.E.W. 1982. Outer membrane permeability in Pseudomonas aeruginosa: Comparison of a wild-type with an antibiotic-rsupersusceptible mutant. Antimicrob. Agents Chemother. 21:299-309. Angus, B.L., and R.E.W. Hancock. 1983. Outer membrane porin proteins F, P, and DI of Pseudomonas aeruginosa and PhoE of Escher i c h i a c o l i : chemical c r o s s - l i n k i n g to reveal native oligomers. J. B a c t e r i o l . 155:1042-1051. Barenkamp, S.J., R.S. Munson, J r . , and D.M. Granoff. 1981. Subtyping i s o l a t e s of Haemophilus influenzae type b by outer membrane protein p r o f i l e s . J. Infect. Dis. 143:668-676. 161 Beneviste, R., and J. Davies. 1973. Mechanism of a n t i b i o t i c resistance in bacteria. Ann. Rev. Biochem. 42:471-506. Benz, R., and R.E.W. Hancock. 1981. Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa i n l i p i d b i l a y e r s . Biochem. Biophys. Acta. 646:298-308. Bessler, W., and U. Henning. 1979. Protein 1 and protein I I * from the outer membrane of Escherichia c o l i are mouse B-lymphocyte mitogens. Z. Immunitatsforsch. 155:398. Blackwood, L.L., and J.E. Pennington. 1981. Influence of mucoid coating on clearance of Pseudomonas aeruginosa from lungs. Inf. Immun. 32:443-448. Bodey, G.P., R. Bo l i v a r , V. F a i n s t e i n , and L. Jadeja. 1983. Infections caused by Pseudomonas aeruginosa. Rev. Infect. Dis. 5_:279-313. Bowman, B.H., B.J. Langford, G.M. F u l l e r , S.D. Carson, A. Kurosky, and D.R. Barnett. 1975. Cystic f i b r o s i s : the c i l i a r y i n h i b i t o r i s a small peptide associated with immunoglobulin G. Biochem. Biophys. Res. Commun. 64:1310-1315. Bradley, S.G. 1979. C e l l u l a r and molecular mechanisms of action of b a c t e r i a l endotoxins. Ann. Rev. M i c r o b i o l . 33:67-94. 162 Braude, A.I., E.J. Z i e g l e r , and J.A. McCutchan. 1978. Antiserum treatment of gram-negative bacteremia. Schweitz. Med. Wochenschr. 108:1872-1876. Braude, A.I., E.J. Z i e g l e r , H. Douglas and J.A. McCutchan. 1977. Antibody to c e l l w all g l y c o l i p i d of gram-negative bac t e r i a : Induction of immunity to bacteremia and endotoxemia. J. Infect. Dis. 136 (Suppl):S167-S173. Braun, V. 1975. Covalent l i p o p r o t e i n from the outer membrane of Escherichia c o l i • Biochim. Biophys. Acta. 415:335-377. Braun, V., V. Bosch, E.R. Klumpp and I. Neff. 1976. Antigenic determinants of murein l i p o p r o t e i n and i t s exposure of the surface of Enterobacteriaceae. Eur. J . Biochem. 62:555-566. Braun, V., and H.J. Krieger-Brauer. 1977. I n t e r r e l a t i o n s h i p of the phage X receptor protein and maltose transport in mutants of Esche r i c h i a c o l i K-12. Biochim. Biophys. Acta. 469:89-98. Brinton, C. 1982. P i l i vaccines f or the prevention of i n f e c t i o n with Pseudomonas aeruginosa. Rev. Inf e c t . Dis. 163 Brokopp, CD., and J. J . Farmer. 1979. Typing methods f or Pseudomonas  aeruginosa In "Pseudomonas aeruginosa: C l i n i c a l manifestations of in f e c t i o n and current therapy". R.G. Doggett (ed.), Academic Press, N.Y. , pp. 89-133. Bryan, L.E. 1979. Resistance to antimicrobial agents: The general nature of the problem and the basis of resistance. In "Pseudomonas  aeruginosa: C l i n i c a l manifestations of i n f e c t i o n and current therapy". R.G. Doggett (ed.), Academic Press, N.Y. , pp. 219-271. Buchanan, T.M., W.A. Pearce, G.K. Schoolnik, and R.J. Arko. 1977. Protection against i n f e c t i o n s with N e i s s e r i a gonorrheae by immunization with outer membrane protein complex and p u r i f i e d p i l i . J . Inf e c t . Dis. 136 (Suppl.) S132-S137. Buchanan, T.M., and J.F. Hildebrandt. 1981. An t i g e n - s p e c i f i c serotyping of N e i s s e r i a gonorrhoeae: c h a r a c t e r i z a t i o n based upon p r i n c i p a l outer membrane protein. Infect. Immun. 32:985-994. Chedid, L., M. Parant, F. Parant, and F. Boyer. 1968. A proposed mechanism f or natural immunity to enterobacterial pathogens. J . Immunol. 100:292-301. 164 Chen, Y.-H. U., R.E.W. Hancock and R.I. M i s h e l l . 1980. Mitogenic e f f e c t s of p u r i f i e d outer membrane proteins from Pseudomonas aeruginosa. Infect. Immun. 28:178-184. Cho, Y., K. Tanamoto, Y. OH and J.H. Homma. 1979. Differences of chemical structures of Pseudomonas aeruginosa lipopolysaccharide e s s e n t i a l f o r adjuvancity and antitumour and interferon-inducing a c t i v i t i e s . FEBS. Let t s . 105:120-122. Costerton, J.W., J. Lam, K. Lam, and R. Chan. 1983. The rol e of the microcolony mode of growth in the pathogenesis of Pseudomonas aeruginosa i n f e c t i o n s . Rev. Infect. Dis. 5_ (Suppl.): S867-S873. Cowan, N.J., D.S. Secher, and C. M i l s t e i n . 1974. I n t r a c e l l u l a r immunoglobulin chain synthesis in non-secreting varients of a mouse myeloma: Detection of inac t i v e l i g h t chain messenger RNA. J. Mol. B i o l . 90:691-701. Cox, CD. 1979. Passage of Pseudomonas aeruginosa in compromised mice. Infect. Immun. 26:118-124. Craven, R.C, and T.C Montie. 1981. M o t i l i t y and chemotaxis of three str a i n s of P. aeruginosa used f or v i r u l e n c e studies. Can. J. Mic r o b i o l . 27:458-460. 165 Darveau, R.P., and R.E.W. Hancock. 1983. A procedure for the i s o l a t i o n of b a c t e r i a l lipopolysaccharides from both smooth and rough s t r a i n s of Pseudomonas aeruginosa and Salmonella typhimurium. J. B a c t e r i d . 155:831-838. Datta, D.B., B. Arden and U. Henning. 1977. Major proteins of the Escherichia c o l i outer c e l l envelope membrane as bacteriophage receptors. J. B a c t e r i d . 131:821-829. De Matted, C.S., M.C. Hammer, A.L. Baltch, R.P. Smith, N.T. Sutphen and P.B. Michelson. 1981. S u s c e p t i b i l i t y of Pseudomonas aeruginosa to serum b a c t e r i c i d a l a c t i v i t y : A comparison of three methods with c l i n i c a l c o r r e l a t i o n s . J. Lab. C l i n . Med. 98:511-518. DeVos, P., and J. DeLey. 1983. I n t r a - and intergenic s i m i l a r i t i e s of Pseudomonas and Xanthomonas ribosomal ribonucleic acid c i s t r o n s . Int. J . Systemat. B a c t e r i d . 33:487-509. Di Rienzo, J.M., N. Nakamura, and M. Inouye. 1978. The outer membrane proteins of Gram-negative b a c t e r i a : Biosynthesis, assembly and functions. Ann. Rev. Biochem. 47:481-532. Fernandes, P.B., C. Kim, K.R. Cundy, and N.N. Huang. 1981. Antibodies to c e l l envelope proteins of Pseudomonas aeruginosa i n c y s t i c f i b r o s i s patients. Infect. Immun. 33:527-532. 166 Frasch, C.E., and J.D. Robbins. 1978. Protection against group B meningococcal disease. I I I . Immunogeneity of serotype 2 vaccines and s p e c i f i c i t y of protection in a quinea pig model. J. Exp. Med. 1A7:629-644. Gabay, J . , and M. Schwartz. 1982. Monoclonal antibody as a probe f o r structure and function of an Escher i c h i a c o l i outer membrane prote i n . J . B i o l . Chem. 257:6627-6630. Galanos, C , M.A. Freudenberg, F. Jay, D. Nerkar, K. Velva, H. Brade, and W. Stritt m a t t e r . 1984. Immunogenic properties of L i p i d A. Rev. Infect. Dis. 6:546-552. Garten, W., and U. Henning. 1974. C e l l envelope and shape of Escher i c h i a  c o l i K-12: I s o l a t i o n and preliminary c h a r a c t e r i z a t i o n of the major ghost-membrane proteins. Eur. J. Biochem. 47:343-352. G i l l e l a n d , H.E., M.G. Parker, J.M. Matthews and R.D. Berg. 1984. Use of a p u r i f i e d outer membrane protein F (porin) preparation of Pseudomonas  aeruginosa as a protective vaccine i n mice. Infect. Immun. 44:49-54. Goding, J.W. 1980. Review a r t i c l e : Antibody production by hybridomas. J.'Immunol. Methods. 39:285-308. 167 Goldman, R.C., and L. Leive. 1980. Heterogeneity of antigenic side chain length in lipopolysaccharide from Escherichia c o l i 0111 and Salmonella  typhimurium LT2. Eur. J . Biochem. 107:145-153. Gulig, P.A., G.H. McCraken J r . , C F . F r i s c h , K.H. Johnston, and E.J. Hansen. 1982. Antibody response of infants to c e l l surface-exposed outer membrane proteins of Haemophilus influenzae type b a f t e r systemic Haemophilus disease. Infect. Immun. 37:82-86. Hancock, R.E.W. 1981. Aminoglycoside uptake and mode of action - with special reference to streptomycin and gentamicin. 1. Antagonists and mutants. J . Antimicrob. Chemother. 8:249-276. Hancock, R.E.W., and A.M. Carey. 1979. Outer membrane of Pseudomonas  aeruginosa: heat and 2-mercaptoethanol modifiable proteins. J . Ba c t e r i o l . 140:902-910. Hancock, R.E.W., and A.M. Carey. 1980. Protein DI - a glucose inducible, pore-forming protein from the outer membrane of Pseudomonas aeruginosa. Fed. Eur. M i c r o b i o l . Soc. L e t t s . 8:105-109. Hancock, R.E.W., CM. Decad, and H. Nikaido. 1979. I d e n t i f i c a t i o n of protein producing transmembrane d i f f u s i o n pores in outer membranes of Pseudomonas aeruginosa PA01. Biochim. Biophys. Acta 554:323-331. 168 Hancock, R.E.W , R.T. I r v i n , J.W. Costeron, and A.M. Carey. 1981. Pseudomonas aeruginosa outer membrane. Peptidoglycan-associated protein. J . B a c t e r i o l . 145:628-631. Hancock, R.E.W., E. Mouat, and D.P. Speert. 1984. Quantitation and i d e n t i f i c a t i o n of antibodies to the outer membrane proteins of Pseudomonas  aeruginosa i n the sera of c y s t i c f i b r o s i s patients. J . In f e c t . Dis. 149:220-226. Hancock, R.E.W., L.M. Mutharia, L. Chan, R.P. Darveau, D.P. Speert, and G.B. Pier. 1983. Pseudomonas aeruginosa i s o l a t e s from patients with c y s t i c f i b r o s i s : a class of serum-sensitive, non-typable s t r a i n s d e f i c i e n t in lipopolysaccharide 0-side chains. Infect. Immun. 42:170-177. Hancock, R.E.W., K. Poole, and R. Benz. 1982. Outer membrane protein P of Pseudomonas aeruginosa: Regulation by phosphate d e f i c i e n c y and formation of small anion-specific channels in l i p i d b i l a y e r membranes. J. B a c t e r i o l . 150:730-738. Hanessian, S., W. Regan, D. Watson, and T.H. Heskell. 1971. 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 antigenic components of a new heptavalent Pseudomonas vaccine. Nature (New Bi o l . ) 229:209-210. 169 Hansen, E.J., C P . F r i s c h , and K.H. Johnston. 1981a. Detection of antibody-accessible proteins on the c e l l surface of Haemophilus influenzae type b. Infect. Immun. 33:950-953. Hansen, E.J. , C F . F r i s c h , R.J. McDade, J r . , and K.H. Johnston. 1981. I d e n t i f i c a t i o n of immunogenic outer membrane proteins of Haemophilus  influenzae type b in the infant rat model system. Infect. Immun. 32:1084-1092. Hansen, E.H., S.M. Robertson, P.A. Gulig, C F . F r i s c h , and E.J. Haanes. 1982. Immunoprotection of rats against Haemophilus influenzae type b disease mediated by monoclonal antibody against outer membrane protein. Lancet 1:366-367. Henning, U., H. Schwerz, and R. Chen. 1979. Radioimmunological screening method f or s p e c i f i c membrane proteins. Anal. Biochem. 97:153-157. Hofstra, H., and J . Dankert. 1979. Antigenic c r o s s - r e a c t i v i t y of major outer membrane proteins in Enterobacteriaceae species. J. Gen. M i c r o b i o l . 111:293-302. Hofstra, H., M.J.D. Van Tol and J. Dankert. 1979. Immunofluorescent detection of the major outer membrane protein I I * i n Escherichia c o l i 0„, K,.. FEMS M i c r o b i o l . L e t t . 6:147-150. 170 Hofstra, H., M.J.D. Van T o l , and J. Dankert. 1980. C r o s s - r e a c t i v i t y of major outer membrane proteins of Enterobacteriaceae. studied by crossed Immunoelectrophoresis. J . B a c t e r i o l . 143:328-337. Holder, I.A., R. Wheeler and T.C. Montie. 1981. F l a g e l l a r preparations from Pseudomonas aeruginosa: animal protection studies. I n f e c t . Immun. 35.: 286-290. I s h i i , J . , and T. Nakae. 1980. Subunit constituent of the porin trimers that form the permeability channels in the outer membrane of Salmonella  typhimurium. J. B a c t e r i o l . 142:27-31. Jahnsson, P-E., A.A. Lindberg, B. Lindberg, an R. Wollin. 1981. Structural studies on the hexose region of the core in lipopolysaccharides from Enterobacteriaceae. Eur. J. Biochem. 115 : 571-577 . Johnston, K.H., K.K. Holmes, and E.C. Gotschlich. 1976. The s e r o l o g i c a l c l a s s i f i c a t i o n of N e i s s e r i a gonorrhoeae. 1. I s o l a t i o n of the outer membrane complex responsible f o r serotypic s p e c i f i c i t y . J . Exp. Med, 143:751-758. Joly , J.R., Y.-Y. Chen, and D. Ramsay. 1983. Serogrouping and subtyping of Legionella pneumophilia with monoclonal antibodies. J . C l i n . M i c r o b i o l . 18:1040-1046. 171 Kabir, S., and P. Mann. 1980. Immunological properties of the c e l l envelope components of V i b r i o cholerae. J. Gen. M i c r o b i o l . 119:517-525. Kamio, Y., and H. Nikaido. 1977. Outer membrane of Salmonella  typhimurium i d e n t i f i c a t i o n of proteins exposed on c e l l surface. Biochim. Biophys. Acta. 464:589-601. Kass, E.H., and S.M. Wolff. 1973. B a c t e r i a l Lipopolysaccharides. The Unive r s i t y of Chicago Press, Chicago. Kay, W.W, T. Buckley, E.E. Ishiguro, B.M. Phipps, J.P.L. Manette, and T.J. Trust. 1981. P u r i f i c a t i o n and d i s p o s i t i o n of a surface protein associated with virulence of Aeromonas salmonicida. J. B a c t e r i o l . 147:1077-1084. Kenimer, J.G., W.H. Habig, and M.C. Hardegoll. 1983. Monoclonal antibodies as probes of tetanus, toxin structure and function. Infect. Immun. 42:942-948. Knapp, J.S., M.R. Tarn, R.C. Nowinski, K.K. Holmes, and E.G. Sandstrom. 1984. Serological c l a s s i f i c a t i o n of N e i s s e r i a gonorrhoeae with use of monoclonal antibodies to gonococcal outer membrane protein I. J. Infect. Dis. 150:44-48. 172 Kohler, G., an C. M i l s t e i n . 1975. Continuous culture of fused c e l l s secreting antibody of predefined s p e c i f i t y . Nature (London) 256:495-497. Kropinski, A.M.B., L. Chan, and F.H. Milazzo. 1878. S u s c e p t i b i l i t y of lipopolysaccharide-defective mutants of Pseudomonas aeruginosa PAO to dyes, detergents, and a n t i b i o t i c s . Antimicrob. Agents. Chemother. 13:494-499. Kuusi, N., M. Nurminen, H. Saxen, M. Valtonen and P.H. Makela. 1979. Immunisation with major outer membrane proteins in experimental Salmonellosis i n mice. Infect. Immun. 25:857-862. Lam, J.S., D.M. Granoff, J.R. G i l s d o r f , and J.W. Costerton. 1980b. Immunogenicity of outer membrane de r i v a t i v e s of Haemophilus influenzae type b. Curr. M i c r o b i o l . 3:359-364. Lam, J.S., L.M. Mutharia, R.E.W. Hancock, N. Hoiby, K. Lam, L. Belk and J.W. Costerton. 1983. Immunogenicity of Pseudomonas aeruginosa outer membrane antigens examined by crossed Immunoelectrophoresis. In f e c t . Immun. 42:88-98. Lambden, P.R., J.E. Heckles, L.T. James, and P.J. Watt. 1979. Vari a t i o n s in surface protein composition associated with virulence properties i n opacity types of N e i s s e r i a gonorrhoeae. J. Gen. Mic r o b i o l . 114:305-312. 173 Lambert, P.A. and B.R. Booth, 1982. Exposure of outer membrane proteins on the surface of Pseudomonas aeruginosa PA01 revealed by l a b e l l i n g with 125 [ I]lactoperoxidase. FEMS. M i c r o b i o l . L e t t s . 14:43-45. Lanyi, B. and T. Bergan. 1979. S e r o l o g i c a l c h a r a c t e r i z a t i o n of Pseudomonas aeruginosa. In Methods of Microbiology. V o l . 10. T. Bergan and J.R. Norris (ed.). Academic Press, N.Y., pp. 93-168. Liang-Takasaki, C.-J., P.H. Makela, and L. Leive. 1982. Phagocytosis of bacteria by macrophages: Changing the carbohydrate of lipopolysaccharide a l t e r s i n t e r a c t i o n with complement and macrophages. J . Immunol. 128:1229-1235. Lieberman, M.M. 1978. Pseudomonas ribosomal vaccines: preparation properties and immunogenicity. In f e c t . Immun. 21:76-86. L i t t l e f i e l d , J.W. 1964. Sele c t i o n of hybrids from mating of f i b r o b l a s t s in v i t r o and t h e i r presumed recombinants. Science. 145:709. Loeb, M.R., and D.H. Smith. 1982. Properties and immunogenicity of Haemophilus influenzae outer membrane proteins. In "Haemophilus inflenzae, epidemiology, immunology and prevention of disease". S.H. S n e l l and P.F. Wright (ed.). E l s i e v i e r Biomedical Press. N.Y. pp. 207-217. 174 Lugtenberg, B., and L. Van Alphen. 1983. Molecular architecture and functioning of the outer membrane of Escherichia c o l i and other Gram-negative b a c t e r i a . Biochim. Biophys. Acta. 737:51-115. L u i , P.V. 1974. E x t r a c e l l u l a r toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130 (Suppl.):594-599. McDade, R.L. J r . , K.H. Johnston. 1980. Characterization of s e r o l o g i c a l l y dominant outer membrane protein of N e i s s e r i a gonorrhoeae. J. B a c t e r i o l . 141:1183-1191. Meadow, P.M., P.L. Wells, M.S. Salonen, and E.L. Nurmiaho. 1978. The e f f e c t of lipopolysaccharide composition on the u l t r a s t r u c t u r e of Pseudomonas aeruginosa. J. Gen. M i c r o b i o l . 105:23-28. Melancon, J . , R.A. Murgita, and I.W. DeVoe. 1983. Ac t i v a t i o n of murine B lymphocytes by N e i s s e r i a meningitidis and i s o l a t e d meningococcal surface antigen. Infect. Immun. 42:471-479. Mile r , M.J., J.F. Spilsbury, R.J. Jones, A.E. Roe and E.J.L. Lowbury. 1977. A new heptavalent Pseudomonas vaccine. J . Med. Mi c r o b i o l . 10:19-27. Mizuno, T. 1981. A novel peptidoglycan-associated l i p o p r o t e i n (PAL) found in the outer membrane of Proteus m i r a b i l i s and other gram negative bacteria. J . Biochem. 89:1039-1049. 175 Mizuno, T., and M. Kageyama. 1978a. Separation and ch a r a c t e r i z a t i o n of the outer membrane of Pseudomonas aeruginosa. J. Infect. Dis. 130 (Suppl.) :S81-S93. Mizuno, T., and M. Kageyama. 1978b. Separation and ch a r a c t e r i z a t i o n of the outer membrane of Pseudomonas aeruginosa. J . Biochem. 84:179-191. Mizuno, T., and M. Kageyama. 1979. I s o l a t i o n and ch a r a c t e r i z a t i o n of major outer membrane proteins of Pseudomonas aeruginosa s t r a i n PAO with s p e c i a l reference to peptidoglycan associated proteins. J . Biochem. 86:979-989. Mo l l , A., P.A. Manning, and K.N. Timmis. 1980. Plasmid-determined resistance to serum b a c t e r i c i d a l a c t i v i t y : A major outer membrane protein, the traT gene product, i s responsible f o r plasmid-specified serum resistance in Escherichia c o l i . I nfect. Immun. 28:359-367. Moorhouse, R., W.T. Winter, and S. Arnott. 1977. Conformation and molecular organization in f i b r e s of the capsular polysaccharide from Escherichia c o l i M41 mutant. J. Mol. B i o l . 109:373-391. Moss, C.W., S.B. Samuels, and R.E. Weaver. 1972. C e l l u l a r f a t t y acid composition of selected Pseudomonas species. Appl. M i c r o b i o l . 24:596-598. 176 Munn, C.B., E.E. Ishiguro, W.W. Kay, and T.J. Trust. 1982. Role of surface components in serum resistance of v i r u l e n t Aeromonas salmonocida. Infect. Immun. 36:1069-107 5. Mutharia, L.M., G. Crockford, W.C. Bogard, and R.E.W. Hancock. 1984. Monoclonal antibodies s p e c i f i c f o r E s c h e r i c h i a c o l i J-5 lipopolysaccharide: Cross-reaction with other gram-negative b a c t e r i a l species. Infect. Immun. 45:631-636 . Nachamkin, I., J.G. Cannon, and R.S. M i t t l e r . 1981. Monoclonal antibodies against N e i s s e r i a gonorrhoeae: Production of antibodies directed against a s t r a i n - s p e c i f i c c e l l surface antigen. Infect. Immun. 32:641-648. Nakajima, K., K. Muroga, and R.E.W. Hancock. 1983. Comparison of f a t t y acid, protein and s e r o l o g i c a l properties d i s t i n g u i s h i n g outer membranes of Pseudomonas a n g u i l l i s e p t i c a s t r a i n s from those of f i s h pathogens and other Pseudomonads. International J. System. B a c t e r i o l . 33:1-8. Nakae, T. 1976b. I d e n t i f i c a t i o n of the outer membrane protein of E. c o l i that produced transmembrane channels i n reconstituted v e s i c l e membranes. Biochem. Biophys. Res. Comm. 71:877-884. Nakae, T. 1975. Outer membrane of Salmonella typhimurium, r e c o n s t i t u t i o n of sucrose-permeable membrane residues. Biochem. Biophys. Res. Commun. 64:1224-1230. Nicas, T.I., and R.E.W. Hancock. 1983. Pseudomonas aeruginosa outer membrane permeability: I s o l a t i o n of a porin protein F - d e f i c i e n t mutant. J. B a c t e r i o l . 153:281-285. Nikaido, H., and T. Nakae. 1979. The outer membrane of gram-negative bacteria. Adv. Microb. Physiol. 19:163-250. <t>rskov, I., F. Orskov, B. Jann, and K. Jann. 1977. Serology, chemistry and genetics of 0 and K antigens of Escherichia c o l i . B a c t e r i o l . Rev. 41:667-710. Osborn, M.J., J.E. Gander, E. P a r i s i , and J. Carson. 1972. Mechanism of assembly of the outer membrane of Salmonella typhimurium. I s o l a t i o n and characterization of cytoplasmic and outer membrane. J. B i o l . Chem. 247:3962-3972. Osborn, M.J., and H.C.P. Wu. 1980. Proteins of the outer membrane of gram-negative b a c t e r i a . Ann. Rev. Mic r o b i o l . 34:369-422 . Ouchterlony, 0. 1959. D i f f u s i o n in gel methods f or immunological analysis. Prog. A l l e r g y . 5_:l-78. 178 Overbeeke, N.-, and B. Lugtenberg. 1980. Major outer membrane proteins of Escherichia c o l i s t r a i n s of human o r i g i n . J. Gen. M i c r o b i o l . 121:373-380. Owen, P. 1981. Immunology of the b a c t e r i a l membrane. In "Organization of prokaryotic c e l l membranes". Vol. 1, B.K. Ghosh (ed.), CRC Press Inc., Boca Raton, F l a . pp. 73-164. P a l l e r o n i , N.J., R.W. B a l l a r d , E. Ralson, and M. Doudoroff. 1973. Nucleic acid homologies in the genus Pseudomonas. Int. J. Systemat. B a c t e r i d . 23:333-339. Pennington, J.E.., and E. Menkes. 1981. Type-specific vs. cross-protective vaccination f o r gram-negative b a c t e r i a l pneumonia. J . Infect. Dis. 144:599-603. P i t t , T.L. (1980). Diphasic v a r i a t i o n in the f l a g e l l a r antigens of Pseudomonas aeruginosa. FEMS Microbiology l e t t e r s 9:301-306. Pollack, M., and L.S. Young. 1979. Protective a c t i v i t y of antibodies to exotoxin A and lipopolysaccharide at the onset of Pseudomonas aeruginosa Septicemia in man. J . C l i n . Invest. 63:276-286. Posner, M.R., D. Antoniou, J. G r i f f i n , S.F. Schlossman, and H. Lazarus. 1982. An enzyme-linked immunosorbent assay (ELISA) for the detection of 179 monoclonal antibodies to c e l l surface antigens on viable c e l l s . J. Immunol. Methods 48:23-31. Pr u i t , B.A., J r . , and R.B. Lindenberg. 1979. Pseudomonas aeruginosa in f e c t i o n s in burn patients. In "Pseudomonas aeruginosa". C l i n i c a l manifestations of i n f e c t i o n s and current therapy. R.G. Dogget (ed.). Academic Press, N.Y., pp. 339-366. Rietschel, E.T., H.W. Wollenwerber, U. Zahringer, and 0. L u d e r i t z . 1983. L i p i d A, the l i p i d component of b a c t e r i a l lipopolysaccharides: r e l a t i o n of chemical structure to b i o l o g i c a l a c t i v i t y . K l i n . Wocherschr. 60:705-709. Robertson, S.M., C F . F r i s c h , P.A. Gulig, J.R. Kettman, K.H. Johnston, and E.J. Hansen. 1982. Monoclonal antibodies d i r e c t e d against a c e l l surface-exposed outer membrane protein of Haemophilus influenzae Type b. Infect. Immun. 36:80-88. Rodriques, V., and C P . Bodey. 1979. Epidemiology, c l i n i c a l manifestation and treatment in cancer patients. In "Pseudomonas  aeruginosa c l i n i c a l manifestations of i n f e c t i o n s and current therapy". R.G. Doggett (ed.), Academic Press, N.Y., pp. 367-407. Rowley, D. 1968. S e n s i t i v i t y of, rough Gram-negative ba c t e r i a to the b a c t e r i c i d a l action of serum. J. Bacterol. 95:1647-1652. 180 Ruitenberg, E.J., P.A. Steerenberg, B.J.M. Brosi, and J. Buys. 1974. Sere-diagnosis of T r i c h i u e l l a s p i r a l i s i n f e c t i o n in pigs by enzyme-linked immunosorbent assay. B u l l . W.H.O. 51:108-109. Sadoff, J.C. and M.S. Artenstein. 1974. The outer c e l l w all membrane of Pseudomonas aeruginosa. J. Infect. Dis. 130 (Suppl.):S81-S93. Schacterle, G.R., and L.R. Pollack. 1973. A s i m p l i f i e d method for the quantitative assay of small amounts of protein in b i o l o g i c a l m a t e r i a l . Anal. Biochem. 51:654-655. Schimpff, S.C., M. Moody, and V.M. Young. 1970. Relationship of colonization with Pseudomonas aeruginosa to development of pseudomonas bacteremia in cancer patients. Antimicrob. Agents Chemother. 240-244. Schindler, M., and M.J. Osborn. 1979. Interaction of d i v a l e n t cations and polymyxin B with lipopolysaccharide. Biochemistry 18:4425-4430. Schindler, P.R.G. and M. Teuber. 1979. Fluorescent l a b e l l i n g of c e l l envelope proteins with 5-Dimethyl-aminonaphthalene-l-sulphonyl-chloride-l e c i t h i n - c h o l e s t e r o l v e s i c l e s upon treatment of Pseudomonas aeruginosa with T r i s (Hydroxymethyl) aminoethane-hydrochloride-ethylene diamine tetraacetate. FEMS Mi c r o b i o l . L e t t s . 6:163-164. 181 Schneider, H., and J.M. G r i f f i s s . 1982. A b a c t e r i c i d a l microassay for testing serum s e n s i t i v i t y of N e i s s e r i a gonorrhoeae. J. Immunol. Methods. 54:101-105. Schulman, M. , CD. Wilde, and G. Kohler. 1978. A better c e l l l i n e f o r making hybridomas secreting s p e c i f i c antibodies. Nature 276:269-270. Sompolinsky, D., J.B. Hertz, N. Hoiby, K. Jensen, B. Mansa, and Z. Samra. 1980a. An antigen common to a wide range of ba c t e r i a . 1. The i s o l a t i o n of a "common antigen" from Pseudomonas aeruginosa. Acta. Path. M i c r o b i o l . Scad. Sect. B. 88:143-149. Su l l i v a n , K.H. and R.P. Williams. 1982. Use of Iodo-Gen and Iodine-125 to l a b e l the outer membranes of N e i s s e r i a gonorrhoeae. Anal. Biochem. 120:254-258. Swanson, J. 1981. Surface-exposed protein antigens of the gonococcal outer membrane. Infect. Immun. 34:804-816. Swanson, J . , L.W. Mayer, and M.R. Tam. 1982. A n t i g e n i c i t y of N e i s s e r i a  gonorrhoaeae. Outer membrane Protein(s) I I I detected by Immunoprecipitation and Western b l o t t r a n s f e r with a monoclonal antibody. Infect. Immun. 38:668-672. 182 Tanamoto, K., C. Abe, J.H. Homma, and Y. Kojima. 1979. Regions of the lipopolysaccharide of Pseudomonas aeruginosa e s s e n t i a l f o r anti-tumour and interferon induction a c t i v i t i e s . Eur. J. Biochem. 97:623-629. Tamamoto, K., and J.H. Homma. 1982. E s s e n t i a l regions of the lipopolysaccharides of Pseudomonas aeruginosa responsible for pyrogenicity and a c t i v a t i o n of the p r o c l o t t i n g enzyme of Horseshoe crabs. Comparison with antitumor, interferon-inducing and adjuvant a c t i v i t i e s . J . Biochem. 91:741-746. Taylor, P.W. 1983. B a c t e r i c i d a l and B a c t e r i o l y t i c a c t i v i t y of serum against gram-negative ba c t e r i a . M i c r o b i o l . Reviews. 47:46-83 . Thomassen, M.J., B. Boxerbaum, D.A. Demko, P.J., Kuchenbrod, D.G. Dearborn, and R.E. Wood. 1979. Inhibitory e f f e c t of c y s t i c f i b r o s i s serum on Pseudomonas phagocytosis by rabbit and human a l v e o l a r macrophages. Pediatr. Res. 13:1085-1091. Towbin, M., T. Sta e h l i n , and J. Gordon. 1979. Elec t r o p h o r e t i c t r a n s f e r of proteins from polyacrylamide gels to n i t r o c e l l u l o s e sheets: procedure and some a p p l i c a t i o n s . Proc. Natl. Acad. S c i . (U.S.A.) 76:4350-4354. Ts a i , C-M., and C.E. Frasch. 1982. A se n s i t i v e s i l v e r s t a i n f o r detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119:115-119. 183 Tsa i , C.-M., C E . Frasch, and L.F. Mocca. 1981. Five s t r u c t u r a l classes of major outer membrane protein in N e i s s e r i a meningitidis. J . B a c t e r i o l . 146:69-78. V i r j i , M., J.E. Heckels and P.H. Watt. 1983. Monoclonal antibodies to Gonococcal p i l i : Studies on antigenic determinants on p i l i from variants of s t r a i n P9. J. Gen. Mi c r o b i o l . 129:1965-1973. Wilkinson, S.G. 1983. Composition and structure of lipopolysaccharides from Pseudomonas aeruginosa. Rev. Infe c t . Dis., 5 (Suppl.):S941-S949. Winter, A.J., E.C McCoy, C S . Fullmer, K. Burda, and P.J. Bier. 1978. Microcapsule of Campylobacter fetus: chemical and physi c a l characterization. Infect. Immun. 22:963-971. Woods, D.E., D.C Straus, W.G. Johanson, J r . , V.K. Berry, and J.A. Bass. 1980a. Role of P i l i in adherence of Pseudomonas aeruginosa to mammalian buccal c e l l e p i t h e l i a l c e l l s . I nfect. Immun. 29:1146-1151. Woods, D.E., J.A. Bass, W.G. Johanson, J r . , and D.C. Straus. 1980b. Role of adherence in the pathogenesis of Pseudomonas aeruginosa lung i n f e c t i o n in c y s t i c f i b r o s i s patients. Infect. Immun. 30:694-699. Wood, R.E.', J.E. Pennington, and N.Y. Reynolds. 1983. Intranasal administration of Pseudomonas lipopolysaccharide vaccine in c y s t i c f i b r o s i s patients. Pediatr. Infect. Dis. 2:367 184 Young, L. 1979. Recent advances in therapy of Pseudomonas aeruginosa i n f e c t i o n . In "Pseudomonas aeruginosa c l i n i c a l manifestations of in f e c t i o n and current therapy". R.G. Doggett (ed.). Academic Press, N.Y., pp. 311-338. Young, L.S. 1980. The role of exotoxins in pathogenesis of Pseudomonas  aeruginosa i n f e c t i o n s . J . Infect. Dis. 142:626-630. Young, L.S., P. Stevens, and J . Ingram. 1975. Functional r o l e of antibody against "core" g l y c o l i p i d A of Enterobacteriaceae. J. C l i n . Invest. 56:850-861. Young, L.S., and M. Pollack. 1980. Immunologic approaches to the prophylaxis and treatment of Pseudomonas aeruginosa i n f e c t i o n . In "Pseudomonas aeruginosa. International Symposium". L.D. Sabath (ed.), Hans Huber Publishers. Bern. pp. 103-108. Young, L.S., P. Stevens, and B. K a i j s e r . 1982. Gram-negative pathogens in septicaemic i n f e c t i o n s . Scad. J. Infect. Dis. Suppl. 31:78-94. Yu, F., S. Ichihara, and S. Mizushima. 1979. A major outer membrane protein (0-8) of Esche r i c h i a c o l i K-12 e x i s t s as a trimer in sodium dodecyl sulphate s o l u t i o n . FEBS Le t t . 100:74-76. 185 Ziegler, E.J., J.A. McCutchan, H. Douglas, and A.I. Braude. 1975. Prevention of l e t h a l Pseudomonas bacteremia with epimerase-deficient E. c o l i antiserum. Trans. Assoc. Am. Physicians. 88:101-106. Zierdt, C.H., and R.L. Williams. 1975. Serotyping of Pseudomonas  aeruginosa i s o l a t e s from patients with c y s t i c f i b r o s i s of the pancreas. J. C l i n . M i c r o b i o l . 1:521-526. Z o l l i n g e r , W.D., R.E. Mandrell, P. A l t i e r i , S. Berman, J . Lowenthal and M.S. Artenstein. 1978. Safety and immunogenicity of a N e i s s e r i a  meningitidis type 2 protein vaccine i n animals and humans. J . Infect. Dis. 137:728-739. Hancock, R.E.W., A.W. Wieczoreck, L.M. Mutharia and K. Poole. Monoclonal a n t i b o d i e s against P. aeruginosa outer membrane antigens: I s o l a t i o n and" c h a r a c t e r i s a t i o n . 1982 I n f e c t . Immun. 37:166-171. Mutharia, L.W., T.I. Nicas and R.E.W. Hancock. Outer membrane pr o t e i n s of P. aeruginosa serotype s t r a i n s . 1982. J . I n f e c t . D i s . U b : //U-//9. Speert D.P., D. Lawton, L.M. Mutharia. Antibody to P. aeruginosa mucoid exopolysaccharide and to sodium a l g i n a t e i n C y c s t i c F i b r o s i s serum. P e d i a t r i c s Research. 1983. 18:431-433. Hancock, R.E.W., L.M. Mutharia, L. Chan, R.P. Darveau, D.P. Speert and G.B. P i e r . Pseudomonas aeruginosa i s o l a t e d from p a t i e n t s with C y s t i c h t b r o s i s ; .A cla.ss of serum s e n s i t i v e , non-typable s t r a i n s d e f i c i e n t i n li p o p o l y s a c c h a r i d e O-antigen s i d e c h a i n s . 1983. I n f e c . Inmun. 42': 170-177. Mutharia, L.M. R.E.W. Hancock. Surface L o c a l i s a t i o n o f P. aeruginosa outer membrane p r o t e i n F using monoclonal a n t i b o d i e s . 1983. I n f e c t . Immun. 42:105-112. Lam, J.S., L.M. Mutharia, R.E.W. Hancock, N. Hoiby, K. Lam, L. Baek, and J.W. Cos t e r t o n . Immunogenicity o f P. aeruginosa outer membrane examined by crosse d immuno-electrophoresis. 1983. I n f e c t . Immun. 42:88-98. Mutharia^ L.L., J.S. Lam and R.E.W. Hancock. Use of monoclonal a n t i b o d i e s i n the study of common antigens of gram-negatiye b a c t e r i a . In "Monoclonal A n t i b o d i e s Against B a c t e r i a " , Ed. A.J.L. Macario and E.C. de Macario. Academic Press. 1984. J.S. Lam., L.M. Mutharia and R.E.W. Hancock. A p p l i c a t i o n of Monoclonal a n t i b o d i e s to the study of the s u r f a c e antigens i n Pseudomonas aeruginosa. In "Monoclonal Antibodes Against B a c t e r i a " , Ed. A.J.L. Macario and E.C. Macario. Academic Press. Mutharia L.M., G. Cro c k f o r d , W.C. Bogard and R.E.W. Hancock. Monoclonal a n t i b o d i e s s p e c i f i c f o r E.col i J-5 li p o p o l y s a c c h a r i d e : C r o s s - r e a c t i o n with other gram-negative b a c t e r i a l s p e c i e s . 1984. I n f e c t . Immun. 45:631-636. Mutharia, L.M. and R.E.W. Hancock. C h a r a c t e r i s a t i o n o f two surface l o c a l i s e d a n t i g e n i c s i t e s on p o r i n p r o t e i n F of P. aeruginosa. Submitted f o r p u b l i c a t i o n . 1984 Sept. Can. J . M i c r o b i o l . Hancock, R.E.W., L.M. Mutharia and E.C. Mouat. Immunotherapeutic p o t e n t i a l of monoclonal a n t i b o d i e s against P. Aeruginosa p r o t e i n F. Submitted f o r P u b l i c a t i o n , 1984 Oct. Eur. J . C l i n . M i c r o b i o l . Mutharia, L.M. and R.E.W. Hancock. A monoclonal antibody s p e c i f i c f o r an outer membrane l i p o p r o t e i n of the Fluorescent group o f the Family Pseudomonandaceae, Submitted f o r p u b l i c a t i o n . 1984 Nov. I n t e r n a t i o n a l Journal of Systematic B a c t e r i o l o g y . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0096736/manifest

Comment

Related Items