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Outer membrane protein OprF of P. aeruginosa Rawling, Eileen Grace 1995

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OUTER MEMBRANE PROTEIN OprF of P. aeruginosa  by  Eileen Grace Rawling B.Sc. (Microbiology), University of British Columbia, 1989  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology)  We accept this thesis as conforming to) required standard  The University of British Columbia August 1995 © Eileen Grace Rawling, 1995  ____  ___________  __________  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of The University of British Columbia Vancouver, Canada  Date  DE-6 (2188)  I i i’iH  Joc0(V ‘I  11  ABSTRACT  An OprF-deficient mutant of P. aeruginosa strain M-2 was constructed by 2 mutagenesis. This strain was unable to grow in low osmolarity media and was 70% the length of the parental strain. These results confirmed that these phenotypes were not strain specific. Consistent with the appearance of OprF-deficient strains in clinical situations, an OprF-deficient strain was shown to not have a major growth disadvantage in an in vivo chamber model. Plasmids encoding truncated and cysteine-to-serine mutants of OprF were constructed by linker-insertion mutagenesis, PCR and subcloning of a previously characterized mutant. Analysis of the resulting proteins indicated that between 102 and 163 N-terminal amino acids of OprF was required for protein expression as determined by Western immunoblotting. All of the truncated mutants expressed were associated with the outer membrane, indicating that the N-terminal 163 amino acids of OprF were sufficient for this association. None of the truncated-OprF mutants tested were associated with the peptidoglycan, indicating that more than 215 N-terminal amino acids of OprF are required for this association. Strains that encoded at least two cysteines were 2-mercaptoethanol modifiable. The full-length cloned OprF and the truncated mutant expressing the N-terminal 290 amino acids were  111  normally heat modifiable. When denatured prior to electrophoresis, the remaining truncated mutants had apparent molecular weights lower than those of untreated samples indicating that between 215 and 290 amino acids were required for the protein to be normally heat modified. Although the truncated-OprF mutants were able to grow in low osmolarity media and were significantly longer than the OprF-deficient strain, the C-terminal half of OprF appeared to be required for the wildtype growth and length. The cysteine-to-serine OprF mutants were associated with the outer membrane and were heat modifiable. Analysis of these mutants with and without 2-mercaptoethanol was consistent with the hypothesis that wild-type OprF has two disulphide bonds Monoclonal antibody reactivity with overlapping octapeptides equivalent to the primary sequence of OprF localized three epitopes. Indirect immunofluorescence and opsonic phagocytosis studies identified the surface location of epitopes binding nine OprF-specific monoclonal antibodies. This information was incorporated into an updated secondary structure model of OprF.  iv TABLE OF CONTENTS Page ABSTRACT  ii  TABLE OF CONTENTS  iv  List of Tables  ix  List of Figures  x  ACKNOWLEDGEMENTS  xiii  INTRODUCTION A. P. aeruginosa  1  B. Gram-negative outer membranes 1. Introduction  2  2. Phospholipids  4  3. LPS  4  4. Proteins a). Lipoproteins  5  b). Porins  6  C. OmpA  9  D. OprF  14  E. Aims of this study  22  V  MATERIALS AND METHODS A. Bacterial strains and plasmids  .  23  B. Media and growth conditions 1. In vitro growth  23  2. In vivo growth  29  C. DNA procedures 1. Plasmid isolation  30  2. Plasmid introduction a). Transformation  30  b). Electroporation  30  c). Biparental mating  31  3. 2 mutagenesis of oprF  31  4. Southern blotting a). chromosomal DNA isolation  31  b). Labeling of the DNA probe  32  c). Southern blotting  32  5. Oligonucleotide synthesis  32  6. Polymerase chain reaction  33  7. DNA sequencing  33  D. Protein procedures 1. SDS-PAGE  34  2. Outer membrane preparation  34  vi 3. Preparation of peptidoglycan-associated proteins....  35  4. Determination of protein concentration  35  E. Immunological techniques 1. Antibodies  35  2. Colony immunoblotting  36  3. Western immunoblotting  37  4. Immunofluorescent labeling  37  5. Opsonic phagocytosis  38  6. Overlapping-octapeptide analysis  39  F. Cell-length measurement 1. Image analysis  39  2. Microscopy  40  G. Growth studies  40  RESULTS CHAPTER 1. CONSTRUCTION AND EXPRESSION OF OprF MUTANTS IN P. aeruginosa. A. Introduction  42  B. Construction of an OprF-deficient mutant of P. aeruginosa strain, M-2  43  C. Construction of truncated versions of OprF  48  D. Site-directed mutagenesis of the cysteines of OprF  54  vii E. Expression of OprF mutants in P. aenosa  .  56  F. Outer membrane and peptidoglycan association of OprF mutants G. Summary  67 72  CHAPTER 2. FUNCTIONAL ANALYSIS OF OprF MUTANTS IN P. aeruginosa. A. Introduction  74  B. Growth of OprF mutants  74  C. Cell-length of OprF mutants  84  D. Summary  89  CHAPTER 3. MONOCLONAL ANTIBODY STUDIES. A. Introduction  91  B. Linear-epitope mapping using overlapping octapeptides  91  C. Monoclonal antibody binding to OprF mutants  96  D. Surface accessibility of epitopes binding OprF-specific monoclonal antibodies  100  E. Conservation of epitopes binding OprF-specific monoclonal antibodies F. Summary: A secondary structure model of OprF  103 104  DISCUSSION A. Aims of this study  111  viii  B. Genetic complementation of (2-mutagenized OprF in P. aeruginosa  111  C. Protein analysis of mutated versions of OprF  113  D. Functional analysis of truncated-OprF mutants  119  E. Analysis of the disulphide region of OprF  120  F. Analysis of epitopes binding OprF-specific monoclonal antibodies G. Conclusion and perspectives  REFERENCES  123 126  128  ix LIST OF TABLES  Table  I. II. III.  Title  Bacterial Strains  24  Plasmids  26  Amino acid residues introduced by linker-insertion truncation of OprF  IV.  VI. VII.  62  Apparent molecular weight of cystreine-to-serine mutants  in P. aeruginosa  65  Relative length of truncated OprF in P. aeruginosa  87  Summary of OprF-specific monoclonal antibody reactivity with truncated OprF in P. aeruginosa  VIII.  52  Characteristics of truncated-OprF mutants in P. aeruginosa  V.  Pane  97  Summary of OprF-specific monoclonal antibody reactivity with cysteine-to-serine mutants of OprF in P. aeniginosa  99  IX. Indirect-immunofluorescence labeling of intact  P. aeruginosa with OprF-specific monoclonal  antibodies  101  x LIST OF FIGURES  Figure  1.  Title  Schematic representation of the outer membrane of a Gram-negative bacterial cell  2.  Map of plasmid pWW2500 and restriction map of oprF  3.  Southern blot of a biotinylated oprF probe hybridized to chromosomal DNA digests of P. aeruginosa strain M-2  4.  58  61  64  SDS-PAGE of outer membrane preparations of H 103/pER, H636/pER and H636/pER326t  9.  50  Western immunoblot of whole cell lysates of cysteine-to serine mutants  8.  47  Western immunoblots of OprF and truncated OprF mutants with and without TCA pretreatment  7.  45  Western immunoblot of whole cell lysates of H636 containing plasmids encoding truncated OprF  6.  3  Map of oprF and OprF showing the restriction endonuclease sites and the length of the mature protein  5.  Page  68  Western immunoblot of outer membrane preparations of truncated versions of OprF  69  xi LIST OF FIGURES (continued).  10.  Western immunoblot of outer membrane preparations of truncated OprF solubilized in Triton-EDTA or Triton lysozyme  71  11.  InvivogrowthofHlo3andH636  76  12.  Growth of OprF-deficient strains in LB no salt or LB 200 mM salt  13.  77  Growth of H636/pER17O-26t and H636/pER with or without the addition of MgC1 2  79  14. Growth of truncated versions of OprF in low osmolarity and high osmolarity media 15.  Western immunoblot of H636/pER29Ot, H636/pER and H103/pER  16.  +  200 mM salt  85  Length of strains of P. aeruginosa containing plasmids encoding full-length or truncated versions of OprF  18.  83  Growth of cysteine-to-serine mutants in LB no salt or LB  17.  81  86  ELISA readings of monoclonal antibodies MA7-1, MA7-2,  and MA5-8 reacting with individual pins derivatized with overlapping octapeptides from OprF  93  xii LIST OF FIGURES (continued).  19.  ELISA readings of mouse arid rabbit polyclonal serum reacting with individual pins derivatized with overlapping octapeptides from OprF  20.  94  Opsonic phagocytosis of M-2 by OprF-specific monoclonal antibodies  102  21.  Secondary structure model of OprF  105  22.  Proposed secondary structure model of OprF  107  xiii ACKNOWLEDGEMENTS  I would like to acknowledge the support and guidance of my supervisor, R.E.W. Hancock, and the members of my supervisory committee. The financial assistance of the Canadian Cystic Fibrosis Foundation and R.E.W. Hancock is gratefully acknowledged. I would also like to acknowledge the friendship and assistance of the members of the Hancock laboratory and the Department of Microbiology and Immunology.  1 INTRODUCTION  A. Pseudomonas aeruginosa. Pseudomonas aeruginosa is a rod-shaped, Gram-negative  bacterium with a polar flagellum and polar p111. Its species name is deduced from one of the extracellular pigments it produces, pyocyanin, which is the colour of oxidized copper. Other extracellular compounds include several enzymes, toxins, and extracellular slime, all of which contribute to its growth versatility and pathogenicity (Young, 1980). P. aeruginosa is a common inhabitant of soil and water that presents little problem in healthy individuals, but is a serious problem in patients that have lowered resistance to infection due to severe burns, immunosuppresive therapy, and for conditions that include cancer, transplants and cystic fibrosis (Blackwood and Pennington, 1981). It is a nosocomial, opportunistic organism that can produce local infections in burn sites, the urinary tract, the respiratory tract, ears and eyes, as well as a generalized septicemia. P. aeruginosa infections are difficult to treat due to their intrinsic resistance to antibiotics (Bryan, 1979; Hancock and Bell, 1988). P. aeruginosa is more resistant to antibiotics than other Gram-negative bacteria, particularly to the f3-lactam antibiotics (Hancock  and Bell, 1988). The in vitro minimal inhibitory concentration (MIC) of these antibiotics is eight to one hundred times higher than that of  2 Escherichia coli (Rolinson, 1986). P. aeruginosa is also resistant to many  other compounds including detergents, disinfectants, bile salts and lysozyme (Hancock, 1984).  B. Gram-negative outer membranes.  1. Introduction The outer membranes of Gram-negative bacteria contain a number of different proteins, including porins, in an asymmetric lipid bilayer composed of an inner layer of phospholipid (Smit et al., 1975; Kamio and Nikaido, 1976) and an outer layer of lipopolysaccharide (LPS) (Figure 1). The outer membrane acts as a molecular sieve, limiting the access of many compounds to the cell. The water-filled porins provide the major pathway for the uptake of small hydrophilic molecules with the specificity and the size of the pore determining the access. Hydrophobic molecules, which can pass directly through the outer membrane of some bacteria such as N. gonorrhea (Martinez de Tejada and MoriyOn, 1993),  are largely excluded from E. coli and P. aeruginosa (Hancock et al., 1994). This resistance is attributed to the structure of the LPS, as described below. A third route into the cell, the self-promoted uptake pathway, is also attributed to the structure of the LPS and allows the uptake of polycationic antibiotics and polycationic peptides (Hancock and Bell, 1988; Vaara, 1992).  3  LPS  Phospholipid  Figure 1. Schematic representation of the outer membrane of a Gram negative bacterial cell. The locations of the LPS, phospholipid, lipoproteins (L), porins (P) and peptidoglycan are indicated.  4 2. Phospholipicls The phospholipids found in the outer membrane are similar to those found in the cytoplasmic membrane, with phosphatidyl ethanolamine being the predominant species (Cronan Jr., 1979; Conrad  and Gilleland, 1981). Acidic phospholipids, including phosphatidyl glycerol, cardiolipin and an unidentified lipid are also present in P. aeruginosa (Conrad and Gilleland, 1981).  3. LPS LPS is a unique molecule found only in Gram-negative bacteria. Lipid A or endotoxin contains the hydrophobic component of the molecule and is highly conserved between bacterial species (Reeves 1994). It is composed of from 5 to 7 fatty acids, which anchor it in the membrane, attached to a diglucosamine disaccharide with phosphates in the 1 and 4’ positions. Attached to Lipid A is the hydrophilic core oligosaccharide which varies from species to species. The core region is composed of 2-keto-3-deoxyoctonate (KDO), heptose, hexose, and phosphate. In smooth strains, between 10-25 % of molecules have the immunodominant, sereotype-specific 0-antigen attached. The 0-antigen is composed of varying numbers of repeating oligosaccharide units which form a capsule like structure over the bacterium. P. aeruginosa contains 2 different types of LPS; B-band which is composed of LPS with 0antigen and A-band LPS which has an antigenically and chemically-  5 distinct, shorter-chain polyrhamnan polysaccharide (Rivera et al., 1988; Rivera and McGroarty, 1989). The negative charges present on the LPS core oligosaccharide and KDO are partially neutralized by the binding of divalent cations such as Mg 2 and Ca . This binding stabilizes the LPS 2 LPS and LPS-protein interactions and limits the uptake of hydrophobic compounds. The addition of divalent-cation chelators such as EDTA destabilizes the outer membrane which allows the uptake of hydrophobic compounds (Hancock and Bell, 1988; Vaara, 1992). This divalent-cation bridging is also involved in self-promoted uptake. It has been proposed that compounds such as polycationic antibiotics and cationic peptides  can displace the divalent cations, allowing not only the uptake of the compounds themselves, but also the uptake of hydrophobic compounds (Hancock and Bell, 1988). 4. Proteins a) Lipoproteins One type of protein which contributes to the stability of the outer membrane is the lipoproteins. The predominant lipoprotein of E. coil is the Braun or murein lipoprotein (110). It is composed of 58 amino acid residues in a primarily alpha-helical conformation, with the hydrophilic and the hydrophobic side chains located at opposite sides of the helix. The fatty acyl chains, linked to the N-terminus of the protein, anchor it in the outer membrane (Braun and Wu, 1994). About 1/3 of these  6  molecules are covalently attached to the peptidoglycan via the Cterminus of the protein and the remaining 2/3 are non-covalently associated (Braun, 1975). Mutants lacking this protein (lpp) (Yem and Wu, 1978) or that were deficient in the bound form of the protein (Fung et al., 1978) were more sensitive to EDTA, had cell surface blebbing,  impaired septum formation (Yem and Wu, 1978) and had leaky periplasms indicating a structural role for this protein. The analogous protein in P. aeruginosa is OprI which has 23-30% amino acid sequence identity with the Braun lipoprotein (Hancock et al., 1990). Although some strains of P. aeruginosa were reported to have both covalent and non-covalent forms, H 103, the wild-type laboratory strain used in this study, does not have the covalently-bound form of this protein (Hancock et al., 1981). E. coli also has a 21 kDa non-covalently associated  lipoprotein, called the peptidoglycan associated protein, or PAL (Lazzaroni and Portalier, 1992). The P. aeruginosa equivalent is OprL which, unlike PAL, is a major constituent of outer membrane. When grown in rich media, OprI, OprL and OprF are the most prevalent outer membrane proteins in P. aeruginosa (Nikaido and Hancock, 1986). b) Porins Proteins of the outer membrane of E. coli include porins as well as proteases, phospholipase A, pili, flagella and inducible receptors for the uptake of iron-siderophore complexes and vitamin B 12. (Martin and  7 Hancock, 1990). The TonB-dependent receptor, FepA, is a high-affinity protein specific for the uptake of ferric enterobactin in E. coli. It appears to be a gated-porin and it has been suggested that other TonB-dependent ligand-specific outer membrane receptors function in the same way (Rutz etal., 1992).  Porins are classified as general or specific porins. Specific porins allow the diffusion of small hydrophilic molecules, but have a specific binding site which is an advantage when the nutrient is at a low concentration. At high nutrient concentrations these sites are saturated. Unlike the general porins, the expression of these porins is co-regulated with a complex transport system (Poole and Hancock, 1984; Benz, 1988). Two examples of this type are LamB from E. coli and OprP from P. aeruginosa which are specific for the uptake of maltose and maltodextrins, and phosphate respectively, and which both function as trimeric proteins (Benz, 1988). LamB has recently been crystallized, showing that each subunit is composed of 18 anti-parallel f3 strands, with 3 of the longer external ioops folding into the barrel (Schirmer et al., 1995). Each subunit has a series of aromatic residues leading from the entrance to the constriction of the pore which have been called a “greasy slide”. These are thought to be a series of sugar-binding sites that align the sugar allowing its guided diffusion through the pore (Schirmer et al., 1995). Although OprP from P. aeniginosa and PhoE from E. coli are both  8 expressed in low phosphate media, they have minimal primary sequence homology (Siehnel et aL, 1989) and differ in their anion-binding preference. PhoE has a weak affinity for anions while OprP has a 100fold preference for the binding of phosphate over other anions (Hancock et at., 1986). Other specific porins include the minor outer membrane  proteins Tsx of E. coli, for the uptake of nucleosides, (Benz, 1988) and TolC of E. coli, which appears to be selective for peptides (Benz, 1994),  and OprB of P. aeruginosa, for the uptake of glucose, (Hancock and Carey, 1980) and OprD of P. aeruginosa, for the uptake of basic amino acids (Benz, 1994) and imipenem in P. aeruginosa (Hancock et at., 1990). General trimeric porins are the most abundant proteins in E. coli (Nikaido, 1993). They allow the passage of small hydrophilic molecules, with an exclusion limit of 6O0 Da. and exclude hydrophobic and/or large molecules. Three trimeric, general porins have been identified in E. coli: OmpF, OmpC and PhoE. OmpF and OmpC are weakly cation selective and PhoE has a slight preference for anions (Benz et al., 1985). OmpF is expressed at higher levels than OmpC in low osmolarity media and the reverse is true in high osmolarity media. PhoE is expressed when the level of phosphate is low. The structures of OmpF and PhoE, which have 63% amino acid sequence identity, have been determined by X-ray crystallography (Cowan et at., 1992). Like the first crystallized porin from Rhodobacter capsulatus, which has low sequence homology  9 with OmpF and PhoE, these porins are comprised of a f3-barrel composed of 16 anti-parallel 13-strands, with short turns at the periplasmic face and longer cell-surface exposed loops. One of the eight external loops, loop 3, folds inside the barrel narrowing its diameter to create the “eyelet” region. Mutants in this region have been shown to have an increased channel size (Benson et al., 1988). Six of these loops partially shield the entrance of the pore. Loop 2 is involved in the subunit-subunit interactions. Another conserved feature is the distribution of charged amino acids in the eyelet region of the channel, with the basic side chains on one side of the channel and the acidic side chains on the other. Lysine 125 of PhoE, which is located on loop 3, has been shown to be primarily responsible for the difference in ion selectivity between PhoE and OmpF (Hancock et al., 1986). P. aeruginosa does not appear to have any general trimeric porins belonging to the porin superfamily (Jeanteur et al., 1994). It does, however, have a general porin OprF which is  related to OmpA of E. coli.  C. OmpA.  OmpA, one of the major outer membrane proteins in E. coli, has a copy number of about 1O per cell. The 325-residue mature protein is synthesized with a 21-residue signal sequence (Chen et al., 1980) and has a high 13-sheet content (Jeanteur et al., 1994). Unlike trimeric porins  10 which require heating in SDS to disassociate into monomers (as determined by the decrease in apparent molecular weight on SDS-PAGE), OmpA has only been observed as a monomer on SDS-PAGE and is thought to be a monomer in its native state (Saint et at., 1993; Nikaido, 1993). OmpA is associated both with the Braun lipoprotein and the peptidoglycan (Braun, 1975). OmpA contributes to the stability of the cell membrane and to the shape of the cell and appears to function as a porin. OmpA-deficient mutants were shown to have a lag in growth when grown in minimal media, but had growth rates only slightly less than wild-type strains (Manning et at., 1977). These strains did not grow as well at 42° C, especially in minimal media, and appeared to have reduced uptake of amino acids (Manning et at., 1977). Unlike either single mutant, double mutants, deficient in both OmpA and in the Braun lipoprotein, described in section 4a, were almost spherical, required cations for growth, but did not require osmotic protection (Sonntag et at., 1978). Electron microscopy showed that the outer membranes of these cells were blebbed. This was also seen in the lipoprotein mutants when they were starved for magnesium. The micrographs also indicated that the peptidoglycan of the double mutants was no longer associated with the outer membrane. OmpA has been shown to be non-covalently associated with both the peptidoglycan and the peptidoglycan-bound Braun  11 lipoprotein (Endermann et at., 1978; Lugtenberg and van Alphen, 1983). The double mutants were also shown to be more susceptible to hydrophobic antibiotics and detergents (Sonntag et at., 1978). Recently, OmpA has been shown to function as a porin by two different groups. Sugawara and Nikaido (1992) showed that OmpA functioned as a nonspecific-porin for small molecules in the osmotic swelling of proteoliposomes. They estimated the size of the channel was similar to that of OmpF, about 1 nm in diameter, but that the rate of diffusion was about 100 times less. Saint et at. (1993), used a black lipid bilayer to show that OmpA has a diameter of about 0.6-0.7 nm and was weakly anion selective. OmpA has also been shown to function as a receptor for several phages, for the action of colicins K and L (Morona et aL, 1984), and to be involved in F’-mediated conjugation (Morona et at., 1984; Ried and Henning, 1987). This has proved to be useful for the construction of a secondary structure model. Morona et al. (1984) determined that mutations in regions containing residues 25, 70, 110 and 154 resulted in phage resistance and proposed a model in which the N-terminal domain of the protein was composed of eight anti-parallel 13-strands with the regions noted above exposed on the surface of the cell. The susceptibility to cleavage in intact cells of trypsin-cleavage sites inserted into these regions is consistent with this model (Freudl et at., 1986; Freudl, 1989).  12 The same approach has been used to confirm the location of the periplasmic turns (Ried et al., 1994). The N-terminal domain of OmpA terminates with a region that is required for insertion of OmpA into the outer membrane (Morona et at., 1984) and has sequence homology with the last strand of the porin super family (Jeanteur et al., 1991). This sequence identity followed by a proline-rich region which resembles the hinge region of immunoglobulins (Chen et at., 1980) and is the proposed site of the characteristic trypsin cleavage site. The C-terminal domain of OmpA has been shown to be inessential for the association of the N-terminus with the outer membrane or with its ability to function as a phage and colicin receptor (indicating that the N-terminus was inserted into the membrane in the same conformation as the wild-type protein) (Bremer et at., 1982). The C-terminal domain has been shown to be the immunodominant portion of the protein in experiments using purified OmpA, or intact E. coli as the antigen (Puohiniemi et at., 1990). In the secondary structure model of OmpA, the C-terminal domain is located entirely in the periplasm. This assignment was made primarily because, unlike the N-terminal domain, which is protected by the outer membrane, the C-terminal domain is completely cleaved by proteases (Chen et at., 1980). In these experiments only the N-terminal region remained associated with the outer membrane (Chen etal., 1980).  13 OmpA-like proteins are present in E. coli as well as other enteric and nonenteric bacteria including P. aeruginosa Studies have used the .  property of heat-modifiability, which is the increase in the apparent molecular weight of the protein due to the unfolding of the protein when boiled in SDS or denatured in trichioroacetic acid (TeA), phenol or urea, to identify these related proteins (Beher et al., 1980; Spinola et al., 1993). Beher et at. (1980) identified heat modifiable proteins of similar molecular weight in twenty three strains of enteric and non-enteric Gram-negative bacteria. Of the strains tested, cleavage of these proteins with trypsin resulted in a characteristic membrane-bound fragment. Also, all of the proteins from the strains of Enterobacteriaceae were antigenically related. Primary structure analysis has shown significant C-terminal homology with a number of proteins including OmpA proteins in strains of E. coli (Bremer et at., 1982), Salmonella typhimurium (Freudl and Cole, 1983), and Shigella dysenteriae (Braun and Cole, 1984), PAL of E. coli (Hardham and Stamm, 1994), P6 of Haemophilus influenzae (Nelson et at., 1988), Pill of Neisseria gonorrhoeae (Gotschlich et al., 1987), a 31 kDa protein of Haemophilus somnus (Won and Griffith, 1993), a 21 kDa protein of Bordetella avium (Gentry-Weeks et at., 1992), TpN5O of Treponema  pallidum (Hardham and Stamm, 1994) and OprF of P. aeruginosa (Woodruff, 1988). The homology of the C-terminal portion of OmpA from E. coli with OprF includes 56 identical amino acid residues and 36  14 conservative substitutions in the 180 C-terminal amino acids of OprF and in the corresponding 168 amino acids of OmpA (Woodruff, 1988). Only two regions in the C-terminal portion of these proteins had no apparent homology: the region located between amino acids 170-200 containing four cysteines in OprF and the region between the two cysteines of OmpA, amino acids 290 and 300.  D. OprF.  As well as being related to the OmpA family, OprF of P. aeruginosa is also part of the OprF family of the fluorescent Pseudomonadaceae. All nine members tested from the rRNA homology group I contain an OprF like protein as determined by Southern blotting with a P. aeruginosa oprF probe (Ullstrom et al., 1991). This group of strains have also been shown, by Western immunoblotting, to bind at least two of the ten OprF specific monoclonal antibodies tested (Martin et al., 1993). Restriction mapping and Southern blotting of 17 serotypes and 42 clinical isolates of P. aeruginosa indicated that one copy of the gene was present in all of  these strains and that the restriction pattern was generally conserved (Ullstrom et al., 1991). The exceptions to this were serotype 12, which had been sequenced by Duchene et al. (1988) and one clinical strain, both of which contained an additional KpnI site in the C-terminal half of the gene (Ullstrom et al., 1991). Comparison of the DNA sequence of  15 H 103, our laboratory wild-type strain which is serotype 5, with that of the serotype 12 strain showed only 16 nucleotide changes, all of which  are silent (Martin et al., 1993). The OprF from two other species of rRNA homology group I have also been sequenced. These are the OprF from the plant pathogen, P. syringae, and from the plant growth-promoting rhizobacteria, P.  fluorescens. Comparison of the DNA sequences of oprF from P. aeruginosa and P. syringae showed 72% identity while the regions  flanking the genes had only 34% identity (Ulistrom et al., 1991). The Cterminal regions had even higher homology with 85% identity and 10% conservative substitutions. Included in this homology is the proline-rich hinge region also present in OmpA, and the region containing the four cysteines. OprF appears to be conserved within strains of P. syringae. Nine strains representing seven different pathovars of P. syringae had an OprF-like protein of the same molecular weight as that of P. aeruginosa  and all were heat and 2-mercaptoethanol modifiable and antigenically cross reactive. Like OprF from P. aeruginosa, OprF from P. syringae reconstituted both large and small channels as determined by black lipid bilayer (Ullstrom et al., 1991). The oprF sequence of P. fluorescens codes for a mature protein of 302 amino acid residues, smaller than the 326 and 320 of P. aeniginosa  and P. syringae, respectively (De Mot et al., 1992). An amino acid  16 sequence comparison of these three OprF molecules showed 71 % identity in the C-terminal half and only 35 % in the N-terminal half. Notable differences are the longer proline-rich region in P. fluorescens OprF and the lack of the cysteine-containing region of P. aeruginosa and P. syringae OprFs. It has been suggested that this region may be related  to the root-adhesive properties of this species (De Mot et al., 1994). Analysis of protease cleaved peptides of OprF from whole cells or outer membrane samples of P. fluorescens located the proline-rich region near the surface of the cell, and retention of the resulting fragments in the outer membrane indicated that the C-terminal domain is not located solely in the periplasm but loops through the membrane (De Mot et al., 1994). Another member of the OprF family appears to be the CD protein from Branhamella catarrhalis, a human pathogen. This 453 amino acid protein is heat modifiable and has surface-exposed epitopes (Murphy et al., 1993). It has significant amino acid homology with the OprF family having -35% identity and 55-61% similarity (varying with the strain compared) (Murphy et al., 1993). It has two cysteines which correspond to cysteines 3 and 4 (numbered from the N-terminus) in P. aeruginosa and P. syringae OprF. OprF, with 200,000 copies per cell, is one of the major outer membrane proteins of P. aeruginosa. OprF is synthesized with a signal  17 sequence of 24 amino acids residues; the mature protein consists of 326 amino acid residues (Duchene et aL, 1988). The oligomeric structure of OprF is controversial. Unlike the trimeric porins which are readily isolated as trimers and which require the native oligomeric conformation to function as porins (Hancock, 1987), the majority of OprF is isolated as monomers which have porin function (Woodruff and Hancock, 1988). Oligomeric OprF have been observed on Western immunoblots, albeit in small amounts (Mutharia and Hancock, 1985), and chemical crosslinking showed that purified OprF could form dimers and trimers (Angus  and Hancock, 1983). It can be expressed in E. coli under control of its own promoter, on a low copy number vector (Woodruff, 1988) due to the similarity of the RNA polymerase binding site sequence to that of the E. coli RNA polymerase binding site consensus sequence (Duchene et al., 1988). In addition to their size, high copy number, similar promoter sequence and high degree of C-terminal homology, as described above, both OprF and OmpA are heat modifiable (Woodruff, 1988), immunologically cross reactive (Woodruff and Hancock, 1989; Martin, 1992), and peptidoglycan associated (although OprF is released from the peptidoglycan at lower temperatures than OmpA) (Lugtenberg and van Alphen, 1983). Both also have a characteristic trypsin cleavage site, although that of OprF appears to be less accessible in outer membranes (Mutharia and Hancock, 1985). Trypsin digestion of outer membrane  18 samples showed that OmpA is easily cleaved (Chen et al., 1980) but, even with ten times more enzyme, OprF is only partially cleaved (Mutharia and Hancock, 1985). Both OmpA and OprF are bifunctional; they are general-diffusion porins and have a role in the structural integrity of the cell. The function of OprF has been studied, in part, by the construction of OprF-deficient strains by chemical mutagenesis (Gotoh et al., 1989a; Nicas and Hancock, 1983) or by c’-cartridge insertional mutagenesis (Woodruff and Hancock, 1989). The effect of the loss of OprF on the structural integrity of these strains was shown in several ways. These strains grew poorly in low osmolarity media (Gotoh et al., 1989a; Woodruff and Hancock, 1989) and were more sensitive to osmotic shock as measured by a decrease in viability (Gotoh et al., 1989a) or by periplasmic leakage as measured by an increase in extracellular f3-lactamase activity (Gotoh et al., 1989a; Woodruff and Hancock, 1989). Electron micrographs of osmotically shocked cells have shown blebbing and plasmolysis of OprF-deficient cells (Gotoh et al., 1989a; Gotoh et al., 1989b). Even in media with an osmolarity optimal for growth, the leakage of 13-lactamase from the periplasm was higher in the OprF-deficient strains than in the wild-type strains (Gotoh et al., 1989a; Woodruff and Hancock, 1989), as was the uptake of the hydrophobic probe, N-phenylnapthylamine (NPN) (Woodruff and Hancock, 1989).  19 The cell shape was also shown to be affected by OprF. OprF deficient cells have been shown to be 67% of the length of the parental strain by image analysis (Woodruff and Hancock, 1989), or to be shorter  and wider than the wild-type strain by electron microscopy (Gotoh et al., 1989a). The expression of cloned OprF in the almost spherical E. coli lpp ompA strain, described in the previous section, resulted in an increase in cell length of 43% (Woodruff and Hancock, 1989). Hardham and Stamm (1994) confirmed these results and also showed that cloned TpN5O, an OmpA-like protein from T. pallidum, expressed in this strain could also substitute for OmpA, resulting in wild-type length cells and growth at 42° C (Hardham and Stamm, 1994). As well as having a role in the structural integrity of the cell, OprF also functions as a general diffusion porin, allowing the uptake of hydrophilic compounds (Hancock et at., 1990). The exclusion limits of both P. aeruginosa and OprF have been controversial. Evidence for a low exclusion limit of P. aeruginosa includes that it has a greater resistance to some antibiotics than E. coli (Gotoh et at., 1989a) and the results of plasmolysis (Caulcott et at., 1984; Yoneyama and Nakae, 1986) and liposome swelling studies using whole membranes (Yoshihara et at., 1988). Evidence for a high exclusion limit include its ability to grow on pentamethionine (Miller and Becker, 1978) and susceptibility to some large antibiotics (Siehnel et at., 1989). Nikaido et at. (1991) showed that  20 tetrasaccharides were able to diffuse across isolated outer membrane by the osmotic swelling of proteoliposomes that had incorporated pieces of outer membrane. Bellido et at. (1992) showed that a wild-type strain provided with the ability to transport and metabolize raffinose and related sugars was able to grow on both a disaccharide and a trisaccharide. This study also demonstrated plasmolysis with a tetrasaccharide of 660 daltons. These experiments indicated that the exclusion limit of P. aeruginosa was greater than that of E. coli which excludes tetrasaccharides (Bellido et at., 1992). The exclusion limit of purified OprF is also controversial. Liposome swelling assays with purified OprF indicated that the exclusion limit was low, excluding even disaccharides (Yoshihara and Nakae, 1989). This work was criticized by Nikaido et al. (1993) for imprecise experimental procedures. Nikaido et al. (1991) showed that tetrasaccharides were able to diffuse through OprF by the osmotic swelling of proteoliposomes. Bellido et al.( 1992) showed that the growth rate of an OprF deficient-strain was three to five fold lower than the wild-type on a disaccharide and a trisaccharide indicating that OprF has a role in the uptake of these sugars. Earlier radioefflux experiments (Hancock et at., 1979) and liposome swelling assays (Yoshimura et at., 1983) also supported a large exclusion limit. Purified OprF’ was shown to have both large (4-7 nS) and small (0.360.38 nS) pores in black lipid bilayer experiments (Benz and Hancock,  21 1981; Woodruff and Hancock, 1988), with the majority of the channels being small (Woodruff and Hancock, 1988). Two hypothesis have been proposed to explain the contradictory evidence of the large channel size of OprF and the general low permeability of the cell, both of which suggest that OprF functions poorly in the uptake of some substrates like antibiotics. Woodruff et al. (1989) proposed that less than 1% of the of OprF formed large channels, therefore limiting the access of larger molecules. In this paper, it was also proposed that the difference in the size of the pore could be due to alternate bonding of the four disuiphides present in OprF. Nikaido et al. (1991) proposed that despite the large exclusion limit of OprF, it was the structure of the channel of OprF that limited the uptake of some solutes. Although the C-terminal domain of OprF has a high degree of amino acid homology with OmpA, calculation of the antigenic index, which is based on measures of secondary structure and on predictive methods, of the N-terminal domain of these two proteins showed a better correlation for these regions than for the C-terminal domains (Martin and Hancock, 1990). A secondary structure model of OprF (Wong et al., 1993) shows the N-terminal domain of OprF composed of eight f3 strands in the same basic configuration as the model of OmpA. The location of C-terminal region of OprF however, differs significantly from the model proposed for OmpA. The model of OmpA located the entire C-terminal  22 domain of the protein in the periplasm, while that of OprF was composed of transmembrane f3 strands and included surface-exposed loops. This conformation was primarily based on the location of permissive sites for linker insertion mutagenesis (Wong et al., 1993), for protection of outer membrane samples from cleavage by trypsin and by surface accessibility of the monoclonal antibody, MA5-8 (Mutharia and Hancock, 1985).  E. Aims of this study.  The aims of this study were to study the structure and function of OprF. The study of the function of proteins often includes mutation of the protein and analysis of the resulting phenotype. This is ideally confirmed by genetic complementation. In this study cloned OprF was returned to an OprF-deficient strain of P. aeruginosa and the resulting phenotype assessed. The conserved sequence of the C-terminal domain of OprF indicates that it has an important function in the cell. To study the role of the C-terminal portion of OprF in the integrity of the outer membrane, truncated mutants of OprF were constructed and expressed in P. aeruginosa. A number of approaches were used to gain a greater understanding of the secondary structure of OprF. These included the mutation of cysteines and the location of the epitopes binding OprF specific monoclonal antibodies both in the primary and secondary structure of OprF.  23 MATERIALS AND METHODS  A. Bacterial strains and plasmids.  The strains of E. coli and P. aeruginosa used in this study are listed in Table I and the plasmids used are listed in Table II.  B. Media and growth conditions.  1. In vitro growth. E. coli strains were grown on Luria Broth (LB) (1% tryptone,  0.5% yeast extract, 1% NaC1, pH 7.0), and P. aeruginosa strains were grown on Mueller-Hinton broth or on Luria Broth High Salt (LBHS) (1% tryptone, 0.5% yeast extract, 200mM NaC1, pH 7.0). The low osmolarity media used in growth experiments was either PP2 (1% proteose peptone no. 2) or Luria Broth No Salt (LBNoS) (1% tryptone, 0.5% yeast extract, pH7.0). Media were solidified with 2% Bacto-agar. All media components were from Difco Laboratories, Detroit, Michigan. Selective media used antimicrobial agents at the following concentrations for E. coli: tetracycline at 25 pg/ml, ampicillin at 75 ig/ml, kanamycin at 25 j.ig/ml; and for P. aeruginosa: tetracycline at 200 pg/mi, kanamycin at 250 jig/mi, streptomycin at 500 jig/ml, and carbenicillin at 300 jig/ml. Plates were spread with 50 jil of dimethylformamide containing 50 jig of  24 Table I: Bacterial Strains. Strain  Characteristics  Reference  E. coliK-12  DHSci.  endAl hsdRl 7 supE44 thi-1 recAl gyrA96 BRL, Burlington, Ont. relAl A(argF-1acZYA)U169 4)80 dlacZzM15  C441  Kans has helper function on ,  Simon et al., 1983  chromosome C441a  C44 1/  pWW2500  This study  P. aeruqinosa  H103  PAO1 Cmr prototroph; wild type  Hancock and Carey, 1979  reference strain H636  H103 oprF::  Woodruff and Hancock, 1988  M-2  strain isolated from GI tract of normal  Stieritz and Holder, 1975  mice M-2F-  M2oprF::)  Thisstudy  PA1O3  pathogenic strain  Mutharia and Hancock, 1983  ATCC-33348- 17 serotype strains of International 64  Antigen Typing Scheme  Benz, R. 1994  25 Table I. Bacterial Strains (continued).  CF832  Clinical isolate from a Cystic Fibrosis Patient Rivera and McGroarty, 1989  CF1452 CF2314 CF3660-1 CF6094 CFLaughlin CF4349 CF1278 CF22 1 CF3790 CF9490 H496-H510 Environmental isolates  Hancock and Chan, 1988  26 Table II: Plasmids. Name  Characteristics! Origin  Reference  pHP45C2-Tc  pHP45 + 2.0 kb fragment coding for TcrQ cartridge  Fellay et al., 1987  pUCP19  pUC19 + 1.8 kb P. aeruginosa stabilizing fragment  Schweizer, 1991  pWW2500  pRZ 102 + 2.5 kb Sail fragment containing Woodruff and Hancock, 1988 the N-terminal half of oprF with the Smr Q cartridge inserted into the Smal site  pWW5  pUC8 + 2.0 kb fragment from pWW4 Woodruff and Hancock, 1989 coding for truncated OprF (a.a. 17 1-300 of OprF were deleted by homologous recombination)  pRW5  pUCP19 + 1.47 kb HinDIII-EcoRI fragment from pRW3 coding for OprF  pRW3O7  linker -insertion at a.a. 188 of pRW3 Wong et al., 1993 (pTZ19R + oprF with a weakened promoter)  pRW3O9  as above, with the linker inserted ata.a. 210  Wong et al., 1993  pRW3 10  as above, with the linker inserted at a.a. 215  Wong et al., 1993  pRW31 1  as above, with the linker inserted ata.a. 231  Wong et al., 1993  pRW3 12  as above, with the linker inserted at a.a. 290  Wong et al., 1993  pER1O2  pRW5 deleted in the 1.1 Kprtl fragment coding for the C-terminal 2/3 of OprF  This study  pERlO2t  pER1O2  2 kb fragment coding for Tcr  This study  pER163  pUCP19 + 0.6 kb HinDIII-EcoRI fragment with a translational stop signal following a.a. 163  +  R. Wong, unpublished results  This study  27 Table II: Plasmids (continued). pERl63t  pER163  +  2 kb fragment coding for Tcr  pER17O-26  pER1O2  +  0.7kb KpnI fragment from pWW5 This study  pER17O-26t  pER17O-26  pER188  pUCP19 + 1.47 kb HinDIII-EcoRI fragment This study coding for the N-terminal 188 a.a. of QprF constructed by linker insertion mutagenesis of pRW3O7  pER 188t  pER 188  pER2 13  pUCP19 + 1.47 kb HinDIII-EcoRI fragment This study coding for the N-terminal 210 a.a. of OprF constructed by linker insertion mutagenesis of pRW3O9  pER2l3t  pER21O  pER215  pUCP19 + 1.47 kb HinDIII-EcoRI fragment This study coding for the N-terminal 215 a.a. of OprF constructed by linker insertion mutagenesis of pRW3 10  pER2l5t  pER215  pER231  pUCP19 + 1.47 kb HinDill-EcoRl fragment This study coding for the N-terminal 231 a.a. of OprF constructed by linker insertion mutagenesis of pRW3 11  pER29O  pUCP19 + 1.47 kb HinDIII-EcoRI fragment This study coding for the N-terminal 290 a.a. of OprF constructed by linker insertion mutagenesis of pRW3 12  pER29Ot  pER29O  +  2 kb fragment coding for Tcr  This study  pER  pUCP19  +  2 kb fragment coding for Tcr  This study  pER326t  pRW5  2 kb fragment coding for Tcr  +  +  +  +  +  This study  2 kb fragment coding for Tcr This study  2 kb fragment coding for Tcr  2kb fragment coding forTcr  2kb fragment coding forTcr  This study  This study  This study  This study  28 Table II: Plasmids (continued). pERC 185s  pRW5 0.7 kb HindIII-SalJ fragment + corresponding fragment with a..a. 185 mutated from a cysteine to a serine  This study  pERC191s  pRW5 0.45 kb SalI-EcoRI fragment + corresponding fragment with a.a. 191 mutated from a cysteine to a serine  This study  pERC185s+ C 19 is  pERl85s SalI-EcoRI fragment + corresponding fragment from pERC 19 is  This study  -  -  -  29 5-bromo-4-chloro-indolyl-J3-D-galactoside when manipulating pUCP 19based plasmids. 2. In vivo growth.  The in vivo chamber model, developed by Day et al. was used for the growth of P. aeruginosa in mice. Briefly, a Millipore filter (0.2 iim pore size) was glued on one end of a chamber made from a 1-cm section of a 1-cc plastic syringe barrel. Bacteria, resuspended in 0.9% saline at 10 cells per ml, as assessed by total count in a Petroff-Hausser bacteria counter, were added to the chambers and a second filter was attached to the open end. Four chambers were surgically implanted in the peritoneal cavity of B6D2 Fl mice. The chambers were removed from the mice after 4, 8, 16, 20, 24 or 48 hours after implantation and plated for a viable count.  C. DNA procedures.  1. Plasmid isolation. Plasmid DNA was routinely isolated by an alkaline lysis method (Sambrook et al., 1989). Plasmid DNA was isolated for sequencing either using Quiawell-8 plasmid purification system (Quiagen Inc., Chatsworth, CA.) or by the modified alkaline-lysis/PEG precipitation procedure described in the manual for the Taq DyeDeoxy terminator cycle sequencing kit from Applied Biosystems Inc. (Mississauga, Ont.).  30 Routine screening of transformants for inserts or for the presence of plasmid DNA was by the slot-lysis method of Sekar (1987). 2. Plasmid introduction. a.) Transformation Competent E. coli were prepared by the CaC1 2 method and transformed as described in Sambrook et al. (1989). P. aeruginosa was transformed by the method of Olsen et al. (1982). This method is similar to the CaC1 2 method used for E. coil, the main difference being the use 0.15 M MgCi 2 for the preparation of the cells instead of CaC1 . 2 b.) Electrop oration The procedure used for the electroporation is described in Farinha et al. (1990). The buffer used for preparation of the cells was 15% (w/v) glycerol in 1mM Hepes- 1mM MgCi . Cells were frozen in 2 aliquots at -70° C until required. The plasmid DNA was prepared by an alkaline lysis method (Sambrook et al., 1989) repeating the rinsing of the DNA in 70% ethanol to reduce the salt content. Either 2.5 or 5 jil of DNA was added to 50 jii of the prepared cells. The samples were gently mixed, and 25 il was added to each chilled cuvette. After 10 mm  incubation on  ice, the samples were electroporated. The settings used were 1.8 kV, 2002, and 25 pP in cuvettes having a 0.1 cm electrode gap. The equipment used was a Gene Pulser (Blo-Rad Laboratories, Richmond, CA). Immediately after the electroporation, high salt media containing 50  31 2 was added to the cuvette. The cells were then gently mM MgCl  suspended, and allowed to grow at 37°C for 1.5-1.75 hr before plating on selective media. c.) Biparental mating The method used for biparental mating was based on the method described in Goldberg and Ohman (1984). Overnight cultures were grown in broth with shaking at 37°C for the donor strain and 42°C for the recipient strain. Aliquots of 0. lml of both the donor and the recipient strains were added to 2 ml of fresh broth. The mixture was then filtered through a 0.45 jim membrane. The membrane was placed cell side up on a non-selective agar plate and incubated overnight at 30°C. The cells were then washed off the membrane with fresh media, diluted, spread onto selective plates and incubated at 30° C for 1 to 2 days. 3.)-cartridge mutagenesis of oprF. The method used for the -cartridge mutagenesis of P. aeruginosa M-2 was basically the same as described in Woodruff and  Hancock (1988) and utilized the plasmid pWW2 500 described in that paper. In this study, biparental mating was done using the donor helper strain C441. 4. Southern blotting. a.) Chromosomal DNA isolation  32 Chromosomal DNA was isolated by the method described in Current Protocols In Microbiology (1987) and quantified by measurement of the absorption at 260nm. b.) Labeling of DNA probe The DNA probe was made by isolating a 1.47 kb HinclIII/EcoRI fragment from pRW5 coding for oprF. This fragment was  labeled with biotin using a Bionick Labeling System from BRL (Burlington, Ont.). c.) Southern blotting Chromosomal DNA was digested with either PstI or Smal and the fragments separated by electrophoresis in an agarose gel. The DNA was transferred to Biotrans nylon membrane (ICN Biomedicals Canada Ltd., St. Laurent, Quebec) by capillary transfer as described in Current Protocols in Molecular Biology (1987). The PhotoGene Nucleic Acid Detection System (BRL, Burlington, Ont.) was used as described in the manufacturer’s instructions for the remainder of the procedure. 5. Oligonucleotide synthesis. Oligonucleotides were synthesized on a Applied Biosystems 392 DNA/RNA Synthesizer (Mississauga, Ont.) exactly as described in the manufacturers instructions.  33 6. Polymerase Chain Reaction. For the construction of the truncated OprF mutants by Polymerase Chain Reaction (PCR), the buffer used was 20 mM Tris, pH 8.3, 1.5mM MgC1 , 25mM KC1, 0.5 %Tween and 1 mg/mi gelatin. To 2 this 5% formamide, 1.25 units Taq polymerase (BRL, Burlington, Ont.), 250 jiM dNTP’s (BRL)  ,  and ijiM each of the 5’and the 3 primers  (described in Chapter 1) were added. DNA was amplified in an MJ Research thermal cycler (MJ Research, Watertown, Mass) at 95° C for 15 seconds, 55° C for 30 seconds and 72° C for 90 seconds and this cycle was repeated 30 times. The product was gel purified and digested with the appropriate restriction enzymes for ligation into pUCP19. Vent DNA polymerase (Fisher Scientific, Vancouver, B.C.) was used for the mutagenesis of two of the cysteines of OprF. The primers used are described in Chapter 1. The procedure followed was exactly as above except for the use of the supplied buffer. 7. DNA seQuencing. DNA was sequenced with an Applied Biosystems Incorporated (ABI, Mississauga, Ont.) automated fluorescent sequencing system, model 373A, and analyzed with an ABI 675 sequence-editor program. The polymerase chain reaction and dye-terminator chemistry was used as described in ABI’s protocols.  34 D. Protein procedures.  1. SDS-PAGE. Proteins were prepared for electrophoresis by solubilization in 2% (w/v) SDS with or without the addition of 2% (v/v) 2mercaptoethanol. Samples prepared from whole cell lysates of P. aeruginosa were then sonicated 3 times for 15 seconds in a sonicating  water bath. Samples were then heated to 1000 C for 10 mm. Heat treated samples were boiled for 30 mm TCA for 30 mm  in SDS or treated with 5% (w/v)  on ice (Hancock and Carey, 1979). Proteins were  separated by electrophoresis with 11%, 12% or 14% polyacrylamide gels as previously described (Hancock and Carey, 1979). The gels were stained with Coomassie Brilliant Blue R250 (Bio-Rad) for visualization of the proteins or used for Western immunoblotting. 2. Outer Membrane Preparation. Outer membranes were prepared by the method of Hancock and Carey (1979). Briefly, after centrifugation, mid-log phase cells were broken with a French pressure cell. The resulting lysate, in 20 % (w/v) sucrose, was loaded onto a 50/60 or 50/70% sucrose gradient and centrifuged in a swinging bucket rotor. The lower band containing the outer membrane was collected, suspended in distilled water and pelleted by ultracentrifugation.  35  3. Preparation of peptidoglycan-associated proteins. Peptidoglycan-associated proteins were prepared by the method of Hancock et al. (1981). Briefly, outer membrane preparations were suspended in 2% Triton X- 100 in buffer and sonicated. The insoluble membrane was pelleted by ultracentrifugation. The pellet was suspended as above with the addition of 10 mM EDTA. This step was repeated resulting in the Triton-EDTA soluble fraction. The remaining pellet was digested with lysozyme and diluted with the Triton-X- 100 in buffer with the addition of 10 mM MgSO , sonicated and pelleted as 4 above. The supernatant was called the Triton-lysozyme soluble fraction. 4. Determination of protein concentration. A modified Lowry assay was used to determine protein concentrations (Peterson, 1977). This method uses SDS to solubilize membrane proteins and also denatures the proteins making the results more reproducible.  E. Immunological techniques.  1. Antibodies OprF-specific monoclonal antibodies MA7-1, MA7-2, MA7-3 (designated IG1 in Pennington et al., 1986) MA7-4, MA7-5, MA7-6, MA77, and MA7-8 were prepared at Oncogen (Seattle, WA) and are described in Pennington et al. (1986). These antibodies were prepared either by  36 injecting mice 4 times with purified OprF in combination with J5 LPS or by extensive immunization over a 6 month period with a variety of antigen preparations which included heat killed P. aeruginosa ATCC 27318, LPS prepared from this strain, live P. aeruginosa ATCC 27317, and then finally injected with outer membranes in combination with purified OprF. Monoclonal antibodies MA4-4 and MA5-8 were made by L. Mutharia et al. (1983) using purified OprF or outer membranes prepared from H 103. FPLC-purified OprF was used to make the polyclonal rabbit and polyclonal mouse sera as described in Rawling et al. (1995). 2. Colony immunoblotting. Bacterial colonies, grown overnight on agar plates, were transferred to nitrocellulose by contact. The filters were hung in a chloroform-vapor-saturated chamber for 30 mm  to allow cell lysis. The  filters were then washed in a buffer containing 3% (w/v) bovine serum albumin (BSA) in phosphate buffered saline (PBS) with 150 mM NaCl, 5 mM MgC1 , 1 jig/mL DNAase I, and 40 j.ig/mL of lysozyme. After rinsing 2 3 times with PBS the filters were incubated with primary antibody (diluted 1:2000 in 1% BSA in PBS) for 2 h at 37° C with shaking. After washing 3 times with PBS, the secondary antibody, goat anti-mouse immunoglobulin G alkaline phosphatase conjugate (BioRad), used at a dilution of 1:2000, was added and incubated for 2 h at 37°C with  37  shaking. After washing 3 times in PBS, the filters were washed in 0.1 M Tris-HC1 pH 9.6 and then developed with the substrate solution (50 pg/ mL 5-bromo-4-chloro-3 -indolyl-phosphate, 10 ig/ mL nitroblue tetrazolium, and 40 jig/mL MgC1 2 in 0.1 M Tris-HC1 pH 9.6). 3. Western immunoblotting. After separation by SDS-PAGE, proteins were electrophoretically transferred to nitrocellulose from the polyacrylamide gel using the BioRad Trans-Blot electrophoretic transfer cell, with a cooling pack, at a constant voltage of bOy for 1 h. The buffer used for transfer contained 25 mM Tris-HC1, 192 mM glycine, and 20% (v/v) methanol, pH 8.3. After transfer, proteins were immunologically detected as described above for colony immunoblots. 4. Immunofluorescent labeling. Cell surface indirect immunofluorescent labeling of cells was performed as described by Martin et al. (1993). Briefly, midlogarithmic cells were collected by centrifugation, washed with PBS, allowed to air dry on a glass slide, and then fixed in 100% ethanol. The fixed cells were incubated with an OprF-specific monoclonal antibody diluted 1:100 in PBS with 1% fetal calf serum for 30 mm  at room  temperature. After washing with PBS, the secondary antibody (fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin (Sigma Chemical Co.) was added at a dilution of 1:20. After washing and  38 the addition of a drop of Sigma Mounting Medium, a cover slip was sealed on the slide with the use of clear fingernail polish. The slides were examined with a Zeiss microscope fitted with a halogen lamp and filters set for emission at 525 nm. 5. Opsonic phagocytosis. Opsonic phagocytosis was performed using a modification of the procedures of Battershill et al. (1987). Mouse peritoneal macrophages from 2-4 month old B6D2(fl) mice were incubated overnight in 8-well chamber slides (NUNC, Naperville, IL) to allow adherence. Antibody concentrations were standardized by an enzymelinked immunosorbent assay (ELISA) with purified OprF. The monoclonal antibody, at a dilution of 1:10, and the bacteria at a ratio of 20:1 (bacteria to macrophage, suspended in 20 jiL bacterial buffer-S mM Hepes and 1 mM MgC1 ), were added to the macrophage monolayer. The 2 plates were then incubated for 90 mm  at 37°C in a CO 2 atmosphere.  After rinsing, cells were stained with Diff-Quick (Canlab, Vancouver, B.C.) and viewed with a Zeiss microscope fitted with a lOOx oil immersion objective. The number of bacteria in 100 macrophages were recorded. Experiments were carried out on 3 different days, and the results from each day were tabulated and subjected  ,  separately, to the  Mann-Whitney test to determine the significant of differences from controls.  39 6. Overlapping-octapeptide analysis. Support-coupled overlapping octapeptides starting at every second amino acid position of OprF were purchased from Chiron Mimotopes (Clayton, Australia). The peptides, attached to polyamide pins in arrays of 96 pins, were used as antigens in ELISA studies in which the pins were inserted into ELISA plates as described previously (Geysen et al., 1987). The ELISAs were performed, in two independent trials, as described in Geysen et al. (1987) except that the absorbance was assessed periodically to ensure that readings were in the linear range of the machine. Positive and negative controls supplied with the kit were successfully performed (data not shown). Antibodies were used at dilutions of 1:10,000 to 1:2500 for the monoclonal antibodies and 1:5,000, 1:500, or 1:350 for the polyclonal antibodies. Antibodies were removed from the pins by sonication of the blocks for 10 mm  in a  solution of 0.1 M phosphate buffer, 1% (w/v) SDS and 0.1 % (v/v) 2mercaptoethanol preheated to 60° C. The blocks were rinsed at in 60° C O and then in methanol at 60°C. 2 dH  F. Cell-length measurement. 1. Image analysis. Bacteria were grown to mid-logarithmic phase (optical density of -0.5 at 600 nm.) in high osmolarity media. A sample was air  40 dried on a microscope slide, heat fixed and stained with crystal violet. Cells were viewed by oil immersion with a Zeiss Universal microscope using x 100 magnification. Images were digitized and analyzed with a SEM-IPS image analysis system (Kontron, Munich, Germany). 2. Microscopy. Bacteria were grown in high osmolarity media with 200 jig/ml tetracycline to mid-logarithmic phase (75 klett units) or to early logarithmic phase (40 klett units). Samples were prepared as described for image analysis. To prevent biased readings, measurement of the samples was done in a single-blinded fashion by S. Farmer. The samples, identified by a code number, were viewed at x 1000 magnification by oil immersion using a Zeiss IIIRS microscope fitted with phase rings and connected to a television monitor. Measurement of the cells was done directly from the monitor. At least 100 cells of each strain, from 3 different experiments, were measured and the results analyzed by Student’s t-test using the Bonferonni correction.  G. Growth studies.  Strains of P. aeruginosa, with wild-type or mutated OprF were assessed for their growth in low osmolarity media. Overnight cultures, grown in high salt media, were diluted 1:100 into high salt and low salt media, with or without the addition of antibiotics. When tetracycline was added, the media was warmed for 15 mm  to allow the evaporation of the  41 ethanol into which the tetracycline was dissolved. Cultures were grown in a shaking water bath at 37° C, and 160-180 rpm. Cell density was determined using a Klett-Summerson photometer with a green filter.  42 RESULTS  CHAPTER 1. CONSTRUCTION AND EXPRESSION OF OprF  MUTANTS IN P. aeruginosa  A. Introduction.  The function of outer membrane proteins can be studied through inactivation of the oprF by chemical mutagenesis, the insertion of transposons or interposons or by gene replacement, and an examination of the resulting phenotypes of the cell. Ideally, the mutation should be confirmed by genetic complementation. Although OprF-deficient strains of P. aeruginosa have been constructed by chemical mutagenesis (Nicas and Hancock, 1983; Gotoh et al., 1989a) and by transposon and interposon mutagenesis (Woodruff and Hancock, 1988), and the cloned gene expressed in E. coli (Woodruff and Hancock, 1989), attempts to complement this mutation in P. aeruginosa had been unsuccessful (Woodruff, 1988). In this study, a cloned variant of oprF, with a weakened promoter, was used to complement an OprF-deficient strain. Various mutants, including deletion mutants, have been used to study regions of OmpA (Maclntyre et al., 1988; Klose et al., 1988a; Freudl  and Henning, 1988; Klose et al., 1989; Ried et al., 1994; Tanji et al., 1991) and PhoE (Bosch et al., 1988) required for translocation across the  43 cytoplasmic membrane and for incorporation into the outer membrane. In this study, deletion mutants of oprF were constructed to determine the role of the C-terminal domain in the incorporation of the protein into the outer membrane and also to study its role in outer membrane stability, as assessed by growth in low osmolarity media and cell length. Cysteine to-serine mutants of OprF were constructed by site-directed mutagenesis and expressed in P. aeruginosa to quantitate the disulphide bond(s) present in the native OprF and to determine the identity of the cysteines that are bonded. The stability of the outer membranes of these strains was also assessed.  B. Construction of an OprF-deficient mutant of P. aeruginosa, strain M-2.  OprF-deficient strains constructed by chemical mutagenesis were shown to have a high frequency of reversion (Nicas and Hancock, 1983) and also exhibited phenotypic variation (Gotoh et al., 1989a). The generation times of three chemically-mutagenized OprF-deficient strains in a low osmolarity medium with 17 mM NaC1 were 452, 1,204 and 1,806 mm  and with 42.5 mM NaC1 was 125, 144, and 301 mm, respectively  (Gotoh et al., 1 989a). Comparison of insertionally mutagenized OprF deficient strains, constructed by Tn 1 mutagenesis or by -cartridge mutagenesis, also showed variation with generation times of 80 and 142 mm  in a low osmolarity medium with 50 mM NaC1 and generation times  44  of 128 mm  and no growth in a medium without added salt, respectively  (Woodruff, 1988). These data were consistent with the suggestion that the more strongly affected a-cartridge insertion mutant had the correct OprF-deficient phenotype and that the other mutants had suffered from phenotypic modulation due to secondary mutations. Woodruff and Hancock (1988) had constructed and characterized only one OprF deficient -cartridge mutagenized strain, H636 (Woodruff, 1988; Woodruff and Hancock, 1988). To ensure that the observed phenotype associated with this method of mutagenesis was not limited to the strain tested, a strain used for mouse pathogenicity studies (Stieritz and Holder, 1975), phagocytosis studies (Battershill et aL, 1987; Speert and Thorson, 1991), and killing assays (Speert and Thorson, 1991; Speert et al., 1994), strain M-2, was selected for mutagenesis. The plasmid, pWW2500, described in Woodruff and Hancock (1988) was used for the mutagenesis procedure (Figure 2A). Plasmid pWW2 500 contains an 2cartridge, which codes for resistance to streptomycin and has stop codons in all reading frames, inserted into the SmaI site of a Sail fragment coding for the N-terminal 60% of OprF. The Sail fragment is flanked by sequence coding for the 1S50 elements of the transposon Tn5 and confers kanamycin resistance. This plasmid is unable to replicate in P. aeruginosa and can undergo homologous recombination (Jorgenson et  al., 1979) between the plasmid oprF:2 and the chromosomal oprF. For  45  A Kn  Sail SmaI Q  pWW2500 16.2kb SmaI Sail  B  4  SmaI  Sail  oprF  Figure 2. A. Map of the plasmid pWW2500 showing the location of the Sail and SmaI sites and the Tn5  E]  oprF  , and 12-cartridge E]  DNA. B. Restriction map of oprF showing the 12 insertion site.  46 the mutagenesis of M-2, pWW2500 was first transformed into the E. coli helper strain C44 1. The resultant strain, selected on the basis of kanamycin resistance, was used for biparental mating with M-2. Transconjugants were first screened for streptomycin resistance, indicating the rescue of the a-cartridge by incorporation into the chromosome, and then for kanamycin sensitivity, indicating the loss of vector sequences, a result consistent with a double cross-over event. The site of the 2-cartridge insertion into oprF is indicated in Figure 2B. The loss of OprF was then determined by colony immunoblot using the OprF specific monoclonal antibody MA7- 1. To ensure that the )-cartridge was only inserted into the oprF gene, chromosomal DNA, isolated from the parent strain and the selected mutants, was digested with PstI and SmaI and analyzed by Southern blot using a biotin-labeled oprF probe. Figure 3 shows the hybridization pattern of three oprF :f mutants and the parent strain M-2. In the PstI digest, the probe hybridized to a 2.4 kb fragment from the strain M-2 (lane 1) and a 4.4 kb fragment from three M-2F- strains (lanes 3, 5, and 7), an increase of 2.0 kb which correlated with the insertion of the 2.0 kb Smal  fragment. In the Smal digest,  which released the -cartridge, the probe bound to identically-sized fragments in all strains (lanes 2, 4, 6, and 8). These results confirmed that the OprF-deficient strains of M-2 constructed by  mutagenesis  47  4.4kb  2.4kb  4  1  2  3  4  5  6  7  8  Figure 3. Southern blot of a biotinylated oprF probe hybridized to chromosomal DNA digests of P. aeruginosa strain M-2 (lanes 1 and 2)  and three M-2F- strains (lanes 3-8). The DNA was digested with PstI (odd numbered lanes), or with SmaI (even numbered lanes).  48 contained one copy of the )-cartridge inserted into the chromosomal oprF gene.  Restriction endonuclease mapping of a 5 kb region surrounding and including oprF in the 17 serotypes of P. aeruginosa has shown that, with the exception of serotype 12, there is strong conservation of the restriction endonuclease sites studied in and upstream of oprF and that there is some heterogeneity of the KpnI sites several kilobases downstream of oprF (Woodruff, 1988). This is consistent with the data presented here. The PstI fragment and the smaller of the two SmaI fragments from M-2 identified in the Southern blot were the same size as the corresponding fragments from H 103 (Woodruff and Hancock, 1988). However, the larger Smal fragment was 1.5 kb smaller than that from H 103 indicating that the downstream Smal sites, as well as the KpnI sites may be heterogeneous. No reversion of M-2F- or H636 was observed after growth in high-salt media for twenty four hours or in low salt media for eight hours.  C. Construction of truncated versions of OprF.  Attempts to clone oprF, and certain other outer membrane protein genes, into high copy number vectors have proven unsuccessful (Duchene et al., 1988; Woodruff, 1988). These constructs are thought to be lethal due to high levels of protein produced as a result of their  49 efficient promoters in combination with the high copy number of the plasmids (Woodruff, 1988). The weakening of the oprF promoter by sitedirected mutagenesis (Wong et at., 1993) permitted the cloning of oprF into a high copy number, 4.5 kb Escherichia-Pseudomonas shuttle vector, pUCP19 (Schweizer, 1991) resulting in the 6.0 kb plasmid, pRW5 (R. Wong, unpublished results). All plasmids constructed in this study were based on pUCP19 and maintained the weakened oprF promoter. Plasmids encoding truncated versions of OprF were constructed using three basic approaches. The first utilized a plasmid previously characterized in this laboratory, pWW5 (Woodruff et at., 1986). Sequencing of this plasmid had shown that amino acids 17 1-300 had been deleted (Finnen et at., 1992). A KpnI fragment, coding for amino acids 103-170 and 30 1-326, from pWW5 was used to replace the wildtype KpnI fragment from pRW5. Screening was done first by slot lysis, to select for insertion of the fragment, and then by colony immunoblot. Colonies selected were reactive with OprF-specific monoclonal antibodies MA7- 1 (N-terminus specific) and MA5-8 (C-terminus specific) (see Chapter 3, B) to ensure the correct orientation of the fragment. The resulting plasmid was designated pER17O-26, referring to the N-terminal 170 and the C-terminal 26 amino acid residues that it encodes (Figure 4). The second approach utilized plasmid constructs made by R. Wong in this laboratory by linker insertion mutagenesis (Wong et at., 1993).  50 100  KpnI  150 200  Sail  250  .‘.:.....:.:.  300  KpnI/EcoRI  mature protein  The encoded length of the truncated OprF are indicated below.  Figure 4. Map of oprF and OprF showing the restriction endonuclease sites and the length of the  pER 170-26  pER29O  pER231  pER215  pER188  pER163  pER1O2  pER3 26  PLASMIDS  H636 oprF:  H103 (wild-type)  STRAIN  Hindlil I  51 Strains constructed by this method were based on plasmid, pRW3, which encoded oprF variants with the mutated oprF promoter described above and had a 12 bp insertion, including a unique PstI site, inserted into different regions of the oprF sequence. An adapter oligonucleotide, with PstI overlaps and stop codons in all three reading frames, was designed  so that upon insertion the PstI site would be lost and an XbaI site introduced. Initial screening was done by colony immunoblotting. Colonies that were reactive with the N-terminus specific monoclonal antibody MA7- 1 and non-reactive with the C-terminus specific monoclonal antibody MA5-8 were selected for restriction endonuclease analysis. HindIII-EcoRI fragments were isolated from plasmids that lacked the PstI site and contained an unique XbaI site and then inserted into pUCP19. Plasmids constructed this way were designated pER188, pER213, pER215, pER231, and pER29O (Figure 4). The plasmid number indicates the number of amino acids that were encoded in the mutant protein. For example, pER 188 encodes the N-terminal 188 amino acids of OprF. This method of truncation resulted in the insertion of a varying number of nucleotides before the intended stop codon. The amino acids which they encoded varied according to the reading frame of the original insertion and are listed in Table III. Insertions at amino acids 188 and 215 added three amino acid residues: aspartate, leucine and histidine. Insertions at amino acids 213, 231, and 290 added six additional amino  52  Table III. Amino acid residues introduced by linker-insertion truncation of OprF.  amino acids  plasmids  insertion sites (amino acid)  pER188  188  DLH  DNV  pER2 13  213  TCTSLD  VQLDVK  pER215  215  DLH  LDV  pER231  231  TCTSLD  YADIKN  pER29O  290  TCTSLD  VNAVGY  a  amino acids inserteda  replaced  The amino acids listed are followed by the stop codon, TAG  53 acids residues: threonine, cysteine, threonine, serine, leucine, and aspartate. As none of the linker mutants described above had insertions near the prospective hinge (proline-rich) region of the protein, polymerase chain reaction was used to produce proteins truncated in this region. The primer for the coding strand included the HindIII site in order to facilitate cloning as well as for maintenance of the mutated promoter. The primers for the non-coding strand were designed to introduce a stop codon after a selected amino acid, followed by an EcoRI site to permit cloning. The resulting HindIII-EcoRI fragments were cut with the appropriate enzymes and cloned into pUCP19. Final selection was made by colony immunoblotting, selecting for colonies that were reactive with MA7-1. Two mutants were constructed in this way, pER158 and pER163 (Figure 4), but after sequencing it was shown that pER 158 contained several unintended mutations. Repetition of the PCR did not produce an accurate fragment and therefore this construction was not pursued further. The insert in pER163 was fully sequenced to ensure that it was free from errors. The addition of carbenicillin to growth media was required to maintain pUCP19-based plasmids in H636. However, the resulting filamentation compromised the study of cell length. To solve this problem, a 2.0 kb fragment coding for tetracycline resistance from  54 pHP45c2-Tc (Fellay et al., 1987) was inserted into the EcoRI site of selected plasmids. The control plasmid, pUCP19, with the added tetracycline resistance cartridge was named pER. The plasmid encoding the entire 326 amino acids of OprF was named pER326t. Other plasmids had a “t” added to their name to differentiate them from the initial constructs.  D. Site-directed mutagenesis of the cysteines of OprF.  The effect of heat and suboptimal concentrations of reducing agents on OprF indicated that the four cysteines of this protein form two disuiphide bonds (Hancock and Carey, 1979; Martin, 1992). SDS-PAGE showed OprF migrating at four different apparent molecular weights. The band with the lowest molecular weight corresponded with OprF that had not been modified by heat or by 2-mercaptoethanol. The protein with the highest apparent molecular weight was the heat-modified, fully reduced OprF. The remaining intermediate molecular weight bands appeared to be OprF that was not heat modified but that had either one or both of the disuiphide bonds reduced. Previous attempts to quantify the disuiphide bonds of OprF were done by biochemical methods. Martin (1992) found that the method of Thannhauser et al. (1984), in which the protein is denatured with guanidine thiocyanate and the broken disuiphide bonds measured in a  55 colourimetric assay, identified only one disulphide bond. The method of Iyer and Klee (1973), which measures the rate of reduction, showed that the disulphide bonds were more available in heat denatured samples and the method of Needleman et al. (1970) indicated that no free thiols were present. These results indicated that two disuiphide bonds may be present but not readily available for assay. In this study, a more direct approach was used to address this problem, the cysteines of OprF were replaced with serines. Site-directed mutagenesis, by PCR, changed the DNA sequence from TGC, encoding a cysteine residue, to TCC, encoding a serine residue. Two of the four cysteines, C 185 and Cl 91, (referring to their relative position from the Nterminus of the protein) were mutated using this method. For the construction of mutant C185S, the coding-strand primer was the same as that used in the construction of pER 163, and included the mutated promoter and the HindIII site (Figure 4). The primer for the noncoding strand contained a one-basepair mutation, changing the cysteine to a serine, and included the existing Sail site (Figure 4). The coding-strand primer for the construction of C 19 iS contained the Sail site and also had a one-base mutation changing the cysteine to a serine. The noncoding strand primer included the wild-type translational stop codon and added an EcoRI site to permit cloning. The resulting fragments were digested with the appropriate enzymes and cloned into a pRW5 derivative in  56 which the corresponding fragment had been removed. These plasmids were named pERC185S and pERC191S, respectively. A plasmid containing both of the mutated cysteines, pERC185S+C191S, was constructed by the replacement of the HindIII-Sall fragment from pERC19 iS with that from pERC185S. Following antibiotic selection, colonies were selected by colony immunoblotting that were reactive with monoclonal antibody MA7- 1, indicating that OprF was produced, and non-reactive with monoclonal antibody MA7-8. It has been shown that disulphide bond formation is required for the binding of MA7-8 (Finnen et al., 1992; Martin, 1992). Selected plasmids were sequenced to ensure  that the desired mutation was the only mutation present.  E. Expression of OprF mutants in P. aeruginosa.  Previous attempts to introduce cloned OprF into P. aeruginosa had been unsuccessful (Woodruff, 1988). This failure may have been due to plasmid copy number, as discussed in Chapter i C, or due to the use of conjugation as the method of plasmid introduction. Like OmpA (Havekes et al., 1976; Achman et al., 1978; Manoil and Rosenbusch, 1982) OprF  may have a role in the stabilization of mating pair formation (Nicas, 1983). In this study, two alternative methods of plasmid introduction were tested. The first utilized a transformation method modified for use with P. aeruginosa (Olson et al., 1982). Despite the high number of  57 transformants obtained with the control strain H 103 and plasmid pUCP19, no transformants were recovered using the OprF-deficient strain H636 and the plasmids pRW5 or pUCP19. There are a number of reasons why this method may have been unsuccessful including the repeated incubations on ice and/or the solution used to make the cells competent which contained only 0.15 M MgC1 . A preliminary study 2 indicated that H636 had a greater decrease in viability than H 103 when held on ice. Also, studies done at room temperature, for 1 hour, indicated that a buffer, pH 7.2, containing salt for osmotic protection as well as MgCl 2 was required to maintain the viability of H636, but not H103. The second method tested was electroporation. Farina et aL (1990) found that water and dilute ionic solutions resulted in extensive cell lysis during the preparation of P. aeruginosa for electroporation, and that the addition of glycerol solved this problem. The solution used for electroporation was composed of 15% (w/v) glycerol in 1mM Hepes- 1mM 2 and the incubations on ice were kept to a minimum. The high salt MgC1 medium used for the recovery period after electroporation included 50 mM MgC1 2 which had been shown to enhance P. aeruginosa recovery (M. Bains, personal communication). Initial selection was made by plating cells on a high osmolarity medium containing the appropriate antibiotic. The resulting colonies were shown to contain plasmids of the correct size by slot lysis. Figure 5 is a Western immunoblot of whole-cell lysates of  58  I  a.  4  1  2  3  4  5  6  7  8  9  10  11  Figure 5. Western immunoblot of whole cell lysates of H636 containing  plasmids coding for truncated OprF. The monoclonal antibody used was MA7-1. Lane 1, molecular weight markers (from top to bottom: 142.9, 97.2, 50.0, 35.1, 29.7, 21.9 kDa), lane 2, H 103/pER; lane 3, H636/pER; lane 4, H636/pERlO2t; lane 5, H636/pERl63t; lane 6, H636/pER17O-26t; lane 7, H636/pERl88t; lane 8, H636/pER2l3t; lane 9, H636/pER2l5t; lane 10, H636/pER29Ot; lane 11, H636/pER326t.  59 recombinant OprF and truncated versions of OprF expressed in H636. The antibody used was MA7- 1 which bound to an epitope localized to amino acids 55-62 (Chapter 3B). Strain H636 containing pERl63t (lane 5), pER17O-26t (lane 6) pERl88t (lane 7), pER2l3t (lane 8), pER2l5t (lane 9), pER29Ot (lane 10) or pER326t (lane 11) and the wild-type control H 103/pER (lane 2) produced protein(s) binding MA7-1 and, with specific exceptions, corresponding in relative mobility to the approximate number of amino acids encoded. These results show that the weakened promoter allowed the expression of the cloned full-length OprF and the truncated-OprF mutants in an OprF-deficient strain of P. aeruginosa. Unexpectedly, two bands reactive with MA7- 1 were seen with the strain H636/pER2 13t (lane 8). One was truncated as expected, but the other appeared to have the same molecular weight as the native protein. In some experiments H636/pER29Ot appeared to have the same molecular weight as the native protein (lane 10). This suggested that these plasmids were able to recombine into the chromosome, although it appeared that the plasmid was also retained in the case of H636/pER2 13t. This apparent recombination was not always observed with these constructs and was rarely observed with the other plasmids. The plasmids pER (lane 3), pERlO2t (lane 4) and pER23lt did not produce a protein detectable by MA7- 1 in the E. coli strain DH5c or in the P. aeruginosa strain H636 indicating either that these proteins had  60 not been synthesized or that they had been degraded. While this was expected for the negative control H636/pER, (H636 has an 2-cartridge inserted at amino acid 102) and H636/pERlO2t, it was surprising that a product was not observed in H636/pER23 it. Restriction mapping of this plasmid indicated that oprF was present and did not appear to be rearranged, but these results were not confirmed by sequencing. These results indicated that between 102 and 163 N-terminal amino acids of OprF are required for the production of a stable protein. The property of “heat modifiability” refers to an increased apparent molecular weight of a protein on SDS-PAGE when pretreated by boiling in SDS or treated with denaturants such as TCA or urea compared to the same protein solubilized at lower temperatures. When pretreated with TCA or boiled in SDS, the recombinant OprF, encoded on pER326t (Figure 6A, lane 3), pER29Ot and the OprF from the control strain, H 103, were all heat modifiable (Table IV). The truncated versions of OprF encoded on pERl63t, pER17O-26t, pERl88t and pER2l5t, however, had a decreased apparent molecular weight when pretreated with TCA or boiled in SDS (Table IV, examples in Figure 6B). A portion of each sample tested retained the apparent molecular weight of the untreated sample (Figure 6). This incomplete modification has been observed previously (Martin, 1992). These results indicated that between 215 and 290 amino acids are required for the protein to be heat modifiable.  61  A.  B.  123  1  234567  Figure 6. Western immunoblots of OprF and truncated OprF mutants with and without TCA pretreatment. Odd numbered lanes pretreated with TCA. A. Lane 1, molecular weight markers; lanes 2 and 3, H636/pER326t (lower—weight bands in lane 2 presumed to be frequently observed degradation products of OprF). B. Lane 1, molecular weight markers; lanes 2 and 3, H636/pERl63t; lanes 4 and 5, H636/pER17O26; lanes 6 and 7, H636/pERl88t.  62 Table IV. Characteristics of truncated-OprF mutants in P. aeruginosa.  strain/plasmid  apparent mwa (kDa)  H103/pER H636/pER  apparent mwa after heating (kDa)  37 —  41.5 —  2-ME modifiableb  +  —  H636/pERl63t  23.5  20  H636/pERl88t  27  23.5  H636/pER17O-26t  28  25.5  H636/pER2l5t  33  28.5  +  H636/pER29Ot  35  39  +  H636/pER326  37  41.5  +  a  as assessed by Western immunoblotting  b  +  —  +  —  indicates an increase in apparent molecular weight after treatment  with 2 —mercaptoethanol  63 These strains were also analyzed for modifiability with 2mercaptoethanol. Full-length OprF from the wild type strain H 103/pER and from the strain H636/pER326t, and truncated OprF encoded on plasmicis pER 188t, pER2 15t, and pER29Ot, all ran at a higher apparent molecular weight with the addition of 2-mercaptoethanol indicating that the cysteines encoded on these plasmids were present and had formed disuiphide bonds (Table IV). The remaining truncated oprF did not contain cysteines and their apparent molecular weights were not affected by the addition of 2-mercaptoethanol (Table IV). The cysteine-to-serine OprF mutants were also introduced into the OprF-deficient strain H636 by electroporation. Colonies, selected by antibiotic resistance, were shown to contain plasmicls of the correct size by slot lysis. Whole cell lysates of these strains were prepared with and without 2-mercaptoethanol pretreatment. Western immunoblotting identified proteins in all of the samples that reacted with the monoclonal antibody MA7-l (Figure 7, Table V). Bands, with apparent molecular weights greater than 50 kDa, observed in the samples untreated with 2mercaptoethanol (lanes 6-8), were not detected in the samples pretreated with 2-mercaptoethanol (examples in lanes 3 and 5) or in the native OprF (lane 3 and 9). These bands may be multimers of OprF stabilized by intra or intermolecular disulphide bonding.  64  1  2  3  4  5  6  7  8  9  10  Figure 7. Western immunoblot of whole cell lysates heated to 1000 C for 10 minutes. The monoclonal antibody used was MA7-1. Lanes 1 and 10, molecular weight markers (from top to bottom: 142.9, 97.2, 50.0, 35.1, 29.7 kDa). The samples in lanes 2-5 were treated with 2mercaptoethanol. Lane 2, H636/pER; lanes 3 and 6, H636/pERC185S; lanes 5 and 7, H636/pERC191S; lane 8, H636/pERC185S+C191S; lane 4 and 9, H103/pUCP19. F* indicates the heat-modified position of OprF from H103/pER.  65 Table V. Apparent molecular weight of cysteine-to-serine mutants in P. aeruginosa.  apparent molecular weighta  strain/plasmid  unheated (kDa)  heated (kDa)  unheated +2-ME (kDa)  heated +2-ME (kDa)  H103/pER  37  41  40.5  43.5  H636/pERC185S+C191S  38  41.5  40.5  43.5  H636/pERC191S  38 39  42  40.5  43.5  H636/pERC185S  8.5 40  42.5  40.5  43.5  a  as assessed by Western immunoblotting.  66 All of the samples appeared to be heat modifiable with and without pretreatment with 2-mercaptoethanol. Bands with apparent molecular weights of 41 (H103/pER, lane 9), 41.5 (H636/pERC185S+C191S, lane 8), 42 (H636/pERC191S, lane 7), 42.5 (H636/pERC185S, Table V) and 43 kDa (all samples pretreated with 2-mercaptoethanol, lanes 3-5, Table V) were not detected in samples heated to 45° C. The apparent molecular weight of the native OprF (lane 9) increased with the addition of 2-mercaptoethanol (lane 4) as did that of the cysteine-to-serine mutants (examples in lanes 3 and 5; Table V) indicating the presence of at least one disulphide bond in all of the samples. With the addition of 2-mercaptoethanol, the apparent molecular weight of all of the samples was similar, indicating that the cysteines located at amino acids 185 and 191 were not essential for the synthesis of a stable, full length protein. Without the addition of 2-mercaptoethanol, a band(s) with an apparent molecular weight higher that of the native OprF (37 kDa) and lower than the native OprF pretreated with 2-mercaptoethanol (40.5) was observed in all of the mutants. Strain H636/pERC185S+C191S had one band with an apparent molecular weight of 38 kDa indicating that the cysteines at amino acid 176 and 205 had formed a disulphide bond. H636/pERC191S had a major band of 39 kDa and a minor band of 38 kDa, and H636/pERC185S had two bands of 38.5 and 40 kDa (Table V).  67 This suggested that more than one combination of the non-mutagenized cysteines may be able to form a disuiphide bond and furthermore, that the residual cysteines in the single mutants pair in such a way to confer a different apparent molecular weight, possibly due to a different configuration of the disuiphide region.  F. Outer membrane and peptidoglycan association of OprF mutants.  An SDS-PAGE of outer membrane preparations of H 103/pER and H636/ER and H636/pER326t is shown in Figure 8. These results indicated that the cloned OprF was associated with the outer membrane when expressed in the OprF-deficient strain, H636. By visual comparison with other proteins in these samples, it appeared that the cloned OprF was produced at roughly the same levels as the native OprF. Accordingly, outer membranes of the truncated OprF strains were prepared. Figure 9 shows a Western immunoblot of these samples. The truncated and full-length OprF expressed in H636 containing pERl63t (lane 6), pER17O-26t (lane 5), pERl88t (lane 4), pER2l5t (lane 3), pER29Ot (lane 2) and the wild-type control (lane 1) were all associated with the outer membrane. The cysteine-to-serine OprF mutants were also associated with the outer membrane (M. Bains, personal  68  4  1  2  3  4  Figure 8. SDS-PAGE of outer membrane preparations of H 103/pER  (lane 1); H636/pER (lane2); H636/pER326t (lane 3); molecular weight markers (from top to bottom: 97.4, 66.2, 45.0, 31.0, 21.5 kDa) (lane 4). The position of OprF is indicated.  69  1  2  3  4  5  6  7  Figure 9. Western immunoblot of outer membrane preparations of truncated versions of OprF. Monoclonal antibody used was MA7- 1.  Lane 1, H 103/pER; lane 2, H636/pER29Ot; lane 3, H636/pER2l5t; lane 4, H636/pERl88t; lane5, H636/pER17O-26t; lane 6, H636/pERl63t; lane 7, molecular weight markers (from top to bottom: 142.9, 97.2, 50.0, 35.1, 35.1, 29.7 and 21.9 kDa).  70 communication). These results indicated that the N-terminal 163 amino acids of OprF were sufficient for outer membrane association and that the wild-type conformation of the disuiphide region was not required. OprF has been shown to be non-covalently associated with the peptidoglycan in strain H103 (Hancock and Carey, 1979). This association has been proposed to contribute to cell shape and stability since the peptidoglycan is involved in both functions. To show peptidoglycan association, Triton-EDTA and Triton-lysozyme soluble fractions were prepared as described in the Materials and Methods section. The Triton-EDTA soluble fraction contained proteins that were stabilized in the outer membrane by LPS association with divalent cations like MgC1 . Incubation of the Triton-EDTA insoluble fraction with 2 lysozyme released outer membrane proteins that are associated with the peptidoglycan. Figure 10 shows a Western immunoblot of Triton-EDTA fractions (lanes 2, 4, 6, 8, 11, and 13) and Triton-lysozyme fractions (lanes 3, 5, 7, 9, 12, and 14) from strains expressing full-length or truncated OprF. OprF from H 103/pER and H636/pER326t was found primarily in the Triton-lysozyme soluble fraction (lanes 3 and 5). A small amount was observed in the Triton-EDTA fraction (lanes 2 and 4) and the pellet remaining after Triton-lysozyme treatment. This may have been due to incomplete digestion of the peptidoglycan. All of the truncated proteins tested were located in the Triton-EDTA soluble fraction (lanes 6,  71  •1  12345  678  9  10  11  12  13  14  Figure 10. Western immunoblot of outer membranes preparations of truncated OprF’ solubilized in Triton-EDTA (lanes 2, 4, 6, 8, 11, and 13) or Triton-lysozyme (lanes 3, 5, 7, 9, 12, and 14). Monoclonal antibody used was MA7-1, Molecular weight markers (lanes 1 and 10), from top to bottom, 112, 84, 53.2, 34.9, 28.7, and 20.5 kDa. H103/pER (lanes 2  and 3); H636/pER326t (lanes 4 and 5); H636/pERl63t (lanes 6 and 7); H636/pER17O-26t (lanes 8 and 9); H636/pER2l5t (lanes 11 and 12); H636/pERl88t (lanes 13 and 14). An arrow indicates the position of H636/pER17O-26t,  72 8, 11, and 13) indicating that they were stabilized by association with LPS and divalent cations in the outer membrane, but were not associated with the peptidoglycan. A small amount was seen in the initial Triton-X 100 solubilization. These data indicated that more than 215 amino acids of OprF were required for association with the peptidoglycan and that, like the wild type OprF, the recombinant OprF was peptidoglycan associated.  G. Summary.  Plasmids encoding truncated mutants of OprF were constructed by PCR, linker-mutagenesis, and use of an existing mutant. The resulting plasmids, and a plasmid encoding full length OprF, were successfully transferred to the OprF-deficient strain H636 by electroporation. Plasmids that encoded at least 163 N-terminal amino acids of OprF expressed proteins detectable by Western immunoblot. These proteins were all associated with the outer membrane, but only the full-length OprF was associated with the peptidoglycan. Plasmids that encoded truncated-OprF mutants with at least two cysteines produced proteins that were 2-mercaptoethanol modifiable. Like the native OprF, the OprF encoded on plasmid pER326t and pER29Ot were heat modifiable, showing an increase in the apparent molecular weight when boiled in SDS or pretreated with TCA. In contrast, the remaining truncated  73 mutants of OprF had a decrease in their apparent molecular weight with these treatments. Two cysteine-to-serine OprF mutants were constructed by PCR and a third mutant containing both of the cysteine-to-serine mutations was constructed by genetic manipulation. These plasmids were electroporated into the OprF-deficient strain, H636. The encoded proteins were associated with the outer membrane of this strain and were heat and 2-mercaptoethanol modifiable. They had an apparent molecular weight higher than than that of the native OprF and lower than that of the native OprF pretreated with 2-mercaptoethanol indicating that mutation of cysteines located at amino acids 185 and 191 had affected disuiphide bond formation. The two single cysteine-to serine OprF mutants produced two bands detected by Western immunoblotting which indicated that the remaining three cysteines may be able to bond in more than one conformation.  74 CHAPTER 2. FUNCTIONAL ANALYSIS OF OprF MUTANTS IN P. aeruginosa.  A. Introduction.  The contribution of OprF to the stability of the outer membrane of P. aeruginosa has been studied with OprF-deficient strains (Woodruff and Hancock, 1988; Gotoh et al., 1989a). These studies have shown that OprF has a role in the shape of the cell and in growth in low osmolarity media. These properties were assessed in the mutants described in Chapter 1.  B. Growth of OprF mutants.  Despite the instability of the outer membrane of OprF-deficient strains, as described in the Introduction, strains with this defect have been observed in clinical situations. This indicated that OprF is not an essential component of the cell and that its deficiency may in fact confer some advantage in vivo (Piddock et al., 1992; Chamberland et al., 1990). To determine if H636 had a growth disadvantage in vivo, H636 and its parent strain H 103 were grown in chambers implanted in the peritoneum of mice. This model is useful because it permits the growth of more than one strain of bacteria per animal, in separate chambers, thus reducing the animal-to-animal variation. Also, because the ends of the chambers  75 are made from 0.2 jim pore size filters, the bacteria are retained in the chamber (permitting their recovery), the host cells are excluded (simplifying the results), and in vivo nutrients are taken up (permitting growth of the bacteria). Results from one experiment are shown in Figure 11. After a lag of 4 hours for H 103 and between 4 and 8 hours for H636, both strains grew, with doubling times of 40 and 53 mm respectively, reaching stationary phase after about 20 hours. This indicated that OprF-deficient strains did not have a major growth disadvantage in vivo. Previous studies have shown that OprF is required for the growth of P. aeruginosa in low osmolarity media (Woodruff and Hancock, 1988; Gotoh et al., 1989a; Nicas and Hancock, 1983). The OprF deficient strain M-2F-, described in Chapter 1, was tested for its ability to grow in high and low osmolarity media. The strains M2F- and H636 as well as their wild-type parent strains were able to grow at a similar rate in the high osmolarity medium (Figure 12B), but only the parent strains were able to grow in the low osmolarity medium (Figure 12A). This indicated that the inability to grow in a low osmolarity medium is due to the 2-cartridge mutagenesis of OprF and is not limited to the strain H636. To determine if the entire OprF was required for growth under these conditions, the truncated OprF mutants, described in Chapter 1, were grown in high and low osmolarity media. Strain H636 had been  ON  •  C  o  o  C  (D  c?i  ITJ  I.,  m  CR 0  0  Ci) 0  1%) 0  0  b-I  0  Ci) .P’ 01  -1  00  O  Viable Count (log cfu/ml)  0  77  A.  B.  400  400  1100  1100  10  10 I  I  I  I  I  I  7  7  I  I  I  I  012345678  012345678  Time (hours)  Time (hours)  Figure 12. Growth of OprF-deficient strains in A) LB no NaC1 or B) LB 200 mM NaC1. The strains are indicated as follows: H 103(0); H636 M2  (a); M2F- (a).  (•);  78 shown to be unable to grow in low osmolarity media (Woodruff and Hancock, 1988). With the addition of 200 mM NaC1, the generation time of H636 had been shown to be similar to its parent strain, H103 (Woodruff, 1988). In the initial growth studies, the addition of tetracycline to ensure plasmid maintenance appeared to inhibit the growth of the controls. H 103/pER had a lag period of 3-4 hours in both high salt and low salt media and H636/pER was unable to grow, in the 6 hours tested, in either medium. When plated with tetracycline at 100 pg/mi, the H 103/pER colonies were small and had rough edges. Normally colonies of H 103 are larger and smooth. As the outer membrane of P. aeruginosa is stabilized by divalent cations, and as tetracycline can act as a divalent-cation chelator (Nikaido and Thanassi, 1993), MgC1 2 was added to the growth media. The addition of 5 mM 2 permitted the expected growth of H 103/pER in both high and low MgCl salt media. The effect of the addition of 5 mM or 15 mM MgC1 2 on the strains H636/pER17O-26 and H636/pER is shown in Figure 13. The addition of 15 mM MgC1 2 resulted in growth of both strains in the high osmolarity medium but did not permit the growth of the strain, H636/pER in the low osmolarity medium. The addition of 15 mM MgC1 2 to the low osmolarity medium permitted growth of the strain H636/pER17O/26 at a rate greater than with the addition of 5 mM . Like H 103/pER, H636/pER17O-26 was able to grow in the high 2 MgC1  79  A.  B.  1  1—  I  I  o o  0 0  o  0 0.1  0.1  I  0.05  0.05  I  I  I  I  I  I  0123456  0123456  Time (hours)  Time (hours)  Figure 13. Growth of H636/pER17O-26t (A) and H636/pER (B) in media with and without the addition of MgC1 . Mueller Hinton broth (0), Mueller 2 Hinton broth  +  15 mM MgCl (), PP2 2  15mM MgC1 (+). 2  (•),  PP2  +  5mM MgC1 2  (a),  and PP2  +  80 osmolarity medium without the addition of MgC1 2 after about three hours. With the addition of 5 mM MgC1 2 to the growth media, all of the strains had smooth colonies. These results suggest that the addition of 2 protected the cells from the divalent-cation chelating (Nikaido and MgC1 Thanassi, 1993) effects of tetracycline. Figure 14 shows an example of the growth of the truncated OprF strains in low and high osmolarity media with the addition of 5 mM 2 and 200 tg/ml tetracycline. All of the strains tested were able to MgC1 grow at about the same rate in the high osmolarity medium. In the low osmolarity medium H636/326t grew at the same rate as the wild type strain and H636/pER showed little or no growth during the 7 hours tested. In the low NaC1 medium, the strains with truncated OprF grew at a rate similar to that of the wild-type strain for between 0 and 3 hours with the length of time of this initial growth varying in independent experiments (N.B. in the growth experiment of strain H636/pER17O-26 shown in Figure 13 there was no period of wild-type growth rate with the addition of 5 mM MgC1 ). This initial ability to grow at a rate near that of 2 the wild-type strain may have been due to carry over from the high osmolarity medium used for the growth of the overnight culture or to the stage of growth of the overnight cultures used in these experiments. After this initial phase, the remaining strains appeared to fall into 2 groups in all experiments. H636/pER29Ot and H636/pER2l5t had growth rates less than that of the full length OprF but  81  A.  B.  400  400  / /  100  1oo  //__4•I 7/ /_/  _—_/ —  •1.  _/  _(  0  10 8  0  0  0  0  0  I  I  I  I  10 8  I  I  I  I  012345678  012345678  Time (hours)  Time (hours)  Figure 14. Growth of truncated forms of OprF in A) LB no salt tetracycline/5 mM MgC1 2 or B) LB  +  200mM  salt  +  +  200 pg/m1  2OOig/ml tetracycline/5  mM MgC1 . The strains are indicated as follows: H636/pER (0); 2 H636/pERl63t(•); H636/pER17O/26t H636/pER2l5t  (s);  H636/ 188t  (•);  (•); H636/pER29Ot (+); and H636/pER326 (.). H103 was  identical to H636/pER326t and was omitted for clarity.  82 greater than the other strains, with the rate of H636/pER29Ot being greater than that of strain H636/pER2 15t. A Western immunoblot of the whole cell lysates of the strain H636/pER29Ot and the control strain H 103/pER taken after 7 hours of growth in the low osmolarity medium is shown in Figure 15. The apparent molecular weight of the truncated OprF expressed in strain H636/pER29Ot was lower than that of the strain H 103/pER indicating that in this experiment the plasmid had not recombined with the chromosome as had been observed previously (Figure 5). The strains H636/pERl63t, H636/pER17O-26t or H636/pERl88t always grew at a rate higher than the OprF deficient strain, H636/pER, but lower than that of the other strains. Although the rate of strain H636/pERl63t was higher than that of H636/pERl88t and H636/pER170-26t in the experiment presented in Figure 14, this was not the case in all experiments. The results from these experiments indicated that the OprF-deficient strain containing the plasmid pER/326t was able to grow at the same rate as the wild-type strain in both high and low osmolarity media. The strains containing the truncated versions of OprF were able to grow in the low osmolarity medium, but not at the same growth rate as the wild-type strain. These strains also appeared to be more susceptible to the chelating effects of tetracycline than the wild-type strain or the strain containing the full length OprF.  83  1  2  3  4  Figure 15. Western immunoblot of H636/pER29Ot (lane 2), H636/pER  (lane 3), and H 103/pER (lane 4) The molecular weight markers (lane 1) are, from top to bottom, 112, 84, 53.2, 34.9, and 28.7 kDa.  84 The three cysteine-to-serine OprF mutant strains were also tested for their ability to grow in a low salt medium. As shown in Figure 16, these strains grew at the same rate as the wild-type strain in both low salt and high salt media, indicating that the apparent disruption of the disuiphide-bond region (Chapter 1 E) did not effect the ability of these strains to grow in a low osmolarity medium.  C. Length of OprF mutants.  Previous studies have shown that OprF-deficient strains were shorter than their wild-type parent strains as judged by image analysis (Woodruff and Hancock, 1988) and by electron microscopy (Gotoh et al., 1989a). In this study, cells were grown with 200 pg/ml tetracycline in LBHS to mid log phase (50 Klett units) fixed, stained and measured directly from a video monitor connected to a phase contrast microscope. The study was conducted blind; the slides were coded and measurements were made by Susan Farmer. At least 100 cells of each strain from 3 different experiments were measured. The exception to this was the strain H636/290t which was assessed twice. All of the cells had similar widths but varied in their length. An example of one of these experiments is shown in Figure 17 and Table VI. The negative control, H636/pER, was 61% the length of its parent strain, H 103/pER. Strain H636 with pERl63t, pERl88t, pER17O—26t, pER2l5t, pER29Ot, or  85  A.  B.  400  z  400  ///  1oo :  ioo  II,  —  II,  1o  1o I  7  I  I  I  I  I  I  7  I  +  I  012345678  012345678  Time (hours)  Time (hours)  Figure 16. Growth of cysteine-to-serine muants in A) LB no salt or B) LB  I  200mM salt. The strains  are indicated as follows:  H103/pUCP19 (0); H636/pUCP19 H636/pERC3  (•); H636/pERC185s  (•); H636/pERC185s+C 19 is (•).  (a);  86  2.50  2.00  E 1.50  -  S -  1.00  0.50  -  -  0.00 1  2  3  4  5  6  7  8  9  10  Figure 17. Length of strains of P. aeruginosa containing plasmids encoding full-length or truncated versions of OprF. The strains are as follows: H 103/pER (positive control) (lane 1), H636/pER (negative control), (lane 2), H636/pERl63t (lane 3), H636/pERl88t (lane 4), H636/pER17O-26t (lane 5), H636/pER2l5t (lane 6), H636/pER290t (lane 7), H636/pER326t (lane 8), M-2 (lane 9), and M-2F- (lane 10). At least 100 cells were measured and the mean normalized to the length of H 103/pER as determined by image analysis.  87 Table VI. Relative length of P. aeruginosa with truncated OprF.  Strain/plasmid  OprFt/OprFa  (%) H103/pER  100± 14  H636/pER  61±20  H636/pERl63t  cfH636 /pER +  P value b cfHlO3 cfH636 cfH636 /pER /pER326t /pER29Ot +  -  -  -  +  +  +  77 ± 19  +  +  +  +  H636/p13R170-26t  86 ± 14  +  +  +  H636/pERl88t  85 ± 14  +  +  +  H636/pER2l5t  88 ± 12  +  +  +  H636/pER29Ot  91 ± 13  +  +  H636/pER326t  97 ± 13  +  a  OprFt (truncated versions of OprF)  b +  -  /  wild-type OprF  -  -  -  -  -  -  -  (%).  indicates strains that are significantly different as assessed by the  Student’s t-test with P0.05.  88 H636/326t were 77, 85, 86, 88, 91 and 97% the length of H 103/pER respectively. All of the strains were statistically larger than H636/pER and smaller than H 103/pER with the exception of H636/pER326t (Table VI). There was no statistical difference between strain H103/pER and strain H636/pER326t. There was also no statistical difference between H636/pER29Ot and H636/pER326t and between the truncated-OprF strains, including H636/pER29Ot, with the exception of H636/pERl63t. H636/pERl63t appeared to be of intermediate size between H636/pER and H636 containing pERl88t, pER17O-26t, pER2l5t and pER29Ot. The strain H636/pERl63t was statistically different from H636/pER and all of the truncated OprF strains. These results indicated that the cloned full-length OprF was able to complement the size defect in the OprF deficient strain, H636. Although the truncated versions of OprF also appeared to affect the size of the OprF deficient strain, with the exception of H636/pER29Ot, the increase in size appeared to be less than that with the full-length protein. Also measured were the OprF-deficient strain, M-2F-, described in Chapter 1 and its parent strain, M-2 (Figure 17). The strain M-2F- was about 70% of the length of the parent strain M-2, indicating that the effect of the a-cartridge mutagenesis of oprF on the size of the cell was not limited to the strain H636. Although the length of the cysteine-to serine mutants were not analyzed in a blinded study, the length of 25  89 cells from one experiment, grown without the addition of carbenicillin, were measured. Filamented cells were not observed and the mutants appeared to be about the same length as the wild-type strain, H 103/pUCP19.  E. Summary.  The c-cartridge mutagenesis of OprF in the strain M-2 resulted in the same phenotype as the previously c-cartridge mutagenized strain H636. M-2F- was unable to grow in a low salt medium and was 70% of the length of its parent strain. In vivo growth of strain H636, in a mouse chamber-model, was similar to that of its parent strain H 103, indicating that OprF was not required for growth of P. aeruginosa in vivo. The strain H636 containing cloned full-length OprF, truncated OprF and cysteine-to-serine mutagenized OprF were able to grow at the same rate as the wild-type strain, H 103/pER, in the high salt medium. In the low salt medium, however, only the strain containing the cloned full-length OprF and the cysteine-to-serine OprF mutants were able to grow like the wild-type strain. The strains of H636 containing the plasmids pER 163t, pER 170-26t, pER 188t, pER2 15t, or pER29Ot were able to grow in the low osmolarity medium, but at a lower rate than pER326 or the wild-type strain, H 103/pER. A similar pattern was observed in the length of the truncated-OprF strains. Although these mutants were longer than the  90 OprF-deficient strain H636/pER, the entire OprF was required for a wild type length cell. The cysteine-to-serine mutants appeared be similar in length to the wild-type strain, H 103/pUCP19, indicating that wild-type conformation of this region is not required for cell length.  91 CHAPTER 3. MONOCLONAL ANTIBODY STUDIES.  A. Introduction.  Although X-ray crystallography of outer membrane proteins is the ideal method for the study of their structure, these proteins are very difficult to crystallize. Sequence analysis (Martin, 1992; Woodruff and Hancock, 1989) and the interaction of OprF-specific monoclonal antibodies with mutant OprF derivatives (Finnen et al., 1992; Wong et al., 1993) and with OprF-derived peptides (Rawling et al., 1995) have contributed to the knowledge of the secondary structure of OprF. In this study, linear epitopes and surface-exposed epitopes were identified. These data were included in an updated secondary-structure model of OprF. The conservation of epitopes in strains of P. aeruginosa was also determined.  B. Linear-epitope mapping using overlapping octapeptides.  Previous studies in this laboratory using truncated forms of OprF, including TnphoA derivatives, partially localized the epitopes recognized by 10 OprF-specific monoclonal antibodies. Unfortunately, the epitopes for most of the antibodies could only be crudely localized by this means and it was not known to what extent the structure of recombinant Cterminal deletion derivatives reflected the structure of the native protein.  92 Overlapping octapeptides, starting at every second amino acid and covering the entire 326 amino acids of OprF were synthesized on 160 pins. ELISAs were performed with 10 monoclonal and 2 polyclonal anti OprF antibodies. Reactivity was observed with only 3 of the 10 monoclonal antibodies as shown in Figure 18. MA7- 1 bound to 3 pins clerivatized with the peptides VRGTYETG (a.a. 53 to 60, pin 27), GTYETGNK (a.a. 55 to 62, pin 28) and YETGNKKV (a.a. 57 to 64, pin 29). The binding of MA7- 1 to pin 28 was approximately three times that of 29  and 10 times that of 27. MA7-2 bound primarily to the peptide NLADFMKQ (a.a. 237 to 244, pin 119) but did bind with low affinity to the peptide IKNLADFM (a.a. 235 to 242, pin 118). MA5-8 bound to the peptide with the sequence TAEGRAIN (a.a. 307 to 314, pin 154) 3 times greater than to the peptide NATAEGRA (a.a. 305 to 312, pin 153). MA73, MA7-4, MA7-5, MA7-6, MA7-7, MA7-8, and MA4-4 showed no reactivity, indicating that they did not recognize linear epitopes. To examine the extent of distribution of linear epitopes on the OprF sequence, the pins were reacted with polyclonal antibodies from pooled mouse and rabbit serum. The mouse serum was tested at a working dilution of 1:5000 and repeated at a dilution of 1:350. The results were similar and those from the 1:350 dilution are presented in Figure 19. The antibodies bound with high affinity to pins 153 and 154, which is the same region as the epitope for MA5-8 and bound more weakly to  93  2  A. MA7-1  U)  Q Cu  Cu  .0 0 Cu .0  0 1  50  100  150  200  250  300  100  150  200  250  300  100  150  200  250  300  2  B. MA7-2 E = U)  Cu 0  = Cu  .0  0 .0  0 1  50  2  C. MA5-8 E U)  Cu 0  =  Cu .0  0 C., .0  I.  0 1  I  50  I  —  Approximate OprF amino acid iocation  Figure 18. ELISA readings of monoclonal antibodies (A) MA7- 1, (B) MA7-2 and (C) MA5-8 reacting with individual pins derivatized with overlapping octapeptides from OprF. Background values were subtracted. The positions of the middle amino acids of the OprF-derived peptides, starting from the N-terminus, are indicated on the X-axis. These data are representative of two independent trials with essentially identical results.  94  3  A.. Polyclonal mouse anti-OprF Ab E to =  2  a’ C.) .0  0  1  C,, .0  0  L 1  .  Iii 50  L 100  150  .  200  U J. 1 250  300  3  B. Polyclonal rabbit anti-OprF Ab E U)  2  0  .0  0  1  C,, .0  0  L I.LIJLhL1J 50  100  150  ..  200  250  300  Approximate OprF amino acid location  Figure 19. ELISA readings of (A) mouse, and (B) rabbit polyclonal serum  reacting with individual pins derivatized with overlapping octapeptides from OprF. Background values were subtracted. The positions of the middle amino acids of the OprF-derived peptides, starting from the N-terminus, are indicated on the X-axis. These data are representative of two independent trials with essentially identical results.  95 peptides composed of a.a. 15 to 22 (pin 8), a.a. 281 to 288 (pin 141), and a.a. 295 to 306 (pins 148-150). This serum bound purified OprF in a standard ELISA assay when used at a dilution of 1:100,000 (R. Wong, personal communication), and specifically recognized OprF on Western immunoblots of whole-cell lysates of wild-type P. aeruginosa, H 103, at dilution of 1:1,000. No high-affinity antibodies were detected in the polyclonal rabbit serum when tested at a dilution of 1:500 (Figure. 19). By increasing the development time, multiple weaker binding sites were observed. Of these sites, those that bound with an absorbance of at least 0.5 included peptides of a.a. 13 to 20 (pin 7), 83 to 90 (pin 42), 117 to 124 (pin 54), 127 to 134 (pin 59), 213 to 220 (pin 107), 231 to 238 (pin 116), 241 to 248 (pin 121), 259 to 266 (pin 130), 277 to 284 (pin 139), 281 to 288 (pin 141), and 289 to 296 (pin 145). Peptides of a.a. 245 to 252 (pin 123) and 295 to 306 (pin 149 and 150) had approximately 2- to 2.5-times greater ELISA reading than the other pins. This serum bound specifically to OprF on Western immunoblots of whole-cell lysates of wild-type P. aeruginosa at a dilution of 1:500 (A. Sukhan, personal communication).  The two polyclonal sera appeared to share weak binding only to pins 141 and 149-150. These results suggested that, as for the monoclonal antibodies, the majority of the polyclonal antibodies produced were directed against conformational epitopes.  96  C. Binding of OprF-specific monoclonal antibodies to OprF mutants.  The location of the OprF-specific monoclonal antibody epitopes in the primary-structure of OprF was useful for the analysis of the truncated-OprF mutants and the disuiphide-bond mutants. The results from colony immunoblotting, and was confirmed in selected cases Western immunoblotting, of the truncated-OprF mutants is shown in Table VII. The strains H636/pER326t and the positive control H 103/pER bound all of the monoclonal antibodies tested confirming that the cloned OprF was expressed in P. aeruginosa and had assumed a conformation similar to that of the native OprF. The negative control strain, H636/pER, truncated by 2 mutagenesis at amino acid 102, did not bind any of the antibodies, including the N-terminal specific MA7- 1 (section B), indicating that no stable protein was produced. All of the remaining truncated-OprF mutants were expressed and stable in P. aeruginosa as shown by their ability to bind MA7-1. The strains H636/pERl63t and H636/pERl88t did not bind any other antibodies, including MA7-8 and MA4-4. The epitopes for these antibodies have been previously located between amino acids 152 and 210 (Finnen et al., 1992). This indicates that part or all of these epitopes lie downstream of amino acid 188. H636/pER2l5t bound both MA7-8 and MA4-4 indicating that this truncated protein was not degraded and, as both of these antibodies  + + + + + +  H636/pER17O-26t  H636/pERl88t  H636/pER2l5t  H636/pER29Ot  H636/pER326t  -  +  +  +  -  -  -  -  -  +  +  +  -  -  -  -  -  +  +  +  -  -  -  -  -  +  +  +  -  -  -  -  -  +  +  +  -  -  -  -  -  +  +  +  -  -  -  -  -  +  +  +  +  -  -  -  -  +  +  +  +  -  -  -  -  +  +  +1-  -  -  +  -  -  +  MA7-1 MA7-2 MA7-3 MA7-4 MA7-5 MA7-6 MA7-7 MA7-8 MA4 4 MA5-8  H636/pERl63t  H636/pER  H103/pER  Strain/plasmid  Monoclonal antibody reactivity  Table VII. Summary of OprF-specific monoclonal antibody reactivity with truncated OprF in P. aeruginosa.  98 require disulphide bonds for binding (Finnen et al., 1992), it appeared that the disuiphide region of this protein had assumed the wild-type conformation. As expected, the strain H636/pER29Ot bound 9 of the 10 monoclonal antibodies tested but also reacted with the monoclonal antibody MA5-8 in some experiments. As the epitope binding this antibody has been located between amino acid residues 305 to 312 (section B), it appears that this plasmid had recombined with the chromosome resulting in a full-length protein. These results are consistent with those presented in Figure 5. The reactivity of the cysteine-to-serine mutants, encoded on plasmids pERC185S, pERC191S and pERC185S+C191S, with the OprF specific monoclonal antibodies was also studied. As shown in Table VIII, these mutants bound all of the antibodies with the exception of MA7-8  and MA4-4. The epitopes for MA7-8 and MA4-4 have been previously localized to the cysteine-containing region of OprF by the analysis of truncated mutants (Finnen et al., 1992) and papain- and cyanogen bromide-cleaved peptides (Rawling et al., 1995). It has also been shown that OprF pretreated with 2-mercaptoethanol no longer binds these antibodies (Rawling et al., 1995). These results indicated that these mutations had affected the conformation of the disuiphide-bond region.  + + +  H636/pERC191s  H636/pERC185S +C1915  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  -  -  -  +  -  -  -  +  +  +  +  +  MA7-1 MA7-2 MA7-3 MA7-4 MA7-5 MA7-6 MA7-7 MA7-8 MA4 4 MA5-8  H636/pERC185S  H636/pER  H103/pER  Strain! plasmid  Monoclonal antibody reactivity  Table VIII. Summary of OprF-specific monoclonal antibody reactivity with cysteine-to-serine mutants in P. aeruginosa.  100 D. Surface accessibility of the epitopes.  The surface accessibility of epitopes binding monoclonal antibodies can provide information useful in making secondary-structure models  and for studying antigenic conservation. In this study, the OprF-specific monoclonal antibodies were used in indirect immunofluorescence studies and as opsonins in phagocytosis to identify surface-localized epitopes. The results of the indirect-immunofluorescent labeling of P. aeruginosa strain M-2, are presented in Table IX. High levels of fluorescence were seen with MA7-1, MA7-3, MA7-4, MA7-7 MA7-8, MA4-4 and MA5-8. Uniform but weaker fluorescence was observed with MA7-5 and MA7-6. No fluorescence was observed with MA7-2 and with the negative control antibody MA 1-3, specific for the lipoprotein, OprI. Another approach to study surface exposed epitopes was to use the monoclonal antibodies as opsonins for phagocytosis of the strain M-2 by mouse peritoneal macrophages. The antibody concentrations used were standardized by ELISA with purified OprF and used at a dilution of 10-2. The results from this study, shown in Figure 20, confirmed previous data which had shown that MA5-8 and MA4-4 were opsonic and that MA5-8 binding resulted in greater uptake than MA4-4 (Battershill et al., 1987). MA7-3, MA7-5, MA7-6, MA7-7 and MA7-8 consistently opsonized strain M-2. MA7- 1 produced significant uptake in 1 out of 3 experiments, although when less diluted antibody was used, significant uptake was consistently  101 Table IX. Indirect immunofluorescence labeling of intact P. aeruginosa strain, M-2, with OprF-specific monoclonal antibodies and the control antibody, MA1-3. Monoclonal antibody  MA7-1  Immunofluorescencea  ++  MA7-2 MA7-3  ++  MA7-4  ++  MA7-5  +  MA7-6  +  MA7-7  ++  MA7-8  ++  MA4-4  ++  MA5-8  ++  MA13b  a  no fluorescence or only slight fluorescence observed, fluorescent, ++ cells highly fluorescent..  b  -  +  cells uniformly  negative-control monoclonal antibody specific for a non-surface localized epitope of outer membrane protein OprI.  102  *  03 C)  * *  i iii *  *  hhh 7-1  7-2  7-3  7-4  7-5  7-6  7-7  7-8  4-4  5-8  Antibody  Figure 20. Opsonic phagocytosis of M-2 by OprF-specific monoclonal antibodies. Results represent the average number of bacteria per mouse peritoneal macrophage. Each different shaded bar represents data from an independent experiment with the control bacteria per macrophage (obtained by using the negative control antibody MA1-3) subtracted. The stars indicate samples that were significantly different from the control (by the Mann-Whitney test P -0.05).  103 observed. An increase in the concentration of MA7-2 and MA7-4 had no effect on uptake; MA7-2 was no better than the control antibody, and MA7-4 produced significant results in only 1 of the 3 experiments. The mean uptake of cells with the negative-control monoclonal antibody MA 1-3 was 1.5 bacteria per macrophage and which was subtracted from the values shown in Figure 20. The combined results from these surface localization studies suggested that the epitopes for nine of the monoclonal antibodies were surface exposed and that the epitope recognized by MA7-2 was not exposed in strain M-2.  E. Conservation of epitopes binding OprF-specific monoclonal antibodies.  Forty six P. aeruginosa strains including the 17 IATS serotype isolates, 15 clinical strains from different countries, 11 environmental isolates, and three laboratory isolates, including M-2, were studied for conservation of epitopes by colony immunoblotting with the 10 monoclonal antibodies. All of the isolates, except for the negative controls H636 and M-2F- reacted with all of the antibodies and in selected cases this was confirmed by Western immunoblotting. These data indicate that these epitopes of OprF were strongly conserved in P. aeruginosa.  104 F. Summary: A secondary structure model of OprF.  Previous secondary structure models of OprF have been derived from experimental evidence and predictive methods based on amino acid sequence analysis. The experimental results include the estimation of 13strand content by CD analysis (Siehnel et al., 1989), the surface localization of sites permissive for linker-insertion mutagenesis (Wong et aL, 1993) and the surface localization of the disulphide-bond dependent epitope of the monoclonal antibody, MA4-4 (Mutharia and Hancock, 1985). CD analysis suggested a 13-sheet content of 62% in the native protein, with 50% in the C-terminal 149 amino acid residues, which is much higher than the 25% predicted by amino acid sequence analysis (Siehnel et al., 1989; Jeanteur et al., 1994). Raman spectroscopy of OmpA predicted 55% 13-sheet, with 60% in the N-terminal 177 amino acids (Vogel and Jahnig, 1986). The original model (Siehnel et al., 1989) was modified by Wong et al. (1993) to include the surface localization of the permissive linker-insertion sites (Figure 21) (Wong et al., 1993). It has been shown that the loop regions of porins are more likely to tolerate the insertion of foreign DNA than the transmembrane strands (Cowan et al., 1992; Ried et al., 1994; Wong et at., 1993). The length of the loops connecting the transmembrane 13-strands were adjusted to conform to the known structures of other porins. The periplasmic loops of these  105  98 ‘ 1N  P V T T VD G DP A T N YG C:CGN E K ioQ LE DN KP Y K S K S D i& A V A T V N D R ?J D E G G H I S L 1J A S V V NQ131G C:CDV*:S31D G  LJ  M N K R 2  D T A C Y  H 290 G ° 31 EN  A  vD2tHEKE  E  D  FG Va  Figure 21. Secondary structure model of OprF. The N-terminus is on  the bottom left-hand side of the model. Sites of linker-insertion mutagenesis indicated by shaded boxes. Predicted regions of transmembrane f3- strands are boxed.  K  106 porins are all shorter than the surface-exposed loops which are of variable length. The updated model presented in this study has been modified from that presented in Rawling et al. (1995) to include the location of 13strands predicted by the method of Jeanteur et al. (1994). These models have incorporated the results from the mimotoping and surfacelocalization studies, presented in section B and D respectively. Unlike other prediction methods, the method of Jeanteur et al. (1994) has been tailored for the analysis of porins. Porins are a unique class of proteins possessing transmembrane strands with f3-strand conformation (Jeanteur et al., 1994). It had been previously believed that transmembrane strands of proteins could only have an ce-helical conformation. The N-terminal domain of the current model is similar to that of the previous models (Rawling et al., 1995; Wong et al., 1993) in that it is composed of eight transmembrane 13-strands connected by short periplasmic loops and longer surface-exposed loops (Figure 22). The location of the 13-strands in the primary structure of OprF was based on those predicted by Jeanteur et al. (1994). This method of prediction results in a conservative estimate in the length of the 13-strands (Jeanteur et al., 1994) and the length of the strands were accordingly extended to include contiguous regions of alternating hydrophobic and hydrophilic  107  <  MA7-4,5,7 <- MA7-6-  E—MA7-3 .  MA7-8, MA4-4  MA71 N  y  ir  NM  R V20 D  K N  R  A D  K K H 70 G  fl T  D S N Iioo  G R Q  r1  V I GETNTTM lli  G F  T  200  D  A H 1YIILI ISDANALPIoIjallEi  L T  0  -  D  ‘ii  N Q 0  -  A N KRD G E 1 40 C:C N P K L G DN 190 A D D G H81 AV MA7-2 GV E QD Q G S V DN “ 210 y ST C:C R E G S  G L iL KiGi Al 1GyA  E  V  A p TD  D  EH  A So  F  v  D 40  U T P  80  iAlIFiIFI  H_jINt 12 K N L° 1 pY  I  I  []T  V K  o P P A  iLl IN I  G  G  YG  GI H T  L!_J  E  G  >  N 2 39 E KK  290 G  T  MA5-8  0  E  a  0210  290 R  GI  ri  El NI  hAl I vi  -  I  20  1 N  [J E  ___j V  R Ij  hr I IA  I  A  N A  IE  LJhA R 300  L!_  A N  A R R N OK LSE270  Figure 22. Modified secondary structure model of OprF. The locations  of epitopes are indicated by outlined letters and by arrows. The Nterminus is on the bottom left-hand side of the model. Predicted regions of transmembrane 13-strands are boxed.  108 amino acid residues. This resulted in a number of minor changes to the previous models primarily affecting strands 6 and 7. The surfaceexposed epitope for monoclonal antibody MA7- 1, identified by mimotoping, was located on the surface exposed loop 2. The concentration of tyrosine and phenylalanine residues at the hydrophobic protein-lipid interface, the excess of negatively-charged residues at the level of the LPS core and the length of the f3-strands were consistent with that of known porin structures (Jeanteur et al., 1994). The f3-sheet content of this region was similar to that predicted by CD analysis. The model of the C-terminal domain of OprF, like that of the Nterminal domain of OprF, is composed of eight transmembrane strands (Figure 22). This is consistent with the number of strands present in the known structures of porins. Interestingly, Jeanteur et al. (1994) predicted only two C-terminal domain 13-strands, the first and seventh J3strands presented in this model. Jeanteur et al.(1994) predicted that the region located between these two 13-strands was composed of amphipathic x-helices. In the model presented in this study, the transmembrane strands in the C-terminal domain have been included as 13-strands to reflect the data from CD analysis, although the total 13-sheet content is about 10 % lower than the predicted value. The epitopes binding nine OprF-specific monoclonal antibodies have been shown to be located in the C-terminal half of OprF (Finnen et al., 1992; Rawling et al.,  109 1995; Wong et al., 1993). In this study, eight of these epitopes were shown to be surface exposed (Table IX, Figure 20). The region binding one of these monoclonal antibodies, MA5-8, localized to eight amino acids by mimotoping (Figure 19), was accordingly located in the loop connecting f3-strands 15 and 16. The epitope for MA7-2 also localized by mimotoping did not appear to be surface located in wild-type cells (Table IX, Figure 20; Martin et al., 1994) and was located primarily in the eleventh transmembrane strand. The conformation and location of the cysteine-containing region from the previous model (Wong et al., 1993) have been retained. The surface exposure of the epitopes binding the 2-mercaptoethanol sensitive monoclonal antibodies support this location (Table VIII and Figure 20) and the results from the analysis of the cysteine-to-serine mutants (Chapter 1E) support the proposal that the cysteines form two disulphide bonds. Also in agreement with the location of this region is the surface location of the proline-rich region of OprF from P. fluorescens which appears to replace the cysteine-containing region of OprF from P. aeruginosa (De Mot et al., 1994).  The region between amino acids 260 and 275 has been located in the periplasm in this model. This region has few nonpolar amino acid residues and is therefore unlikely to be a transmembrane strand. It has been highly conserved in OmpA-related outer membrane proteins with 7  110 of the 14 amino acid residues being perfectly conserved in the 10 proteins analyzed (De Mot et al., 1992). The C-terminal region of OprF (amino acid residues 163-326) has homology with the B. subtilis protein, MotB (not an outer membrane protein) and with the non-covalently peptidoglycan associated lipoproteins (PALS) and has been proposed to be a site for peptidoglycan interaction (De Mot and Vanderleyden, 1994).  111 DISCUSSION  A. Aims of this study.  The aims of this study were to confirm the function OprF by genetic complementation and to assess the role of the C-terminal domain of the protein in cell length and growth on low osmolarity media. Another aim was to assess the disulphide bond region of OprF including the determination of the number of disulphide bonds present in the native protein and the identification of the cysteines bonded. It was hoped that these studies and those with the OprF-specific monoclonal antibodies would increase the understanding of the structure and function of this protein.  B. Genetic complementation of Q-cartridge mutagenized OprF in P. aeruginosa.  The function of OprF has been studied by the construction of OprF-deficient strains by chemical mutagenesis (Gotoh et al., 1989a; Nicas and Hancock, 1983) and by transposon and interposon insertional mutagenesis (Woodruff and Hancock, 1988). Although the length of these strains and their ability to grow in a low osmolarity medium was generally the same, there was some phenotypic variation (described in Chapter 1-B). To ensure that the more extreme results observed with 2-  112 cartridge mutagenesis of OprF were not strain specific, this method was used to mutagenize the oprF gene in strain M-2. The resulting strain, M 2F-, was unable to grow in a low osmolarity medium (Figure 12) and was about 30% shorter than its parent strain (Figure 17). This confirmed that the phenotype previously observed was not strain-specific and that OprF has a significant role in cell length and growth on low osmolarity medium. The expression of certain outer membrane proteins, which can have copy numbers of 10 per cell, from high copy number plasmids has proven lethal (Duchene et al., 1988; Woodruff, 1988). In order to facilitate the genetic complementation of an OprF-deficient strain of P. aeruginosa, the promoter of oprF was weakened by site directed  mutagenesis (Wong et aL, 1993). The less efficient promoter permitted the cloning of oprF into a E. coli-P. aeruginosa shuttle vector, pUCP19, which has a copy number of about 100 in E. coli and 10-25 in P. aeruginosa (Schweizer, 1991). Although the control plasmid, pUCP19, was introduced into the wild-type strain by transformation and by electroporation, only electroporation resulted in the successful uptake of plasmid DNA by the OprF-deficient strain, albeit at a low frequency. This underscores the contribution of OprF to the cell. OprF appears to provide protection from the potentially damaging procedures of  113 centrifugation, low temperatures and incubation of the cells in low osmolarity buffers, all of which are required in transformation. Visual comparison indicated that the level of expression of OprF, encoded on plasmid pER326t, in the OprF-deficient strain H626 was similar to that of the parent strain, H 103/pER (Figure 8). Like the native OprF, the recombinant OprF had the same apparent molecular weight, was heat and 2-mercaptoethanol modifiable (Table IV), was associated with the outer membrane (Figure 8) and the peptidoglycan (Figure 10), and bound all of the OprF-specific monoclonal antibodies tested (Table VII). This strain was able to grow at the same rate as the wild-type strain in the low osmolarity medium (Figure 14) and regained the wild-type cell length (Figure 17, Table VI). The genetic complementation of the OprF deficient strain, H636, confirmed the role of OprF in the growth of P. aeruginosa in low osmolarity media and in the determination of cell  length.  C. Protein analysis of mutated versions of OprF.  The construction of plasmid-encoded truncated and cysteine-to serine mutants of OprF has enabled the assessment of the role of the Cterminal domain and the disulphide-containing region of OprF in the expression and conformation of the encoded protein. In this study, it was shown that between 102 and 163 N-terminal amino acids were  114 required for the stable expression of OprF as detected by Western immunoblot (Figure 5). This correlated with the minimum number of amino acids required for the expression of OmpA in E. coli. Bremer et at (1982) constructed truncated-OmpA mutants by exonuclease digestion. A construct encoding the N-terminal 133 amino acids produced a truncated protein detected 45 s after radioactive labeling. No protein was detected 1 hr after labeling which indicated that the protein was unstable and had been degraded. A protein truncated at amino acid 193 was stable (Bremer et at., 1982) as was a protein that contained a frame shift after amino acid 188 (188fs+1) (Klose et at., 1988b). It should be noted that this latter mutation resulted in the addition of 18 non-OmpA amino acids which could have affected the conformation of this protein or its insertion into the outer membrane. Recently, Ried et at (1994) constructed an OmpA mutant truncated at amino acid residue 171 by site directed mutagenesis which also produced a stable protein. All of the truncated versions of OprF expressed in P. aeruginosa were associated with the outer membrane (Figure 9). These results indicated that the N-terminal 163 amino acids were sufficient for association with the outer membrane. Secondary structure models of OprF (Figures 21 and 22) and OmpA (Morona et at., 1984; Klose et at., 1989; Ried et at., 1994) predict an N-terminal domain composed of 8 transmembrane (3-strands, terminating at amino acids 160 and 170  115 respectively. A series of plasmids containing overlapping deletions of OmpA were examined by immunoelectron microscopy for their association with the outer membrane (Kiose et al., l988b). All of the constructs containing amino acids 154-180, including the frameshift mutant 188fs+ 1 and the mutant truncated at amino acid 193 described above, were associated with the outer membrane and all of those missing this region were located in the periplasm. Although association with the outer membrane was not determined, the mutant truncated at amino acid residue 171 was associated with the cell envelope (Ried et al., 1994). The importance of this region was confirmed by Kiose et al (1989). In that study, the eighth transmembrane strand, predicted to be composed of amino acids 158-170, was shown to be required for association with the outer membrane by the mutation of leu’ 64 to pro and val’ 66 to asp. The resulting full-length, double mutant was located in the periplasm. These results indicate that the proposed eighth transmembrane strand is essential for association with the outer membrane. Unfortunately, in this study, a mutant designed to terminate within the proposed eighth transmembrane strand of OprF was found to be rearranged. I hypothesize that the observed rearrangement in the PCR-amplified region of this mutant was required to permit non-lethal production of a fragment expressing the epitope binding the monoclonal antobody, MA7-  116 1. Mutants, truncated within the proposed eighth 13-strand would determine if the results observed in OmpA are applicable to OprF. The outer membranes of many Gram-negative bacteria contain OmpA- or OprF-like proteins which have been identified in part by the distinctive property of heat modifiability (see Introduction). This characteristic has been proposed to be due to the resistance of the f3barrel structure to denaturation (Cowan et at., 1992), requiring boiling in SDS for unfolding. The extended structure of the unfolded protein has a higher apparent molecular weight in SDS-PAGE than the folded form since the latter is less compact. This property has also been used to assess the conformation of truncated mutants of OmpA. It has been suggested that non-heat modifiable mutants are not folded correctly (Klose et at., 1988b; Ried et at., 1994). An OmpA mutant truncated at amino acid 193 and the frameshift mutant l8Ofs+ 1 were heat modifiable as well as being able to bind phages and colicins indicating that these mutants were correctly assembled into the outer membrane (Kiose et at., 1 988b). The increase in the apparent molecular weight of the frameshift mutant upon heating was slight compared to that of the wild-type strain and was attributed to the loss of the C-terminal portion of the protein rather than to an effect of the additional 18 non-OmpA amino acids (Klose et at., 1988b). Interestingly, not all OmpA mutants have both the property of phage sensitivity and of heat modifiability indicating that  117 these mutated proteins may be inserted into the outer membrane in the wild-type conformation without the property of heat modifiability. Double mutants of OmpA, with amino acid changes in the proposed first or eighth f3-strand, conferred phage sensitivity and produced a trypsin fragment of the expected-size but were not heat modifiable (Klose et al., 1988a). Without heating, these mutants had the apparent molecular weight of the heat-modified wild-type protein. OmpA mutants truncated at amino acids 228 or 274 had a similar degree of heat modifiability as the wild-type protein (Ried et al., 1994). Trypsin-cleaved fragments derived from mutants truncated at amino acid 228 and 274 and from the wild-type strain had the same apparent molecular weight, indicating correct membrane insertion. It has been proposed that insertion into the outer membrane protects the N-terminal domain of OmpA from digestion by proteases (Chen et al., 1980). When heat treated, the tryptic fragments derived from the native protein and from the mutants truncated at amino acid 228 and 274 had a decrease, instead of the expected increase, in their apparent molecular weight when compared to untreated samples (Ried et at., 1994). Another OmpA mutant, truncated at amino acid 171 also exhibited this aberrant mobility (Ried et at., 1994). These studies indicated that although mutations in the N terminal domain of the protein can affect heat modifiability, the entire protein is not required for this property and that the portion of OmpA  118 that is required for the property of heat modifiability terminates between amino acids 171 and 193. In the study presented here, OprF truncated at amino acid 290  and the full-length plasmid-encoded protein were heat modifiable, showing the expected increase in their apparent molecular weight (Table IV). The truncated-OprF mutants encoded on pERl63t, pER17O-26t, pER 188t or pER2 15t had a decreased apparent molecular weight when denatured. These mutants did not appear to be degraded and the mutants truncated at amino acid 188 and 215 remained 2mercaptoethanol modifiable. These results indicated that between 215 and 290 amino acids of OprF were required for the wild-type heat modifiability. The amino acid alignment of OmpA and OprF indicated that regions located between amino acids 197 and 204 and between amino acids 215 and 222 respectively have significant homology with the eighth f3-strand of these proteins and has been predicted to form a f3strand in both proteins (Jeanteur et at., 1994). This strand, like the proposed eighth 13-strand of both proteins, has strong homology to the sixteenth (terminal) 13-strand of the crystallized nonspecific porins, and in these proteins is required for the correct membrane insertion of these porins (Jeanteur et at., 1994). The contribution of this region to the formation of an SDS-stable (heat modifiable) structure in OmpF is compatible with the experimental data, but further studies are required  119 to test this hypothesis. This region does not appear to be required for this property in OmpA. Ried et al. (1994) suggested that the decreased mobility of the non-heat modifiable OmpA mutants could be due to the disassociation of a protein dimer. The relative change in the apparent molecular weight observed with the OprF mutants is not consistent with dimer disassociation. It is also inconsistent with a heat/TCA induced cleavage of the protein since the change in apparent molecular weight was similar for all mutant proteins and no common fragment was observed. Another explanation is that these truncated versions of OprF may be partly denatured by SDS before the heat treatment and the further treatment with heat or TCA may actually induce secondary structure, resulting in the increased mobility on SDS-PAGE.  D. Functional analysis of truncated OprF mutants.  The conservation of the sequence of the C-terminal domain of OmpA-like proteins, including OprF, is consistent with the hypothesis that it has an important function in the cell. C-terminal deletion mutants were analyzed for their ability to grow in a low salt medium and for their effect on cell length. Interestingly, the plasmid pERl63t, which encoded only the N-terminal region of OprF, produced a significant increase in cell length of the OprF-deficient strain (Figure 18) and permitted its growth in a low salt medium, although at a rate lower than  120 the wild-type OprF (Figure 14). This suggested that this protein was not only associated with the outer membrane but was inserted into the membrane and may have folded into a wild-type conformation. There appeared to be a further increase in cell length with the expression of portions of the C-terminal region. A similar increase in length was observed with truncated versions of OprF encoded on pER17O-26t, pER 188t, and pER2 15t which suggested that the proline-alanine region may have a role in cell length. Without further testing, it was not possible to draw any conclusions about the length of strain H636/pER29Ot. These results indicated that the entire OprF was required for wild-type cell length. The growth of the truncated mutants in a low osmolarity medium also appeared to be affected by the number of amino acids encoded. When grown with tetracycline to maintain their plasmids, the mutants truncated at amino acids 215 or 290 grew at a higher rate than the others. The remaining mutants all grew at a rate higher than the negative control strain. But, as with cell length, the entire protein appeared to be required for wild-type growth in a low osmolarity medium. These experiments indicate that the C-terminal region of OprF had a significant, though not complete, role in the contribution of OprF to the growth of P. aeruginosa in low osmolarity media.  121 E. Analysis of the disuiphide region of OprF’.  The cysteine-containing region of OprF is highly conserved in P. aeruginosa and P. syringae. Thirty-two of the 34 amino acids that  compose this region are perfectly conserved and 1 of the remaining 2 amino acids is a conservative substitution. Amino acid alignment of OmpA and the OprFs from P. aeruginosa and P. syringae locate this sequence within the proline-rich so called hinge region of OmpA. Similarly, alignments with OprF from P. fluorescens, which lacks this cysteine region, also suggest that its insertion occurred in the proline rich region. The titration of OprF with 2-mercaptoethanol, resulting in a three-step increase in apparent molecular weight, led to the proposal that the four cysteines of OprF form 2 disulphide bonds (Hancock and Carey, 1979; Martin, 1992). Previous attempts to measure the number of disulphide bonds by chemical methods identified one bond but also indicated that there were no free thiols present (Martin, 1992). In this study, another method was used in an attempt to unambiguously quantify the number of bonds and also to identify the cysteines involved in each bond. The cysteine-to-serine mutagenesis of amino acid residues 185 and 191 appeared to result in the disruption of a single disulphide bond as assessed by the change in apparent molecular weight of these mutated proteins under non-denaturing conditions and by their failure to react with the 2-mercaptoethanol  122 sensitive monoclonal antibodies, MA7-8 and MA4-4. In the absence of 2mercaptoethanol treatment, the apparent molecular weights of these proteins were higher than that of the native OprF but were still modifiable with 2-mercaptoethanol to a position identical to that of OprF. This was consistent with the presence of one disulphide bond in each of the mutants and two in the native protein. An alternative explanation is that these mutations altered the conformation of the protein resulting in the bonding of free thiols. However, this explanation is not favored by the observation that no free thiols have been detected (Martin, 1992). The apparent ability of the mutant proteins to bond in more than one configuration (Figure 7) prevented the definitive identification of the cysteine residues involved in each bond. The mutation of the remaining two cysteine residues has been hampered by technical problems (Manjeet Bains, personal communication), but may provide further insight into the analysis of this region. I suggest that the best method of resolving this dilemma would be to insert a methionine residue between amino acid residues 185 and 191. Cleavage with cyanogen bromide and the analysis of the resulting peptides, with and without the addition of 2mercaptoethanol, would unambiguously identiir one of the three possible combinations of bonding between the four cysteine residues (i.e. that equivalent to the model in Figure 22). If required, the insertion of  123 methionines before and after these amino acids would distinguish between the two remaining combinations. No function has been assigned to the disuiphide region of OprF. Woodruff et al. (1986) proposed that alternate disuiphide bonding could account for the two porin sizes identified by black lipid bilayer methods. The results with the cysteine-to-serine mutants and the double mutants  are consistent with this possibility. This region has a high degree of homology with the calcium binding repeats present in the eukaryotic extracellular matrix protein, thrombospondin and with the CD protein of Branhamella catarrhalis, a respiratory tract pathogen (De Mot and Vanderleyden, 1994). However, the significance of this observation is unknown. This study has shown that the native conformation of the disuiphide region is not required for association with the outer membrane, heat modifiability (Figure 6), wild-type growth in low osmolarity media (Figure 16) or for wild-type cell length, but has not identified a clear role for this region.  F. Analysis of epitopes binding OprF-specific monoclonal antibodies.  Previous analysis of truncated mutants of OprF (Finnen et al., 1992), and protease- and cyanogen bromide-cleaved peptides (Rawling et  124 al., 1995) localized the epitopes binding 10 OprF-specific antibodies to regions of OprF that were 42-90 amino acids long. These regions were as follows: MA7-1, amino acids 24-112; MA7-2, amino acids 250-273; MA7-3, amino acids 188-230; MA7-4, MA7-5 and MA7-7, amino acids 188-278; MA7-6, amino acids 198-240; MA7-8 and MA4-4, amino acids 152-2 10; MA5-8, amino acids 30 1-326. In this study, three of these epitopes, binding MA7- 1, MA7-2 and MA5-8, were localized to regions of eight amino acids by overlapping peptide methodology (Figure 22). Nine of these epitopes appeared to be surface exposed in strain M-2 as assessed by opsonic phagocytosis studies (Figure 20) and by indirect immunofluorescence labeling (Table IX). The remaining epitope binding monoclonal antibody MA7-2 did not appear to be surface exposed. The results of indirect-immunofluorescence labeling of the strain H692 (Martin et al., 1993), which has a rough LPS, with the OprF-specific monoclonal antibodies were in agreement with the results from strain M 2, with the exception of the binding of MA7-2. Unlike strain M-2, strain H692 showed a moderate level of fluorescence when reacted with monoclonal antibody MA7-2 (Martin et al., 1993). This difference might be due to the masking of this epitope in strain M-2 by the smooth LPS (Martin et al., 1993). The surface localization of the epitope binding MA58, (amino acids 307-314) is in agreement with recent studies (Hughes et al., 1992; Von Specht et al., 1995). In both of these studies, polyclonal  125 sera raised against peptides composed of amino acids 308-326 (Von Specht et al., 1995) and 305-318 (Hughes et al., 1992) bound to surface exposed epitopes as determined by immunofluorescence of intact bacteria or by opsonic-phagocytosis assays, respectively. The surface localization of a region composed of amino acids 188-216 (Von Specht et al., 1995), and the lack of reactivity to serum against a peptide composed of amino acids 189-203 (Hughes et al., 1992) is also consistent with the data in this study. In this study, the truncated OprF encoded on pER2 15t (Table VII) bound the surface localized 2-mercaptoethanolsensitive monoclonal antibodies MA7-8 and MA4-4 (Figure 20, Table IX), while the truncated OprF encoded on pER188 did not (Table VII). One may speculate that a disuiphide bond involving the cysteines located at amino acids 191 and/or 205 may be required for the production of antibodies reacting with this area of OprF. Polyclonal sera raised against peptides composed of amino acid residues 166-189, 2 15-226, 260-292 Von Specht et al. (1995), 7-19, 177-190 or 242-56 (Hughes et al., 1992) did not bind surface-exposed epitopes. These results are in agreement with the assignment of these regions in the proposed model of OprF (Figure 22). However, polyclonal sera raised against peptides composed of amino acid residues 54-67, 98-111, or 136-149 did not appear to bind surface exposed epitopes (Hughes et al., 1992). In the present study, the epitope binding monoclonal antibody MA7- 1 was located between amino  126 acid residues 55 and 62 (Figure 18) and appeared to be surface exposed as assessed by indirect immunofluorescence labeling and by opsonic phagocytosis studies (Figure 20, Table VIII). Cells were highly fluorescent when labeled with MA7- 1. Unlike the other monoclonal antibodies binding surface-exposed epitopes, a higher concentration of MA7- 1 was required for the consistent uptake of opsonized bacteria by macrophages. The reason for the discrepancy between the two studies is not clear but it may be a reflection of the different methodologies used. Other conflicting data are the surface (Hughes et al., 1992) and non-surface (Von Specht et al., 1995) localization of the region between amino acid residues 26 1-274 and 260-292, respectively. Further studies are required to unambiguously determine the location of surface exposed loops in OprF.  G. Conclusions and perspectives. This study has shown that the C-terminal domain of OprF has a role in the outer membrane stability of P. aeruginosa as assessed by cell length and growth in low osmolarity media and is required for peptidoglycan association and is partially required for the property of heat modifiability. It would be of interest to determine if this region is also required for the porin function of the protein. The truncated mutants constructed in this study could be assessed for pore forming ability using black lipid bilayer methodology. The analysis of the pore  127 function and pore size of the cysteine-to-serine mutants of OprF would indicate whether wild-type conformation of the disuiphide region is required for porin function and may also be useful in testing the alternate-disuiphide bonding hypothesis proposed by Woodruff et al., (1986) to account for the two sizes of pore measured. As well, the analysis of cyanogen bromide-cleaved peptides, as described in the discussion, may unambiguously determine the number and configuration of the disuiphide bond(s). This study has also shown that several regions of OprF, binding OprF-specific monoclonal antibodies, are surface exposed and highly conserved in strains of P. aeruginosa. 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