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Molecular studies of the structure-function relationships of Psuedomonas aeruginosa OprM : an outer membrane… Wong, Kendy Kit-Ying 2001

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MOLECULAR STUDIES OF THE  STRUCTURE-FUNCTION  RELATIONSHIPS OF PSEUDOMONAS AERUGINOSA OPRM: AN OUTER MEMBRANE PROTEIN ASSOCIATED WITH EFFLUX by K E N D Y KIT-YING WONG B.Sc. (Hons.), University of New Brunswick, 1994 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A May 2001 © Kendy Kit-Ying Wong, 2001  I  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.  Department The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  MarcA  ' 3-1 , Zoo I-  ABSTRACT  Pseudomonas aeruginosa demonstrates high intrinsic resistance to multiple classes of structurally unrelated antimicrobial agents. This broad-spectrum resistance is primarily due to a combination of low outer membrane permeability coupled to secondary resistance mechanisms such as the resistance-nodulation-division (RND) efflux systems.  The outer  membrane protein OprM is involved in intrinsic and mutational multiple-antibiotic resistance as part of two R N D efflux systems in P. aeruginosa. To study  structure-function  relationships for OprM, optimal conditions for oprM overexpression were determined; it appeared that excessive production of the protein was lethal to cells.  In addition,  overexpression of oprM alone in wild-type P. aeruginosa did not increase the resistance of the cells, suggesting OprM could not function independent of the pump and linker proteins of the efflux systems. OprM was demonstrated for the first time to have channel-forming activities like porins, and was shown to be cation selective like the related TolC protein of Escherichia coli. Based on the functional and sequence similarity between OprM and TolC, and their similar content of a-helical and P-sheet structures determined by circular dichroism spectroscopy, a three-dimensional model of OprM was constructed by threading its sequence to the TolC crystal structure.  This suggested that, like TolC, OprM has a distinctive architecture  comprising outer membrane p-barrel and periplasmic helical-barrel structures. Analyses of OprM insertion and deletion mutants in the context of this model indicated that the helical barrel is critical for both function and integrity of OprM, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Unlike the classic ii  porins, extracellular loops appear to play a minimal role in substrate specificity.  There  appears to be a correlation between the change in antimicrobial activity for certain OprM mutants and the channel size determined by planar bilayer analysis, supporting the "iris" mechanism of action first suggested for TolC.  This study provids information on the  structure-function relationships of OprM in a three-dimensional context, and will allow more focused hypotheses and studies about the functional domains of OprM and its related family of efflux proteins.  iii  TABLE OF CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST OF FIGURES  x  LIST OF TABLES  xii  LIST OF ABBREVIATIONS  xiii  ACKNOWLEDGMENTS  xvi  DEDICATION  INTRODUCTION  xvii  1  1. Pseudomonas aeruginosa  1  2. Pseudomonas aeruginosa outer membrane  2  2.1 Outer membrane structure  2  2.2 Porins  6  3. Antibiotic uptake across the outer membrane  7  3.1 The hydrophilic pathway  7  3.2 The hydrophobic pathway  8  3.3 The self-promoted uptake pathway  8  4. Mechanisms of antimicrobial resistance in P. aeruginosa 5. Efflux systems  9 10  5.1 Families  10  5.2 Resistance-Nodulation-Division (RND) efflux systems in P. aeruginosa  11  iv  5.3 Assembly of the R N D efflux systems in the cell envelope 6. Structural analysis of outer membrane proteins  13 14  6.1 Techniques  14  6.2 Crystal structures of porin channels  14  6.3 Structure prediction of outer membrane proteins (porins)  15  6.4 Functional studies for outer membrane proteins  16  6.5 Crystal structure of E. coli TolC  18  7. OprM of P. aeruginosa  19  8. Aims of this study  21  METHODS AND MATERIALS  23  1. Strains, plasmids and growth conditions  23  2. Genetic manipulations  23  2.1 General techniques  23  2.2 Transfer of D N A into E. coli and P. aeruginosa  29  2.3 Oligonucleotide synthesis and purification  30  2.4 D N A sequencing....  30  3. General protein and immunological techniques  30  3.1 Electrophoresis  31  3.2 Western immunoblotting  31  3.3 Indirect immunofluorescence  32  4. Prediction of the topology model of OprM 4.1 Sequence alignment  32 32  v  4.2 Structural characteristics  33  5. Carboxyl-terminal deletion of OprM  34  6. Insertional mutagenesis  34  6.1 Linker insertion mutagenesis  34  6.2 PCR site-directed insertion mutagenesis  37  7. PCR-based site-specific deletion mutagenesis  46  7.1 Direct extension  46  7.2 Overlap extension  46  7.3 Polymerase chain reaction (PCR)  48  8. Expression of oprM and oprM mutants  48  9. Purification of OprM and mutants  48  9.1 Outer membrane isolation  49  9.2 Detergent solubilization  50  9.3 Fast protein liquid chromatography (FPLC)  50  9.4 Gel elution  50  10. Planar lipid bilayer analysis  51  11. Circular dichroism (CD) analysis  52  12. Three-dimensional modeling  52  13. Assays  52  13.1 Protein assay  52  13.2 Minimal inhibitory concentration (MIC) determination  53  RESULTS  54  vi  CHAPTER ONE: Expression of oprM* 1.1 Introduction  54 •.  54  1.2 Construction of pKPM-2 and pKW35M  54  1.3 Effect of overproduction of OprM  55  1.4 Overexpression of oprM* in E. coli  58  1.5 Overexpression of oprM* in P. aeruginosa  58  1.6 Summary  61  CHAPTER TWO: Functional characterization of OprM 2.1 Introduction  63 63  2.2 In vivo functional characterization of OprM i n / , aeruginosa: antibiotic 3  susceptibility 2.3 In vitro functional characterization of OprM: channel-forming activity  64 66  2.3.1 Purification of OprM  66  2.3.2 Planar lipid bilayer analysis of OprM  68  2.4 Summary  CHAPTER THREE: MexAB-OprM homologues from the P. aeruginosa genome 3.1 Introduction  73  .74 74  3.2 Phylogenetic analysis of the MexA-MexB-OprM efflux components with their homologues from the genome  74  3.3 The OprM family in P. aeruginosa  80  3.4 Summary  87  CHAPTER FOUR: Structural characterization of OprM - insertion mutagenesis  88  4.1 Introduction  88  4.2 Prediction of an OprM topology model  88  4.3 C-terminal deletion of OprM  93  4.4 Insertional mutagenesis of OprM with the malarial epitope N A N P  99  4.5 Characterization of the oprM insertion mutants 4.5.1 Expression of oprM insertion mutants in an E. coli background  100 100  4.5.2 Surface exposure of insertion mutant proteins in E. coli  103  4.5.3 Expression of oprM insertion mutants in P. aeruginosa  103  4.5.4 Antimicrobial susceptibilities of P. aeruginosa cells carrying the insertion mutants  108  4.6 Summary  110  CHAPTER FIVE: Three-dimensional model of OprM and its structure-function relationships  112  5.1 Introduction  112  5.2 Secondary structure of OprM  113  5.3 Three-dimensional model of OprM  115  5.4 Insertion mutagenesis of oprM.  119  5.5 Deletion mutagenesis of oprM and characterization of mutants  123  5.5.1 Expression and in vivo function of the oprM deletion mutants  123  5.5.2 In vitro function of the oprM mutants: channel-forming activities  126  5.6 Summary  128  DISCUSSION  129  General  129  Difference at the C-termini of OprM and OprM*  130  Overexpression of oprM  130  Influence of OprM on multiple-antimicrobial resistance  131  Analysis of MexA-MexB-OprM homologues from the P. aeruginosa genome  132  Prediction of an OprM membrane topology model  133  Insertional mutagenesis of oprM  135  A three-dimensional molecular model of OprM  139  Antibiotic passage through the MexAB-OprM efflux system  145  Design of inhibitory methods  148  REFERENCES  152  i,  ix  LIST OF FIGURES  Figure 1.  Schematic representation of the cell envelope of P. aeruginosa  Figure 2.  Schematic representation of semi-random malarial epitope-insertion mutagenesis with a kanamycin resistance cassette  Figure 3.  3  35  Nucleotide and encoded amino acid sequences of the oligonucleotides used for the insertion of malarial epitope repeats into OprM via a K m cassette adaptor  Figure 4.  Primer design strategy for site-directed insertion mutagenesis of OprM using P C R  Figure 5.  38  39  Schematic diagram showing two PCR strategies for defined-deletion mutagenesis of oprM  47  Figure 6.  Map of pKPM-2  56  Figure 7.  Effect of OprM* overproduction on the growth of P. aeruginosa OprMdeficient strain K613  Figure 8.  Expression of oprM* in the outer membrane of E. coli  Figure 9.  Overexpression of oprM* in the outer membrane of P. aeruginosa wild-type strain  Figure 10.  57 59  60  Effect of heat and p-mercaptoethanol on OprM* in the outer membrane of P. aeruginosa  62  Figure 11.  Differences at the 3' end between oprM* and oprM  67  Figure 12.  Purification of OprM from E. coli CE1248 strain carrying pKW3 5 T M . . . .  69  Figure 13.  Planar lipid bilayer analysis of purified OprM  Figure 14.  Phylogenetic analysis of MexA with its homologues from P. aeruginosa.. 76  Figure 15.  Phylogenetic analysis of MexB with its homologues from P. aeruginosa.. 77  Figure 16.  Phylogenetic analysis of OprM with its homologues from P. aeruginosa.. 78  Figure 17.  Multiple-sequence alignment of OprM with homologues from P. aeruginosa  70  83 f ,  Figure 18.  Multiple-sequence alignment of OprM with several homologous proteins.. 90  Figure 19.  Amphipathicity profile of OprM  91 x  Figure 20.  Predicted membrane topology model of the OprM monomer  92  Figure 21.  Sequence alignment of OprM with P. putida TodU  Figure 22.  Primers for constructing a deletion mutant of oprM at the 3' end  96  Figure 23.  Western immunoblot of the C-terminal truncated OprM mutant  97  Figure 24.  Expression of oprM malarial epitope insertion mutants in E. coli  Figure 25.  Expression of oprM malarial epitope insertion mutants in P. aeruginosa..  Figure 26.  Circular dichroism spectral analysis of purified wild-type OprM  114  Figure 27.  Alignment of the sequences of TolC and OprM  116  Figure 28.  Three-dimensional model of an OprM homotrimer  117  Figure 29.  Three-dimensional model of an OprM monomer  118  Figure 30.  p-barrel domain of the three-dimensional model of OprM  120  Figure 31.  Three-dimensional model of an OprM homotrimer with malarial epitope  94  101 105  insertion sites highlighted  121  Figure 32.  Top and bottom views of the three-dimensional model of OprM  122  Figure 33.  Single channel conductances of wild-type OprM and its mutants  127  Figure 34.  Proposed mechanism of action for RND efflux systems  149  xi  LIST OF TABLES  Table I.  Bacterial strains  24  Table II.  Plasmids  25  Table III.  Oligonucleotides for the amplification and mutagenesis of oprM* and oprM  Table IV.  Antibiotic susceptibility of P. aeruginosa strains over-producing OprM* to antibiotics  Table V .  102  Expression and characterization of the oprM epitope insertion mutants in P. aeruginosa  Table XII.  98  Expression and characterization of the oprM epitope insertion mutants in E. coli  Table XI.  82  Antibiotic susceptibilities o f f . aeruginosa OprM-deficient strain OCR03T over-producing OprM* or a C-terminal truncated OprM  Table X .  81  The OprM family deduced from analysis of the Pseudomonas aeruginosa genome  Table IX.  79  Percent identity and similarity among known P. aeruginosa efflux components  Table VIII.  72  The possible efflux components present in the Pseudomonas aeruginosa genome  Table VII.  65  Average single channel conductance of the wild-type OprM protein as a function of different salt solutions  Table VI.  41  107  Antimicrobial susceptibilities of P. aeruginosa OprM-deficient strain OCR03T carrying plasmids expressing wild-type or insertion mutants of oprM  Table XIII.  109  Antimicrobial susceptibilities of P. aeruginosa OprM-deficient strain OCR03T carrying plasmids expressing wild-type or deletion mutants of oprM  124  xii  LIST OF ABBREVIATIONS  ::number (e.g. ::23)  indicates insertion of malarial epitopes at amino acid e.g. 23 of OprM  aa  amino acid  amp  r  ampicillin-resistant  bp  base pair  BSA  bovine serum albumin  Car  carbenicillin  CCCP  carbonyl cyanide w-chlorophenylhydrazone  CD  circular dichroism  CF  cystic fibrosis  Cfp  cefepime  Cfpm  cefpirome  Cfs  cefsulodin  Cml  chloramphenicol  Ctx  ceftriaxone  Ctz  ceftazidime  EDTA  ethylenediamine tetraacetic acid  FPLC  fast protein liquid chromatography  h  hour(s)  HPLC  high performance liquid chromatography  IgG  immunoglobulin G  IPTG  isopropyl thio-P-D-galactopyranoside  kb  kilobase pairs  kDa  kilodalton  Km  kanamycin  LB  Luria broth  LP  linker protein of RND efflux systems  LPS  lipopolysaccharide  mAb  monoclonal antibody  MCS  multiple cloning site  ME  malarial epitope; insertion of a sequence containing N A N P  Mer  meropenem  min  minute(s)  MOPS  3-N-morpholino propane sulfonic acid  Nal  nalidixic acid  Nfx  norfloxacin  nS  nano Siemen  Octyl-POE  octyl-polyoxyethelene  OD  optical density  OMPs  outer membrane proteins  Opm  probable outer membrane protein - OprM family  oprM*  originally cloned oprM gene with an incorrect 3' end which yielded a functional protein OprM* in which the final 22 amino acids from native OprM were replaced with 14 amino acids derived from the phagemid used to clone the gene  xiv  ORF  open reading frame  PBP  penicillin binding protein  PBS  phosphate-buffered saline (0.14M NaCl/2.7mMKCl/l .47mM K H P O / 2 0 m M N a H P 0 pH7.4) 2  4  4  PCR  polymerase chain reaction  PVDF  polyvinylidene difluoride  RND  resistance-nodulation-division efflux family  rpm  revolutions per minute  SDS  sodium dodecyl sulfate  SDS-PAGE  SDS polyacrylamide gel electrophoresis  Spe  spectinomycin  Sm  streptomycin  Tet  tetracycline  wt  wild-type  ACKNOWLEDGMENTS  I am grateful to my supervisor Dr. Robert E . W. Hancock for his guidance and support. I would like to thank the members of my supervisory committee, Drs. Davies, Gold and Warren, for their time and advice. Special thanks to the past and present members of the Hancock lab, for their technical assistance, support and friendship during my study. I would also like to acknowledge Dr. Roland Benz for his technical advice on planar lipid bilayer studies, and Dr. Fiona S. L . Brinkman especially for her guidance in genome analysis and three-dimensional modeling.  Last but not least, I am deeply grateful to my family and  friends for their support, encouragement and understanding. The financial support of the Canadian Cystic Fibrosis Foundation is also gratefully acknowledged.  xvi  DEDICATION  T o my beloved husband.  xvii  INTRODUCTION  1. Pseudomonas aeruginosa  Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium. It has very simple and flexible nutritional requirements and so is a ubiquitous microorganism. However, soil and water are the most common habitats of this organism. It is encountered very often by humans through inhalation or ingestion from vegetables and water.  P.  aeruginosa can also enter the skin through abrasions but it does not adhere well to normal epithelium and thus has little effect on healthy individuals. However, various infections can occur in weakened or immunocompromised hosts. For example, systemic septicemia can be caused by a P. aeruginosa infection in burn patients, while local pneumonia can be caused in cystic fibrosis (CF) patients. Since P. aeruginosa does not normally cause disease in healthy individuals, it is considered an opportunistic pathogen. Spread of this organism is very easy in hospitals and it is a major cause of nosocomial infections. P. aeruginosa has a number of virulence factors which include the polysaccharide slime layer for antiphagocytosis, the polar pili for adherence and colonization, and the polar flagella for motility and dissemination. Exotoxin A , exoenzyme S, elastase, alkaline protease, and phospholipase C are secreted to cause tissue damage in the host. In CF patients, mucoid strains secrete copious amounts of the antiphagocytic exopolysaccharide alginate through the action of many regulatory and structural genes of the alginate pathway (Chitnis and Ohman, 1990; Pier et al, 1994). P. aeruginosa is becoming a major clinical problem since it has high intrinsic resistance to many commonly used antibiotics, including first and second generation 1  penicillins and cephalosporins, tetracycline and chloramphenicol (Bryan, 1979). This high level of intrinsic antibiotic resistance is partly due to the possession of an outer membrane with low permeability. B y comparing the hydrolytic rates of various hydrophilic compounds by enzymes located in the periplasm, it was determined that the outer membrane permeability of P. aeruginosa is about 100 fold lower than that of E. coli (Yoshimura and Nikaido, 1982). Other resistance mechanisms include the hyperexpression of chromosomal P-lactamases, expression of plasmid-encoded P-lactamases, and the decreased affinity of penicillin-binding proteins (PBP) for p-lactams. The organism can also synthesize aminoglycoside-modifying enzymes, produce altered ribosomes, and decrease the uptake of aminoglycosides. Mutations in D N A gyrase or alterations of outer membrane proteins can lead to quinolone resistance. Alternatively, various drugs can also be pumped out actively through efflux systems (Li et al, 1994a & 1994b; Quinn, 1992). This latter mechanism of antibiotic resistance is now receiving much attention and recognition. Often, several of the above mechanisms work synergistically to yield the multidrug resistance phenotype.  There are also increasing  incidences of multiple-antibiotic resistance, especially in hospitals where the organism is constantly under high selection pressure.  2. Pseudomonas aeruginosa outer membrane  2.1 Outer membrane structure The cell envelope of P. aeruginosa (Fig. 1) consists of two membranes separated by a layer of peptidoglycan and a compartment called the periplasm.  The inner cytoplasmic  membrane is a typical phospholipid bilayer membrane with a wide variety of polypeptides.  2  Figure 1.  Schematic representation of the cell envelope of P. aeruginosa.  The outermost layer of the cell envelope is the outer membrane (OM) which contains lipopolysaccharides (LPS) on the outer surface and porins that are water-filled channels for the entry of small hydrophilic molecules. The inner layer is the cytoplasmic or inner membrane (IM) which carries a membrane potential and contains different proteins (e.g. penicillin-binding proteins PBPs) with various functions. Between the two membranes are the periplasmic space and the peptidoglycan. The three components of ResistanceNodulation-Division (RND) efflux systems are presumably arranged as shown, where the pump is located in the cytoplasmic membrane and is linked to the outer membrane component (presumably a porin) by the linker protein (LP) that is also anchored to the cytoplasmic membrane. 3  The major functions of cytoplasmic membrane proteins are in cellular energization, transportation of nutrients and export of toxic byproducts (Cronan et al, 1987). Beneath the outer membrane is the peptidoglycan responsible for cell shape and osmotic stability (Oliver, 1987). The periplasm includes systems for processing and traffic of molecules entering or leaving the cell. The outer membrane of P. aeruginosa, like those of other Gram-negative bacteria, is an asymmetric lipid bilayer, with the unique component lipopolysaccharide (LPS) on the outer leaflet.  It is noncovalently associated with the underlying peptidoglycan through  certain peptidoglycan-associated proteins. The basic LPS molecule consists of three regions: (i) the hydrophobic, biologically active endotoxin, Lipid A , (ii) the rough core, and (iii) the immunodominant O-antigen region (Lugtenberg and van Alphen, 1983; Nikaido and Nakae, 1979; Rietschel et al., 1984). The Lipid A region is antigenically and chemically conserved, with a single backbone of glucosaminyl-P-(l-»6) glucosamine substituted with six or seven saturated or hydroxyl fatty acid residues (Karunaratne et al, 1992). Covalently bound to Lipid A is the rough core which in P. aeruginosa contains eleven heterogeneous sugar residues including glucose, rhamnose, galactosamine residues, and a unique octose (2-keto-3deoxyoctulosonic acid [KDO]). This fraction also contains alanine and a large number of phosphate residues (Drewry et al, 1975; Kropinski et al, 1979). The rough core may be capped by the O-antigen, with repeating of tri- to pentasaccharide units, containing sugars such as glucose, rhamnose, glucosamine, fucosamine and quinavosamine (Kropinski et al, 1985). This portion is one of the most immunogenic antigens of smooth Gram-negative bacteria and determines the O-serotype of such bacteria (for reviews, see Nikaido and Hancock, 1986; Hancock et al, 1994). LPS molecules are anchored in the outer membrane  4  by the fatty acyl chains of the Lipid A portion (Morrison, 1985), and its asymmetric distribution and chemical characteristics give many unique barrier properties to the outer membrane. The LPS molecules are also stabilized in the outer membrane by non-covalent cross-bridging with divalent cations (Rottem and Leive,  1977), together with the  hydrophobic interactions between Lipid A and the outer membrane proteins (Nikaido and Vaara, 1985). The large number of phosphate residues in the core region of P. aeruginosa LPS results in a strong surface negative charge (Sherbert and Lakshmi, 1973). The crossbridged LPS molecules combined with this negative surface charge make P. aeruginosa, as well as other Gram-negative bacteria, resistant to hydrophobic antibiotics, detergents, bile salts, proteases, lipases and lysozyme (Nikaido and Vaara, 1985). Various proteins of the P. aeruginosa outer membrane contribute to different functions.  Two lipoproteins, OprI and OprL, inserted in the inner phospholipid monolayer  are structural proteins that stabilize the outer membrane by non-covalent association with the underlying peptidoglycan (Mizuno, 1979; Hancock et al, 1981a). The major protein OprF is also strongly but non-covalently associated with peptidoglycan and plays an important role in outer membrane stabilization and maintenance of cell shape (Gotoh et al, 1989; Woodruff and Hancock, 1989). One major role of the outer membrane is to serve as a selective barrier. The lipid bilayer is hydrophobic and excludes most hydrophilic compounds. However, small hydrophilic molecules including many nutrients can pass through the outer membrane via specific or nonspecific porins. In general, porins are proteins with a high content of P-sheet structure that form trans-outer membrane, water-filled channels.  They are often non-  covalently associated with the peptidoglycan and with LPS, and are usually present in the membrane as trimers (Hancock et al, 1990). Certain recently discovered outer membrane  5  proteins have been shown to be associated with multiple-antibiotic efflux in P. aeruginosa (Poole etai, 1993b).  2.2 Porins Porins are proteins that form water-filled channels across the outer membrane and are generally divided into two classes: non-specific or general porins and specific porins. General porins are responsible for the exclusion limit of the outer membrane by allowing the passive diffusion of hydrophilic molecules under a certain size. The rate of passage of molecules through general porins also depends on the electrical charge and hydrophilicity of the molecules (Nikaido and Vaara, 1985). In P. aeruginosa, OprF is a major non-specific porin with a channel diameter of approximately 20A (Benz and Hancock, 1981; Bellido et al, 1992) and can allow the passage of saccharides with molecular weights of about 3 kDa (Nikaido and Hancock, 1986). The channel diameter of OprF is about twice of that of E. coli porin channels; however, the outer membrane permeability of P. aeruginosa is about 100 fold lower than that of E. coli (Yoshimura and Nikaido, 1982). It was demonstrated that less than 1% of the 200,000 OprF molecules could form active functional channels while the rest appear to form small channels that are presumably antibiotic impermeable (Hancock, 1985; Woodruff et al, 1986). OprC and OprE are also general porins with small channel size (Yoshihara and Nakae, 1989). The low permeability of the P. aeruginosa outer membrane was proposed to contribute to the high level of intrinsic drug resistance for this bacterium (Nikaido and Hancock, 1986); it is now believed that multidrug active efflux plays a major role in the intrinsic antibiotic resistance of P. aeruginosa, in combination with the permeation barrier (Nikaido, 1994).  6  Nutrients are often available at low concentrations in the medium. Thus, the presence of specific porins, with specific substrate-binding sites, can allow effective uptake of essential nutrients from the surrounding. The uptake of the substrate for a specific porin is accelerated when the solute concentration is low, but the uptake rate is slowed when the concentration is high. In P. aeruginosa, OprB prefers D-glucose and D-xylose (Trias et al, 1988) and is induced by growth in the presence of glucose (Hancock and Carey, 1980). OprD is specific for the uptake of basic amino acids but it was originally discovered to facilitate the uptake of imipenem, a carbapenem with excellent activity against P. aeruginosa (Trias and Nikaido, 1990a & 1990b). OprP is induced by growth under phosphate starvation (Hancock et al, 1982) and shows 100-fold preference for phosphate over other anions due to a phosphate-binding site (Hancock and Benz, 1986). OprO is highly homologous to OprP and forms pyrophosphate-specific channels (Siehnel et al, 1992; Hancock et al, 1992).  3. Antibiotic uptake across the outer membrane  3.1 The hydrophilic pathway Hydrophilic antibiotics which include a number of P-lactams, chloramphenicol and tetracycline (Foulds, 1976) can pass across the outer'membrane of Gram-negative bacteria through their water-filled channels, the porins. Porin-deficient mutants were shown to have significantly higher MICs for some but not all P-lactams (Hancock and Bell, 1988), as well as a 10- to 100-fold decrease in permeation rates of p-lactams compared to their isogenic porin-sufficient parent strains (Hancock, 1987). Certain hydrophilic antibiotics can utilize specific porins to enhance their uptake by resembling the natural substrates of the channel.  7  3.2 The hydrophobic pathway Moderately hydrophobic antibiotics, including macrolides, novobiocin, some more hydrophobic P-lactams and actinomycin D (Nikaido et al, 1983) generally diffuse slowly into Gram-negative bacteria due to the outer membrane which is an unusual asymmetric lipid bilayer with LPS cross-bridged by divalent cations. Although the outer membrane was once thought to be a barrier for uptake of such antibiotics, it is their efficient efflux that gives rise to the high observed MICs.  Permeabilization to hydrophobic antibiotics can therefore be  achieved when the outer membrane is modified by compounds which remove (e.g. EDTA) or displace (e.g. cationic peptides) divalent cations from their LPS binding sites (Hancock, 1984; Nikaido and Hancock, 1986), or by alterations of LPS due to mutations (Nikaido and Vaara, 1985).  3.3 The self-promoted uptake pathway The self-promoted uptake pathway was postulated for the uptake of polycationic antibiotics such as aminoglycosides and polymyxin across the outer membrane of P. aeruginosa (Hancock, 1981; Nicas and Hancock, 1983). These polycationic antibiotics are believed to displace the divalent cations from LPS to destroy the cross-bridging and thus destabilize the outer membrane (Hancock et al, 1981b; Nicas and Hancock, 1983). This can lead to the enhanced uptake of lysozyme, P-lactams and hydrophobic fluorescent dyes across the outer membrane, and is also thought to promote the uptake of the interacting polycationic antibiotics themselves.  The divalent cation chelator E D T A that removes M g  2 +  from.outer  membrane sites also causes similar enhanced uptake of lysozyme and P-lactams (Nicas and Hancock, 1983) and enhanced killing by polycationic antibiotics (Sykes and Morris, 1975).  8  4. Mechanisms of antimicrobial resistance in P. aeruginosa  P. aeruginosa has a high level of intrinsic antibiotic resistance due in part to the possession of an outer membrane with low permeability (Nikaido and Hancock, 1986; Hancock, 1997; Hancock, 1998) in addition to secondary defence mechanisms such as an inducible P-lactamase and active efflux that take advantage of this low passage of antibiotics through the outer membrane. mechanisms  It can also acquire antibiotic resistance through different  such as the transfer  of plasmids harboring antibiotic-resistance genes  (Markowitz et al, 1978). There are increasing incidences of multiple-antibiotic resistance, especially in hospitals where the organism is constantly under high selection pressure. Other antimicrobial resistance mechanisms include the expression of P-lactamases and inducible cephalosporinases and the decreased affinity of penicillin-binding proteins (PBPs) for Plactams (Matsumoto and Terawaki, 1981; Gotoh et al., 1990).  The organism can also  synthesize aminoglycoside-modifying enzymes, produce altered ribosomes, and decrease the uptake of aminoglycosides by altering LPS and porins (Pitt et al, 1990; Gotoh and Nishino, 1990; Neu, 1989). Mutations in D N A gyrase subunit A or alterations of outer membrane proteins can lead to quinolone resistance (Cambau and Gutmann, 1993). Alternatively, various antimicrobial agents can be actively pumped out to the medium through efflux systems (Li et al, 1994a & 1994b; Quinn, 1992). This latter mechanism is now receiving much attention and recognition because a broad range of antimicrobial agents can often be expelled through a single system. Usually, more than one of the above mechanisms work synergistically to yield a multidrug resistance phenotype.  9  5. Efflux systems  5.1 Families Gram-negative organisms are intrinsically resistant to antimicrobial agents. In the past, this multiple-antibiotic resistance property was mainly attributed to decreased uptake of drug due to low outer membrane permeability. However, the outer membrane alone does not seem to be sufficient for high-level drug resistance (Nikaido, 1994). For instance, it is only a matter of seconds for imipenem to equilibrate across the outer membrane of P. aeruginosa, much shorter than the generation time of the organism (Quinn, 1992). A n efflux mechanism contributing to drug resistance was first demonstrated in E. coli (McMurry et al, 1980). Multidrug resistance in bacteria was first reported in Staphylococcus, in which the QacA protein was shown to pump out quaternary ammonium compounds, ethidium bromide, and other drugs (Littlejohn et al, 1990 & 1992; Rouch et al, 1990; Tennent et al, 1989). Active efflux systems are indeed common in bacteria (Levy, 1992; Lewis, 1994; M a et al, 1994; Nikaido, 1994, 1996, 1998a, 1998b) and they usually have a broad range of substrates, consistent with the high intrinsic multidrug resistance in species like P. aeruginosa. Generally, efflux systems are classified into five families based on sequence homology, assembly and the mechanism of action. Four of the families are the Major Facilitator  family  (MF), the  Resistance-Nodulation-Division  family  (RND),  the  Staphylococcal or Small Multidrug Resistance family (Smr), and the ATP-Binding Cassette (ABC) transporters (Lewis, 1994; M a et al, 1994; Nikaido, 1994 & 1998a; Saier et al, 1994; Zgurskaya and Nikaido, 2000b). The M F family can be divided into two subfamilies: the 14 transmembrane-segments (TMS) and 12-TMS subfamilies. The S M R transporters contain 4  10  TMS and function as oligomers (Paulsen et al, 1996). The R N D system is unique for Gramnegative bacteria and contains three components (Saier et al, 1994). The A B C transporters are homologous to the human P-glycoprotein for anticancer drug efflux and the mouse M D R 2 protein for phosphatidylcholine translocation (Homolya et al, 1993; Ruetz and Gros, 1994). A fifth family has been identified recently and is referred to as the Multidrug And Toxic compound Extrusion (MATE) family (Brown et al, 1999). Different efflux systems have different properties.  Some are chromosomally encoded while some are encoded by  plasmids. They utilize varying energy sources and have different spectra of substrates, some being more non-specific than others (Levy, 1992; Lewis, 1994; M a et al, 1994; Nikaido, 1994 & 1998b). In general, the M F and S M R transporters have a more limited range of substrates, with a preference for cationic hydrophobic molecules, while the R N D systems can extrude almost all lipophilic or amphiphilic antibiotics, dyes, detergents, and solvents (Nikaido, 1996).  However, there are significant similarities between multidrug efflux  transporters and specific antibiotic extrusion pumps, suggesting that multidrug resistance pumps might have evolved from more specific drug efflux pumps through the loss of substrate specificity (Levy, 1992; Lewis, 1994).  5.2 Resistance-Nodulation-Division (RND) efflux systems in P. aeruginosa Four multidrug resistant R N D efflux systems have been identified in P. aeruginosa, and numerous homologues could also be identified from the complete genome sequence. MexAB-OprM was the first one identified and it is produced constitutively in wild-type cells, resulting in intrinsic resistance to various antimicrobial agents including quinolones and different P-lactams, dyes, solvents, detergents, and disinfectants (Poole, 2000; Gotoh et al,  11  1995; Poole et al, 1993a & 1993b). The P. aeruginosa 7V-(3-oxo)-dodecanoyl-L-homoserine lactone was also suggested to be a substrate for MexAB-OprM (Evans et al, 1998). In nalB mutants, mutations in the upstream gene mexR change it from a repressor to an activator and cause hyperexpression of mexAB-oprM that leads to increased resistance levels to a wide spectrum of antimicrobial agents (Srikumar et al, 2000). Highly resistant mutants with increased expression of the operon independent of mutations in the mexR gene were also identified (Srikumar et al, 2000). Two other operons, mexCD-oprJ and mexEF-oprN, are not expressed in wild-type P. aeruginosa cells, but they are found to be highly expressed in nfxB and nfxC mutants respectively. Similar to mexR, the nfxB gene is located upstream of the operon and encodes a repressor. Multidrug resistant mutants with increased expression of mexCD-oprJ were found to have mutations within nfxB (Okazaki et al, 1991; Okazaki and Hirai, 1992). This system contributes to increased resistance to dyes, detergents, organic solvents, and to various antibiotics including quinolones and cephems, but cells are hypersusceptible to P-lactams and aminoglycosides (Poole et al, 1996). It is not definitively known which mutations lead to increased expression of mexEF-oprN in nfxC strains, but the mexT gene located upstream of the efflux genes encodes a positive regulator and is also responsible for hyperexpression of the system (Kohler et al, 1999; Ochs et al, 1999). The MexEF-OprN system contributes to elevated resistance levels to organic solvents and antibiotics including quinolones and carbapenems, but cells overexpressing the system are still hypersensitive to cephems, carbenicillin and aminoglycosides (Kohler et al, 1997). The recently described mexXY (also called amrAE) system does not have a linked gene for an outer membrane component, but either the OprM protein or the E. coli TolC  12  protein appear to be necessary for the functioning of the system (Aires et al, 1999; Mine et al., 1999). In contrast to the MexCD-OprJ and MexEF-OprN systems, the M e x X Y system seems to play an important role in the intrinsic resistance of P. aeruginosa to aminoglycosides (Aires et al, 1999). Upstream of mexXY is the mexZ (or amrR) gene that encodes a putative repressor for the transcription of the system (Aires et al, 1999; Westbrock-Wadman etal, 1999).  5.3 Assembly of the R N D efflux systems in the cell envelope The RND efflux systems consist of three components. The transporter (e.g. MexB) is located in the cytoplasmic membrane and is presumably driven by proton-motive-force for the efflux of substrates. Inhibition by uncouplers such as CCCP is consistent with this drugproton antiporter mechanism (Li et al, 1995), which was also confirmed for AcrB and CzcA (Goldberg et al, 1999; Zgurskaya and Nikaido, 1999). MexB was predicted to have 12 transmembrane helices, with 2 large periplasmic loops that may interact with the other components (Guan et al, 1999). The linker protein (e.g. MexA) is largely located in the periplasm while anchored to the inner membrane, probably via the lipid moiety at the N terminus. The E. coli AcrA protein, a homologue of MexA, was shown by chemical crosslinking to form oligomers, probably trimers that interact with the transporter AcrB independently of substrate and the outer membrane component TolC (Zgurskaya and Nikaido, 2000a). In an efflux complex, the linker protein is presumed to link the pump protein to the outer membrane component (e.g. OprM) that has been assumed to form channels in the outer membrane, like porins. However, the recently released crystal structure  13  of TolC, a homologue (20% identity) of OprM, revealed a unique and different structure (see below) for the outer membrane components of the RND systems (Koronakis et al, 2000).  6. Structural analysis of outer membrane proteins  6.1 Techniques To obtain the structural information of outer membrane proteins, the highest resolution can be obtained by X-ray crystallography that can solve the structure at the atomic level. This is a very useful tool, however, there are many limitations. X-ray crystallography requires specific training and expertise, large amounts of highly purified protein, and it is very time consuming with no guarantee of success. Without a crystal structure, structure analysis can be started with the building of a structural model based on the sequence, followed by genetic, biochemical and immunological approaches to test and revise the predicted model (see below).  6.2 Crystal structures of porin channels The crystal structures of several porins have been published (Cowan et al, 1992; Weiss and Schulz, 1992; Schirmer et al, 1995; Koebnik et al, 2000). Porins generally form trimers from identical monomers, each monomer consisting of a (3-barrel with usually 8-18 anti-parallel amphipathic f3-strands, in which side-chains of the hydrophobic and hydrophilic residues face the lipid bilayer and the water-filled channel respectively.  The P-strands,  usually 7 to 11 residues long, are often conserved among homologues and connected by short periplasmic turns and long surface loops. Loop 3 is usually one of the longest and folds into  14  the pore to constrict the channel size at the "eyelet" or constriction zone. For some channels, like OmpF and PhoE of E. coli, the pores of the three monomers are separated (Cowan et al., 1992). On the other hand, the pores of the three monomers in the Rhodobacter capsulatus porin merge into one channel at the periplasmic side (Weiss et al, 1991). The monomeric proteins vary in size, from the smallest P-barrel formed by OmpA with 8 P-strands and a diameter of 26 A (Pautsch and Schulz, 1998) to the largest 22 P-stranded barrels formed by FhuA and FepA, with diameters of 39-46 A (Locher et al, 1998; Buchanan et al, 1999). At the base of porins, there is usually a ring of aromatic residues, mainly Tyr and Phe, to occupy the lipid-water interface.  Structures of specific porins (e.g. E. coli LamB) have general  similarity to the structures of nonspecific porins, but often contain specific binding sites to favor the transport of certain substrates.  6.3 Structure prediction of outer membrane proteins (porins) Many porins have been identified in Gram-negative bacteria but the threedimensional structures of most are unknown. Prediction of their topologies is important for further investigations as they play key roles in antibiotic resistance and could be potential candidates for vaccines.  A number of methods can be used for the prediction of porin  topology. As porins generally adopt a P-barrel structure comprised of anti-parallel P-strands traversing the membrane, the P-barrel motif evident in previously published porin models is maintained during structure prediction. The method of Paul and Rosenbush (1985) can be used to predict turns. This method divides amino acids into turn blockers (A, Q, I, L , M , F, W, Y), turn promoters (N, D, E, G, P, S) and the other residues. Turns are recognized as 3 or more consecutive residues with at least one turn promoter and no turn blockers. As proposed  15  by Jeanteur et al. (1991), though porins do not share significant homology based on overall sequence, their transmembrane segments are usually more conserved. Therefore, multiple sequence alignments with homologous proteins, especially with those of known structures, provide an approach to identify transmembrane segments.  The fact that p-strands are  amphipathic (Vogel and Jahnig, 1986) also helps the structure prediction of outer membrane proteins by identifying segments with alternating hydrophobic and hydrophilic amino acids from their amphipathicity profiles (Gromiha et al, 1997). In addition, residues with both hydrophobic and hydrophilic properties, such as tyrosine, are favorable at the polar-nonpolar interface; and aromatic residues often occupying the base of porins can protect the porin conformation from adverse membrane fluctuations.  6.4 Functional studies for outer membrane proteins The predicted topology can then be tested and revised by different approaches, such as insertion mutagenesis or deletion mutagenesis (see Materials and Methods).  As the  characteristic alternating hydrophobic/hydrophilic motif of the P-barrel is fairly conserved, changes within the P-barrel will likely disrupt its proper folding and lead to degradation of the protein.  On the other hand, alterations within the more flexible surface loops or  periplasmic turns are usually permissive.  Tolerance levels of insertions or deletions at  different regions of an outer membrane protein can therefore help to refine its structure. In addition, epitope insertions can be utilized to identify surface exposed regions by indirect immunofluorescence assay with antibodies against the epitope. The liposome-swelling assay (Nikaido and Rosenberg, 1981; Hancock, 1986) and planar lipid bilayer assay (Hancock, 1986 & 1987) are two systems commonly used to  16  investigate the physical properties of outer membrane proteins. Both methods can be used to demonstrate the channel-forming function of porins in vitro. Our laboratory utilizes the latter system in which outer membrane proteins in detergent solution are added to an aqueous salt solution bathing a lipid bilayer.  Protein molecules will spontaneously insert into the  membrane and, i f they form channels, cause step-wise increases in the conductance between the two electrodes on either side of the membrane. This method is highly sensitive and can measure a single channel-forming unit; it can also provide an estimate of the channel diameter, the ion selectivity of the channel, and evidence for the presence of substrate binding site(s); it can also be utilized to determine whether the channel is voltage regulated. One major disadvantage of this method is that only the permeability of charged ions can be studied (Hancock, 1986). In the liposome-swelling assay, purified protein molecules are incorporated into phospholipid and the reconstituted proteoliposomes are added to a test solution adjusted to be isotonic with the impermeant solute (e.g. the sugar stachyose) inside the liposomes. The permeant solute (e.g. maltose) in the test solution creates a concentration gradient across the membrane and flows into the liposomes through the incorporated proteins, i f they form porins that are permeable to the solute. This uptake of solute is followed by an influx of water into the liposomes to maintain the osmotic equilibrium. The liposomes therefore swell and their abilities to scatter light are reduced, as indicated by a decrease in optical density. Similar to the planar lipid bilayer assay, this method can provide an estimate of the channel diameter, but it can only provide a qualitative estimate of the charge selectivity of the channel. Contrary to the planar lipid bilayer method, this assay can study the permeability of different classes of substances including saccharides and antibiotics; it can also provide an  17  estimate of the relative diffusion rates of a variety of solutes, and an estimate of the relative efficiency of individual porins.  Some possible complications of this technique include the  influence from light scattering by the solutes and the interactions between the liposome and the solute, which have to be controlled or regulated carefully (Hancock, 1986).  6.5 Crystal structure of E. coli TolC The E. coli TolC X-ray structure was released towards the end of this thesis work, and revealed a unique architecture (Koronakis et al, 2000). Solved at 2.1 A , the TolC trimer forms a 140 A long cylinder that consists of a 100 A long a-helical tunnel linked to a 40 A long p-barrel traversing the outer membrane. Instead of each monomer forming a p-barrel like the porins, three monomers of TolC form the 12-stranded p-barrel linked to the 12 stranded a-helical barrel spanning the periplasm. Each of the three monomers contributes to one-third of this continuous channel - 4 P-strands and 4 a-helical strands. Proline residues between the P-barrel and the helical tunnel may be important for introducing abrupt turns for the transition from the right handed P-strands into the left handed a-helices. There is a mixed a/p structure at the equatorial domain of the helical barrel that was hypothesized to be involved in interactions with the other efflux components. The size of the channel appeared to be restricted or controlled by the periplasmic ends of the helices, which apparently bring the channel to a virtual close. The cross sectional area of TolC is more than 10-fold larger than the  64A  2  of the general porin OmpF, which can allow diffusion of molecules with sizes  up to 600 Da (Cowan et al, 1992). This architecture, especially the a-helical tunnel portion, was not seen previously in cell membrane proteins, but reflects a very efficient structure for the direct extrusion of a wide spectrum of substrates across the two membranes and the 18  periplasmic space through efflux systems. This unique structure also gives new insights into the three-dimensional structures of proteins homologous to TolC, such as OprM, whose most similar homologue in E. coli is in fact TolC.  7. OprM of P. aeruginosa  The outer membrane protein OprM was first identified in 1992 from multidrug resistant mutants of P. aeruginosa isolated on medium containing ofloxacin and cefsulodin (Masuda and Ohya, 1992). These mutants were 4- to 8-fold more resistant to a variety of structurally  unrelated  drugs  like  meropenem,  cephems,  carbenicillin,  quinolones,  tetracycline, and chloramphenicol. A 50-kDa protein was found to be overproduced in the outer membrane of these mutants and was named OprM to stand for "outer membrane protein responsible for multiple-drug resistance" (Masuda and Ohya, 1992; Gotoh et al, 1994).  OprM is present in the outer membrane as oligomers, probably trimers with an  apparent molecular weight of about 100 kDa, that is heat-modifiable to the monomelic form of 50 kDa in SDS-PAGE (Masuda et al, 1995). The OprM overproduction phenotype and the multidrug-resistant phenotype were cotransferable by transduction to other P. aeruginosa strains, and OprM-deficient mutants isolated by transposon insertion mutagenesis were more susceptible when compared to wild-type P. aeruginosa (Masuda and Ohya, 1992; Gotoh et al, 1994). These observations suggested that OprM is associated with multidrug resistance as well as the intrinsic resistance in P. aeruginosa.  The R N D efflux systems probably  operate as tripartite pumps. In fact, OprM was shown to form functional chimeric systems  19  with components from other R N D efflux systems (Srikumar et al, 1997; Yoneyama et al, 1998). The 1458 base-pair oprM gene was cloned as part of the mexA-mexB-oprM operon, which was the first multidrug efflux operon shown to contain genes for all three of the components. The oprM gene was initially named oprK when it was cloned and sequenced from a siderophore (ferripyoverdine) receptor-deficient mutant of P. aeruginosa that could restore growth on iron-deficient medium. Increased resistance to multiple antibiotics and the overproduction of a 50 kDa outer membrane protein were also observed in this mutant (Poole et al, 1993b). Since the product of oprK shares many characteristics with the previously identified outer membrane protein OprM, efforts were made to determine i f they were actually the same protein. The transposon insertion sites of several OprM-deficient mutants were mapped to the mex operon. Multiple-antibiotic resistance and OprM production were both restored when the mexAB-oprK operon was introduced into OprM-deficient mutants. Immunological studies also indicated that OprM-specific antiserum did not react with OprK. These data confirmed that OprK and OprM are indeed two distinct proteins and oprK of the mex operon actually codes for OprM, and the operon was renamed mexAB-oprM (Gotoh et al, 1995; Hamzehpour et al, 1995). On the other hand, OprK was subsequently shown to share identity with OprJ, as confirmed with trypsin digestion and N-terminal sequencing of trypsin fragments from both proteins (Poole et al, 1996). The N-terminus of the 485 amino acids coding for OprM has the characteristics of a signal sequence with a putative cleavage site for lipoprotein signal peptidase (Poole et al, 1993b; Wu and Tokunaga, 1986). The cloned oprM sequence (encodes OprM*) was later found to contain errors at the 3' end when compared with the completed genome sequence, which encodes the native protein sequence  20  (OprM) containing 22 amino acids that were replaced with 14 residues derived from the phagemid used to clone the gene. Fortunately, this difference at the C-terminus did not seem to affect the expression or function of the protein, as determined in this study.  8. Aims of this study  OprM is the outer membrane component of the P. aeruginosa MexA-MexB-OprM efflux system and is homologous to numerous proteins associated with export of harmful materials in other Gram-negative bacteria. The MexA-MexB-OprM system was shown to contribute to the intrinsic resistance of P. aeruginosa against a wide spectrum of structurally unrelated antimicrobial agents plus other harmful agents, and increased susceptibility to drugs and increased accumulation of drugs were demonstrated in OprM-deficient mutants. OprM is therefore clearly involved in multidrug resistance via an efflux mechanism. However, at the start of this study, its mechanism of action was still unknown and there remained many questions. For instance, the physical, structural, and functional natures of the protein itself were still unclear. Little was known about the structure of OprM besides its primary sequence.  Could OprM function alone, without the other two components of the  RND system? Did OprM form channels in the outer membrane like porins as proposed? If it did form channels, what was the selectivity of the pore? What was the folding pattern of OprM in the outer membrane and how did function relate to structure in OprM? Attempts to solve these questions made the goals of this thesis: (1) to investigate the role of OprM in efflux by overproducing the protein and examining its effect on antimicrobial susceptibility of cells; (2) to predict an OprM topology model and verify it by mutagenesis; (3) to purify  21  OprM and investigate its channel-forming ability and other physical properties by planar lipid bilayer analysis; and ultimately (4) to elucidate the molecular architecture of OprM based on results from the genetic approaches, structural and functional studies in order to understand the mechanism of efflux through the protein.  22  METHODS AND MATERIALS  1.  Strains, plasmids a n d growth conditions  A l l bacterial strains used in this study are listed in Table I and all plasmids used are listed in Table II. Strains were routinely grown at 37°C in Luria Broth (LB) medium {1.0% (w/v) Tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl for E. coli or 0.05% (w/v) NaCl for P. aeruginosa} or on L B agar containing, in addition 2% (w/v) agar. components were obtained from Difco Laboratories, Detroit, Michigan.  A l l media  When plasmids  were present, media were supplemented with antibiotics at the following concentrations, for E. coli: ampicillin 100 p-g/ml; spectinomycin 30 p-g/ml; tetracycline 10 p.g/ml; kanamycin 50 p-g/ml; for P. aeruginosa: HgCL. 15 p-g/ml; streptomycin 45 tig/ml; and tetracycline 50 Lig/ml for OprM-deficient strains and 200 ng/ml for the wild-type strain.  2. G e n e t i c m a n i p u l a t i o n s  2.1 General techniques General D N A techniques such as D N A isolation and agarose gel electrophoresis were performed as described in Sambrook et al. (1989). Other methods included slot lysis gel electrophoresis (Sekar, 1987).  D N A restriction and modifying enzymes purchased from  Bethesda Research Laboratories (BRL, Burlington, O N , Canada) or Boehringer Mannheim (Mannheim, Germany) and Vent D N A polymerase purchased from New England Biolabs (Beverly, M A ) were used according to the manufacturers' methods. D N A fragments were 23  Table I . Bacterial strains.  Strain  Description  Reference/Source  E. coli DH5oc  supEAA hsdR 17 recA 1 endA 1 gyrA96 thi-1 relA 1 A(lacZYA-argF)Ul  CE1248  Hanahan, 1983  69rfeoR(<|)80d/acZAMl 5)  Porin deficient strain: OmpF", OmpC", PhoE"  Van der Lay et. al. 1985  P. aeruginosa H103  P A O l prototroph: wild-type reference strain  Hancock & Carey, 1979  K372  PAO6609(me?9011 am/E200 rpsL pvd9) Pch"  Poole et. al., 1991  deficient in production of pyochelin and the ferripyochelin receptor K613  K372 oprM::QHg, OprM-deficient  Poole et. al., 1993b  OCR1  P A O l M D R mutant overproducing  Masuda & Ohya, 1992  MexABOprM, selected on ofloxacin and cefsulodin OCR03T  OCR3 transduced with oprM::QHg, OprM-  Hamzehpour et. al.,  deficient  1995  24  Table II. Plasmids.  Plasmid  Description  Reference/Source  pPV20  carrying oprM* on a 4-kb Pstl fragment derived  Poole etal, 1993b  from the phagemid p P V l  pT7-7  pBR322 derivative carrying a M C S downstream of a  Tabor & Richardson,  strong phage T7 gene 10 promoter and a ribosome  1985  binding site; amp pUC4KAPA  r  a vector containing a K m Q interposon flanked by r  Pharmacia  symmetrical restriction sites pVLT31  broad-host-range expression vector; ladplacMCS, Tet  Lorenzo et al., 1993  r  pVLT35  a derivative of pVLT31, Spe7Sm  pKPM-1  pT7-7: :oprM*  Wong etal, 1997  pKPM-2  pVLT31::oprM*  Wong etal, 1997  pXZL33  pT7-7::o/?rMwt  L i & Poole, 2001  pXZL34  pVLT31::oprMwt  L i & Poole, 2001  pKW35M  pVLT35v.oprM*, pVLT35 with a 1.5 kb  This study  r  Lorenzo et al., 1993  Xbal/Hindlll insert carrying the entire oprM* gene with the upstream ribosome binding site from pT7-7 To be continued...  25  T a b l e II.  Plasmids. (continued)  Plasmid  Description  Reference/Source  pKW35TM  pVLT35:: oprMwt, pVLT35 with a 1.5 kb  This study  XballHindlll insert carrying the entire oprM gene with the upstream ribosome binding site from pT7-7  pKWINl  pVLT35::oprM* with malarial epitope insertion  This study  G P A P N A ( N P N A ) G H A G P at site ME1 (::23) 2  pKWIN2  pVLT35::oprM with malarial epitope insertion  This study  K R K ( N P N A ) N P N at site ME2 (::37) 2  pKWIN3  pVLT35 v.oprM* with malarial epitope insertion  This study  G P A P N A ( N P N A ) G H A G P at site ME3 (: :72) 2  pKWIN4  pVLT35 v.oprM* with malarial epitope insertion at  This study  site ME4 (::77); insertion generated translational stop site  pKWTN5  p V L T 3 5:: oprM with malarial epitope insertion  This study  K R K N P N A N P N at site ME5 (:: 103)  pKWTN6  pVLT35::oprM* with malarial epitope insertion  This study  TC(NPNA) CRS at site ME6 (::130) 3  To be continued..  26  T a b l e II.  Plasmids. (continued)  Plasmid  Description  Reference/Source  pKWTN7  pVLT35 v.oprM* with malarial epitope insertion at  This study  site ME7 (:: 143); insertion generated translational stop site  pKWIN8  pVLT35 v.oprM* with malarial epitope insertion  This study  G P A P N A ( N P N A ) G H A G P at site ME8 (::159) 2  pKWIN9  pVLT35::oprMwith malarial epitope insertion  This study  K R K ( N P N A ) N P N at site ME9 (::241) 2  pKWINlO  pVLT35 v.oprM* with malarial epitope insertion  This study  GTC(NPNA) CRS at site ME10 (::251) 3  pKWTNll  pVLT35::oprM* with malarial epitope insertion  This study  GTC(NPNA) CRS at site ME11 (::315) 3  pKWIN12  pVLT35 v.oprM* with malarial epitope insertion  This study  D L Q ( N P N A ) N A L D V Q V at site ME12 (::377) 2  pKWTN13  pVLT31 v.oprM with malarial epitope insertion  This study  K R K N P N A P N A N P N at site ME13 (::393) To be continued...  27  Table II. Plasmids. (continued)  Plasmid  Description  Reference/Source  pKWDl  pVLT35::oprMwt with a deletion of 24 bp in oprM  This study  (AG29-A36)  pKWD2  pVLT35::oprMwt with a deletion of 24 bp in oprM  This study  (AS93-P100)  pKWD3  pVLT35::o/>rMwt with a deletion of 12 bp in oprM  This study  (AA375-T378)  pKWD4  pVLT35::o/?rMwt with a deletion of 24 bp in oprM  This study  (AQ382-L389)  pKWD5  pVLT35::oprMwt with a deletion of 24 bp in oprM  This study  (AI429-L436) pKWDC  pVLT31 v.oprM* with a deletion of 186 bp in oprM*This study (i.e. 210 bp in oprMwt) at the 3' end to construct a C-terminal deletion mutant  28  isolated by using the G E N E C L E A N kit (BIO 101 Inc. L a Jolla, C A ) or the QIAprep spin miniprep kit (Qiagen Inc., Chatsworth, California), and P C R fragments were purified using the Qiaquick P C R purification kit (Qiagen Inc., Chatsworth, California) following the manufacturers' protocols.  For oligonucleotides used in ligations, the sense and antisense  strands of each set were annealed by heating an equal amount of each strand (100 uM) in 2 m M MgCl /50 m M NaCl/20 m M Tris-HCl pH7.5 at 90°C for 15 min, followed by gradual 2  cooling to room temperature.  2.2 Transfer of D N A into E. coli and P. aeruginosa Competent E. coli cells were prepared using the CaCl method (Hanahan, 1983) and 2  transformations with plasmid D N A or ligation products were performed as described in Sambrook et al. (1989). Competent P. aeruginosa cells and transformation were performed as described by Olsen et al. (1982). Briefly, cells to be transformed were grown to an OD o 55  of 0.2 - 0.6 in L B with 0.05% w/v NaCl (LBLS). Cells were pelleted and resuspended in half culture volume of.cold, 0.15 M M g C l , and placed on ice for 5 min. This step was 2  repeated except that the cells were kept on ice for 20 min. The cells were then pelleted and resuspended in one-twentieth culture volume of ice-cold 0.15 M M g C l . 2  For each  transformation, about 100 ng of D N A was added to 0.2 ml cells and the mixture was placed on ice for 60 min followed by a 3 min heat pulse at 37°C. L B L S (0.5 ml) was added and the mixture was incubated at 37°C for 2 h to allow the expression of the plasmid's antibiotic resistance gene(s). Aliquots (0.1 ml) of cells were plated on selective medium and grown for 24 to 48 h at 37°C.  29  2.3 Oligonucleotide synthesis and purification Oligonucleotides were synthesized on an Applied Biosystems Incorporated (ABI, Foster City, C A ) 392 D N A / R N A synthesizer according to the manufacturer's instructions. Oligonucleotides were incubated at 55°C overnight and purified by precipitation with the addition of an equal amount of 1-butanol and centrifuged for 10 min. This step was repeated and the oligonucleotides were air-dried or dried in the Speed Vac Concentrator (Fisher Scientific, Ottawa, ON). The oligonucleotides were then resuspended in 0.5 ml dFbO before quantification by OD 6o absorbance. 2  2.4 D N A sequencing Primers annealing to different regions of oprM were synthesized as described in the previous section.  D N A plasmids for sequencing were isolated using a QIAprep spin  miniprep kit (Qiagen Inc., Chatsworth, California) following the manufacturer's protocol. Sequencing reactions were set up according to the manufacturer's method, containing at least 200 ng of D N A , 3.2 pmol of primer and components from an Applied Biosystems Inc. (ABI, Foster City, California) Ampli Taq FS DyeDeoxy Terminator Cycle Sequencing kit or a BigDye Terminator Cycle Sequencing kit. Sequencing reactions were carried out using a thermocycler (MJ Research, Inc., Massachusetts, USA) (96°C for 30 sec, 50°C for 15 sec, 60°C for 4 min; 26 cycles), run on an ABI 373XL automated D N A sequencer, and analyzed using A B I 373 Data Collection and Analysis programs.  3. General protein and immunological techniques  30  3.1 Electrophoresis Proteins were separated by electrophoresis through SDS-polyacrylamide gels as previously described (Hancock and Carey, 1979). The percentage of acrylamide used was 8.5% w/v unless otherwise stated. The amount of protein loaded per lane was normalized by protein assay (see section 13.1). Proteins were incubated in solubilization buffer (2X: 4% v/v SDS, 20% v/v glycerol, 125 m M Tris-HCl pH 6.8, 40 m M EDTA) with or without pmercaptoethanol at 100°C or room temperature before loading. Proteins were stained with Coomassie brilliant blue solution (0.25% w/v Coomassie Brilliant Blue R250 [Sigma, M O , USA], 10% v/v glacial acetic acid, 45% v/v methanol, 45% v/v distilled water) followed by destaining the gels with 7.5% v/v acetic acid, 20% v/v methanol, 72.5% v/v distilled water.  3.2 Western immunoblotting Proteins from unstained gels were transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, M A ) in cold transfer buffer {25 m M Tris, 0.2 M glycine, 20% (v/v) methanol} at 100 V for 1 h. Proteins were then detected as described in Mutharia and Hancock (1985).  The P V D F membranes were air-dried  completely after protein transfer and no blocking was necessary; they were wetted with methanol, followed by incubation with primary antibody (an • OprM-specific murine monoclonal antibody, kindly provided by N . Gotoh) for 1 h at 37°C. After 3 x 10 min washes in l x PBS, secondary antibody (an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G) was added and incubated for 30 min at 37°C, followed by PBS washing and color development with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium.  31  3.3 Indirect immunofluorescence Surface-exposed proteins were detected by the method of Hofstra et al. (1979). Briefly, aliquots of cells were incubated at room temperature for 1 h with an anti-OprM or anti-malarial epitope monoclonal antibody (p£2A.10, obtained from R. Wirtz) (both at 1/100 dilution) in PBS containing 1% (w/v) bovine serum albumin (BSA) (Boehringer Mannheim, Germany), followed by incubation in the dark with a fluorescein isothiocyanate-conjugated goat anti-mouse IgG secondary antibody (Gibco/BRL) at 1/20 dilution in PBS/1% B S A for 30 min at room temperature. Washings after each incubation were done with 1 x PBS. Cells were spread, air-dried on poly-L-lysine-coated slides (Sigma, M O , USA), and covered with a drop of mounting reagent {50%(v/v) glycerol, 50%(v/v) PBS} and a coverslip. Fluorescence was monitored with a Zeiss microscope fitted with a halogen lamp, a condenser and filters set for emission of fluorescein isothiocyanate at 525 nm.  4. Prediction of the topology model of O p r M  4.1 Sequence alignment The first criterion utilized for modeling was based on multiple-sequence alignment of P. aeruginosa OprM with several proteins with highest homology.  The online ClustalW  Multiple Sequence Alignment program at Baylor College of Medicine (BCM) was used to perform the alignment, the gap penalty was adjusted to optimize the alignment and minimize gaps in the more conserved regions. The regions with more variations were assigned to be surface loops or periplasmic turns. Conversely, the more conserved regions were predicted to be transmembrane segments.  32  4.2 Structural characteristics The primary model was adjusted to the structural characteristics of porins as OprM has been presumed to form a channel in the outer membrane of P. aeruginosa (Ma et al, 1994) and it showed some homology to, and was predicted to function similarly as the E. coli outer membrane protein TolC, which had been shown to form channels (Benz et al, 1993). 4.2.1 (Kleffel  Transmembrane strands.  et al., 1985).  Porins have high content of P-sheet conformation  The more conserved transmembrane  P-strands are typically  amphipathic and are composed of alternating polar and non-polar amino acid residues which are exposed to the aqueous porin channel and hydrophobic lipid membrane interior respectively.  The amphipathicity profile of OprM was calculated using the method as  described (Gromiha et al, 1997). The identified amphipathic and more conserved regions of OprM were mostly contained in the transmembrane P-strands. 4.2.2  Surface loops. The external loops are long and less conserved, and of variable  length and they contain many polar residues and are hydrophilic. The long flexible regions between conserved segments identified in multiple-sequence alignment of OprM with its homologues were predicted as surface loops. 4.2.3  Periplasmic turns.  The P-strands are connected by short turns at the  periplasmic space and turns are mainly identified according to the method of Paul and Rosenbush (1985). Briefly, amino acids are classified into turn promoters (D, E, G, N , P, S), turn blockers (A, F, I, L , M , Q, W, Y ) and other residues. And turns are recognized as 3 or more consecutive residues with at least one turn promoter and no turn blockers.  33  5. Carboxyl-terminal deletion of OprM  A deletion of the last 70 amino acid residues at the C terminus of OprM was accomplished  by  PCR.  The  oligonucleotide  GCGACGAGTACTACCAGZ4GTAGAAGCTTATGC  complementary  to  [5'  3'] containing two stop codons  (italicized) and a Hindlll site (underlined) was synthesized and used to amplify oprM up to amino acid residue 405 with a primer annealing to the 5' end of oprM. The PCR product was purified, digested with Ndel and Hindlll, and ligated to pT7-7.  After confirmation by  sequencing, the truncated oprM was cloned into pVLT31 for expression studies and antibiotic susceptibility assays.  6 . Insertional mutagenesis  6.1 Linker insertion mutagenesis A schematic diagram of the procedures as described in Wong et al. (1993) is shown in Fig. 2. Insertion of 12-bp linkers into the oprM* gene was done by partial digestion of pT7'-7v.oprM* with frequently cutting blunt-end restriction endonucleases Rsal or Haelll (Boehringer Mannheim). The singly cut plasmids were purified using the G E N E C L E A N kit (BIO 101 Inc. L a Jolla, C A ) and ligated to a 1.3 kb Hincll fragment isolated from the kanamycin resistance plasmid p U C 4 K A P A . After transformation of the ligation products to E. coli DH5a, clones were selected for both ampicillin and kanamycin resistance.  The  resultant colonies were screened by PCR assay for insertion of the K m gene into oprM*, r  using an internal primer for the K m gene and a forward primer annealing to the 5' end and a r  34  Figure 2.  Schematic representation of semi-random malarial mutagenesis with a kanamycin resistance cassette.  epitope-insertion  A. The kanamycin (Km) resistance cassette used for generating a unique Pstl site for the insertion of malarial epitope repeats. The solid box represents the region that encodes a gene conferring kanamycin resistance. The relative position of the symmetric restriction enzyme sites Hindi and Pstl are indicated. B . Procedures for linker-insertion mutagenesis to generate the unique Pstl site for malarial epitope insertions. The solid and hatched boxes represent the K m cassette and the oprM* gene respectively. Screening for the insertion of Km cassette into oprM* was by PCR using primer pair a,b (T7rbs, oprmHind). One copy of Km into oprM* gives one product of 2.8 kb (1.5kb oprM* + 1.3kb Km), OprM -ve. C . The orientation and position of the Km cassette inserted in oprM* were determined by PCR using primer pairs a,c (T7rbs, Pkmrev) and b,c (oprmHind, Pkmrev). A PCR product can be obtained from only one primer pair which also indicates the orientation of Km in oprM*. The size of the PCR product reflects the position of the insertion in oprM*.  35  A. Hindi  Hindi  1.3  I  B.  kb  1  oprM  pT7-7 Linearized with oiunt-ena enzymes (/faem, foal) Insert Hindi fragment of Km Select for Km resistance (PCR screening with primers a,b)  oprM + ve fTTTv  OR  a  oprM - ve  V  Cut with _  p f] S  to remove K m and religate OprM linker mutant: 12bp insert contains Pstl site for malarial epitope insertion  c.  1.3kbKm  36  reverse primer annealing to the 3' end of oprM*. The kanamycin resistance gene was then removed by Pstl digestion, leaving a 12-bp linker sequence containing the unique Pstl site in oprM*. After religation, the plasmids were transformed back into DH5oc and sequenced. The  linker mutant plasmids were then linearized by Pstl and ligated to synthetic  oligonucleotides (Fig. 3) encoding the malarial epitope with the corresponding reading frame to ensure correct translation.  The resultant insertion mutant forms of oprM* were then  excised by Xbal and Hindlll and cloned into pVLT35 for expression experiments.  6.2 PCR site-directed insertion mutagenesis When pXZL33 (pT7-7:: oprMwt) became available, PCR was used for site-directed insertion mutagenesis of the malarial epitope into oprM. The primer design strategy for this approach is shown in Fig. 4 and the primers are listed in Table III. Primers containing a D N A region encoding the amino acid sequence K R K N P N A N P N A N P N were designed to introduce the malarial epitope (NANP) at the chosen insertion sites. The K R K motif was used to ensure that insertion into the amphipathic P-strands would interrupt the correct folding of the protein.  The PCR products were then phosphorylated, religated, and  transformed into E. coli DH5a cells; and the oprM insertion mutant forms, confirmed by sequencing, were eventually cloned into pVLT35 for expression experiments.  37  A.  Phase 1  P  N  A  N  P  N  A  N  P  N  A  G  H  C C G A A C G C C A A C C C G A A C GCC A A C C C G A A C GCC G G G ACGTGGC  B.  A  CATGCA  T T G C G G T T G GGC T T G C G G T T G GGC T T G C G G C C C G T  Phase 2  N  P  N  A  N  P  N  A  N  P  N  A  C  A C C C G A A C G C C A A C C C G A A C G C C A A C C C G A A C G C A TGC A  ACGTTG GGC T T G C G G T T G GGC T T G C G G T T G GGC T T G CGT  C.  Phase 3  N  A  N  P  N  A  N  P  N  A  L  D  V  Q  G A A C G C C A A C C C A A A C G C G A A T C C G A A T G C C C T A G A C TTG CA  ACGTC T T G C G G T T G GGT T T G CGC T T A GGC T T A C G G G A T C T G A  Figure 3.  Nucleotide and encoded amino acid sequences of the oligonucleotides used for the insertion of malarial epitope repeats into OprM via a Km-cassette adaptor.  Each set of the three pairs of oligonucleotides encodes the malarial epitope sequence in one of the three possible reading frames at the Pstl cleavage sites. The Pstl compatible ends for the ligation into the Pstl sites generated in the 12 bp linker by removing the Kanamycin resistance cassette are in italics. Unique restriction enzyme sites in the oligonucleotides are underlined (Sphl in frames 1 and 2, Bsml in frame 3). For each pair of oligonucleotides, equal amounts (50^) of each were mixed, heated to 100°C for 2 min and cooled to room temperature for annealing. The annealed oligos were phosphorylated with T4 polynucleotide kinase for the insertion into the Pstl cleavage site in the 12 bp linker.  38  Figure 4.  Primer design strategy for site-directed insertion mutagenesis of OprM using PCR.  A. D N A sequence encoding the shown amino acid sequence for introducing malarial epitope (NANP) repeats at desired sites in OprM. The K R K motif was used to ensure that insertion into the amphipathic P-strands would interrupt the correct folding of the protein. B. The forward and reverse complementary primer pairs. Each primer contains half of the sequence to be inserted at the 5' end and approximately 18 bp of oprM sequence at the 3' end for annealing to the desired site of insertion. A list of the oligos is shown in Table III. The template for PCR was pXZL33 (pT7-7::oprMwt). The PCR products were then phosphorylated and religated.  39  2 I—  5  O CD CD O (3  ^  o  I-  s I-  < CD  o  CD  IS O  ^5  CD CD <  E  ^ *  CD CD O CD  * <  c  O O CD O  E CD  5  H  Q  o  a.  E o u  0)  & 2 DO  40  Table III. Oligonucleotides for the amplification and mutagenesis of oprM* or oprM.  Name  Description  oprmNde  5'-AGCGCAT^rGAAACGGTCCTTCC-3' Anneals to 5' end of oprM(*), introducing Ndel site (underlined), start codon for oprM italicized  oprmHind  5'-GCATAAGCTTGGCTCTAGCCX4TCC-3' Complementary to the 3' end of oprM*, introducing Hindlll site (underlined), stop codon for oprM italicized  oprm2F  5'-AATCCAAACGCTAATCCGAATATTGGCTGGCGCGAGTTCTTCC-3' N P N A N P N I G W R E F F Mutagenic primer for introducing part of the malarial epitope repeats at M E 2  oprm2R  5'-TGCGTTCGGGTTTTTCCGCTTGTCGGCGGCCGGAACGG-3' A N P N K R K D A A P V Mutagenic primer for introducing part of the malarial epitope repeats at M E 2  opim5F  5'-AATCCAAACGCTAATCCGAATTCGACCACCGGCAGTCCGG-3' N P N A N P N S T T G S P Mutagenic primer for introducing part of the malarial epitope repeats at M E 5  oprm5R  5' - T G C G T T C G G G T T T T T C C G C T T C A G G T C G C C C G G C A A A C G - 3 ' A N P N K R K L D G P L R Mutagenic primer for introducing part of the malarial epitope repeats at M E 5  oprm9F  5'-AATCCAAACGCTAATCCGAATATTCCGGCGAACCTGCCGC-3' N P N A N P N I P A N L P Mutagenic primer for introducing part of the malarial epitope repeats at M E 9 To be continued..  41  Table III. Oligonucleotides, (continued)  Name  Description  oprm9R  5'-TGCGTTCGGGTTTTTCCGCTTCCCGGAGCCCAGCAGC-3' A N P N K R K G S G L L Mutagenic primer for introducing part of the malarial epitope repeats at M E 9  oprml3F  5'-AATCCAAACGCTAATCCGAATTCCGACGAGTACTACCAGCTCG-3' N P N A N P N S D E Y Y Q L Mutagenic primer for introducing part of the malarial epitope repeats at M E 13  oprml3R  5' - T G C G T T C G G G T T T T T C C G C T T G G C C T T G A C C A G A T C G C - 3 ' A N P N K R K A K V L D Mutagenic primer for introducing part of the malarial epitope repeats at M E 13  MEP1F  5'-CCGAACGCCAACCCGAACGCCAACCCGAACGCCGGGCArGG4-3' P N A N P N A N P N A G H A M E phase 1 forward primer; Sphl site underlined; Pstl compatible end italicized  MEP1R  5'-TGCCCGGCGTTCGGGTTGGCGTTCGGGTTGGCGTTCGGrGC4-3' M E phase 1 reverse complementary primer; Pstl compatible end italicized  MEP2F  5'-ACCCGAACGCCAACCCGAACGCCAACCCGAACGCArGC4-3' N N A N P N A N P N A C M E phase 2 forward primer; Sphl site underlined; Pstl compatible end italicized  MEP2R  5'-TGCGTTCGGGTTGGCGTTCGGGTTGGCGTTCGGGTrGG4-3' M E phase 2 reverse complementary primer; Pstl compatible end italicized  MEP3F  5'-GAACGCCAACCCAAACGCGAATCCGAATGCCCTAGACT7/GC4-3' N A N P N A N P N A L D V Q M E phase 3 forward primer; Bsml site underlined; Pstl compatible end italicized To be continued... 42  T a b l e III. Oligonucleotides, (continued)  Name  MEP3R  Description  5'-AGTCTAGGGCATTCGGATTCGCGTTTGGGTTGGCGTTCJGC4-3' M E phase 3 reverse complementary primer; Pstl compatible end italicized  kmrev  5'-GACCTTTCCCGTTGAATATGGC-3' Complementary to the 5' end of the Km cassette, for screening insertion & orientation of the Km cassette  nanp  5'-AACGCC A A T C C G A A T G C G A A C C - 3 ' N A N P N A N Anneals to the malarial epitope for screening of insertions with T7rbs/ tmHIIIr  oprmDIF  5'-CAGGCCTACGGGCAGAACACC A GACATCGGCTGGCGCGAGTTC-3' Q A Y G Q N T D I G W R E F Mutagenic primer with a deletion of 24 bp at A for AG29-A36 mutant D l  oprmP2r  5 '-T A C T G G C T G G A A A T C G C C G G A C T G C - 3 ' Used to amplify a portion of oprM with oprmDIF in constructing mutant D l  oprmDlR  5'-GAACTCGCGCCAGCCGATGTCGGTGTTCTGCCCGTAGGCCTG-3' Complementary to oprmDIF for overlap extension  T7rbs  5' - C C C T C T A G A A A T A A T T T T G T T T A A C T T T AAG-3' Anneals to upstream of ribosome binding site in pT7-7; Xbal site underlined; used to amplify 5' end of oprMfrompT7-7: :oprM for constructing deletion mutants D1,D2 To be continued...  43  Table III. Oligonucleotides, (continued)  Name  Description  oprmD2R  5'-GCCGGTGGTCGACAGGTCGCC A ACCGTCCACGCCGATCCGCGG-3' Mutagenic primer with a deletion of 24 bp at A for AS93-P100 mutant D2; its complementary sequence codes for PRIGVDG A G D L S T T G  oprmD3F  5'-GAAGTCGCCGACGGCCTGGCC A TTCACCGAGCAGTTGCAGGCG-3' E V A D G L A F T E Q L Q A Mutagenic primer with a deletion of 12 bp at A for AA375-T378 mutant D3  tmHIIIr  5' -GCGGAAGCTTAJC4y4 GCCTGGGGA  TCTTCC-3'  Complementary to 3' end of oprM (italicized), with Hindlll site (underlined) after the stop codon (bold face); for constructing mutants D3, D4, D5 oprmD3R  5'-CGCCTGCAACTGCTCGGTGAAGGCCAGGCCGTCGGCGACTTC-3' Complementary to oprmD3F for overlap extension  oprmP5f  5'-CCAGACCCTGCTGACCGAAGTGC-3' Used to amplify a portion of oprM with oprmD3R in constructing a AA375T378 mutant D3  oprmD4F  5 ' - G C G C G C G G T A C C T T C A C C G A G A G T C A A G G C C A G C G A C G A G T A-3' A R G T F T E V K A S D E Mutagenic primer with a deletion of 24 bp at A for AQ382-L389 mutant D4  oprmD5F  5'-TTCACCGCGCAGCAGCAACTG A ACCAGCGAGGTCAACCTGTAC-3' F T A Q Q Q L T S E V N L Y Mutagenic primer with a deletion of 24 bp at A for AI429-L436 mutant D5 To be continued... 44  Table III. Oligonucleotides, (continued)  Name  Description  oprmD5R  5'-GTACAGGTTGACCTCGCTGGTCAGTTGCTGCTGCGCGGTGAA-3' Complementary to oprmD5F for overlap extension  oprmP6f  5'-GTTCGACGCCGGTTCGGGTTCC-3' Used to amplify a portion of oprM with oprmD5R in constructing a AI429-L436 mutant D5  45  7. P C R - b a s e d site-specific deletion mutagenesis  7.1 Direct extension The method described by Vallette et al. (1989) was applied to the regions of oprM with convenient (e.g. unique) restriction sites adjacent to the nucleotide sequences to be deleted. This procedure involved 2 synthetic oligonucleotides as the primers to amplify the nucleotide sequence of interest. deletion.  Primer "a" contained the restriction site and the desired  Primer "b" annealed at another end of the targeted sequence and was in the  opposite orientation as shown in Fig. 5.  7.2 Overlap extension The method described by Ho et al. (1989) was applied for regions without convenient restriction sites located near the sequence to be deleted. This method (Fig. 5) required 2 pairs of primers which included an external pair "a" and "d" hybridizing at each end of the targeted sequence, and an internal complementary pair "b" and "c" hybridizing to either side of the desired site of the mutation and containing the desired deletion. Two PCR reactions were carried out. The first reaction involved separate amplification of the oprM regions with primers a/b or c/d to obtain fragments A B and CD, which overlap with each other. These two products were mixed with primers a and d for the second reaction to yield the extension product of the overlap fragments, and resulted in the mutant product. The primers were designed by the PRIMER program to minimize the chance of non-specific annealing and primer-dimer formation. Each of the mutagenic primers contained at least 20 nucleotides  46  I. Direct Extension  II. Overlap Extension Deleted Sequence Primer a  Deleted Sequence REI  :  •v  •  Primer a  REI  ^  RE2 -I  VPrimer c  2  < •-  Primer  Primer d  <ZZ1 Primer b  Primer a REI  AB  RE2  CD  Overlap  Schematic diagram showing two mutagenesis of oprM.  ^ . . Primer d  Extension  REl  Figure 5.  RE  PCR strategies  RE2  for defined-deletion  R E l and RE2 were two unique restriction sites flanking the mutated site and they were used for subsequent cloning procedures. I. Direct extension: the deleted sequence is shown by the broken circle, primer "a" was the mutagenic oligo which contained R E l and the deletion. II. Overlap extension: the deleted sequence is shown as the dotted lines in the middle, the solid and the broken lines are the template sequences on either side of the deletion site. Primers "b" and "c" were designed such that their 5' ends were complementary to the template sequence on one side of the deletion and their 3' ends were complementary to the template sequence on the other side of the deletion. The first step PCR products A B and CD thus overlapped at the deletion site.  47  from each side of the deletion site.  A list of the primers designed and used in both  approaches is also shown in Table III.  7.3 Polymerase chain reaction (PCR) The reaction mixture (50 ul total volume) contained: 5 ul of 10 X Vent reaction buffer, 400 u M of each dNTPs, 10 ng of D N A template, 2 u M of each primer, and 2 units of Vent D N A polymerase (New England Biolabs, Beverly, MA). The reactions were carried out for 24 cycles using a thermal cycler (Ericomp Inc.) and each cycle included a denaturation step (94°C for 1 min), followed by an annealing step (50-55°C for 2 min) and an extension step (72°C for 1-1.5 min).  8. Expression of oprM and oprM mutants  For expression of oprM and mutant forms of oprM from plasmid pVLT31 or pVLT35 in E. coli CE1248 strain or different P. aeruginosa strains, fresh L B broth was inoculated at 1/100 dilution with an overnight stationary phase culture. When the cell cultures reached mid-log phase (OD oo ~ 0.4 - 0.6), isopropylthio-P-D-galactoside (IPTG) was added to a final 6  concentration of 0.1 m M and cells were induced for 2 h before harvest.  9. Purification of OprM and mutants  48  9.1 Outer membrane isolation 9.1.1  Sucrose  gradient centrifugation.  Cultures overexpressing  oprM were  harvested and the cell pellet was resuspended in cold 20% (w/v) sucrose in 10 mM Tris-HCI pH 8.0. DNase I (50 p.g/ml) was added, followed by incubation at room temperature for 15 min. Cell lysis was achieved by two passages through a French pressure cell at 15,000 psi. The lysed cells were centrifuged at 1,700 x g for 10 min to remove cell debris.  The  supernatants were then subjected to a 2-step, 50% (w/v)/70% (w/v) sucrose gradient and centrifuged at 5 °C for at least 6 h at 100,000 x g with a SW28 rotor (Beckman Instruments Inc., C A , USA). The lower band that formed at the interface of the 50% and 70% sucrose layers was collected and the sucrose was diluted with at least two volumes of distilled water, followed by centrifugation at 200,000 x g in a 70Ti rotor (Beckman, CA) at 5°C for 1 h. The final pellets were resuspended in a small volume of distilled water or 10 m M Tris-HCI pH 8.0. 9.1.2  Sarkosyl extraction.  Harvested cells were resuspended in 0.01 M 4-(2-  Hydroxyethyl)-l-piperazinethane-sulfonic acid (HEPES) pH 7.4 for French pressing. After removal of unbroken cells and debris, samples were incubated with 0.7% (w/v) sarkosyl solution for 25 min at room temperature.  Outer membranes were extracted as insoluble  pellets after 1 h of high speed centrifugation (200,000 x g) in the 60Ti rotor (Beckman, CA) at 5°C.  The membranes were then washed with distilled water and pelleted by an hour of  high speed centrifugation.  49  9.2 Detergent solubilization Large quantities of OprM could be extracted from outer membrane proteins by solubilization  with  Philadelphia, PA).  octyl-polyoxyethelene  (octyl-POE)  (Bachem  Bioscience  Inc.,  Briefly, the outer membrane pellets obtained from sucrose density  gradients were resuspended in 10 mM Tris-HCl pH8.0 with 0.5% (v/v) octyl-POE, followed by incubation at 37°C for 1 h and centrifugation at 200,000 x g for 1 h in a 60Ti rotor (Beckman, CA) at 5°C. The supernatant was reserved and the pellet was extracted with 10 mM Tris-HCl (pH 8.0), 3% (v/v) octyl-POE followed by centrifugation as above. A high concentration of OprM was obtained in the supernatant with many other membrane components removed.  9.3 Fast protein liquid chromatography (FPLC) The solubilized fraction containing OprM was dialyzed into buffer A {10 m M TrisH C l (pH 8.0), 1% (v/v) octyl-POE} at 4°C overnight and then passed through an FPLC anion-exchange MonoQ column (Pharmacia, Sweden) (bed volume=T.O ml, flow rate=0.5 to 1.0 ml/min) that had been equilibrated with buffer A. The protein was eluted by applying a linear gradient of buffer B (buffer A with 1.0 M NaCl). A fraction that contained native 110kDa  OprM with the least contaminants, as determined by SDS-PAGE and Western  immunoblotting, was retained.  9.4 Gel elution The fraction from FPLC containing native OprM with the least contaminants or the OprM-containing supernatant from detergent solubilization with octyl-POE was subjected to  50  SDS-PAGE without heating of the sample. The OprM band was then excised from the gel and eluted in 10 m M Tris-HCI (pH8.0) with 0.1% (v/v) SDS at 4°C overnight to renature the protein. Purified OprM was stored in small aliquots at -20°C to prevent inactivation due to frequent freezing and thawing, and the protein remained in the native state for at least a month.  10. P l a n a r l i p i d b i l a y e r a n a l y s i s  The techniques and instrument for this analysis were described in details by Benz and Hancock (1981) and Benz et al. (1985). The apparatus includes a Teflon chamber divided into two compartments by a Telfon wall that contains a hole (0.1 mm diameter). Electrodes were dipped into the aqueous solution in the two compartments. A membrane was formed across the hole by painting a solution of 1.0% (w/v) diphytanoyl phosphatidylcholine in ndecane. The membrane turning optically black to incident light indicated bilayer formation. Purified protein was diluted into 0.1% (v/v) Triton X-100 (usually 1:100) prior to addition to the solution in the front compartment. For single channel conductance measurements, one electrode was connected to a millivolt voltage source. The other electrode was connected to a Keithley 427 current amplifier to boost the output by 10 -fold. 9  A Tektronik 5111A storage  oscilloscope and a chart recorder (Huston Instrument, Texas, USA) were used to monitor and record the amplified output. For each experiment, more than 100 events were recorded.  51  11. Circular dichroism (CD) analysis  The C D spectrum of OprM was obtained with a J810 spectropolarimeter (Jasco, Tokyo, Japan) using a quartz cell with a 1-ram path length. C D spectra were measured at 25°C, between 190 and 250 nm at a scanning speed of 10 nm/min in 10 m M Tris buffer (pH 8.0) with 0.1% w/v SDS. After subtracting the background spectrum generated with buffer alone from the sample spectrum, the spectrum for OprM was deconvoluted with the K2D program (Andrade et al, 1993) to determine the percentages of P-pleated sheet and a-helical structure.  12. Three-dimensional modeling  The OprM sequence was threaded (or passed) into the TolC crystal structure using the Insight II (version 97.2) molecular modeling program "Homology" (Molecular Simulations Inc., San Diego, Calif), constraining regions that aligned with the a-helical regions or Pstrands of TolC and allowing more freedom in the loop regions. The entire structure was then subjected to energy minimization using the "Discover" program of Insight II.  13. Assays  13.1 Protein assay Protein concentrations were estimated using a modified Lowry assay (Sandermann and Strominger, 1972). A 1 mg/ml solution of BSA was used to generate the standard curve.  52  13.2 Minimal inhibitory concentration (MIC) determination MICs were determined by serial twofold dilution in L B or Mueller-Hinton medium using the method described by Amsterdam (1991). Results were determined after incubation at 37°C for 24 h. The MICs were taken as the lowest antibiotic concentration at which cell growth was inhibited and MICs were determined from at least three experiments. Antibiotics were purchased from Sigma (MO, USA).  53  RESULTS CHAPTER ONE: Expression of oprM*  1.1 Introduction  To investigate the role of OprM in efflux and multiple antimicrobial resistance, and to study the structure-function relationship of the protein, it is important to have a good expression system for the gene.  It is also necessary for the protein to be localized and  function properly. This chapter describes the construction of a plasmid for the expression of oprM*, some physical properties of the protein, and the effect of its excessive production.  1.2 Construction of pKPM-2 and pKW35M  Overexpression of oprM from a 4 kb Pstl fragment containing the oprM* sequence in various P. aeruginosa expression vectors was not successful. A n attempt was then made to subclone the 1.5-kb oprM* coding sequence, without any flanking regions. Two synthetic oligonucleotides oprmNde and oprmHind (see Table III) were used to amplify oprM* from plasmid pPV20 (see Table II) and to incorporate Ndel and Hindlll restriction sites at the 5' and 3' ends, respectively. The ca 1.5-kb fragment was first cloned into the MCS of plasmid pT7-7, and the gene, together with the ribosome binding site on pT7-7, was excised by Xbal and Hindlll and ligated to the broad host range expression vector pVLT31 to create pKPM-2 54  as shown in Fig. 6.  Sequencing confirmed the sequence of the subcloned oprM* gene.  Similarly, pKW35M was constructed; with the 1.5-kb Xbal/Hindlll fragment cloned into the MCS of pVLT35, a derivative of pVLT31 with the streptomycin/spectinomycin resistance gene replacing the tetracycline resistance gene.  1.3 Effect of overproduction of O p r M  Expression of oprM* from pKPM-2 in Escherichia coli DH5a or various P. aeruginosa strains was induced by isopropylthio-P-D-galactoside (IPTG).  However,  excessive production of OprM* from pKPM-2 seemed to be harmful to cells, as revealed by growth studies. The growth of a P. aeruginosa OprM-deficient strain K613 carrying pKPM2 are shown in Fig. 7. Cell densities of strains carrying pKPM-2 started to decline after 2 h of induction with 0.1 m M or higher concentrations of IPTG, at which point OprM* was already substantially overproduced, as shown by SDS-PAGE of isolated outer membranes. A concentration of 0.05 m M IPTG led to no change in growth rate for at least 3 h and a normal yield of cells with OprM* substantially overproduced (see below). It is possible that excess OprM* perturbed the outer membrane and led to cell lysis. Therefore, a lower IPTG concentration (0.05 - 0.1 mM) was used in further experiments for the expression of oprM* from pKPM-2 or its derivatives and to obtain a normal yield of cells.  55  Xbal  Figure 6 .  MapofpKPM-2.  The black bar represents the 1.5 kb XballHindlll fragment containing the oprM* gene coding region and the ribosome binding site (rbs) from pT7-7 cloned into the MCS of pVLT31. The positions and orientations of the oprM* gene, the tac promoter (Ptac), and the tetracycline resistance gene (Tet ) are indicated. The lacP gene and the location of the replication and mobilization functions (rep/mob) of the replicon are also indicated. Plasmid pKW35M is similar, with the 1.5 Vb XballHindlll fragment cloned into the MCS of pVLT35, a derivative of pVLT31, with the streptomycin/spectinomycin resistance gene (Sm/Spe ) replacing the Tef~ gene. R  R  56  10  0.1  -I  1  1  1  1  1  1  1  1  1  1  4.5  5  5.5  6  6.5  7  7.5  8  8.5  9  9.5  Time (hours after inoculation  Figure  7.  Effect of OprM* overproduction on the growth of P. aeruginosa OprMdeficient strain K613.  Growth of K613/pKPM-2 (pVLT31 v.oprM*) in L B medium induced with different concentrations of IPTG as indicated in the figure legend. IPTG was added to the cell cultures at 5.5 h after inoculation (1:100 from overnight culture). 1 ml samples were taken at 30-min intervals for optical density measurements.  57  1.4 Overexpression of oprM* in E. coli  OprM* was overproduced in E. coli DH5oc carrying pKPM-2 (Fig. 8, lane 5) and confirmed by Western immunoblotting with a murine monoclonal antibody against OprM. Surface exposure of OprM* was also confirmed by indirect immunofluorescence (Hofstra et al, 1979). It was observed that heating the protein samples at 100°C in solubilization buffer alone did not give any noticeable change in the intensities of the 100 kDa oligomer band of OprM*.  Both the monomeric 50 kDa and the native oligomeric 100 kDa forms were  associated with the outer membrane under such conditions (see also below).  1.5 Overexpression of oprM* in P. aeruginosa  The control vector pVLT31 and the construct pKPM-2 were transformed into P. aeruginosa wild-type strain HI03, and two P. aeruginosa OprM-deficient QHg interposon r  mutants, K613 and OCR03T.  IPTG-induced expression of oprM* from pKPM-2 was  confirmed by SDS-PAGE and Western immunoblotting with a murine monoclonal antibody against OprM. Strain H103/pKPM-2 produced significantly larger amounts of OprM* (Fig. 9, lane 6) than the wild-type strain, Hl03; the vector control strain H103/pVLT31 (Fig. 9, lanes 1 through 4); and the nalB mutant OCR1 (Fig. 9, lane 7). The amount of OprM from the OCR1 outer membrane (Fig. 9, lane 7) appeared slightly lower than that from strain H103 (Fig. 9, lane 1). This could be due to a slight underloading of sample in the OCR1 lane, as observed by comparing the intensities of the OprF band at about 40 kDa in the two lanes.  58  Figure 8.  Expression of oprM* in the outer membrane of E.coli.  SDS-PAGE of outer membrane proteins from E. coli DH5cc/pVLT31 (lanes 2 and 3) and DH5a/pKPM-2 (lanes 4 and 5). Samples were heated at 100°C for 10 min in sample buffer without P-mercaptoethanol before loading. The gel was stained with Coomassie blue after electrophoresis. Each lane contained -25 p.g protein from each sample. Molecular mass standards are shown in lane 1 and are as follows: phosphorylase B, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; and carbonic anhydrase, 30 kDa. -, samples from cultures without IPTG induction; +, samples from cultures with 0.05 m M IPTG induction for 2 h. Positions of the OprM* monomer and oligomer are indicated by arrowheads on the right.  59  Figure 9.  1  2  3  4  5  6  -  +  -  +  -  +  7  8  Overexpression of oprM* in the outer membrane of P. aeruginosa wild-type strain.  SDS-PAGE of outer membrane proteins from P. aeruginosa wild-type strain HI03 (lanes 1 and 2), H103/pVLT31 (lanes 3 and 4), H103/pKPM-2 (lanes 5 and 6), OprMoverproducing strain OCR1 (lane 7), and OprM-deficient strain K613 (lane 8). PMercaptoethanol (5% v/v) was included in the solubilization buffer. Molecular masses in kDa are indicated on the left. -, samples from cultures without IPTG induction; +, samples from cultures with 0.05 m M IPTG induction for 2 h. The gel was stained with Coomassie blue after electrophoresis. Each lane contained -25 p.g protein from each sample. Position of the OprM* monomer is indicated by an arrowhead on the right.  60  However, using an identical gel subjected to Western immunoblotting, OprM in the OCR1 lane reacted much stronger with a monoclonal antibody against OprM than the OprM protein from the HI03 lane (data not shown).  As in E. coli, it was also observed that heating the  samples from P. aeruginosa strains carrying pKPM-2 in solubilization buffer alone did not lead to noticeable changes in the intensities of the oligomeric band of OprM* (Fig. 10, lanes 1 to 3). Only when P-mercaptoethanol was included did the 100 kDa band shift to the 50 kDa monomeric form (Fig. 10, lanes 4 to 6), and this occurred even when solubilization was performed at room temperature or 37°C (Fig. 10, lanes 4 and 5). Surface exposure of OprM* was also confirmed by indirect immunofluorescence. The fluorescence signal from cells carrying pKPM-2 and induced by IPTG was the strongest. The wild-type strain HI03 and the vector control strain H103/pVLT31 gave weak signals due to OprM expressed from the chromosomal gene (results not shown).  1.6 Summary  From the construct pKPM-2 (pVLT31::oprM*), OprM* was overproduced and the protein was properly localized to the surface of E. coli and P. aeruginosa cells. However, a low concentration of IPTG, the inducer for the expression vector pVLT31, was required for the proper growth and normal yield of the cells. lysis.  High concentrations of IPTG led to cell  It was also observed that OprM* was P-mercaptoethanol-modifiable,  even when  solubilization of protein samples was performed at room temperature or 37°C.  61  1  2  3  ^  ~~ ........... 5- i.. j  4  _  4  3  30 -  Figure 10.  6  I j g A jUto J M | ^HHMflU^  M  5  ^HMftN0^  HJHMM^  p P P » IHi  mm*  Effect of heat and p-mercaptoethanol on OprM* in the outer membrane of P.  aeruginosa. SDS-PAGE of outer membrane proteins from H103/pKPM-2 induced with 0.05 mM IPTG. P-Mercaptoethanol (5% v/v) was included in the solubilization buffer in lanes 4, 5, and 6. Samples in lanes 1 and 4 were left at room temperature, samples in lanes 2 and 5 were left at 37°C for 10 min, and the samples in lanes 3 and 6 were heated at 100°C for 10 min before being loaded into the wells. The gel was stained with Coomassie blue after electrophoresis. Each lane contained -25 ug protein from each sample. Molecular masses in kDa are indicated on the left. The positions of the 50 kDa and 100 kDa OprM* forms are shown by arrowheads on the right.  62  CHAPTER TWO: Functional characterization of OprM  2.1 Introduction  Products from the P. aeruginosa efflux  operon mexA-mexB-oprM have been  demonstrated to contribute to the high intrinsic antibiotic resistance of this organism as well as lead to multiple-antibiotic resistance after overexpression in nalB mutants (Poole et al., 1993b). It has been suggested that the relatively hydrophilic and often negatively charged Plactams, which have targets in the periplasm, can also be extruded directly from the periplasm or from the surface of the cytoplasmic membrane through this system (Li et al, 1994b). Therefore, it was of interest whether the outer membrane component OprM could function independently in vivo. OprM has also been assumed to be a porin-like protein present in the outer membrane of P. aeruginosa that assists in the efflux of multiple antibiotics (Ma et al, 1994; Nikaido, 1994). OprM is homologous to the outer membrane components of other efflux systems in P. aeruginosa and in other bacteria; and similar to E. coli TolC, it was shown to be able to act as the outer membrane component of other efflux systems (Srikumar et al,  1997;  Yoneyama et al, 1998; Mine et al, 1999). However, of the efflux outer membrane proteins, only the E. coli TolC protein has been shown to have porin activity (Benz et al, 1993). Therefore, it was of interest to determine whether OprM could form channels in lipid bilayers, like porins and TolC. This chapter describes the influence of the presence of increased amounts of OprM on multiple-antibiotic resistance in P. aeruginosa using  63  antibiotic susceptibility assay. In addition, this chapter describes the purification of OprM and analysis of its in vitro functions in the planar lipid bilayer system.  2.2  In vivo  functional characterization of O p r M in  P. aeruginosa:  antibiotic  susceptibility  To examine the influence of oprM expression on multiple-antibiotic resistance, including resistance to P-lactams, the protein was overproduced from pKPM-2 in wild-type strain H103 and two P. aeruginosa OprM-deficient strains K613 and OCR03T. Antibiotic susceptibilities of the various strains carrying pKPM-2 were compared to those carrying the vector pVLT31 by MIC determinations (Amsterdam, 1991). Controls demonstrated that the growth of cells carrying pKPM-2 was not affected by oprM expression induced with 0.05 mM IPTG.  As shown in Table IV, overproduction of OprM* in the two OprM-deficient  strains led to complementation of their mutations and restoration of resistance to the antibiotics tested. However, the excess OprM* molecules produced from pKPM-2 did not increase the resistance of these two strains for the different antibiotics to levels higher than those of their corresponding parents. Tetracycline resistance was the selective marker on pVLT31; thus, strains carrying pVLT31 or pKPM-2 were highly resistant to tetracycline (data not shown). The MICs of some antibiotics for control vector strains K613/pVLT31 and OCR03T/pVLT31 were increased compared to the MICs of those antibiotics for the OprMdeficient parents of those strains. This is possibly due to the Tef gene on pVLT31 and the requirement to include tetracycline to maintain the plasmids in growing bacteria to seed the MIC plates. As tetracycline is a substrate for various efflux systems (Nikaido, 1994 & 1996), 64  oo ro  NO  in  oo r©  oo  CN  ©  ©  CN  ©  ON  cn o  cn cn  CN ©  ON  NO  ©  ,—1  cn  in  o H  PH HH  E CN  U  u  CN  .  ©  a  in  CN  IT)  CN  o  ©  o  o  CS X  in ©  m  o o fl  IT)  o  in ©  in  CN O  cn  in  ©'  © o  m CN ©  cn  NO  ©  CN  o ©  + + + +  in  CN 1—" ©  /  oo  i—i  CN cn  NO  in  m  o  in  m  o  ©  m  CN ©  in  CN ©  CN  in ©  o 'x '•fl cS  c  in  CN ©  o ON  cn  cn  ©  o  ON  cn ©  ON  cn ©  CN ©  r—  O  CN ©  \o  in  in  00  o  ^ H  ©  ©  ©  OH  O  o < u  (D T3  CN  o  IT)  in CN  <n CN  ON  ON  ©  ©  cn  cn  in CN  c • i-H  O  o o.  O  CN o  — i< O  ON  cn o  in  ©  0>  CN  •A  CN O  CN ©  © ©  ON  ON  ©  ©  cn  cn  CN  oo r©  in  © ©  V  i—i  ©  © ©  ON  cn  IU  fl "c3  IT)  CN  in  CN  m CN  >n  IT)  in  CN  VO  in  *—i  NO  in  CN  in CN  © ©  oo  in  ©  *o  CN  © ©  "3 IT)  a u <+H  o  oo  o  CN  CN  CN vd  in  CN r  +  +  + + + + +  "~  oo  m <—I  ©  cn  CN  in  l  +  •  i  + + + + +  + + +  ©  i  in  i  ©  + + + + +  fl B o W P •H  O  fl  C (H  fl  r3 -fl  T3  oo fl  GO  i-c  CN  <^  *rH  > cn ©  .OH  cn ©  ^OH  cn © 1—H  o fl 3 CN cn  I~H  cn  CN  H cn NO  OH  >  .OH  cn  cn  ^o  ^o  ^ H  I—I  + + +  1~H  o  u o  cn  H  & > H  OH  cn ©  H  U  u o  o  cn ©  CN  a  ^OH  (U  oo CL) fl G O . .  O  > -c  O  G in ai O  i  iH  <D (L)  c fl •o ^ fl o  a s"  'S .-2  c3 £  •fl  o  c  H—'  ^-T  0  1 ^  OH  **H  to J3 fl § -S CJ  O.  cS  -S o o* 00 00 ca —  <D O  )H  I 8  O  cn  OH  H  oo O  o  a A  fl  ao? -fl  fl •A  i—*  CN  a  OH & <^  I  1/3  ID  J3  ©  l-l  > o  u  'S a s .a  C fl  e  o cs U in  o  '<&  CO  ON  s H—I  cS  c o O  ©  O  If  60 VH ^—» CO  *  •fl  T  CS  C/3  o  • i-H  IH  -4—»  a  U fl  ^  o u 2  8  I B ^ ° c 3 > •A 2 0) -»H  H  cn o  Pi  U  o  )-i oo OH CS  O  ^  l-H  ^  the tetracycline included might have induced the expression of other efflux operons that are also involved in the extrusion of additional antimicrobial agents (George, 1996). Nevertheless, when comparing isogenic strains with the oprM*-expressing plasmid or with the vector plasmid alone, complementation was clearly observed.  On the other hand,  overproducing OprM* from the cloned gene in the wild-type P. aeruginosa PAO strain HI03 did not significantly alter the MICs of any of the antibiotics tested (only 2-fold difference for norfloxacin, cefepime and cefpirome, considered insignificant as observed for many other bacteria). The same results were obtained with overproduced wild-type OprM containing the native sequence at the C-terminus (Fig. 11), after the native oprM sequence was revealed in the completed genome sequence and the gene became available on pXZL34 (data not shown). The overproduced wild-type OprM protein, like OprM*, complemented the OprMdeficiency in the two mutants; but overproduction of OprM did not increase the resistance levels to any of the antibiotics tested.  2.3 In vitro functional characterization of OprM: channel-forming activity  2.3.1 Purification of OprM Wild-type OprM was purified from both E. coli and P. aeruginosa containing plasmid pKW35TM (pVLT35v.oprM) as described in Materials and Methods. The sequence at the 3' ends of native oprM and the originally cloned oprM* gene are shown in Fig. 11. For the purification of OprM from E. coli, the porin deficient strain CE1248 carrying pKW35TM was grown in the presence of IPTG to induce expression of oprM. Various detergents at different concentrations were combined with different salt concentrations to optimize the 66  published sequence: L W G G D C F D T C Q K R A G C T G T G G G G A GGT G A C TGT T T T G A C A C A T G C C A A A A G A G G G C G G G A T A G  true sequence: L G G G W N Q Q T V T Q Q Q T CTC GGC GGC GGC TGG A A C C A G C A G A C C GTG A C C C A G C A G C A G A C C A K K E D P Q A G C G A A G A A G G A A GAT CCC C A G GCT TGA  Figure 11.  Differences at the 3' end between oprM* (the published sequence) and oprM (the native sequence).  The native sequence of oprM encodes for 22 amino acids that are replaced by 14 residues in the protein encoded by oprM*. The originally cloned oprM* gene had an incorrect 3' end derived from the phagemid used to clone the gene.  67  solubilization conditions for OprM from outer membranes isolated from sucrose density gradients. The maximum amount of OprM was extracted with subsequent solubilization in 0.5% and 3% (v/v) Octyl-POE in 10 m M Tris-HCl (pH 8) (Fig. 12B, lanes 1-2; Fig. 12C, lanes 1-2).  This soluble fraction was dialyzed into buffer A (10 m M Tris [pH8], 1% [v/v]  octyl-POE) and then applied to an anion-exchange MonoQ column. When subjected to a NaCl gradient with buffer B (buffer A with I M NaCl), OprM was eluted in a fraction along with one other major protein at about 70 kDa and traces of other proteins of lower molecular weight (Fig. 12A; Fig. 12B, lanes 3-4; Fig. 12C, lanes 3-4). This fraction was then subjected to SDS-PAGE without heating of the sample, and OprM was excised from the gel and eluted in 10 mM Tris (pH8) with 0.1% SDS at 4°C overnight. OprM was purified similarly from P. aeruginosa, with oprM expressed from pKW35TM in the OprM-deficient strain OCR03T.  2.3.2 Planar lipid bilayer analysis of OprM Analysis of the pore-forming ability of OprM was performed with the planar lipid bilayer technique described in Materials and Methods.  The purified OprM protein still  retained its heat- and P-mercaptoethanol-modifiability (Fig. 13A).  The native oligomeric  form of OprM was added at nanomolar concentrations to the aqueous solution bathing a lipid membrane and the changes in membrane conductance were recorded. The stepwise-increase fashion in membrane conductance was presumably due to the incorporation of individual porin units into the membrane (Benz and Hancock, 1981).  Initially, an average single  channel conductance of approximately 82 pS was obtained for OprM in I M KC1 (pH7) bathing a membrane composed of 1.5% (w/v) oxidized cholesterol in n-decane at an applied  68  B.  C.  1  o-  ,  19  2  3  21 " 2 3 " '25 27  Fractions  Figure 12.  Purification of OprM from E. coli CE1248 strain carrying pKW35TM.  A . Mono-Q FPLC elution profile with a NaCl gradient and absorbance A at 280nm. The peak containing OprM is indicated by an asterisk. B . SDS-PAGE of outer membrane proteins solubilized with 3% (v/v) octyl-POE (after the 0.5% octyl-POE solubilization step) (lanes 1,2), and the OprM-containing fraction from FPLC (lanes 3,4). Samples were incubated with solubilization buffer at room temperature (lanes 1,3) or heated at 100°C with 5% v/v p-mercaptoethanol for 10 min (lanes 2,4). Molecular masses (kDa) for the prestained markers in lane M are shown on the left. The gel was stained with Coomassie blue after electrophoresis. Each lane contained -25 u.g protein from each sample. C . Western immunoblot of a gel with the same samples and treatments in (B), using an anti-OprM monoclonal antibody.  69  Figure 13.  Planar lipid bilayer analysis of purified OprM.  A . SDS-PAGE of gel purified OprM following solubilization of outer membranes with Octyl-POE and FPLC with MonoQ anion-exchange column. Protein samples were untreated (lane 1) or heated to 100°C and treated with 5% v/v P-mercaptoethanol for 10 minutes (lane 2). The molecular masses of the marker (in kDa) are shown on the left. B. Chart recording of stepwise increases of the membrane current formed by purified oligomeric wild-type OprM in 1% w/v diphytanoyl phosphatidylcholine dissolved in n-decane at 25°C. The aqueous phase was 1M KC1 (pH 7.0) and the applied voltage was 50 mV. The arrow indicates a conductance of 1 nS.  70  voltage of 50 mV (Wong and Hancock, 2000). This level was very similar to that obtained from oligomeric TolC (80 pS) at the same voltage and in the same salt concentration (Benz et al, 1993). However, later experiments using several fresh samples of OprM gave a single channel conductance of approximately 1 nS (mean = 0.85 nS for over 100 events) in I M KC1 (pH7) at 50 mV (Fig. 13B).  This indicated that the age of the OprM preparation was  important since older samples tended to give noisy current tracings and the apparent channel size varied considerably.  In addition, the phospholipid diphytanoyl phosphatidylcholine,  rather than oxidized cholesterol, was employed in the later experiments as recommended by Dr. Roland S. Benz (as in the case with TolC, Benz et al., 1993), since cholesterol is very sensitive and can give rise to artefacts even with the detergent Triton X-100 used for diluting the protein samples.  From the examination of current tracings, OprM molecules tended to  form transient channels that were not very stable in the lipid membrane; they migrated out of the membrane bilayer quickly, as reflected by decreases in membrane conductance (Fig. 13B).  OprM also appeared to form substates in the bilayer membrane, as if the channel  partly opened and closed (Fig. 13B, the smaller steps).  Studying the single channel  conductance of OprM as a function of voltage indicated that OprM did not form voltagedependent channels. The single channel conductances of freshly eluted OprM in different salt solutions were also measured (Table V).  They appeared to be a linear function of the KC1  concentration between 0.3 and 3.0 M , indicating that the channel is water filled. Changing the bathing salt solution to I M LiCl (where L i is a highly hydrated bulky ion) resulted in a +  strong decrease in single channel conductance.  In contrast, there was little change in the  71  Table V.  Average single channel conductance of the wild-type OprM protein as a function of different salt solutions. a  salt  concentration  voltage applied  Single channel  (M)  (mV)  conductance" (nS ± SE)  0.3  50  0.20 ± 0 . 0 1  1  50  0.85 ± 0.03  3  50  1.78 ± 0 . 0 9  LiCl  1  50  0.23 ± 0.01  KCH3COO  1  50  0.76 ± 0.02  KC1  a  Single channel conductances were obtained with gel-purified oligomeric OprM at 25°C. The different solutions were all at pH7.0. Membranes were composed of 1.0% (w/v) diphytanoyl phosphatidylcholine dissolved in n-decane. Purified protein was diluted into 0.1% (v/v) Triton-XlOO prior to addition to the solution bathing the planar bilayer membrane.  b  All results (± standard error SE) were obtained from over 100 events.  72  single channel conductance when the bulky anion acetate replaced the chloride ion. These data suggested that OprM is cation selective, similar to TolC.  2.4 Summary  To determine the role of OprM in antibiotic efflux, especially for p-lactam extrusion, the protein was overproduced in the wild-type P. aeruginosa strain and two OprM-deficient strains. The complementation of the OprM-deficiency in the two mutants clearly showed that OprM was important for a functional MexA-MexB-OprM efflux system, and it was subsequently demonstrated in other studies that the pump proteins and the linker proteins anchored in the cytoplasmic membrane do not work in the absence of an outer membrane component (Srikumar et al., 1997).  However, the excess molecules of OprM produced in  these two pKPM-2-containing strains did not increase the MICs for any antibiotic tested to levels higher than those of their corresponding parents. Similarly, extra copies of OprM from pKPM-2 in the wild-type strain HI03 did not increase its resistance. These data suggested that OprM could not function independently for the extrusion of antimicrobial agents, including P-lactams whose targets are in the outer leaflet of the cytoplasmic membrane. OprM was purified and was able to reconstitute channels in lipid bilayer membranes, with an average single channel conductance of 0.85 nS in I M KC1 at 50 mV. The results suggested that the channel was voltage-independent, water-filled, and cation-selective.  In  addition, it was observed that OprM appeared to form substates in the lipid membrane.  73  CHAPTER T H R E E : MexAB-OprM homologues from the P. aeruginosa genome  3.1 Introduction  The complete 6.3 million base-pair genome sequence of P. aeruginosa was completed recently (Stover et al, 2000).  It is the largest published bacterial genome  sequenced, with 5570 predicted ORFs, most of which have a high G+C content of 66.6% as that of the genome. The successful environmental adaptability of P. aeruginosa is probably due to its large and complex genome, with genes involved in diverse functions and with a high proportion of regulatory genes.  Analysis of this complex genome sequence will  definitely provide many insights for the better understanding of the organism. This chapter describes information related to the MexA-MexB-OprM efflux system from analysis of the P. aeruginosa genome.  3.2 Phylogenetic analysis of the MexA-MexB-OprM efflux components with their homologues from the genome  Analysis of the P. aeruginosa genome to identify homologues for the components of the MexA-MexB-OprM efflux system was performed when contigs of the sequence were available. The sequence-alignment algorithm T B L A S T N (Altschul et al, 1997) was used to perform the search.  Many homologues were obtained for each of the three efflux  components and each group of homologues was subjected to phylogenetic analysis.  74  Phylogenetic analysis and tree display for the three groups of homologues were conducted using the PHYLIP program (Felsenstein, 1989) and the TreeView program (version Win32; Page, 1996) respectively.  There were twenty-one, thirteen, and eighteen members in the  MexA (Fig. 14), MexB (Fig. 15), and OprM (Fig. 16) phylogenetic trees respectively. Putative proteins in the OprM family have been temporarily named OpmA, OpmB, and so on to stand for "probable outer membrane protein - OprM family". Members homologous to proteins previously shown to be involved in efflux are noted to cluster with OprM, while those homologous to outer membrane proteins involved in secretion are within the other cluster. These two clusters might reflect some functional variations. Similar clustering can be observed from the phylogenetic tree for the MexA family but it is not noticeable in the tree for the MexB homologues, which apparently only contains a cluster of proteins involved in efflux. In the three phylogenetic trees, ORFs or members for all three RND components (pump, linker, and OMP) can be observed in the corresponding clusters for some operons, as in the cases with PA4026-PA4027-PA4028 and the characterized Mex systems. On the other hand, there are members which do not have related ORFs in the corresponding clusters. Table VI shows the possible efflux operons from the genome, encoding products similar to the R N D MexA-MexB-OprM system. Some of these putative operons have the same gene organization as mexA-mexB-oprM; some have the gene for the outer membrane component upstream of the genes for the pump and the linker proteins; others do not have the gene for the outer membrane component, as for the AcrA-AcrB system in E. coli (Fralick, 1996); while a couple have one of the genes duplicated. This large number of possible efflux systems in the genome suggested that efflux is an important mechanism for the organism. Components of the few systems characterized from P. aeruginosa in recent years were  75  MexE  F i g u r e 14.  Phylogenetic analysis of MexA with its homologues from P. aeruginosa.  The tree was constructed using the Neighbor-joining matrix method from PHYLIP (Felsenstein, 1989) and TreeView (version Win32; Page, 1996). The identification for the ORF encoding MexA or its homologues is indicated as P A followed by a number. The scale bar stands for nucleotide substitutions per site.  76  F i g u r e 15.  Phylogenetic analysis of MexB with its homologues from P. aeruginosa.  The tree was constructed using the Neighbor-joining matrix method from PHYLIP (Felsenstein, 1989) and TreeView (version Win32; Page, 1996). The identification for the ORF encoding MexB or its homologues is indicated as P A followed by a number. The scale bar stands for nucleotide substitutions per site.  77  O p m J (PA1238) OpmE  Figure 16.  I  Phylogenetic analysis of OprM with its homologues from P. aeruginosa.  The tree was constructed using the Neighbor-joining matrix method from PHYLIP (Felsenstein, 1989) and TreeView (version Win32; Page, 1996). Some putative proteins in this family have been temporarily named OpmA, OpmB, and so on to stand for "probable outer membrane protein - OprM family". The identification for the ORF encoding OprM or its homologues is indicated as P A followed by a number. The scale bar stands for nucleotide substitutions per site. 78  The possible efflux components present in the Pseudomonas aeruginosa genome.  Table V I .  Range  3  Orientation  Possible efflux components'  (Alternate) Protein Names / P A #  0  3  472024-477790  -  ABM  MexA-MexB-OprM / PA425-PA426-PA427  5155399-5149632  +  ABM  MexC-MexD-OprJ / PA4599-PA4598-PA4597  2808742-2814611  -  ABM  MexE-MexF-OprN / PA2493-PA2494-PA2495  4706409-4712083  -  ABM  PA4206-PA4207-PA4208 (OpmD)  3943806-3938019  +  ABM  PA3523-PA3522-PA3521 (OpmE)  4362983-4358166  +  ABM  PA3892-PA3893-PA3894 (Opml)  1337941-1339079  -  MAB  PA1236-PA1237-PA1238 (OpmJ)  2843304-2837333  +  MAB  PA2522 (OpmN)-PA2521-PA2520  5805679-5809896  -  MAB  OpmG-PmrA-PmrB / PA5158-PA5159-PA5160  2212512-2208169  +  AB  AmrA-AmrB / MexX(G)-MexY(H) / PA2019PA2018  a  b  0  1562813-1567077  -  AB  PA1435-PA1436  4903465-4907702  -  AB  PA4374-PA4375  4120372-4116187  +  AB  PA3677-PA3676  177307-182569  -  AAB  PA156-PA157-PA158  2855290-2846282  +  ABBM  PA2528-PA2527-PA2526-PA2525 (OpmB)  The coordinates of the ORF in the complete P. aeruginosa genome. A: possible components homologous to MexA; B: possible components homologous to MexB; M : possible components homologous to OprM. PA# is the identification of the ORF in the genome.  79  compared against one another and their percent identity and similarity are shown in Table VII. It is noted that the pump proteins, with an average percent similarity of 63.8% among themselves, are slightly more conserved than the other two components, with average percent similarities of 54% and 52.6% for the linker and outer membrane proteins respectively. In addition, MexA-MexB-OprM is more closely related to MexC-MexD-OprJ than to MexEMexF-OprN, as observed also in the phylogenetic trees (Fig. 14, 15 & 16).  3 . 3 The O p r M family in P. aeruginosa  Table VIII shows the members of the OprM family deduced from analysis of the P. aeruginosa genome, with their relative positions in the genome indicated by their ranges and identification numbers. The information indicates these ORFs are scattered throughout the genome sequence.  As shown in the last column, some of the ORFs are most similar to  putative or identified efflux components in other organisms. Among these ORFs, the most similar homologue of OprM in E. coli is TolC, which is 54% similar to OpmH. Multiplesequence alignment of all eighteen members in the family is shown in Fig. 17.  The  sequences range from 425 (OpmL) to 498 (OpmB) residues long and there were regions more conserved than the other parts of the sequences.  Five residues (one leucine, one  proline, and three glycines) were shown to be completely conserved among the eighteen ORFs in this alignment, while three others were highly conserved.  80  Table V I I . Percent identity and similarity among known P.  aeruginosa efflux components.  T3  ay 3  O  s  a.  ©  £ 8  iH  C  o o in  fa  J3  c  •4—>  R  -a  u  •SP  o ex  "3  s  eg  s  Cx  3 o  M  C3  I! <  0)  ex O o  to  O  c  I§  S  ro  H  I< & Is  PH  1  o  ON ID  h  N ON  r~r-Tt CO  ro  CO NO  Tt  m TJ-  < < + + PH PH CN CO  CN  m 00  < i ON  CN  in ^  +  in Tt oo NO CN o NO CO Tt Tt ON Tt i—H1—H 1-H oo in in CO CN CO o oo CO r-CN o in rTt o r7-H m Tt ON 0Tt0 » — < l—H in m CO CN NO ON  CO  ir>  OS Tt  io  oS —'  ov  1/1  ON  CO  oo  o CN  ON  Tt  Tt 5 < < fe fe 1  r-  i  ca  ••N?  Tt  NO  ON Tt  oo  I i  >N  O  cx  .g ON  P. 5 . ( i  o o o o a,  Tt Tt oo oo Tt in Tt CN in ON ON ro Tt Tt oo oTt CN 00 CN CN CO in CO »n Tt Tt ro <3 <^ <N  00  <; fe << < fe fe fe < fe fe i + t + 1 1  << PH  1  oo CO ON Tt ON t-in Tt O ON m CO oo NO CO CO m in Tt  CN Tt CN r>CN i in in m ro Tt CO NO o >— Tt CN o CO C^00 o CN ON ON o CN CN ON Tt NO NO ON 00 ro »—( Tt m 0 0 CN NO CN o CO in o CO ON in Tt ro 0 0 NO CO o m CO Tt < ON Tt Tt NO CO CO CO NO o oo oo in CN Tt CN CO in in Tt < —1 •—i Tt CN CO  .g  ID  <  ON  00 in \—t NO ro o < NO CN Tt oo rCO o Tt 1 NO r~- oo ON 0 0 CN Tt CN CO in CO o CO Tt ON  CN  o  «j c>  a  O O O o O  o  c>  .g  Cx  O  c  a, a. •*o-»  O < B  >  w cN  .g  B  w a m^ ^ S S1 s s  cx O  ON  Tt m o Tt  CN </">  N©  Tt  cx ex  o  W)  03  oo  PH  ID  3  vu  t-i  CX  >  NO  Tt  o a  o CX  xu  K  o H  O o  So  nilar  o  ID  <4-(  "o H o  +3  BBS  B  2  D  y  —  fl  R  nilar  o *o  '53 -*-»  T3  o  o oj  -4-»  o  O  c  P  O 60  <a  13  C3  o  nilar  £  R  nilar  NU  B  o  fl o  CzcC  O  s o S -§ s  o  •a  4) O  S  u  I  <4H  AprF  c  (D 00  MM  CyaE  a o  o R  p  AprF  X p E  ID  i  CN  1  PH  PH  + +  m CN  oo  o CN Tt oo oo CO CN ON  o  CN  Tt  T—I o  ro o ro — f0 0( Tt 00 NO  CO  CN  82  Figure 17.  Multiple-sequence alignment of OprM with homologues from P. aeruginosa.  The sequences include OprM and 17 homologues from the P. aeruginosa genome. The alignment was obtained by the ClustalW Multiple Sequence Alignment online program at B C M (Baylor College of Medicine). Highly conserved residues are shaded in black, with a star above the completely conserved ones, while similar residues are in different shades of gray. The putative proteins in this family have been temporarily named OpmA, OpmB, and so on to stand for "probable outer membrane protein - OprM family".  83  AprF OpmH OprM OprJ OpmQ OpmB OpmA OprH OpmD OpmE OpmJ OpmG Opml OpmH OpmK OpmH OpmL OpmF  AprF OpmH OprH OprJ OpmQ OpmB OpmA OprH OpmD OpmE OpmJ OpmG Opml OpmH OpmK OpmH OpmL OpmF  AprF OpmH OprH OprJ OpmQ OpmB OpmA OprH OpmD OpmE OpmJ OpmG Opml OpmH OpmK OpmH OpmL OpmF  MTHRRLHTW L FGAFL L L L RE DAFAL G HHRLRACLLSSAILSAS SAQALG HKRS F L S L AVAAWL S GC S LIPDYQ RP E APVAAAYP Q GQAYGQNT GAAAVP AADIGWR HRKP A FCVSALLIALT L GACSHAPT YE R P AAP VADS WSGAAAQ RQ GAAID—TLDWK HSHKNL S LI SAC L L L GAC GS T P AP LD S GL AAP S QWRYLAAGRSD ASDIR QW HKHT P S L L AL AL VAAL GGC Al GPDYQ RPD LAVPAE FKS AS GWRRAE P RD VF Q RG—AWW HKGT P L L L IAS L AL GAC S L GPD FTRPDRPAP GE WS L QAAAGHP SHL AAAP - L AAQ WW HIHAQ SIRS GLAS AL GL F S L L AL SAC TVGPDYRT PD T AAAKID AT ASKP YD RS RF E S LWW - -HKRSYPNL S RLALALAVGT GLAAC SVGPDYQ RPQSPPP RVAS EHL GE F S GE RRE AP WW HKP YL RS S L SAL ILL GGC AAVGPDYAP P S AS AP AS F GAHPAGID GS—G VEIEWW HP L ASHL RCVALAL GIS T AL GC AHRHQ PAP RAE S LD P GL S RVAGT RGD AL P AQWW -HP F P L LHPWP Q RLALASAIL LAAGCVT SEGLEPHARLQ PAGAL QAGRS LD GVAL S P AAWP RQDWW HVGS FVGF L WF S Al S GC VS T GDI AP K AAT LDAHALATDHAIQAAARE AG-WP QAQBW HL RRL S LAAAVAAAT GVAWAAQ P T P LP MRALAGLLCGLL GLVP GAAAYE PD VF GT T GQVAGQAVYD L GGS GL P CRGG HP IL RP L ASAGKRACWL LHGL C L GL PAL AHEAP VS FH G HRGRRQYARKGRRHGKGAIWL LSLGLP HHRWGLGVLWLVTAL PVAASVHPAL S PDVP SHARE QGRSVLLS  EFFRDPQ SFIVDAE KAFGAPE ELYGDQT TLFDDAQ KQFDDPT SFFDDPQ RGFDEP. TLYQDPG TGLGDRQ KVYADPQ - T K T D PPPTELS T S I S HFASAHP EQVIDLS  SYHRGRSWSDVT Q T T T j i Q EHEAGRQYRAL GRAAL Ll 'LHERRAGSENRAIGRAGLL] 50RYDYHKARHDS TVSQ ILNVEAFRAQYRIQPADL "GVDGSGTRQRLPGDLSTTJRQTLLDIEAARAQYRIQRADRVRG TAAAT GNRQ RQ P AD L SAGJGA0VARVRQAQASAVIAGAP L L ATLGASRQKLLRDSGYSG S.Q SVAQ F RQAEALVRGARAAF F TGHVGKTRSGQGGGDSTVL UALQQRVQRAIILDQRSQAARLQQSRAIRRSLGGDALJS A S GHYQ RQRTTSAGLFDP JRVIFARLRAARALRDDVAHDR: TSRASADIGKGQQPG JRESRAHLRSARALFDDRWLDQLQQ |TSQAGYSRSIEQQLDYDGkLwGARLD E ARAL LREHREEFLJJRGGPAFDYQARRRGEVE T P — AHA PALLGHLRGAQ GERWHRT EVGYGYQYGRDGDD QT LAE E A R A R Q A A A T A Q A Q D A A R Q B T B D A K A S Y S GI RAPT SVAP AP RVRQAKSHAGLVESIESHQF E GKG £LVRHRWPDDYFYGP QADYLARKEWPQARAGLLBQ GAGA RVGD T RlAFD E )GRLDASRGYSDHDYRDAP RLSWAHAKAQAAQVGIGKSAYLB -VGRET EIAS GARQQAGLIPHHDGSWSVEDT RQGHRQ TS— RSgHAEADRAGT E V E H A K G G Y Y S S | T H S G G P Q E F D F GEIVYRS 0 Y L Q RIAQKFD L RVAAD A F H B K § V V R G D YRAHRAT EDRTRTS  R  T DA LPGGSTVSS S  C  V  AT G Y  GDFKEDRDYDSYVSTLSLQQPL YEAFSRYRKGVAQA GDARVERDYRSYASTLSLIQPL YEAYARYRQ GEAQA GSPAISSQYGVTLGTTAWELDL RLRSLRDQJjjJLEQY HRS EVAS SYQVGLAL P E Y I LD L - R V K S L T D A J J L Q Q Y T SDHDAVD SFSAGL SASYIVD F Jg-GRQ AAYRS0L ESL GGS GAIS T SYS THL SVSWEVD Lfflj-KLRRQ L EAHQ AS L KAGKGHYHHALAGFDASWILD F jg-RVRRELEAjjDATV TEDRVHSERYDLGLDSAW1LDL RIRRQ LESSDALS EPRRRLAESYRAGFDAQWEIDL RLGRLSDA0LARA AGQ Q RDIETYRGALDASWEID L SVRRSVIJ DEDLHSQWKHTVRLDLSYQLDL -EVRARIi LGGRYSAIKYLSLGFHYDFD: GERAAWEi DLARTTSWHHSTEIGLHYKLDL RDRSDSEI RPATVKRHSQVVQATLSQPL RADRWFQWQi LSGDGHRHRRGASLQLSWVLFD RRSAALI WSIAQPLEL KRGARVE\  DLTASQHLYDjng-RVTSKVDS HVS P TAT L L GE YGT RF S LAWVKQ FRTADEAGRYRSDGLDL T W Q P L L RDAGWD VT T AP L RL  84  TGALLAQDQIEQASAQKRSYREQFQQKTQRQFE] WGTRTDTLET SETLFAREQOT JAEAQ RRAL E T QLAFNQRAF E EQ EGTRTDLLET LTLKADQAQLQJTKDTLGTYQKSFDFFLTQRSYDI (/ASALDLRQA LSYDGALRRLA JT RQ T LVS RE YS F AMID Q RI AATALDYQEA L Q VL AL RE Q Q R jjARLHLDNAEHVL RIJVE T1 SATALEVAQQ LQLRVHDEQIR|LHD TVTAYE RS] IVTRADVAQA IQLRGEQNRAAglRDNLETARRSLEgTRTRLi VATDLEVAQA VGAELDVLRA GQLRGAQLREK3ALSHLENQKESRQ|TIQLRDJ SGLPEDVEHA FQLQGI«AELA|VHDIAGNQRI>SLISVERLVS. SAHEFDRLRA RACALARRAEGQ RRSVGLLDASLAGSIRQLAAH LSSELQRRRL |SD LAHAF TVRD |AEEELKRSQRHTEHSQKRHSA1: LDSKVQLQQT fQLSLQYAEHDSAKAMLQQQRDILASAQRRLRGI:IGTHFEVSQA JF TVLRAQDHLAT SKAE E AAFKRQ LI> QAHE RFD LSDKTDVLEA AAALSDRLQA SISSVERVRA YADRSELDRA RHAEFEIVQT  189 182 226 223 224 23S 227 226 226 220 226 233 227 188 220 187 181 241  AprF OpmM OprH Opr J OpmQ OpmB OpmA OprH OpmD OpmE OpmJ OpmG Opml OpmH OpmK OpmH OpmL OpmF  LLSDERFRSQSQEJLVRVLEJ LFADEQFRGRSQEJAVRLFJ LAT E QAQ RSAQ T TWASVATJ L AS E EAARAARIA WAEVS QJ KASEYDRATVELT|LSGVAHSJ HASAADLAAVRLSQQSQLAQ: EAS EN E LRDVQVS|LAEAARDi EAAEADLQQLQVs|lAELVD EAADADL RLVRL SHAAD TA: GS REAL L RHVQ A S | A A T VAHSJ EAAQAARDLL RVS jjjjAS QTTL, NAARIDSQAARIGfflSASIAr HHAAAEARQAQLEGEGHIV: DQARLEFSATQQD|ILRSAETJ LAANASQDAT LQHTFALAAQ, EIAWTQLEVRRAESJRAQVRG. RKL S E AVL VARDD AALDIVE TJ DAHRLQ LKASVS QTISQVIG,  AprF OpmH OprH Opr J OpmQ OpmB OpmA OprH OpmD OpmE OpmJ OpmG Opml OpmH OpmK OpmH OpmL OpmF  QARFHLAQAQEIEARD SQDAALRE E RIJVSAP L EI AD LAP L GE RF QVRP L S P ASYT AWRD Li RARgjS L T RAE EIAASD RAAAARRT E A T & p QAL ED RELAAPIERFPALRLQPATFE GWRQVA SG IPANLPQGLGLDQTLLTEVPAGLPSDLFL Q TA|EGARAT LAQYTRLVAQDQH SDD--AAQAIPRSPGQRPKLLQDIAPGTPSELJERR L GL|E QARAB Q ERHLRQKQQAFH. EP VQAL QVAERPFDSLRWPETGAGLPSELJSRR SSLgASQRKQLPLLEQQAHEALIT 'LP PAQ FHL P P VAS VPKL PD L P A W P S Q LQERR RTQfflKS T QAQ A I D LKYQ RAQ L E: 3PG-SLLAELGPARAIPRPPGSVPVGLPSELAQRR LAQSASHEARLPEVEKNQAHLVNAPG JQRP—EELTVDLSPRDLPAITKALPIGDPGEL0 DARfflAATAASVPQ L QAEAE RARH7 EAP—GSGAPILDGGAAAP LAKHLP L GDVD RLJJ QAHGLRSEAAIPPLTTALESARYR EAP-QAFS P PVARASGERLTLRTLGVGD PAGLJ EALQHNVEAAVPD L E RRRAAT RH. SERSP-RQLDAPAATCAGIPQLRRALPTGDGWSL LAL RE RT RAALPHLEARRRAALYE :GP—DRGLELQRPQPLHPASLSLPSVLPAELI QTQRATARQQLSAAEQDIASARI :GP—GEGRTIRRPSLHLAAQPSLPSALPAEL[ EVPJPETERRIEVIDEEIQLTRHL JTNRDY--SAIEGHRHTLPWPPAPHDAKAWVDTA3(QQN Q ASYD T ARAHRLIAE Q RVDDAF Q. L RBJJJL APDTPLRLSGELEAQPDTGFVKAIDEHLAEARREH QTAJJSQASLAQVRDEGALSHALG QP GFARVCGALDAVP A S I T RGAL L RBflfo E S QVLADHAQLDLSQAELEQQRTYVQ SSTTOEP QEP AD-LVEPBPHSLQRYLAASDMARWORES HLEQSRAQEQLSL EKGNL QDARHQYAI LD LSTQIRASDALAAT PIEVD RQ QAlRTATJQ QQ EADSASQBLNVEESTNQVDSARLA0LQ  255 248 289 287 287 297 292 290 290 285 291 297 291 252 286 249 241 304  AprF OpmH OprH Opr J OpmQ OpmB OpmA OprH OpmD OpmE OpmJ OpmG Opml OpmH OpmK OpmH OpmL OpmF  QEIJASL ASTGKSKSG S EHTYHQ RYE TD SVGI •VARYEJEQ AERGAQ ASSSKTHSA SESTYEQKYDTDSVGL JEAAAYEgE |LEAEHQ[ rAsgc. TAHAGTHSRQ LSGLFDAGSGSWLFQP LAAEH ARNADMG. TGSFGTSSAE HSGLFDGGSRSWSFLP fAEAQ LQAD|QV SASLSSGAHR AADTFRH—PYYHLGA SAE SANAQMGV, SAAGGYRSGS L SNWIS T PNRFWSIGP IRRAEA: iTASHGW GHFGFESLQ LSSLGDWDHRQFAIGP IRAAERI .STADSGVATl SGFLGFTAGR GSQIGSSAARAWSVGP JVSAERQ ISTED|GAAT|JEL GGFIGFFALR SGDLGSAS-RAFELAP ADHAAAE IT AR[JGVE T GL EQRGSIGLVAGH LDALDESGTS FHVLHP 3D|RAAER ARRALAE"EL SFAVGAETSAAT LAGLGGSGALAYAAGP ADBVAARW E A A R R W J J J D S J IGAHAGLAALH TSDULQAPSRFIQVAP ARRWQ .LAKGgDVAfgDF' SVGFSAVGGGHLEFFRSAKYTYSACP rAAE E TfflRQ RwGHL LASHY. AVAQYQKGDHDAL GFAHSAAHPLVHYGKYVDE RSIGL LAAQA: .SSEESSAGRJ: [SAHLARSHSD QAHAFHGD TRERDRSIGL RLAAQE RGEAQSDLEJRQRIJ SIGSKYDQTAR DGRGERVNLIGL SLL IEASALRREIG GHPESDSWSL |LQRKALEDAHVAEAESRE1 GGASQIRDR YSEGGGDNSRSWDSYA "EYLQRLIGSRQADLHSJVLJ JQRLWDi  311 304 345 343 341 353 348 346 345 341 347 353 349 318 344 302 292 360  1  85  AprF OpmH OprH OprJ OpmQ OpmB OpmA OprH OpmD OpmE OpmJ OpmG Opml OpmH OpmK OpmH OpmL OpmF  JRsGGE T L A A T R Q A T H R H E K S H Y D L D D K ^ R E T L H Q V R K H Y H Q S S S S A A K I R A Y E H T V D S - A ^ E  G G R V S A A T RQ A G D K Y A Q AQ A E LDAQWASVIND L H S Q F D L T A S S L A K V R A Y E H A V A A - A T A G S L RAS LD  YAKIQKDIHVAQYEKAy  Q TA F  QEVAD G L A A R G T F T E Q L Q A Q R D L V K A  - S  369  410  S A V A A Y E G T jj Q T A F R E V A D A L A A S D T L R R E E K A L R A L A N S - S  408  RLRAERDRSLARQEELLETYRKAMLTAFADTERSLHSIDGLDRQLHWQQQELEQ-A  406  G G R N R A H L S L A E A RKD  GGLIGSQVDQABATYDQTVATYRQTRLDGFREVEDYLVQLSVLDEESGVQREALES-A  418  E G G R L R G R L E L R E AQ Q Q E A A I D Y Q R T W L R A W Q E V D D A H H D Y A A H Q R R Q E R L G E A V A Q - H  413  YE Q Q A L L A L E I S ABAF S D Y G K R Q E R L V S L V R Q S E A - S SWPAQRLGHVRARLRAVEAQ SDAALARYQRSJJLLAQEDVGRALMQ LAEHQ RRLVAL F Q SATH- G  411 410  L C S V R A R L R G A K A D A DA A L A S  RWAQLD R G R V W A R I A A S E A R A Q E A L I L Y D R T A L R A L Q E T D D A F H G Y G A A A D RL RL RL L E A T A - H  406  SHRF^HRESARGPJJ>SAAAERDAALARFDGAALGALRJVERALALYAGERQRRADLQRALDE-Q  412  )GGRRRAHLAERDADYDLAVGQYHKTJJVQALGEVSDDLGKLRSLEQQVIDQRQARDI-A  418  )CCRLRSQLGEAAAGYDAAVBQYHQTBVDALKHISDQLIRLHSVDIQKDFAAQSWAS-A  414  S G G L T S S QV R E S Y Q R L H Q S E Q S R E G Q R R Q W Q D T R H L H R A V H T D V E QV Q A R R Q A I I S - H QE  CF E R T Y Q V R H A L A R R E A S E A E L A D T E Q Q V S L E V W H N Y Q S L SVE  T RS L A R T R E L V E Q - S  )—RHQGHIYAAQSRADQARDLQRATBLRLRSEAVQAYDQLRTSEQELALVRRDLLPGA RF RED T Q Q C L S H F R R P G  376  QVE  TAAQ Q R L E S A K W S A D A H Q R D I R R Q L Q N L  FDNGD TL RUREQ S L TQ QVT E - S  PIGDLSRRQAEVRAQVDVEHQKILIEDARQTLEQNVIDAVRDLGTRWRQYQIAQRATALS  AprF OpmH OprH OprJ OpmQ OpmB OpmA OprH OpmD OpmE OpmJ OpmG Opml OpmH OpmK OpmH OpmL OpmF  RTLVHATRKSI RE QVTAT RRS VAi DEYYQLADKRSRT HEALKLAKARRES QRAFDLSDSRSQ. REALRLAEHQ RRALQSAREQg: RAAAQ Q A A I RyRE| ANALEIAHE >" REAARLARE RHAYRLARS: RSHFDLAHRRgGEl QKTYDIATLARQ QSSLEATEI&SQ RQSLEWQGRSRS| QSALDSHTRGQE  AprF OpmH OprH OprJ OpmQ OpmB OpmA OprH OpmD OpmE OpmJ OpmG Opml OpmH OpmK OpmH OpmL OpmF  HFVS GE T PARRRD CATTDCPAP LHT L SKTD T E EHRSALH YFGAGEGRAQVTAAIR TAKKEDPQA RGGRS KD ERLGRVEEGLPPSP  481 451 485 479 474 498  LAAGE TAGAHR GVATDDTSPGVARQRDS RS EHGQ ATAKAPAE RKLAP EHVPVRAVS S R YLKQDYDPDKDF L P PD LAKAAAE QLQSKPRQQY  487 491 482 492 496 482  GS H  425 493  T  EQVGELYRBQ"E'  RRKLEIEREKLR  1  QALYSAHHE L SKAKYDYLTAHA A[jQ Q F YGARRD LAB ARYAYLHA1 JRSLFTAQQQLITDRLHQLTSEJ AWRSSFLHlIAFIDGSTQRQIALl "SRTLYAAQDAAVQLRLARLQAS|G[ TATALSHIRTVLTLLGSRLTASSQI JRQLLDHQEQQVASDEAVSLTLl lAgRE Q L SAEDAQAQAE VE LYRGI EHMRALYQIREELAQAETAS F— RSDYL S RRAL SIART E Q RLAVgGJ RSLVADRARLVDAEHRVAERQ|Ej QQLLVAERQLASLESQQIDLS'Q TRLFQQQLVQEQVQAARLAAHAS" ,Q LYAAVRD YHHS RYD Y I LD T J L TAYASAXDQHIRAL GHWQTS RP ET LVGVRAQYVRALDAAAQARJJSJERLP RERFE AS RQLIHLRIERKRIEYRAAAQG TDLRHVEHTQLHALISFLHAQTQBDLI'  J  LDEADLELVAA LEDRDLAVLAA GGWHQQTVTQQQ GGHDEGRSLWH GG¥QSDRQGLAR GGWDSADIE RTD GGWSPTSDPASG GGWQPSA SGDLAPGAGQ GGWEACAGARRC GGTOQAASSPSHQ GGFQPDSRSAAL GGVGAGAD T PAQ TLSPADLEALSA RLGFWSLR EDIGHLGQ LLGPLLEHRLHH TLDSWEISLHD  383 409 366  3£7 426  442 435 476 474 472 484 479 472 476 472 478 484 480 449 471 428 423 492  86  3.4 S u m m a r y  Four RND multidrug efflux systems have been reported in P. aeruginosa; however, analysis of the genome revealed a larger number of putative RND efflux systems scattered around the genome.  Most of these putative systems contain the genes for all three  components adjacent to each other while a few lack the gene for the outer membrane component.  Phylogenetic analyses showed that the efflux components are generally divided  into two clusters: one with members homologous to those involved in efflux, while the other cluster contains members homologous to proteins involved in secretion.  This was clearly  observed in the phylogenetic tree for the OprM family. The observation that not all the members in a cluster (e.g. the OprM family) have related ORFs in the corresponding clusters (e.g. the MexA and MexB clusters) suggested that P. aeruginosa did not always acquire or duplicate all the genes in an operon as a whole package.  Comparison of the efflux  components already reported in the literature indicated that the pump proteins are more conserved among one another than the other two components. Multiple-sequence alignment of the members in the OprM family showed some conserved regions, and a few residues that were highly conserved among the eighteen members.  87  CHAPTER FOUR: Structural characterization of OprM - insertion mutagenesis  4.1 Introduction  Although the proton motive force-driven pump proteins and the linker proteins have been shown to be responsible for substrate selectivity (Srikumar et al, 1997), they do not work ih the absence of an outer membrane component, which must therefore be important for direct passage of the wide range of antimicrobial agents into the medium. Understanding the mechanism whereby OprM is involved in facilitating the extrusion of antibiotics will help to develop appropriate inhibitory methods for these multidrug efflux systems. A knowledge of the molecular structure of OprM is a starting point for studying its structure-function relationships. OprM has been assumed to be a porin-like protein in the outer membrane of P. aeruginosa that assists in the efflux of multiple antimicrobial agents (Ma et al, Nikaido, 1994). sequence  1994;  In this chapter, an OprM topology model was constructed by multiple-  alignments,  structure prediction methods  such as  P-turn prediction and  hydrophobicity calculation for outer membrane proteins, together with the results from insertion mutagenesis and a C-terminal deletion.  4.2 Prediction of an OprM topology model  OprM has been presumed to form a channel, like porins, in the outer membrane of P. aeruginosa for multidrug efflux (Ma et al, 1994; Nikaido, 1994).  OprM showed some  88  homology to and thus was predicted to function similarly to the E. coli outer membrane protein TolC, which had been previously shown to form channels (Benz et al, 1993) and was predicted, based on studies with two-dimensional crystals, to form a trimer of P-barrel monomers (Koronakis et al, 1997). Data from planar lipid bilayer experiments using the purified oligomeric form of OprM showed that it had channel-forming activity with a singlechannel conductance of 0.85 ± 0.03 nS (mean ± standard error) in 1 M KC1 (pH7) at an applied membrane potential of 50 mV (see Chapter 2).  Hence, a topology model of OprM  was predicted based on the P-barrel motif evident in previously published porin models. OprM shows limited homology to either general porins or substrate-specific  porins.  However, it was possible to identify conserved regions from its multiple-sequence alignment with several highly homologous proteins that are also involved in efflux (Fig. 18).  In  addition, possible p turns were identified and the amphipathicity profile of OprM was calculated using previously published methods (Huang et al, 1995; Gromiha et al, 1997). The amphipathicity profile of OprM is shown in Fig. 19.  The stretch of hydrophobic  residues at the N-terminus is a typical feature of a signal sequence.  The stretch of  hydrophilic residues at the C-terminus includes 19 of the final 22 amino acids of native OprM which were replaced by 14 residues derived from the phagemid used to clone the gene originally.  This highly hydrophilic C-terminus was presumed to be located within the  periplasm, where variations are usually tolerable.  Therefore, the substitutions at this C-  terminal region of OprM should not alter the expression and function of the protein. Using the above methods, an OprM topology model with 16 P-strands was predicted and revised slightly with data from insertion mutagenesis (Fig. 20, see also sections 4.4 and 4.5), though it lacked the consensus sequence in the 16th C-terminal P-strand of the porin superfamily,  89  O (Tl  T  V  CO ^* N  »H ^ N  TT CO (M  O  <0 N  W  £3 ©  fl fl  H  00  ft st+H CU  «o  H  w  CJ  &  SE  Q  ^ .s  H  o  60  ft  cn CU  -o  Cfl  fl  CU  3 ,i  ,!  U op  ^  3  •^i  C/J  -9 cu  </j  Ia  ###  O -2 ~,  fl 3  «  S  t!  M  O  +cu o-»  f. m  *t •< ft O >•  «  « -J •=>,  M  -  o ^ -« -«  cn  ,J J J a 3  O  O  43  13 SM  cu > <u cn  s-.  PH  § gse s  7 ¥ g ? S  o  w K o « 2 H L ; |z rt p »>. • J -J H H (fl h a ci ci  H-H  o  c cu  fl  a>  a <L>  cr <u ca a  * - •< tn  H  >  >  « .< n i ft in H n i  W ft J  i_3  iJ  _3  ft,  •fl  cn C  £  CCJ  3  «  O CU  •S O  u  •fl +-  1  £ S 1 «I cu • 1 cn2 c &  s  43  l>  fl  a ° .2 c s  S3 -  S  cn cu 3 T3 at) cu  g  fl  LH  VH  ^M  CU Cn  "S ap •a £ u -fl > CCJ c S cn ccS  CU <U  43  H 2 a u  i3  l •S xs  OH  cu ^ fl 3  •8 tn  J  °0  1a  i  cu  •~q W O* P< i-3  M 4!  ft, O S-> •<  J9  01}  O  tn  c3  cn <U S cU fl 'cn cu OH  CJ cU i f l Cn  ^a  5c ^ H  S <H  C+H  PH  cu rfl cu°0  fl o IO O3 § eg u « s s  6 M  •rl  H kt  H  p, tfi  o  H  »-5  M P. -P  Ou  u 53  Kh O & ft  H  •H ~H O U  o en M  tL-3 w I s-t fl. JJ o n  S U  s •rl H  14 P, O  90 ^ W kl  k4  > - -H >-3 m Li < p. O Li Li  S3  cn £3 >> CU ccS fl  •a 2ffl w  3 ox)  TT;  PH  o  U  CQ  a  T3  CU  ti  M  D  90  Residues (1-97)  Residues (389-485)  f  3 £ 1 ra  1-1  E -3 a.  <  -5  Figure 19.  Amphipathicity profile of OprM.  The profile was calculated with the method of Gromiha et al. (1997). Positive values indicate hydrophobicity and negative values indicate hydrophilicity. The stretch of hydrophobic residues at the N-terminus is a typical feature of a signal sequence. The stretch of hydrophilic residues at the C-terminus includes 19 of the final 22 amino acids of native OprM which were replaced by 14 residues derived from the phagemid used to clone the gene. Dotted lines indicate proposed transmembrane regions in the first OprM topology model. 91  Figure 20.  Predicted membrane topology model of the OprM monomer.  The model was slightly revised according to results obtained from insertion mutagenesis. The top of this model is proposed to face the exterior of the cell and the bottom is proposed to face the periplasmic space. Rectangles enclose the proposed transmembrane (3-strands. Circles indicate insertion sites M E 1 to 3, 5, 6, and 8 to 13 for malarial epitopes. Completely tolerated insertions are indicated by solid triangles, partially tolerated ones are indicated by shaded triangles and non-tolerated ones are indicated by open triangles.  92  which includes a conserved phenylalanine residue believed to be critical for protein stability and proper folding (Jeanteur et al, 1991 and 1994; De Cock et al, 1997). The segments identified with the most conserved regions among OprM homologues, and with good amphipathicity (e.g. strands 3, 5, 10, 12, 13, and 15), were mostly contained in the transmembrane P-strands in this slightly revised topology model, connected by long, surfaceexposed loops and shorter, periplasmic loops. contained  poor  amphipathicity  and  However, it was noted that some P-strands  fewer  conserved  residues,  especially  those  transmembrane strands in the central region. These were initially ignored since this is true of the p-strands in the central region of porins (Jeanteur et al., 1991 and 1994). OprM had been predicted to have a putative lipoprotein signal peptidase cleavage site (Poole et al., 1993b), and the homologous OpcM protein in Burkholderia cepacia was experimentally shown to be a lipoprotein (Burns et al., 1996). For simplicity, the predicted OprM topology model is shown as that of the mature protein after modification by signal peptidase at the N-terminus but without any possible lipid modification.  4.3 C - t e r m i n a l deletion of O p r M  An initial B L A S T search for OprM homologues from GenBank identified a P. putida 64 amino acid-long TodU sequence highly homologous to the C-terminus of OprM, with 54.7% identity (Fig. 21).  TodU was predicted to have no transmembrane region and was  encoded by a gene downstream of a two-component regulatory system of the toluene degradation pathway in P. putida. It was therefore of interest to examine the localization and importance of the C-terminus of OprM.  This was studied by creating a C-terminally 93  OprHt  TodU  :  :  SGL FDAGS GSUL FQ F SINL PIFTAGS LRAS LD YAK IQKDIHVAQYEKAIQTAFQEVAD GLAARGTF  _____________  OprHt TodU  TE Q L QAQRD LVKASDEYYQ LRDKJ3SRT0  OprHt TodU  QQTVTQQQTAKKEDPQA NISSQPLTAQN  Figu re 21.  HgY0®)ri  : :  :  .  396  462 47  485 64  Sequence alignment of OprM with P. putida TodU.  The whole TodU sequence, with 64 amino acids, aligned to the C-terminus of OprM. Identical residues are shaded in black while similar residues are shaded in gray.  94  truncated OprM. If the C-terminus of OprM had no transmembrane region, as indicated for TodU, this part would probably be more flexible and located within the periplasmic space and a deletion of this region might not alter the expression or function of the protein. Since there was no convenient unique restriction site, the deletion of 70 amino acids at the C-terminus of OprM (or 62 amino acids of OprM*) was accomplished by PCR with the two primers indicated in Fig. 22.  The PCR product was cloned into pVLT31 to create  pKWDC, and the construct was transformed into the P. aeruginosa OprM-deficient strain OCR03T and wild-type strain HI03 for expression and antimicrobial susceptibility assays. The truncated mutant was expressed (Fig. 23) but its oligomeric form could not be observed in isolated outer membranes.  In addition, the C-terminal deletion mutant was not surface  exposed when assayed by indirect immunofluorescence with an anti-OprM monoclonal antibody followed by a fluorescein isothiocyanate-conjugated goat anti-mouse IgG secondary antibody, even after treatment with 0.1 M NaCl to destabilize the outer membrane (Table IX).  Furthermore, OCR03T cells carrying pKWDC and induced by IPTG did not restore  resistance to any antibiotics tested as in cells carrying pKPM-2 (Table IX).  The data  suggested that the 70-residue region at the C-terminus of OprM was important for the proper assembly, localization and function of the protein. It was later shown that TodU was the Cterminal sequence of a larger ORF now named SepC (personal communication from Dr. Peter Lau whose laboratory sequenced the clone further upstream and downstream).  SepC  was encoded by the last gene of an operon sepA-sepB-sepC, which was shown to be homologous to mexA-mexB-oprM.  95  Forward primer: Xba  5' CCCTCTAGAAATAATTTTGTTTAACTTTAAG 3'  —  D  E  Y  Y  Q  3 9 8  Hindlll  oprM- 5' GC GAC GAG TAC TAC CAG TAG TAG A A G CTT ATG C 3'  Reverse complementary primer: 5' GCATAAGCTTCr^Cr^CTGGTAGTACTCGTCGC 3'  Figure 22.  Primers for constructing a deletion mutant of oprM at the 3' end.  The forward primer, with Xbal site, anneals to a region of pKPM-2 (pT7-7::oprM*) just upstream of the ribosome binding site in pT7-7. The reverse complementary primer contains sequences encoding two stop codons (italicized) followed by a Hindlll site.  96  12  47 -  3 4  -4  33 -  Figure 23.  Western immunoblot of the C-terminal truncated OprM mutant.  The PVDF membrane was reacted with an anti-OprM monoclonal antibody. Samples were outer membranes from OprM-deficient strain OCR03T carrying pVLT31 (lane 1) or pKWDC (lane 2), and from wild-type strain HI03 carrying pVLT31 (lane 3) or pKWDC (lane 4). A l l samples were from cultures induced with 0.05 m M IPTG for 2 h, and heated at 100°C with 5% v/v p-mercaptoethanol for 10 min in solubilization buffer before loading. Relevant molecular masses (kDa) are shown on the left. The position of the OprM monomer is indicated by an arrowhead and the C-terminal truncated OprM is indicated by an arrowhead with asterisk on the right. pKWDC, pVLT3Xv.oprM* with a deletion of 186 bp at  the 3' end of oprM*.  97  m  u  CN ©  s  co  co  ©  ©  V  V  co ©  co  & u  o  ©  V 1 =1.  ©  co O  o  o o  V  ©  ©  in ©  O  V  CN  CN O  CN ©  m  co  co  co  co  CN  fc  V  IT)  V  E  ©  o o  o  o o  ©  V  U l-H  CO  V  V  in o  in ©  ©  ©  V  V  CN  o  ©  V  CN ©  © o  V  "fl  a  ' fl +->  O g? •  oo o  —i  in  o  CN CS fl  •SP  U  53 MH  a O  H  co  U O  o.  CO  +  +  J<  U Q  a  I  13 to ,<  w  4.4 Insertional mutagenesis of OprM with the malarial epitope N A N P  Epitope insertion mutagenesis was done to determine if insertion of a large stretch of amino acids was tolerated in various regions of OprM.  The malarial epitope from the  circumsporozoite form of Plasmodium falciparum was chosen because previous studies in this laboratory have shown that this epitope is permissive and antigenic when inserted into the loop regions of the outer membrane proteins OprF (Wong et al, 1995) and OprP (Sukhan and Hancock, 1995). On the other hand, the epitope is not tolerated when inserted into the transmembrane P-strands.  Initially, insertion mutants were created using a Km-resistance  cassette as described in Materials and Methods; nine oprM insertion mutants were created by random insertion of the malarial epitope (NANP) repeats into oprM* and these constructs were named pKWIN plasmids. The protein encoded by oprM* contained a substitution of 14 unrelated amino acids for the 22 C-terminal residues of native OprM, but was capable of reconstituting OprM function (section 2.2; Wong et al, 1997). To supplement these mutants, a PCR approach described in Materials and Methods was used to create four additional epitope insertion mutants of oprM with the native 3' sequence when pXZL33 became available. When the complete oprM gene from these mutants was sequenced, some PCRintroduced errors were discovered. Except for the Y18C and Q20C changes in pKWIN2, the other three plasmids contained conserved changes (A110V in pKWIN5, S152T in pKWIN9, A400V in pKWIN13). Plasmid pKWIN2 was retained for further studies since subsequent analysis demonstrated efficient expression in both E. coli and P. aeruginosa, indicating the relatively benign effects of the observed mutations. The insertion sites were named ME1 to  99  ME13 from N to C terminus, corresponding to the pKWIN plasmid number (i.e. pKWINl carries insertion at site ME1).  4.5 Characterization of the oprM insertion mutants  4.5.1 Expression of oprM insertion mutants in an E. coli background The various pKWIN plasmids were transformed into the porin-deficient E. coli strain CE1248 to allow overexpression of the cloned insertion oprM mutants.  Outer membranes  were isolated for analysis by SDS-PAGE and Western immunoblotting with both anti-OprM and anti-malarial epitope antibodies.  Plasmid pKW35TM (pVLT35 v.oprM with native  sequence) and the control vector pVLT35 were also transformed into the E. coli CE1248 strain as controls. Insertions at sites ME4 and ME7 generated translational stop sites and as expected, did not produce mutant proteins (Fig. 24A - 24C, Table X). Three of the mutant plasmids, with insertion sites at ME3, ME9 and M E 13, led to expression of the mutants at greatly reduced levels in comparison with wild-type OprM. Mutant proteins with insertions at ME8 and M E 12 were expressed at intermediate levels but were strongly detected by the anti-OprM antibody. higher  The other 6 plasmids directed expression of the mutants at levels similar to or  than  that  from pKW35TM,  as  determined  by  SDS-PAGE  and Western  immunoblotting with an anti-OprM antibody (Fig. 24 A, 24B). Most of these mutant proteins exhibited slightly lower electrophoretic mobility compared to that of wild-type OprM. Similar results were obtained from Western immunoblotting with an anti-malarial epitope  100  M  1 2 3  C  4 5 6 7  8 9 10 11 121314  TM ME ME ME ME ME ME ME ME ME ME ME ME 1 2 3 4 5 6 8 9 10 11 12 13  B. 1 2  c  4  5  6  7  8  7  8  9  10 11 12 13 14  47 -  1 47 -  Figure 24.  3  2  3  4  5  6  w  9 10 11 12 13 14 ~ t iar  Expression of oprM malarial epitope insertion mutants in E. coli.  A . SDS-PAGE of outer membranes from E. coli CE1248 strain carrying control vector pVLT35 (lane 1, C), native oprM-expressing plasmid pKW35TM (lane 2, TM), and OprM-encoding plasmids with malarial epitope insertions at sites ME1 to ME6 (lanes 3 - 8), ME8 to M E 13 (lanes 9 - 14). OprM and OprM insertion mutants are indicated by triangles. Due to low level of expression, proteins expressed by pKWIN3 and pKWIN13 (lanes 5 and 14) were localized by Western immunoblotting. The gel was stained with Coomassie blue after electrophoresis. Each lane was loaded with -20 ug of protein, treated with 5% (v/v) Pmercaptoethanol and heated at 100°C for 10 min. The molecular masses (kDa) for the prestained markers shown in lane M are indicated on the left. Expression of oprM and the mutants was confirmed by Western immunoblotting with B) an anti-OprM antibody and C) an anti-malarial epitope antibody. Samples were in the same order as in the gel shown in panel A , and the corresponding regions of the blots are shown, with the relevant molecular mass indicated on the left.  101  Expression and characterization of the oprM epitope insertion mutants in  Table X .  E. coli  Plasmid  Insertion  3  Amino acids inserted  site (aa)  b  -  None (control vector)  -  Protein  Surface  expression  reactivity*  (SDS-PAGE)  (anti-malarial)  C  1  -  -  None (wild-type OprM)  +++  -  ME1(23)  GPAP(NANP) NAGHAGP  +++  +++  ME2(37)  KRKNP(NANP) N  +++  +++  pKWIN3  ME3(72)  GPAP(NANP) NAGHAGP  +  -  pKWIN4  ME4(77)  - (stop)  -  -  ME5(103)  KRKNPNANPN  +++  +++  pKWIN6  ME6(130)  TCNP(NANP) NACRS  +++  +++  pKWIN7  ME7(143)  - (stop)  -  -  pKWIN8  ME8(159)  GPAP(NANP) NAGHAGP  ++  +++  ME9(241)  KRKNP(NANP) N  +  +  pVLT35 pKW35TM pKWINl pKWIN2  f  pKWTN5  pKWIN9  f  f  e  2  2  2  2  2  2  pKWTNlO  ME10(251)  GTCNP(NANP) NACRS  ++++  +++  pKWTNll  ME11(315)  GTCNP(NANP) NACRS  +++  +++  pKWIN12  ME12(377)  DLQNPNANPNANALDVQV  ++  +  ME13(393)  KRKNPNAPNANPN  +  -  pKWIN13  a  b  c  d  e  f  2  2  pKWINl to pKWTN12 contain the insertion mutants cloned into pVLT35, pKWIN13 contains the insertion mutant cloned into pVLT31 (a derivative of pVLT35). Position 1 is the N-terminal amino acid (aa) of the mature OprM amino acid sequence. Results obtained using outer membranes of E. coli CE1248 containing the various plasmids. Expression level ranging from undetectable (-) to strong (++++). Expression was confirmed by Western immunoblot using a monoclonal antibody against OprM. Fluorescence level ranging from no fluorescence (-) to strong (+++), assessed by indirect immunofluorescence. pKW35TM contains wild-type oprM with native sequence cloned into pVLT35. These plasmids contain native oprM sequence at 3' end, M E insertion generated by PCR; the other plasmids contain insertion, via Km-cassette, into the previously published oprM sequence (oprM*). 102  antibody, except that the mutant proteins with insertions at M E 12 arid M E 13 became less detectable and undetectable respectively (Fig. 24C).  4.5.2 Surface exposure of insertion mutant proteins in E. coli To determine if the OprM mutants were properly inserted into the outer membrane of the E. coli host cells, clones were subjected to indirect immunofluorescence for surface exposure analysis of the mutant proteins. Cells carrying the pKWIN plasmids or the control plasmids pVLT35 and pKW35TM were incubated with anti-malarial epitope antibodies followed by a goat-anti-mouse fluorescein isothiocyanate-conjugated antibody and examined under a fluorescence microscope. The 7 mutant proteins detected strongly in Western immunoblot with an antibody specific for the malarial epitope also bound the antibody on the surface of the cell, demonstrating that these mutant proteins were surface exposed and that insertions in these sites were similarly surface exposed, or that enough surface disruption occurred to permit access of the antibody to internal sites (Table X). Mutants with insertions at ME9 and M E 12 fluoresced very weakly. The other two mutants, with insertions at ME3 and M E 13, did not fluoresce. These clones also showed reduced or undetectable levels of the mutant proteins. Thus, these four sites (ME3, 9, 12, and 13) were probably not well exposed on the surface of E. coli CE1248.  4.5.3 Expression of oprM insertion mutants in P, aeruginosa To determine if the oprM mutants were also expressed in the native host for OprM, P. aeruginosa, the pKWIN plasmids and the negative control plasmids pVLT35 and pVLT31 and the native oprM-expressing plasmid pKW35TM were transformed into P. aeruginosa.  103  The strain OCR03T, an OprM-deficient mutant of P. aeruginosa was utilized to ensure that the modest level of OprM constitutively produced in wild-type cells would not interfere with detection of the mutant proteins. Interestingly, the expression pattern of the oprM mutants in P. aeruginosa OCR03T strain was somewhat different from that obtained in the E. coli CE1248 strain. Among the three plasmids which produced reduced levels of mutant proteins in E. coli, pKWIN3 did not produce any of the mutant protein in P. aeruginosa OCR03T while plasmids pKWIN9 and pKWIN13 led to medium expression levels of the oprM mutants they carried (Fig. 25A - 25C). The result for these latter two plasmids could reflect the ability of MexAMexB, present only in P. aeruginosa, to stabilize these mutant forms of OprM, or could be due to differences in proteases that degrade mutant proteins in these two organisms. On the other hand, expression of oprM mutants from three other plasmids (pKWIN6, pKWIN8 and pKWTN12) was undetectable in P. aeruginosa (Fig. 25 A - 25C, Table XI). We assume this to be due to enhanced susceptibility of these mutant OprM forms to proteolytic degradation in P. aeruginosa. P. aeruginosa OCR03T cells carrying the remaining five plasmids (pKWINl, pKWIN2, pKWIN5, pKWINIO and p K W I N l l ) showed varying expression levels of the oprM mutants (Fig. 25 A - 25C, Table XI). When reacted with the anti-malarial epitope antibody, most of the results with the OprM mutant proteins were similar to that obtained with the anti-OprM antibody, except with the OprM mutant from pKWIN13, which became undetectable (Fig. 25D). This was probably due to inaccessibility of the epitope when inserted at site ME13.  Wild-type OprM  is heat modifiable and partially exists as an oligomeric form of about 100 kDa on SDSP A G E (Masuda et al, 1995). This oligomeric form was also observed for the mutant forms  104  Figure 25.  Expression of oprM malarial epitope insertion mutants in P. aeruginosa.  A. SDS-PAGE of outer membranes from P. aeruginosa OCR03T carrying control vector pVLT35 (lane 1; C), native o/?rM-expressing plasmid pKW35TM (lane 2; TM), and OprM-encoding plasmids with malarial epitope insertions at sites ME1 to ME6 (lanes 3 to 8) and ME8 to ME13 (lanes 9 to 14). Each lane was loaded with -20 u.g of protein, treated with 5% (v/v) P-mercaptoethanol and heated at 100°C for 10 min. The molecular masses (kDa) of the prestained markers in lane M are indicated on the left. The arrowhead on the right indicates the position of some stable oligomeric forms of the proteins. B. SDS-PAGE of the same samples incubated in solubilization buffer at room temperature for 10 min and loaded in the same order as in panel A. The arrowhead on the right indicates the position of the oligomeric forms of the proteins. Relative molecular masses (kDa) are indicated on the left. Expression of insertion oprM mutant forms was confirmed by Western immunoblotting with C) an anti-OprM antibody and D) and E) an anti-malarial epitope antibody. The corresponding regions of the blots are shown, with the relevant molecular mass (kDa) indicated on the left. Samples are in the same order as in the gel shown in panel A. Proteins for the Western immunoblots were E) untreated or C) and D) treated with 5% (v/v) Pmercaptoethanol and heated at 100°C for 10 min.  105  A  M  1  B  2  1 2  3  4  5  3  4  5  6  7  8  6  9  8 W  110  9*/  10  11 12 13 14  10 IV 11  •fit mm - B  12 13 14 1 -C I w IT ;  » IB  It  47  mm  m-» mm  *«***<•»«•*  w  33  1  2  3  4  MMk\  47-  5  6  110  8  9  10 11 12 13  14 m  mmw -  47  7 mmy  •*  mm  i  1  2  3  4  5  6  7  8  9  10 11 12 13 14  1  2  3  4  5  6  7  8  9 10 11 12 13 14  C  TM  ME  ME  1  2  -  ME ME ME ME ME  3  4  5  6  8  ME ME  9  10  ME  ME  ME  11  12  13  106  Table XI.  Expression and characterization of the oprM epitope insertion mutants in P. aeruginosa.  Plasmid  3  Insertion site (amino acid)  +++  M E 1(23)  ++  +++  ME2(37)  +++  +++  pKWIN3  ME3(72)  -  pKWIN4  ME4(77)  -  pKWIN5  ME5(103)  +++  pKWIN6  ME6(130)  -  pKWIN7  ME7(143)  -  pKWIN8  ME8(159)  -  pKWIN9  ME9(241)  ++  +++  pKWINl 0  ME10(251)  ++  +++  pKWINl 1  ME11C315)  +  ++  pKWINl 2  ME12(377)  -  ME13(393)  ++  pKWIN2  e  e  e  pKWINl 3  d  e  anti-malarial epitope  - (wild-type OprM)  pKWINl  c  anti-OprM  -  pKW35TM  b  0  - (control vector)  pVLT35  a  b  Western immunoblot with antibody  e  d  +++  pKWINl to pKWIN12 contain the insertion mutants cloned into pVLT35, pKWIN13 contains the insertion mutant cloned into pVLT31 (a derivative of pVLT35). Position 1 is the N-terminal amino acid of the mature OprM amino acid sequence. Results obtained using outer membranes of P. aeruginosa OprM-deficient OCR03T strain containing the various plasmids. Expression level ranging from undetectable (-) to strong (+++). pKW3 5TM contains wild-type oprM cloned into pVLT3 5. These plasmids contain native oprM sequence at 3' end, M E insertion generated by PCR; the other plasmids contain insertion, via Km-cassette, into oprM* (the previously published oprM sequence). 107  of OprM that were expressed (Fig. 25A, 25B). When reacted with the anti-malarial epitope antibody, these expressed mutant proteins gave results similar to those for their monomelic forms (Fig. 25E).  Surface exposure could not be assessed because P. aeruginosa cells  always gave false positives due to cell clumping.  4.5.4 Antimicrobial susceptibilities of P. aeruginosa cells carrying the insertion mutants The P. aeruginosa clones carrying the various pKWIN plasmids were subjected to antimicrobial susceptibility assays to determine if the malarial epitope insertions affected the function of the OprM mutants. MICs for various antimicrobial agents were determined for the P. aeruginosa clones (Table XII).  Antimicrobial susceptibility assays performed on  clones carrying the oprM or oprM* sequence in a P. aeruginosa OprM-deficient background strain did not show any significant differences (Table XII). Apparently, variation at the 3' end of oprM did not affect the function of the protein and so should not have affected the results in this study. All non-expressing clones failed to influence antibiotic resistance.  In contrast, P.  aeruginosa cells carrying the expressed mutants showed variable patterns in their resistance profiles. Cells with pKWIN2, pKWTN9 and pKWINl 3 had resistance profiles identical to those with wild-type OprM.  Cells carrying pKWINl and pKWIN5 had resistance profiles  very similar to that of the control with wild-type OprM, except for only partial restoration of resistance to some of the P-lactams tested. Cells with pKWINl 0 and pKWINl 1 restored resistance only partially to all of the various antimicrobials tested, with at least a 4-fold reduction in MIC levels when compared with those obtained from cells carrying wild-type OprM (Table XII).  The variation in expression levels of oprM mutants was not solely  108  c3  U  o  © in CN  o  oo r-  © V  o  co O © V  in CN ©  o  o o  ©  u  o  fN O V  © © © in  o  in CN  © © V m  N  o  © V 03 fc  o  CN ©  O  CN ©  o  ON  O  co ©  NO  (N  o o  o ©  © ©  ON CO  o  IT, CN  V) O vd T—1 in  CN  © © © in  o o o o  ©  fN  ON CO  o  ©  ^o  oo  ^o  ©  ©  ©  ©  ^  ©  .—I  co  ro  CO  CO  © o  in CN ©  o  m ©  © m ©  ©  O*  ©  o o  ir>  ©  ©  © ©  © CN ©  O  CN ©  CN  © ©  ©  o  IT)  CO  ©  NO  CO  co  NO  CN  NO  H  o  ©  ©  fN  ©  ©  © ©  ©  ©  IT; O  O  ©  ©  ©  ©  CN ©  ©  IT)  IT; CN  CO  ©  CN  NO  CO  IT)  ©  © in CN  in CN  CO  CO  CO  CO  IT;  in  V  © © CN  © O CN in 1—1 CN IT)  O  © © CN  V  © O in CN  NO IT)  © in CN 1—1  CN  II  © in CN  fN  o  o  fN  © ©  o  oo  ^d i-H  ON CO  ON CO  ON CO  ©  ©  o  o  © CN ©*  ON  CO o  •3 *  1—1  V  ON  CO  i—1  ©  00  ON  ©  ©  o  ©  © in CN  © © O* m  © © © © CN  o  © © in CN  © in CN  OH  o  H  c3  C  *  -i  S  GO 1/3  k x  <N  o  co  CO  m  in i—1  ^o  CO  r—1  VO IT) i-H  CO  •o  1  + + + + +  o (D >  + +  + + +  + +  60  /—s  ,—  CO CN  w O CM  U  in CO  H  H  in  CO  CN  N  CO  i-H  T-H  CS  ©  ^ — ^ IHO  ON  *—1  CN  CO  i—i  8  CS  CO ON CO  in i—(  © i—i  W  2 ^  (H  o  Cf'  "° i •o i  CN  £ 2^ ^  i  n  CO  > OH  OH  109  responsible for this result (cf. pKWIN9 and pKWINIO; Table XII). Therefore, the variations in antimicrobial susceptibility by the different mutant proteins was likely due to different effects asserted by insertion of the malarial epitope at specific sites.  These experimental  data, together with results from the expression studies, were used to generate a revised OprM p-barrel topology model shown in Fig. 20.  4.6 Summary  An OprM topology model was proposed based on previously published porin models, multiple-sequence alignment with highly homologous proteins, P-turn prediction and amphipathicity calculation. Sixteen transmembrane p-strands were predicted, connected by short periplasmic turns and longer surface-exposed loops. obtained using data from the OprM insertion mutants.  A slightly revised model was  Completely or partially tolerated  insertion sites were localized to the flexible loop regions while the non-tolerated ones were localized to the transmembrane P-strands. Variations of the last 22 amino acids at the Cterminus of native OprM did not affect the expression or function of the protein. Therefore, results obtained with the OprM insertion mutant proteins should not have been affected by the native or the incorrect (due to cloning) OprM C-terminal sequence. On the other hand, a deletion of the last 70 residues at the C-terminus completely abolished the function of the protein and apparently affected the proper folding since no oligomeric or surface-expressed form was obtained from the truncated monomer. Therefore, it seems that the last 70 residues contain amino acids or regions that are indispensable for the expression of oprM. Although this OprM topology model subsequently proved to be incorrect (see Chapter 5), these 110  attempts to test the model provided useful tools for analysis of structure-function relationships in OprM.  Ill  CHAPTER FIVE: Three-dimensional model of OprM and its structure-function relationships  5.1 Introduction  The most similar homologue of OprM in Escherichia coli is outer membrane protein TolC, which is also involved in multiple-antimicrobial resistance through an energydependent efflux mechanism. It was shown to function with AcrA and AcrB (Fralick, 1996) to extrude a wide spectrum of antimicrobial agents. The amino acid sequences of TolC and OprM are 21% identical and 40% similar, and both proteins were shown to form oligomers (Benz et al, 1993; Koronakis et al, 1997; Masuda et al, 1995) and appeared to be able to interact with different components from other efflux systems to form functional chimeric complexes (Mine et al, 1999; Srikumar et al, 1997; Yoneyama et al,  1998). Channel  activities have also been observed for both proteins (Benz et al, 1993; Wong and Hancock, 2000; section 2.3).  Therefore, the two proteins, as well as their numerous homologues in  Gram-negative bacteria, are likely to share a similar structure.  As the outer membrane  proteins of the RND efflux systems appeared to form trimers and channels similar to porins, a structure was predicted for OprM (Chapter 4; Wong and Hancock, 2000) based on that identified for porins, whereby each monomer of the trimer forms a P-barrel.  Interestingly,  early topology models for TolC likewise suggested a P-barrel conformation (18-stranded) (Koronakis et al, 1997). The recent publication of the crystal structure for TolC, however, provided a very different vision. It has a distinctive, and novel structure for a cell membrane protein, comprising three monomers making up one long continuous channel spanning both 112  the outer membrane and the periplasm, where each monomer supplies strands required for channel formation.  The outer membrane is traversed by a 12-stranded P-barrel (each  monomer supplying 4 strands to this barrel) and this domain sits atop a 12-stranded coiled ahelical channel that spans the periplasm (again, 4 helices are supplied per monomer; Koronakis et al, 2000).  As OprM and TolC are similar in sequence and function, this  chapter describes a revised OprM topology model obtained by threading its sequence into the TolC crystal structure. In addition, the defined deletion mutants and data from previously isolated insertion mutants were used to provide information on functional domains in the model.  5.2 Secondary structure of O p r M  To obtain further evidence that the OprM structure is similar to that of TolC, the purified oligomeric OprM protein was subjected to circular dichroism (CD) analysis (Fig. 26). A n estimate of secondary structure was obtained by the K2D method (Andrade et al, 1993), yielding estimates of 19% P-sheet and 32% a-helical structure. These data were more comparable to the values of 14% P-sheet and 56% a-helix determined for the TolC crystal structure, than the high proportions (>50%) of P-sheet observed for several outer membrane proteins, including porins, and the predicted topology in Fig. 20.  113  I  I  190  200  I 210  I  I  220  230  I I 240 250  W a v e l e n g t h (nm)  Figure 26.  Circular Dichroism (CD) spectral analysis of purified wild-type OprM.  CD analysis was performed with a J-810 spectropolarimeter using a quartz cell with a 1mm path length. C D spectra were measured at 25 °C, between 190 and 250nm at a scanning speed of 10 nm/min in lOmM Tris (pH8.0) with 0.1% (w/v) sodium dodecyl sulfate.  114  5.3 T h r e e - d i m e n s i o n a l m o d e l o f O p r M  To assist in the modeling of the three-dimensional OprM structure, the sequence of OprM was first aligned with that of TolC (Fig. 27).  The elements of secondary structure  from the TolC crystal structure are indicated as boxes.  Most of the similar or identical  residues were observed to be within these structural elements.  There was a significant  sequence gap at the position of a TolC extracellular loop (between S4 and S5) and another lay between p-strand S2 and helical strand H3, while variable extensions were observed at the termini.  Interestingly, the 43 residues at the C-terminus of TolC were shown to be  dispensable for its function (Koronakis et al, 2000), as were the 22 amino acids at the Cterminus of OprM (Wong et al, 1997), and the TolC crystal structure was actually obtained without these 43 amino acids.  Overall, the two sequences share 40% similarity, which is  adequate for modeling purposes. Using the molecular modeling program "Homology" of the Insight II (version 97.2) program (Molecular Simulations Inc., San Diego, Calif), the OprM amino acid sequence was threaded into the TolC crystal structure based on the alignment shown in Fig. 27. The first 52 residues at the N-terminus of OprM were excluded, as there are no corresponding residues in the TolC structure. After the entire structure was subjected to energy minimization using the "Discover" program of Insight II, a model of the architecture of the OprM trimer was generated as shown in Fig. 28. In this model, the OprM channel, like TolC, consists of three monomers (Fig. 29).  Each apparently comprises two tandem structural repeats and  contributed 4 P-strands to the p-barrel plus 4 a-helical strands to the helical barrel. Notably, a ring of aromatic residues was evident along the base of the P-barrel at the proposed 115  Dl  2  OprM TolC  H I T AVVLSGCSLIPDYQRPEAPVAAAYPQGQAYGQNTGAAAVPAADIGWREFFRDPCLQQLIG F.MT.MOVYO  OprM TolC  H2 ^ SI D2 S2 VALEKNRDLRVAALNVEAFRAQYRIQRADLFE R I G V D G S G T R Q R [ J P G D L S T T 5SPAISSQ QARLsMPELRKSAADRDAAFEKINEARlsPLLpbLGLGADYTYSNbYRDANGIINSNATSAS  OprM TolC  H3 5ZYGVT LGTTAWELDL FGRLRSLRDQALEQYLATEQAQRSAQTTLVASVATAYLTLKADQAQ LOLTPS IFuMSKWRALTLOEKAAGIODVTYO-TDOOTLILNTATAYFNVLNAIDV  S7  I  '6 '  OprM TolC OprM TolC OprM TolC  H4 LQLTKDTLGTYQKSFDLTQRSYdVGVAaALDLRQAQTAVEGARATLAQYTRLVAQDQNAL LSYTQAQKEAIYRQLDQTTQRFfr|VGLVA|ITDVQNARAQYDTVLANEVTARNNLDNAVEQL . * .. . * • 9 10 H5 H6 S3 • VLLL|3SGIPKNLPdGLG^DQTLLTEVPAGLPSDLLQPjR|PDILEAEHQLMAANASIGAARt\ ROITbNYYPELAALINVENFKTDKPOPiVNALLKEAEKRlNLsLLOARLSODLAREOIROAob 11 S4 V AFFP SISLTANAGTiy 3RQLSG  -LFDAGjSGSWLFQPSIK LPIFTAG SLRASL  GHT.T^T.nTTA.STGT.^TSY.SGSKTRGAAGTOYnnRlJMGONKVGT.SFApiYOGGlMVNSOV ***• 12 13 ' D3 D4 H7  OprM TolC  DYAKIQKDINVAQYEKAIQTAFQEVADGLAARGTFTEQLQAQRDLVKASDEYYQLADKRY KQAQYN FVGAS EQLESAHRSVVQTVRS S FNNINASIS SINAYKQAVVSAQS S LDAMEAGY  OprM TolC  H8 D5 H9 RTGVDNYLTLLDAQRSLFTAQQQLITDRLNQLTSEVNLYKJ'LGGGHWQQrvrQQQr^KKE sIvGTRdlVDVLDATTTLYNAKOELANARYNYLINOLNIKSaLGTLNEODLLALNNklJsKP *** .*. *.*.* * * * . .*. *** S6  OprM TolC  VSTJNPENVAPQTPEQNAIADGYAPDSPAPVVQQTSARTTTSNGHNPFRN  Figure 27.  +  * +  DPQA  Alignment of the sequences of TolC and OprM.  Identical residues (*) and similar residues (: and .) are noted, using the similarity defaults of ClustalX. Elements of secondary structure from the TolC crystal structure are boxed, HI to H9 are helices, SI to S6 are p-strands where SI, S2, S4 and S5 (thicker borders) are within the P-barrel. The helical barrel is comprised of 2 long helices (H3 and H7) and 2 pairs of shorter helices stacking to form pseudocontinuous helices (H2 + H4; H6 + H8) from each monomer. The equatorial domain is made up of strands S3 and S6 and the 3 short helices H I , H5 and H9. Sites of malarial epitope insertion into OprM are indicated by solid triangles (fully tolerated), grey triangles (partially tolerated), and open triangles (nontolerated). Deletions D l to D5 in OprM are underlined and bolded. Residues at the Cterminus of wild-type OprM that are different from those in previously published OprM*, contained within H9 and S6, are italicized. 116  p-barrel  a-helical barrel  Figure 28.  Three-dimensional model of an OprM homotrimer.  The structure was constructed by threading the sequence of OprM on a crystal structure of TolC.  The p-barrel at the top anchors the protein in the outer membrane while  the a-helical barrel spans the periplasmic space.  117  Figure 29.  Three-dimensional model of an OprM monomer.  The structure was constructed by threading the sequence of OprM on a crystal structure of TolC.  The P-strands at the top contribute to the p-barrel. The N - and C-termini  are labeled.  118  interface between the lipid bilayer and periplasm (Fig. 30). This feature was consistent with both the structure of TolC and all outer membrane proteins structures characterized to date, supporting the validity of the proposed model. Proline residues also form a ring at the base of the P-barrel (Fig. 30). As for TolC, these prolines are probably important for disrupting secondary structure for the transition from right-handed p-barrel into left-handed a-helices.  5.4  I n s e r t i o n m u t a g e n e s i s o f oprM  A number of insertion mutants have been generated in oprM (Chapter 4; Wong and Hancock, 2000).  The sites of insertions at ME3, ME5, ME6, and ME8 to ME13 are  indicated in both the alignment (Fig. 27) and the three-dimensional model (Fig. 31). The top and bottom views of the model with the insertion sites are shown in Fig. 32. Malarial epitope insertion sites at ME1 and ME2 are not shown in the three-dimensional model because they were within the first 52 amino acids excluded from the threading. However, according to the model, malarial epitopes inserted at these two sites would probably be located within an N terminal extension in the periplasm and this might explain why they were tolerated.  As  shown in Fig. 31 and Fig. 32, all the insertions that prevented expression of the mutant (black, at sites ME3, ME6, ME8 and M E 12) were located within the conserved helical structure (ME3 in H2, ME6 and ME8 in H3, and M E 12 in H7), suggesting that the a-helical barrel core was important for the proper expression and function of OprM. The insertion at ME13 was, however, fully tolerated.  Perhaps insertion of 13 residues at ME13 might be  permissive owing to its location close to the more flexible end of helix H7. Another fully tolerated insertion, at ME9, together with a partially tolerated insertion at M E 10, were 119  Figure 30.  p-barrel domain of the three-dimensional model of OprM.  Proline residues (black) and phenylalanine residues (gray) are shown with their sidechains. They predominantly form rings at the base of the barrel.  120  Figure 31.  Three-dimensional model of an OprM homotrimer with malarial epitope insertion sites highlighted.  The structure was constructed by threading the sequence of OprM on a crystal structure of TolC. The P-barrel at the top anchors the protein in the outer membrane while the a-helical barrel spans the periplasmic space. Amino acids at which the malarial epitopes were inserted are highlighted: shaded balls (ME 9,13), tolerated; gray (ME 5,10,11), partially tolerated; black (ME 3,6,8,12), non-tolerated. One copy of each insertion in the trimer is labeled for clarity.  121  Figure 32.  Top and bottom views of the three-dimensional model of OprM.  A. Top view of the three-dimensional model of OprM. B. Bottom view of the structure. The three-dimensional structure was constructed by threading the sequence of OprM on a crystal structure of TolC. The amino acids at which the malarial epitopes were inserted are highlighted (shaded balls, tolerated; gray, partially tolerated; black, nontolerated).  122  located within the equatorial domain of the oc-helical tunnel, suggesting that this domain is more amenable to disruption.  Two partially tolerated insertions (at ME5 and ME11  respectively) were each located within the proposed two external loops of the P-barrel in the three-dimensional model.  This indicates that these loops are also amenable to some  disruption.  5.5 D e l e t i o n m u t a g e n e s i s o f  oprM and  characterization of mutants  5.5.1 Expression and in vivo function of the oprM deletion mutants A series of defined deletion mutants of OprM (Fig. 27) were created by PCR as described in Materials and Methods and introduced into the P. aeruginosa OprM-deficient strain OCR03T for expression studies and antimicrobial susceptibility assays (Table XIII). Expression of the OprM deletion mutants was only slightly less than that of wild-type OprM. These deletion mutants exhibited similar behavior to the wild-type OprM protein in that a portion of each protein ran as an oligomer on SDS-PAGE and the remainder ran as the monomeric form even without heat denaturion or p-mercaptoethanol treatment.  OCR03T  cells expressing mutants with deletion D l or D2 showed no significant difference in antimicrobial resistance compared to cells expressing wild-type oprM (Table XIII). Deletion D l overlapped the permissive insertion site ME2 and removed 8 amino acids at the N terminus of OprM. As mentioned above, the N-terminal region of OprM is probably located within the periplasm and this might explain why both mutations were tolerated. Deletion D2 removed 8 amino acids, some of which formed the upper half of P-strand SI. 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This is probably why this particular deletion was tolerated. In addition, this deletion fortuitously permitted a continuity of amphipathic sequence probably pulling part of the following external loop into the P-barrel structure and shortening the length of the loop over the pore. On the other hand, cells expressing the other three oprM mutants with deletions D3, D4 or D5, notably all located in the helical barrel, showed varying resistance profiles. Cells expressing the oprM mutant with the deletion D4 (removed 8 amino acids of helix H7) had resistance levels similar to those from the cells carrying the control plasmid pVLT35. Cells expressing oprM mutants with deletions D3 (removed 4 amino acids straddling insertion site M E 12 of H7) and D5 (removed 8 amino acids of helix H8) did not increase resistance levels to some antimicrobial agents but partially restored resistance levels to others, with deletion D3 having a relatively larger impact on the function of OprM. In particular, resistance to the tested P-lactams (meropenem, cefotaxime, cefepime, cefsulodin, and to a somewhat lesser extent carbenicillin) was eliminated by deletions D3 and D5. A n OprM truncation mutant, DC70, with the deletion of 70 amino acids at the C-terminus abolished the proper localization and function of the protein (Table XIII, Chapter 4).  This deletion removed a  mixed a/p domain (helix H9 and strand S6), the complete helix H8, and part of helix H7 of the helical barrel.  These data again suggested the a-helical barrel was important for the  proper expression and function of OprM. In addition, the TolC crystal model was obtained with a mutant deleted for the 43 amino acids at the C-terminus which were shown to be dispensable for the function of TolC (Koronakis et al, 2000). The C-terminus of OprM has also been shown to be non-essential and deletion and partial replacement of the last 22 residues (Fig. 27), removing a mixed a/p  125  domain that was shown in the TolC structure to form part of an equatorial band around the helical barrel segment, did not affect the function of OprM (Table XIII; Wong and Hancock, 2000).  Therefore, the C-termini of both TolC and OprM might be structurally and  functionally non-essential.  5.5.2 In vitro function of the oprM mutants: channel-forming activities The five deletion mutants, together with two insertion mutants, were purified from OCR03T for planar lipid bilayer analysis, and results are shown in Fig. 33.  The ME5  insertion mutant in one of the extracellular loops failed to restore resistance to some of the Plactams, but increased MICs to all the other antimicrobial agents.  The ME11 insertion  mutant in the other extracellular loop was expressed relatively poorly and thus only partly reconstituted resistance levels to all tested antimicrobial agents were observed. The single channel conductances of both of these insertion mutants were similar and were only slightly smaller than that of wild-type OprM. Mutants with the fully tolerated deletion mutants D l and D2 gave similar single channel conductances to that of wild-type OprM.  Deletion  mutant D5 restored resistance levels partially and had a single channel conductance that was only slightly smaller than wild-type OprM. Deletion mutants D3 and D4 gave rise to lower resistance levels than mutant D5 and had small single channel conductances in 1 M KC1 that were half to a third of that of OprM.  Thus, the results from the planar lipid bilayer  experiments correlated with changes from the antimicrobial susceptibility assays.  126  — CO  Oi C c S o ra SZ c 0  0 ra 4-1  11 01 u  (0 o  o  1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0  0.85  0.86  °p  8  I 0.73 0.75 fH—ir~^"  0.73  0.34  OprM  M E 5 ME11 D1  D2  D3  0.39  D4  D5  Samples  Figure 33.  Single channel conductances of wild-type OprM and its mutants.  Mean single channel conductances (from 101 to 145 events) of purified oligomeric wild-type OprM and its mutant forms after addition to the aqueous phase (1M KC1, pH7.0) bathing a membrane formed from 1% (w/v) diphytanyol phosphatidylcholine dissolved in ndecane. Experiments were performed at 25°C with applied voltage of 50mV. The error bars indicate standard errors. The OprM mutants included two insertion mutants with malarial epitopes inserted at site ME5 and ME11, and the 5 deletion mutants D l to D5.  127  5.6 Summary  TolC is the most similar homologue of OprM in E. coli. In addition, OprM and TolC are similar functionally and the two proteins share significant sequence similarity. In this chapter, circular dichroism spectroscopy of OprM indicated that OprM has a similar content of a-helical and p-sheet structure as TolC.  A three-dimensional model of OprM was  therefore constructed by threading its sequence to the recently released TolC crystal structure.  Residues thought to be important for the TolC structure (e.g. prolines) were  conserved in space in this OprM model. Five deletion mutants were created in OprM and the overall results with these deletion mutants agreed with those of the previously isolated insertion mutants. Analyses of the deletion and insertion mutants of OprM in the context of this model indicated that the helical barrel was critical for both function and integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel was dispensable. Extracellular loops appeared to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appeared to be a correlation between changes in antimicrobial activity for OprM mutants and pore size.  128  DISCUSSION  General  OprM is an outer membrane component of the Resistance-Nodulation-Division (RND)  systems associated  mechanism.  with multiple-antimicrobial resistance  through an efflux  Phylogenetic analysis of the recently completed P. aeruginosa genome  suggested RND efflux systems are very important for the organism. The influence of oprM over-expression alone on multiple-antibiotic resistance and growth of cells was examined here. In vitro functional studies combined with planar lipid bilayer analysis demonstrated the channel-forming activities of OprM. Although OprM did not show very high homology to either general porins or substrate-specific porins, a prediction for a membrane topology model of OprM was attempted in this study based on its multiple-sequence alignment with several highly homologous, efflux-associated outer membrane proteins and other structure prediction methods. Insertion mutagenesis with the malarial epitope (NANP) was used to generate useful information for the topology. A three-dimensional molecular architecture for OprM was later constructed, based on the crystal structure of TolC, the most similar homologue of OprM in E. coli. Results from deletion mutagenesis of oprM, combined with insertion mutagenesis data in the context of this three-dimensional model of OprM, provided insights into structure-function relationships of OprM that could eventually contribute to the development of inhibitory methods for these efflux systems.  129  Difference at the C-termini of OprM and OprM*  The last 22 amino acids at the carboxyl-terminal end of native OprM were different from those of the sequence previously published (termed OprM*), due to substitution with 14 residues from the phagemid used to clone the gene. This variation apparently did not affect the expression and function of the proteins. Both oprM sequences were expressed similarly and restored resistance to various antimicrobial agents without any noticeable difference in P. aeruginosa strains. In addition, the C-terminus of native OprM contained a large stretch of hydrophilic residues and is presumably located in the periplasm, where variations are usually tolerable. Therefore, the results obtained in this study should not have been affected by the presence of the native or the incorrect OprM C-terminal sequence.  Overexpression of oprM  Using the vector pVLT31, oprM was overexpressed in E. coli and P. aeruginosa by induction with IPTG. However, excessive production of OprM seemed to be harmful to the host, as revealed by the decline of cell densities in growth studies. It is possible that excess OprM perturbed the outer membrane and led to cell lysis, as observed in the case of overexpression of a mutant OmpA precursor protein in E. coli (Freudl et al, 1985). In this study, it was also observed that only when P-mercaptoethanol was included did the 100 kDa band of OprM shift to the 50 kDa monomelic form, and this could occur even with solubilization at room temperature or 37°C.  Many porins exist as oligomers in the outer  membrane (Koebnik et al, 2000), OprM probably exists as an oligomer in its native form.  130  Overproduction of OprM could have overwhelmed the ability of the cells to correctly form the oligomer, or most oligomers formed might have been less SDS stable and were reduced to the monomelic forms by P-mercaptoethanol even at low temperatures.  Influence of OprM on multiple-antimicrobial resistance  Overproduction of OprM in the two P. aeruginosa OprM-deficient strains (K613 and OCR03T) led to complementation of their mutations, but only brought the resistance levels for multiple antibiotics to the same levels as their corresponding parents.  In addition,  overproducing OprM alone from the cloned gene in wild-type P. aeruginosa PAO strain HI03 did not increase the MICs for any of the antibiotics tested. This indicated that OprM cannot function independently as an antibiotic efflux channel. In strains K613 and OCR03T, only the most distal gene, oprM, of the operon was interrupted and mexA and mexB could still be expressed. Thus, OprM produced from pKPM-2 could function with these MexA and MexB molecules to complement the OprM-deficiency.  The excess molecules of OprM  produced in these pKPM-2-containing strains might not be able to function properly, since there would be too little MexA and MexB available to reconstruct additional complete efflux systems (assuming that efflux systems involved stoichiometric amounts of the three components). Consistent with this view, there are small amounts of MexA, MexB, and OprM produced in the wild-type PAO strain HI03 which assemble into an efflux apparatus and contribute to intrinsic antibiotic resistance (Poole et al, 1993b).  The lack of influence of  OprM overexpression in strain HI03 is consistent with the explanation that extra copies of OprM expressed from pKPM-2 would presumably not have any MexA and MexB molecules  131  available to form additional efflux complexes.  This would explain why, in this genetic  background, there was no significant change in antibiotic susceptibility.  The data did not  provide concrete proof that OprM required MexA and MexB to function properly. However, these results indicated that OprM cannot function independently. It was subsequently shown that the cytoplasmic membrane-associated proteins are involved in substrate selectivity, but an outer membrane component was indispensable for extrusion of antimicrobials (Srikumar, et al., 1997).  In addition, the fact that efflux systems with chimeric outer membrane  components were less effective (Srikumar, et al., 1997) suggested that these outer membrane components, including OprM, more effectively associate with their native pump and linker proteins.  Analysis of M e x A - M e x B - O p r M homologues from the P. aeruginosa genome  The 6.3 million base-pair genome sequence of P. aeruginosa was recently completed (Stover et al., 2000). Analysis of this complex genome sequence can reveal a lot of useful information for the better understanding of the organism, such as what might contribute to its successful adaptation in diverse environments and its intrinsic resistance to many front-line antimicrobial agents. Only four RND multidrug efflux systems had previously been reported in P. aeruginosa (Westbrock-Wadman et al., 1999; Nikaido, 1998a), but it was determined that the genome encoded a larger number of putative RND efflux systems that were scattered around the genome.  It was observed that all of these putative RND efflux systems contain  genes encoding both putative pump protein and linker protein.  However, not all of the  putative RND loci contain genes for outer membrane components of the OprM family.  132  Overall, these observations suggest that active efflux is likely a very important mechanism for multiple-antimicrobial resistance in P. aeruginosa and the outer membrane component might be recruited to form functional extrusion complexes for some systems. As in the case of the E. coli protein TolC, OprM was shown to be recruited to form functional chimeric complexes with linker and pump efflux components from some other RND systems, with the substrate specificity being associated with the latter two components (Mine et al., 1999; Srikumar et al, 1997; Yoneyama et al, 1998). Phylogenetic analyses were also performed with homologues of MexA/MexB/OprM identified from the genome. The two clusters of homologues in the OprM family (one with members homologous to those involved in efflux while the other cluster contains members homologous to outer membrane proteins involved in secretion) might reflect some functional variations.  Similar clustering could be observed from the phylogenetic analysis for the  MexA family but it was less noticeable in the MexB family. Interestingly, comparison of the efflux components from the four P. aeruginosa RND systems already described in the literature indicated that the pump proteins are more conserved than the linker proteins and the outer membrane components.  Consistent with this was the observation here of more  variation within the OprM and MexA families than in the MexB family. These suggested that P. aeruginosa did not acquire or duplicate all the genes in an operon as a package.  Prediction of an O p r M membrane topology model  OprM had been predicted to function like a porin (Ma et al, 1994; Nikaido, 1994). Results in this study, from planar lipid bilayer analysis with purified OprM oligomers,  133  showed that OprM had channel-forming activities and suggests that the protein might indeed function as a channel in the outer membrane, like porins.  Furthermore, planar lipid bilayer  results using different salt solutions suggested OprM is probably water-filled and cation selective, and might be more efficient for the efflux of substrates with an overall positive charge. OprM and E. coli TolC are both outer membrane proteins involved in RND efflux systems.  Phylogenetically, OpmH, a homologue of OprM in the P. aeruginosa genome,  showed highest homology to TolC but OprM still demonstrated reasonable similarity to TolC. Functionally, TolC also reconstituted channel-forming activities in planar lipid bilayer experiments (Benz et al, 1993), and both proteins could form functional chimeric complexes with other linker and pump components for the extrusion of a wide spectrum of antimicrobial agents (Mine et al, 1999; Srikumar et al, 1997).  Studies with two-dimensional crystals  (purified proteins crystallized in two-dimensional lattices by reconstitution in phospholipid bilayers with detergents and buffers) showed that the structure of TolC was trimeric, similar to that of the non-specific, channel-forming porins (Koronakis, et al, 1997). Hence, it was originally proposed that both TolC (Koronakis et al, 1997) and OprM (Chapter 4) had a structure similar to that of the porins. The published crystal structures of the general and substrate-specific porins reveal certain consensus structures.  They usually form trimers of identical subunits, with each  monomer consisting of 16 or 18 anti-parallel p-strands forming a stable barrel. The strands are connected by short loops on the periplasmic face of the porin whereas the surface loops are in general longer and of variable length (Cowan et al, 1992; Koebnik et al, 2000). Similar structures are suggested for all of the general porins in the porin superfamily by analysis using sequence alignment and structure predictions (Jeanteur et al, 1991). OprM  134  accommodates a wide spectrum of antimicrobial agents and thus might be expected to be structurally similar to general porins.  However, OprM did not show any significant  homology to either the general or substrate-specific porins; although it was possible to identify conserved regions of amino acids based on its multiple-sequence alignment with various highly homologous outer membrane proteins that are also involved in efflux.  In  the absence of crystallographic data for OprM or any of the RND outer membrane efflux components at the time, a topology model of OprM was initially predicted (Fig. 20) based on the classic 16-stranded P-barrel motif observed in the crystal structures of porins (Cowan et al., 1992; Koebnik et al., 2000). The P-strands contained the most amphipathic stretches of amino acid residues identified from amphipathicity calculations and the majority of the conserved regions from multiple-sequence alignment of OprM with highly homologous proteins. Similar to the classic porin structures, most of the surface loops in the predicted OprM topology model were long. However, some of the predicted periplasmic loops were longer than those observed in traditional porin structures.  Furthermore there was no  consensus C-terminal P-strand as observed in many porins (Jeanteur et al., 1991 and 1994), and generally speaking the transmembrane p-strands were less amphipathic (viewed as alternating bars above and below the line in Fig. 19) than observed in other porins. Also, subsequent C D analysis was inconsistent with this P-barrel configuration.  Insertional mutagenesis of o p r M  Sequence comparison studies have revealed that the sequences in the surface loop regions of outer membrane proteins are hypervariable, and these regions are probably less  135  spatially constrained due to their surface location.  Therefore, the loop regions are more  likely to tolerate insertion of extra amino acids. To verify the accuracy of the first predicted OprM model, insertion mutagenesis with the malarial epitope (NANP) was employed. This approach had been successfully used before with P. aeruginosa porins OprF and OprP (Wong et al, 1995; Sukhan and Hancock, 1995), in which the epitope was permissive and antigenic when inserted into the putative loop regions but non-tolerated when inserted into the putative transmembrane regions.  Similarly, insertion mutagenesis of E. coli PhoE and  LamB has shown that insertion sites located in loop regions are permissive (Agterberg et al, 1990; Charbit etal, 1988). The predicted OprM topology model was revised slightly with data from insertion mutagenesis (Fig. 20).  Mutants of oprM with insertions of malarial epitope at sites ME1,  ME2, ME5, ME9, ME10, ME11 and ME13 were expressed in P. aeruginosa and the proteins still maintained their abilities to form oligomers that were heat- and P-mercaptoethanolmodifiable like the wild-type protein. These insertion sites seemed to be permissive for OprM and were placed in the surface loops.  It should be noted however that such an  interpretation depended on the validity of the proposed membrane topology model, which subsequently proved to be incorrect. For example, in the new model, insertion into a flexible periplasmic region would also be expected to be permissive. Moreover, there were variations in antimicrobial susceptibilities from cells carrying the pKWIN plasmids with these insertions. Although it has been shown that the cytoplasmic membrane-associated proteins (MexA and MexB) in this efflux system were involved in determining the substrate specificity including P-lactams (Srikumar et al, 1997), an outer membrane component was indispensable for efflux of antimicrobials. The observation that efflux systems with chimeric  136  outer membrane components were less efficient suggested that these outer membrane proteins may more readily associate with their native cytoplasmic membrane-associated components (Srikumar et al, 1997).  Conversely, the outer membrane components might  also have some influence on substrate passage. For instance, it was shown that OprM could contribute to resistance to certain agents in the absence of MexAB (Zhao et al, 1998), though it appears likely from subsequent information that other efflux components are involved. Consequently, the insertion of a long stretch of amino acids into these particular sites might have caused sufficient disruption in the protein conformation of the OprM mutants, such as changes in the size and charge distribution of the pore, to affect the passage of antibiotics, a conclusion that remained valid even when it was obvious that the original model was inaccurate. In the crystal structures of E. coli OmpF and LamB, loop 3 is folded entirely into the P-barrel to form the eyelet and some other loops also fold over the channel to different extents (Cowan et al, 1992; Schirmer, et al, 1995). Similarly, the loop regions in OprM might fold partly over the exterior opening of the channel to restrict the channel diameter at the surface, and to provide partial gating or some selectivity on substrate passage. P. aeruginosa cells carrying plasmids with insertions at 4 sites (ME3, ME6, ME8 and ME12) did not express the mutant forms of oprM and did not show any noticeable increase in resistance to the various antimicrobial agents tested.  These four insertion sites were  proposed to be located within P-strands of the 16-stranded p-barrel topology model, which subsequently proved to be incorrect. Insertion of the malarial epitope repeats into the Pstrands of traditional porins would disrupt the proper folding patterns of the proteins and render them more susceptible to proteolytic degradation in the periplasm (Misra et al., 1991; Wulfing and Pluckthun, 1994). However, in the three-dimensional model, these sites were  137  located in the coils of the helical barrel spanning the periplasm.  Insertions into the  periplasmic regions of classic porins would be permissive but these four insertions were not tolerated in the context of the three-dimensional model, suggesting the proper folding patterns of the RND efflux outer membrane components are different from those of the traditional porins. There were also differences observed in the expression of some oprM mutants ' between E. coli and P. aeruginosa carrying the same plasmids. OprM mutants with malarial epitope insertions at ME6, ME8 and M E 12 were expressed in an E. coli CE1248 background but not in P. aeruginosa OCR03T strain.  Initially, it was presumed that these mutant  proteins could be more readily degraded in P. aeruginosa by its different proteolytic enzymes and therefore these three OprM mutants were not recovered in this organism. However, the different structure and genetic context (presence of MexAB) would provide another explanation.  It was demonstrated that the content of P-sheet structure in the three-  dimensional model of OprM was much less than that in a traditional porin. Consistent with this, instead of the usual porin configuration of a trimer comprising three monomers of 16 to 18 P-strands each* the three-dimensional molecular model of an OprM homotrimer is proposed to consist of a 12 P-stranded barrel only, with 4 strands from each of the three monomers that are likely linked by disulfide bridges. This would make the protein less stable than classic porins and would also explain why OprM was shown to be modifiable by Pmercaptoethanol even at low temperatures (Chapter 1). In addition, since P. aeruginosa is the native host for the MexAB-OprM  and MexXY-OprM systems, OprM was probably  stabilized by the inner membrane complexes MexAB and MexXY in the P. aeruginosa background and possibly required interaction with these components to be stably expressed.  138  Thus some mutants expressed in E. coli but not P. aeruginosa may have not "fitted" with MexAB or MexXY and thus were degraded in the latter organism.  For the insertion at  ME13, which was also located within the helical barrel in the periplasm, the mutant protein was expressed and detected by an anti-OprM antibody in both E. coli and P. aeruginosa and this protein also reconstituted the function of wild-type OprM.  The complete tolerance of  this particular insertion was probably due to its proximity to the periplasmic end of the helical coils, which in TolC was suggested to be a more flexible region for controlling the passage of substrates but may also be unnecessary for functional reconstitution as indicated here.  A three-dimensional molecular model of O p r M  P. aeruginosa OprM and E. coli TolC are both outer membrane proteins involved in RND multidrug efflux systems, and are members of a large, phylogenetically-related set of efflux and secretion proteins (Saier, 2000). Their primary sequences share 21% identity and 40% similarity. The two proteins are also functionally similar (Wong and Hancock, 2000; Mine et al., 1999). Studies with two-dimensional crystals showed that the structure of TolC was trimeric, similar to that of the non-specific, channel-forming porins (Koronakis et al., 1997).  A P-barrel configuration was proposed (Koronakis et al,  1997).  Hence, it was  originally proposed that TolC and OprM shared a similar structure to the porins and a topology model of OprM (Wong and Hancock, 2000) was predicted based on the classic p~ barrel motif observed in the crystal structures of porins (Cowan et al, 1992; Koebnik et al., 2000). However, this was clearly inconsistent with the recently-released crystal structure of  139  TolC, which revealed a unique architecture (Koronakis et al, 2000), comprising a 12stranded P-barrel spanning the outer membrane bilayer and a helical barrel spanning the periplasm, in which each member of the trimer contributed strands used to build the resulting barrel structure. Consistent with the architecture of TolC, circular dichroism spectroscopy data showed that OprM consists of a mixture of a-helical and p structure.  OprM was thus  remodeled by threading its sequence into the published crystal structure of TolC (Fig. 28 & 29). In this model, only the regions that aligned with TolC were included, and thus the N and C termini that differ in length between the two proteins were excluded. The N-terminus of OprM is extended by more than 30 residues relative to TolC, a feature that exists in other members of the R N D efflux outer membrane proteins with higher sequence similarity to OprM. Deletion of 8 of the N-terminal residues in mutant D l or the insertion of 14 residues at M E 2 had no effect on the ability of the protein to reconstitute a functional R N D efflux system, while the insertion of 19 residues at ME1 was relatively benign, only slightly reducing protein expression and ability to reconstitute a functional efflux system. exclusion of this portion of OprM from the model was therefore justified.  The  In addition,  according to this proposed OprM model, the N-terminus would be in the periplasm, outside of the more constraining regions which shaped the TolC structure. Similarly, the C-terminus of TolC is extended when aligned with OprM. Interestingly, the TolC crystal model was obtained with a mutant deleted for the 43 amino acids at the C-terminus which were shown to be dispensable for the function of TolC (Koronakis et al, 2000). The C-terminus of OprM has also been shown to be non-essential and deletion and partial replacement of the last 22 residues (as in OprM*), removing a mixed a/p domain that was shown in the TolC structure  140  to form part of an equatorial band around the helical barrel segment, did not affect the function of OprM (Wong and Hancock, 2000). Therefore, the N and C-termini of both TolC and OprM might be considered structurally and functionally non-essential.  However, a  longer deletion of 70 residues at the C-terminus of OprM (DC70) that removed the complete helix H8 and part of helix H7 in addition to the apparently non-essential mixed a/p domain (helix H9 and strand S6) was not tolerated, suggesting the helical barrel might be important for OprM. Analysis of the new three-dimensional model of OprM indicated that it had similar specific features to TolC, with the proposed OprM p-strands as amphipathic as those in TolC, with predominantly hydrophobic residues in the P-barrel facing towards the hydrophobic core of the bilayer.  Also, a ring of aromatic residues (phenylalanine) at the lipid-water  interface between the outer membrane P-barrel and the periplasmic a-helical barrel were clearly evident. Such rings of aromatic residues are found at predominantly the periplasmic side of all outer membrane P-barrels examined to date (Cowan et al, 1992; Koebnik et al., 2000) and may act to stabilize the barrel. In addition, in this model, proline residues form a notable ring between the P-barrel and the a-helical barrel, consistent with the needed promotion of turns in this region to handle the transition from P-strands into a-helices. Analyses of the OprM deletion mutants and previously obtained insertion mutants, in the context of this model, provided some insights into the functioning of proposed structure of OprM.  Apparently, the external loops of the P-barrel (regions between SI and S2 and  between S4 and S5) and the mixed a/p structure at the equatorial domain of the helical barrel (region between H4 and H6) are flexible, since the insertion of 10 and 18 residues into these external loops (at ME5 and ME11 respectively) were almost fully or partially tolerated, and 141  had only a modest effect on the single channel conductance of the protein. The insertion at ME5 decreased the resistance of the cells to only one of the tested P-lactams, the bulky anionic compound cefsulodin, whereas the ability of ME11 to reconstitute resistance was reduced resulting from its reduced expression.  Presumably the extension of the external  loops in ME5 and ME11 might permit them to fold over and partially block the outer membrane channel consistent with the observed small decrease in channel size. The lack of an apparent significant role of these external loops in the function of OprM contrasts with that for other outer membrane protein porins which are noted for constricting their P-barrel channels by a long external loop (usually loop 3) that folds into the channel (Cowan et al, 1992; Koebnik et al, 2000). No such external loop is observed in the OprM model or in TolC. However, the OprM surface-exposed loops still apparently contribute to some control of the passage of substrates, according to the functional analysis of the ME5 and M E 11 loop mutants in this study. It has been suggested the equatorial domain is a possible recognition site for the recruitment of TolC by the inner membrane translocase, or that it might be involved in stabilization of the TolC structure (Koronakis et al, 2000). Results from this study appear to indicate that at least part of this domain is unnecessary and that the other part has some structural versatility. The deletion/replacement in the entire C-terminal region contributing to a proposed mixed a/p structure (H9 + S6, i.e. C-terminus of OprM*, Fig. 27) at the equatorial region had no influence on expression or function. Two insertions (at ME9 and M E 10) were within the other proposed mixed a/p region (S3 + H5) at the equatorial domain of the helical barrel.  The insertion mutant ME9 was fully tolerated while the insertion  mutant ME10 was partially tolerated, with good levels of expression but only partial  142  restoration of resistance to various antimicrobial agents tested. The results with the insertion at ME10 could be explained if the portion of the protein (the flexible domain between S3 and H5) is involved in interaction between OprM and the linker protein MexB, as suggested for the equatorial domain in TolC (Koronakis et al, 2000). The two large periplasmic loops predicted in the MexB topology model may be involved in this interaction (Guan et al, 1999). A l l of the 17 to 19 amino acid insertions that prevented expression of OprM (ME3 in H2; M E 6 and M E 8 in H3; ME12 in H7) were located at sites within the a-helical barrel of the proposed OprM model. Notably, three new OprM deletion mutants that were positioned within the helical barrel were well expressed, but either failed to restore resistance (deletion D4 removing 8 amino acids of H7), or partially restored resistance to a few antimicrobial agents (deletion D3 of 4 amino acids straddling insertion site ME12 of H7 and deletion D5 removing 8 amino acids of H8), when compared with cells expressing wild-type oprM. A deletion of the last 70 amino acids at the C-terminus of OprM (DC70) was also performed due to the very high homology of this region with the 64 residue-long P. putida TodU sequence identified from B L A S T search at the time. TodU was later discovered to be the Cterminus of a larger ORF encoding SepC of the SepABC system that is homologous to MexAB-OprM and this explained the high percent identity.  The 70-residue deletion,  removing H8 and H9 and S6 in the new OprM model, abolished the proper localization and function of the protein. A l l these data indicated that the a-helical barrel core was important for the proper expression and function of OprM. The upper portions of (3-barrels close to the cell surface are usually less constrained than the rest of the barrel, as indicated by their comparatively higher atomic displacement  143  parameters in X-ray crystallography (i.e. an element gives signals at different locations relative to certain reference point) that suggest conformational mobility.  This is probably  the reason for the tolerance of deletion D2, which removed the upper part of [3-strand SI. In addition, this deletion fortuitously permitted a continuity of amphipathic sequence by probably drawing part of the following surface loop into the P-barrel structure and shortening the length of the loop over the pore. Results from the planar lipid bilayer experiments of the deletion mutants and the two mutants with insertions at the external loops agreed with results from the antimicrobial susceptibility assays.  The mutant proteins containing changes that were well tolerated and  did not affect the resistance of the cells showed no or only slight changes in the single channel conductance when compared with wild-type OprM. On the other hand, the mutants that led to decreased resistance of the cells to antimicrobial agents also showed a large decrease in single channel conductance. This indicates that the permeability of the channel is important and changes in the protein that affect its channel size also affect the proper functioning of the efflux complex. In general, the three-dimensional model of OprM based on that of TolC appears to be plausible and trends in the locations of certain residues, such as proline and phenylalanine residues, support the model. This OprM model, like TolC, also contains several structural features that are consistent to those identified by Johnson and Church (1999) for these OMPs. These features include a periplasmic domain and two tandem repeats in the protein sequence. In addition, the model is in accordance with the results of L i and Poole (2001).  These  authors also constructed a series of insertion and deletion mutants of OprM and assessed their ability to reconstitute functional efflux pathways in P. aeruginosa. The superimposition of  144  these results on an alignment of OprM and TolC was consistent with, and adds further support for, the three-dimensional OprM model constructed here. . Such three-dimensional modeling for OprM could also be performed for other related members of the extended OprM-TolC family identified in the genome sequence of P. aeruginosa. The insertion and deletion data obtained in this study have provided some insights into structure-function relationships of OprM. Further mutagenesis studies, guided by the current model would help to refine the structure and determine amino acid residues that are essential for its proper function or for its interaction with the other components.  Antibiotic passage through the M e x A B - O p r M efflux system  The  proton  motive  force-driven pump  protein  (e.g. MexB)  contains  12  transmembrane segments (TMS), with two large hydrophilic periplasmic loops (between TMS 1 and 2; between T M S 7 and 8) that are presumably for interaction with the linker protein (MexA) (Guan et al., 1999). As almost all substrates for RND systems are lipophilic or amphiphilic, though structurally unrelated and with different charges, it was proposed that they insert at least partially into the lipid bilayer through their hydrophobic domains and are then captured by the transporters, as in the case of some A B C transporters (Nikaido, 1996; Van Veen et al, 1998; Homolya et al, 1993; Ruetz and Gros, 1994). This would also explain the efflux of P-lactams whose targets are on the outer layer of the cytoplasmic membrane. The positive correlation obtained between the hydrophobicity of the substituent at the 6 position of penicillins (or position 7 for cephalosporins) and their ability to become substrates of the Salmonella typhimurium AcrAB pump, homologue of MexAB, was  145  consistent with the lipid bilayer insertion model (Nikaido et al, 1998). However, this model requires further investigation, which could be difficult with the presence of the outer membrane in Gram-negative bacteria. The linkers (e.g. MexA), largely located in the periplasm and anchored to the inner membrane through the linked lipids at the N-terminus, were proposed to link the pumps and outer membrane components through interaction with the two hydrophobic domains identified near the N - and C-termini of these linker proteins. These linkers are long enough to span the periplasm, with a large a-helical region in the centre predicted to form a coiledcoil that could fold back to form an a-helical hairpin (Johnson and Church, 1999). AcrA, a linker protein in E. coli, was proposed to either simply block the efflux channel by interacting with the O M P and the pump; or to simply bring the two membranes into close proximity by folding back on itself to form the hairpin (Zgurskaya and Nikaido, 2000b). However, the TolC crystal structure reveals the a-helical portion that can span the whole periplasm, it does not seem necessary for the close association of the two membranes in order to bring the pump and OMP close to each other.  The fact that the linkers are not  interchangeable (Yoneyama et al., 1998) suggested that they interact specifically with their corresponding pump proteins and these complexes are stable, as indicated in cross-linking experiments (Zgurskaya and Nikaido, 2000a). The TolC crystal structure obtained by Koronakis et al. (2000) indicates that these outer membrane proteins involved in export and efflux are designed to provide a very efficient mechanism for the direct extrusion of substrates across two membranes and the periplasmic space, and these principles would appear to also be true for OprM. It has been hypothesized that the helical barrel in TolC is coiled in such a way as to form an iris  146  diaphragm at the periplasmic end that controls substrate passage, by dilating under appropriate conditions to permit the extrusion of substrates (Koronakis et al., 2000). These conditions probably include the recruitment of TolC by the AcrA:AcrB linkenpump complex which has been activated by engagement with substrate, and possibly energy from the proton-motive force. This interaction is probably cooperative since chemical cross-linking has demonstrated that the linker protein AcrA of E. coli forms oligomers, probably trimers, as does TolC (Zgurskaya and Nikaido, 2000a). These postulations could also be logically applied for OprM. As OprM was also shown to be able to form functional chimeric efflux complexes with cytoplasmic membrane-associated components of other systems (Mine et al, 1999; Srikumar et al, linkenpump complex.  1997; Yoneyama et al,  1998), it is probably recruited by the  However, OprM appears to more readily associate with its native  efflux complex MexAB, probably through more specific and stable interaction with the cytoplasmic membrane complex at the periplasmic end or even the upper equatorial domain of OprM.  This recruitment is likely activated by the linkenpump complex after substrate  engagement, providing the substrate specificity for the system.  This would be consistent  with the observations that substrate specificity was associated with the inner-membrane components of these RND efflux systems (Srikumar et al, 1997), though the mechanism of substrate recognition remains unclear. Other than being lipophilic or amphiphilic, additional properties of the substrates could also be important for the recognition by different inner membrane complexes.  The OprM  channel would then be triggered  to undergo  conformational changes, probably by movement of the helical coils, to allow a continuous passage of the substrate to the external environment. No long external loop is observed in the OprM model or in TolC.  However, the OprM surface-exposed loops apparently  147  contribute to some control of substrate passage, according to the results from the ME5 and ME11 loop mutants in this study.  Insertion of extra residues in the surface loops might  permit them to fold over and partially block the outer membrane channel, which was consistent with the observed small decrease in channel size.  However, in native OprM  without the insertions, these loops probably do not have a major role. The single channel conductance measured in planar lipid bilayer experiments for native OprM was 0.85 nS, i.e. larger than that of the E.coli porin OmpF monomer (0.6 nS, i.e. one third of the trimer conductance; Benz et al., 1978), and this presumably represents the open configuration of OprM. In contrast, TolC had a single channel conductance of only 0.08 nS and could be in the closed configuration. The planar bilayer data also indicated that OprM underwent rapid switching from open to closed states and exhibited several substates (smaller channel sizes), whereas aged OprM stored at 4°C for a month demonstrated channels of 0.08 nS. These data are then consistent with a dynamic channel in which the permeability is controlled by the helical diaphragm.  The large channel size of OprM (0.85 nS) is  consistent with the established ability of this protein to efflux a wide spectrum of antimicrobial agents, some of which are very bulky. A proposed mechanism of action for the RND efflux systems is shown in Fig. 34.  Design of inhibitory methods  The sequence similarity between TolC and OprM, which has family members that are homologous to many bacterial efflux pumps for the transport of proteins, antibiotics and other molecules, suggests they probably have conserved structural features and similar  148  closed OMP  O  IM complex  Figure 34.  <y substrate  Proposed mechanism of action for RND efflux systems.  An outer membrane protein (OMP) for the efflux system is shown on the left, with its p-barrel portion embedded in the outer membrane (OM) and the a-helical barrel closed at the bottom. This closed OMP could be free in the O M or in close proximity with weak association to the inner membrane (IM) complex. The IM complex contains the pump protein and the linker protein (LP) which is anchored to the IM through its lipophilic N terminus. The LPs are proposed to form trimers and associate with the pump protein stably. The IM complex probably recruits an OMP after capturing a substrate, thus providing the substrate specificity. The equatorial domain of the OMP and the large loops of the pump proteins were predicted for the interaction between the two components. The OMP is presumably triggered to open for the direct extrusion of the substrate to the outside by the proton motive force-driven pump. The surface loops of the OMP P-barrel could have some effect on substrate passage as well. The pump might capture substrates such as lipophilic Plactams from within the inner membrane.  149  mechanisms of action. Similar modeling could be performed for other related members of the extended OprM-TolC  family to identify these conserved regions in their three-  dimensional molecular architectures.  Further targeted mutagenesis studies, guided by the  current three-dimensional model, would help to refine the structure and to pinpoint the domains or residues that are essential for the function of these outer membrane proteins or for their interaction with the other components. This information could eventually contribute to the development of inhibitory methods for these efflux systems, such as designing inhibitors to target the essential domains or residues identified among the outer membrane components. For instance, as the decrease in pore size appeared to reduce the antimicrobial activity of OprM mutants, inhibitors could be designed to partially or completely block the channel. Since an open stage of the channels was apparently important for the function of the outer membrane components, and the channels appeared not to function independently, this suggested that active efflux systems require the recruitment of the outer membrane proteins to the inner membrane complexes followed by the opening of the channels. Since the outer membrane components were able to form chimeric efflux systems with non-native inner membrane components, there could be important domains that allow the more general interaction of these outer membrane channels with different inner membrane complexes. Inhibitors could be designed to target these regions to prevent the recruitment of outer membrane channels and thus prevent the formation of functional efflux systems. Similarly, inhibitors could be designed to prevent the formation of inner membrane complexes, but this may require more specificity due to the higher stability of these transporter:linker complexes. On the other hand, broad-spectrum efflux pump inhibitors could be identified by a different approach - the screening of natural and synthetic compounds for inhibition of various RND  150  efflux systems.  A compound called MC-207,110, discovered by screening of a small  molecule library, has been recently shown to inhibit multiple RND systems from P. aeruginosa (Lomovskaya et al, 2001), but substantial efforts are needed before it can be applied clinically. Understanding the mode of action of this compound against the efflux systems, such as how and where it interacts with the systems, will also help in the development and improvement of efflux pump inhibitors.  151  REFERENCES  Agterberg, M . , H. Adriaanse, H. Lankhof, R. Meloen, and J. Tommassen. 1990. Outer membrane PhoE protein of Escherichia coli K-12 as an exposure vector: possibilities and limitations. Gene. 88:37-45. Aires, J. R., T. Kohler, H. Nikaido, and P. Plesiat. 1999. 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