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Two novel outer membrane proteins involved in intrinsic aminoglycoside resistence in Pseudomonas aeruginosa Jo, James T. H. 2002-10-05

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Two novel outer membrane proteins involved in intrinsic aminoglycoside resistance in Pseudomonas aeruginosa by James T. H. Jo B. Sc. (High Honours) in Biology, 1999. University of Regina, Regina, Saskatchewan A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (MICROBIOLOGY PROGRAM) We accept this thesis as conforming to the required standard UNIVERSITY OF BRITISH COLUMBIA June 2002 © James T. H. Jo, 2002 UBC Rare Books and Special Collections - Thesis Authorisation Form Page 1 of 1 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 of IAICTO\>(.OIQ^ T^iv^u-tno/c^ The University of British Columbia Vancouver, Canada Date 02/12/2002 ABSTRACT The expression of tripartite multi-drug efflux pumps such as MexA-MexB-OprM in Pseudomonas aeruginosa contributes to intrinsic resistance to a wide variety of antimicrobials, including (3-lactams, chloramphenicol, macrolides, quinolones, and tetracycline. MexX-MexY are the only linker and pump efflux system components in P. aeruginosa that have been shown to confer intrinsic resistance to aminoglycosides. While a number of studies suggest that OprM, the main efflux outer membrane protein (OMP), forms a functional channel with the MexX-MexY proteins, other data suggests that another OMP is the native channel for the MexX-MexY efflux system. Fifteen functionally uncharacterized OprM-homologues identified in the recently-sequenced genome of P. aeruginosa were possible candidates for the role of the native outer membrane channel for MexX-MexY. Insertional inactivation of OpmG resulted in an 8-fold decrease in MIC to streptomycin, kanamycin, and gentamicin, while inactivation of OpmH resulted in 4- to 8-fold decreases in MIC to kanamycin and streptomycin. When reintroduced into P. aeruginosa on multicopy plasmids, both OpmG and OpmH were able to complement the susceptibility of their respective mutants. Changes in MIC due to pseudo-reversion through compensatory mutations were not a factor, as demonstrated by mini-microarray hybridization analysis of the OprM-homologues. This study demonstrates that the two novel outer membrane proteins OpmG and Opmh play a role in aminoglycoside resistance, and that OpmG is likely the main aminoglycoside efflux channel. n ABSTRACT ii TABLE OF CONTENTS hLIST OF FIGURES vi LIST OF TABLES viLIST OF ABBREVIATIONS viii ACKNOWLEDGEMENTS x 1. INTRODUCTION 1.1. Pseudomonas aeruginosa 1 1.2. Multi-Drug Efflux systems of Gram Negative Bacteria 2 1.3. RND-type Transport Systems 4 1.4. Aminoglycoside Uptake and Efflux 11 1.5. MexX-MexY and Intrinsic Aminoglycoside Resistance 13 1.6. Outer Membrane Proteins of Pseudomonas aeruginosa 14 1.7. Aims of this Study 16 2. METHODS AND MATERIALS 2.1. Strains, Plasmids, and Growth Conditions 18 2.2. General Techniques 12.3. PCR 22 2.4. DNA Sequencing 22.5. Transfer of DNA into E. coli and P. aeruginosa 22 2.6. Determining Minimal Inhibitory Concentration 25 2.7. Mini-microarrays 26 iii 2.7.1. Micro-array Construction 26 2.7.2. RNA Isolation 27 2.7.3. Reverse Transcription 8 2.7.4. Radiolabelling DNA Probe and Hybridization 29 2.7.5. Autoradiographic Imaging 30 3. RESULTS: OprM-family outer membrane proteins from the P. aeruginosa genome 3.1. Introduction 31 3.2. Homology of Multi-Drug Efflux Pumps of Pseudomonas aeruginosa 31 3.3. The OprM Family of Outer Membrane Proteins 43 3.4. Conclusions 48 4. RESULTS: OpmG- and OpmH-mediated intrinsic aminoglycoside resistance 4.1. Introduction 49 4.2. Screening miniTn5 insertion mutants 44.3. Complementation of the opmG, opmH, and oprM mutants 51 4.3.1. Complementation of an oprM mutant susceptibility phenotype 54 4.3.2. Complementation of opmG and opmH mutant susceptibility phenotypes.56 4.4. Use of DNA mini-microarrays to assess compensation 58 4.5. Conclusions 60 5. DISCUSSION 5.1. Introduction 66 5.2. Phylogenetic analysis of OprM homologues 67 5.3. Complementation of an OprM" defect by OpmG and OpmH 68 5.4. Complementation of OpmG" and OpmH" defects by OpmG and OpmH 69 iv 5.5. Mini-microarray analysis 69 5.6. Summary 71 6. REFERENCES 3 v LIST OF FIGURES Figure 1. Model of the structure of the tripartite Gram negative RND efflux system 6 Figure 2. Topological model of an inner membrane RND pump 7 Figure 3. Phylogenetic analysis of the 18-member OprM family 15 Figure 4. Gene organization of the OprM homologues 35 Figure 5. An alignment of the amino acid sequences of OprM and its seventeen homologues 4 Figure 6. Comparison of expression of OprM homologue genes in wildtype PAK and the opmG mutant using mini-microarrays 61 Figure 7. Comparison of expression of OprM homologue genes in wildtype PAK and the opmH mutant using mini-microarrays 63 vi LIST OF TABLES Table I. Bacterial strains used in this study 20 Table II. Plasmids used in this study 21 Table III. Oligonucleotide primers used for the amplification of oprM homologues 23 Table IV. Homology of the OprM homologues 32 Table V. Efflux genes of the MexA-MexB-OprM homologues 41 Table VI. Compounds tested in initial MIC screen of miniTn5 insertion mutants 52 Table VII. MICs to aminoglycosides, carbenicillin and tetracycline of P. aeruginosa mutants lacking selected outer membrane channel proteins 53 Table VIII. Complementation of an OprM" mutant with OpmG and OpmH 55 Table IX. Complementation of OpmG" and OpmH" mutants with OpmG and OpmH....57 Table X. Quantitated spot densitometry values for strains H911 and H958 62 Table XL Quantitated spot densitometry values for strains H911 and H966 64 vii LIST OF ABBREVIATIONS ABC ATP-binding cassette Acr acriflavin ApR ampicillin-resistant bp base pair Cb carbenicillin CCCP carbonyl cyanide m-chlorophenyl hydrazone Cm chloramphenicol Clin clindamycin Ctax ceftriaxone Ctzd ceftazidime CV crystal violet DEPC diethylpyrocarbonate Ery erythromycin Fus fusidic acid Gm gentamicin HP hypothetical protein Imi imipenem Km kanamycin LB Luria broth LBLS Luria broth (low salt) LBNS Luria broth (normal salt) MDR multi-drug resistant Mer meropenem MF(S) major facilitator (superfamily) MIC minimal inhibitory concentration Nai nalidixic acid Nor norfloxacin OD optical density OMP outer membrane protein Vlll Opm outer membrane protein of the OprM family ORF open reading frame PCR polymerase chain reaction Pmb polymixin B RND resistance/nodulation/cell division RT reverse transcription Sm streptomycin SDS sodium dodecyl sulfate ssc sodium acetate and sodium chloride Tc tetracycline TcR tetracycline-resistant Tm tobramycin TMD transmembrane domain UV ultraviolet IX ACKNOWLEDGEMENTS I would to thank my supervisor Dr. Robert E. W. Hancock for allowing me the opportunity to work with him and for his guidance and support during my studies. I would also like to thank my supervisory committee, Drs. Lindsay Eltis and Rachel Fernandez for their time and advice. Special thanks goes out to the past and present members of the Hancock lab for both technical advice and friendship. The financial support of the Canadian Cystic Fibrosis Foundation was greatly appreciated. Last but not least, I must thank my family and friends for helping me maintain a thin veil of sanity over the past three years. x INTRODUCTION 1.1 Pseudomonas aeruginosa Pseudomonas aeruginosa is a common Gram negative bacterium, found ubiquitously in the environment and often in association with plants and animals (Beinlich et al, 2001). In healthy humans, P. aeruginosa presents no significant threat to an individual's health; however, in immunocompromised persons, P. aeruginosa has been known to cause severe infections (Hancock and Speert, 2000). Almost all patients with Cystic Fibrosis (CF), in whom P. aeruginosa often colonizes the thick mucous layer in the lungs, eventually suffer from chronic respiratory tract infections that end in death (Hancock and Lam, 1998; Hancock and Speert, 2000). It is also a common pathogen of patients with burn wounds or those who have received immunosuppressive drugs for surgery (Hancock and Lam, 1998). Indeed, P. aeruginosa is responsible for about 10% of all nosocomial (hospital-acquired) infections (Hancock and Speert, 2000). P. aeruginosa possesses several virulence factors that aid in colonizing the human host, including mucous production, exotoxins (such as exotoxin A), extracellular proteases, extracellular lipases, endotoxin, and antibiotic resistance determinants (Hancock and Lam, 1998). Antibiotic resistance in particular is a major obstacle for treatment of P. aeruginosa infections. This bacterium has been shown to have high-level intrinsic resistance to nearly all major classes of antibiotics, including quinolones, aminoglycosides, macrolides, (3-lactams, tetracyclines, and chloramphenicol (Masuda et al., 2000b). Antibiotic-modifying enzymes, such as an inducible chromosomally-encoded periplasmic (3-lactamase, contribute to this resistance (Hancock and Woodruff, 1998; Nakae et al., 1 1999; Ciofu et al, 2000). However, the broad-spectrum resistance seen in this organism is conferred largely by a combination of low outer membrane permeability and multi drug efflux systems (Nikaido, 1996). The multi-drug efflux systems of P. aeruginosa, and indeed of many Gram negative bacteria, are noted for their ability to transport several structurally-unrelated compounds as substrates for efflux. 1.2 Multi-Drug Efflux Systems of Gram Negative Bacteria It was originally proposed that the intrinsic resistance to antibiotics seen in P. aeruginosa and other Gram negative bacteria was solely a result of low outer membrane permeability. However, a poorly permeable outer membrane cannot account for this resistance on its own, since internal drug concentrations would nevertheless reach equilibrium levels without the aid of another compensatory mechanism (Li et al, 1994; Nikaido, 1996). One such mechanism is the multi-drug efflux system, which functions to transport the antibiotic from the cell back into the extracellular environment. Thus, the low rate of drug influx due to an outer membrane of low permeability acts synergistically with active antibiotic export in maintaining a low intracellular drug concentration. Energy-dependent efflux in Gram negative bacteria uses a proton antiport system where the energy from protons diffusing across the cytoplasmic membrane down their concentration gradient is coupled to the active movement of the drug from the intracellular to the extracellular environment. Therefore, an energy uncoupler, such as carbonylcyanide m-chlorophenylhydrazone (CCCP), which dispels the energy gradient by shuttling protons back into the cell, serves to abolish efflux, leading to the accumulation of antibiotic inside the cell (Li et al, 1994; Nikaido, 1996; Poole et al, 1996;Kohlere/a/., 1997). 2 Escherichia coli possesses one constitutively expressed multi-drug efflux system, AcrA-AcrB-TolC, which was initially implicated in resistance to the dye acriflavin, and now known to play a role in resistance to dyes, detergents, erythromycin, and fusidic acid (Nikaido, 1996). The constitutively expressed MexA-MexB-OprM multi-drug efflux system of wild-type P. aeruginosa is chromosomal ly encoded as a three-gene operon, and accounts for the broad-spectrum resistance to many antibiotics including P-lactams (but not imipenem), chloramphenicol, macrolides, quinolones, and tetracycline (Li et al., 1995; Nikaido, 1996, Masuda etal, 2000b). It is known that P. aeruginosa possesses at least two other multi-drug efflux systems, MexC-MexD-OprJ and MexE-MexF-OprN, both of which are also encoded chromosomally as three gene operons. Although neither of these systems account for intrinsic resistance in this organism since they are not normally expressed in wild-type P. aeruginosa. They can nevertheless account for mutational and acquired resistance. MexC-MexD-OprJ is negatively regulated by the NfxB repressor, and nfxB mutants show increases in resistance to fourth-generation cephalosporins, chloramphenicol, macrolides, quinolones, and tetracycline (Nikaido, 1996; Poole et al, 1996; Masuda et al., 2000b). MexC-MexD-OprJ was later shown to be inducible by subinhibitory concentrations of ethidium bromide, rhodamine 6G, and acriflavin (Morita et al, 2001). In contrast, MexE-MexF-OprN is positively regulated by the MexT protein, and nfxC mutants, which express MexE-MexF-OprN, are resistant to chloramphenicol, quinolones, and trimethoprim, as well as the carbapenem subclass of P-lactams, (Nikaido, 1996; Kohler et al., 1997) due to an incident downregulation of the carbapenem porin OprD (Ochs et al., 1999). In addition, mutations in the mexR gene, encoding the repressor for the mexA-3 mexB-oprM operon, can result in overexpression of the MexA-MexB-OprM proteins and confer the hyper-resistant nalB phenotype (Saito et al, 1999; Ziha-Zarifi et al, 1999; Srikumar et al, 2000). However, the mechanisms by which these additional pumps are regulated in vivo are not yet well understood. Interestingly, it has been shown that the inactivation of one efflux system can result in the upregulation of another. Indeed, mexA-mexB-oprM and oprM deletion mutants were found to upregulate expression of both MexC-MexD-OprJ and MexE-MexF-OprN (Li et al, 2000). Since the outer membrane components of an efflux system are interchangeable (Srikumar et al, 1997; Gotoh et al, 1998), and since many efflux systems have overlapping substrates specificities, this sort of compensation might mask phenotypic differences between mutants, particularly in terms of drug resistance and susceptibility. 1.3 RND-type Transport Systems Transport of compounds across the Gram negative double membrane is significantly more complex than across the Gram positive single membrane (Nikaido, 1996). Not only are there two biological barriers to be traversed, but the energy needed for transport is localized to the cytoplasmic membrane. The efflux pumps of Gram negative organisms overcome this difficulty by utilizing a multi-protein transport system that facilitates movement of the compound across both membranes in a single step (Nikaido, 1996; Zgurskaya and Nikaido, 2000b). E. coli AcrA-AcrB-TolC and the three P. aeruginosa systems Mex-A-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN, all belong to this class of transport systems, called the Resistance/Nodulation/Cell Division (RND)-4 family, so named for the original proteins placed in this group (for review see Saier Jr. et al, 1994), and whose members share a common tripartite structural make-up (Figure 1). The first and defining component of the RND-type transport system, designated AcrB in E. coli and MexB/MexD/MexF in P. aeruginosa, is the RND-pump. It resides in the inner membrane and acts as a proton antiporter, actively transporting compounds using the proton motive force. Analysis of the amino acid sequence suggests a characteristic transmembrane topology for these inner membrane transporters (Saier et al., 1994; Zgurskaya and Nikaido, 2000b). It was later experimentally confirmed (Gotoh et al, 1999; Guan et al, 1999) that this topology consisted of twelve transmembrane domains (TMD), where the first and fourth extracytoplasmic loops (between TMD's 1 and 2, and TMD's 7 and 8) are largely hydrophilic and extend into the periplasmic space (Figure 2). It is believed that these two extended loops may form the basis of the interaction between the inner membrane component of the system and the other components of the efflux system (Zgurskaya and Nikaido, 2000b). A second component (TolC and OprM/OprJ/OprN) is the outer membrane protein (OMP), and is the pore through which the drug finally exits the cell into the extracellular milieu. Porins play several roles in bacterial outer membranes, and the existence of a large number of characterized OMPs and uncharacterized homologues in P. aeruginosa is suggests that they collectively have many functions, such as the export of toxic compounds and virulence factors and the import of catabolites such as amino acids and alternative carbon sources (for a review, see Hancock and Brinkman 2002). Many porins present in the bacterial outer membrane are substrate-specific, such as FhuA of E coli, a gated porin that specifically mediates the transport of iron-siderophores, or are general 5 G G PH CD CS OH o o s 0) & o 03 o 03 l-i 03 o 60 g IS '3 . 00 3 03 z 9 2 S c o3 SH HO -a c 03 CN S o3 SH ,—I C5 P 03 -a c o o S ^ 03 O 03 60 ^ ° E o 2 O c/) 3 o E CN pj diffusion porins, like OmpF of E. coli, which are selective on the bases of size and charge. Multi-drug efflux pumps, by their nature, accommodate many, often structurally dissimilar, antimicrobial agents, and thus the OMP component must also accommodate a broad range of substrates. This includes compounds that are both hydrophobic and hydrophilic, are of many different sizes, including proteins in the case of TolC and other porins involved in type I secretion, which is .a substrate range that is greater than that of general diffusion porins but which is still able to distinguish those substrates from normal and necessary cellular constituents (Zgurskaya and Nikaido, 2000b). Although it has been suggested that the channel-forming efflux OMPs are gated and contain multiple substrate binding sites, so that engagement of the substrates and/or the inner membrane components of the pump opens the gate, allowing the substrate to pass through (Andersen et al, 2000; Koronakis et al, 2000), the mechanism of substrate selectivity is still unknown (Zgurskaya and Nikaido, 2000b). It is believed that the general mechanism involves the recognition of broad physical characteristics of the substrates, such as charge or hydrophobicity, instead of a recognition of structure (Paulsen et al, 1996). A third component of the tripartite RND efflux systems, is a linker protein, which in some manner facilitates a one-step transport event across both the inner and outer membranes of the Gram negative bacteria (Zgurskaya and Nikaido, 2000b). Linker proteins were once referred to as membrane fusion proteins, so named for sharing C-terminal sequence homology with a group of paramyxoviral fusion proteins (Zgurskaya and Nikaido, 2000b), and it was once thought that they fulfilled their role by fusing the inner and outer membranes of the bacterial cell envelope. Recent crystallization of the outer membrane protein TolC of E. coli (Koronakis et al, 2000) has forced researchers to 8 re-evaluate this hypothesis (Putman et al, 2000). It is now believed that the linker somehow facilitates interaction between inner and outer membrane components. Designated AcrA in E. coli and MexA, MexC, or MexE in the P. aeruginosa multi-drug efflux systems, the linker is largely periplasmic. A number of linkers contain a lipid modification consensus sequence in their N-terminal regions, followed by an invariant cysteine residue that marks the site of cleavage and modification for lipid attachment (Dinh et al, 1994). Experimental evidence has shown that both MexA of P. aeruginosa (Yoneyama et al, 2000) and a cell division linker protein, EnvC, of E. coli (Seiffer et al, 1993) are in fact lipoproteins each anchored in the inner membrane by their lipid components. Each of the three components has previously been shown to be necessary for efflux since the absence of any one of the three components of the efflux system abrogates efflux activity. It has been shown through cross-linking studies that the inner membrane RND-pump and the linker protein are closely associated in complexes (Zgurskaya and Nikaido, 2000a) and are non-interchangeable, such that MexA forms a functional complex only with MexB, but not with MexD, for example (Yoneyama et al, 1998). In contrast, engineering P. aeruginosa to express chimeric MexA-MexB-OprJ and MexC-MexD-OprM efflux pumps has demonstrated that outer membrane components are interchangeable (Srikumar et al, 1997; Gotoh et al, 1998). Since the MexA-MexB-OprJ chimera retains the substrate specificity of the native MexA-MexB-OprM pump, and similarly, MexC-MexD-OprM mediates the efflux of the same antibiotics as MexC-MexD-OprJ, it is in fact the inner membrane components that determine the substrate selectivity of the efflux systems (Srikumar et al, 1997; Gotoh et al, 1998). 9 The crystal structure of TolC was recently determined (Koronakis et al, 2000). It was originally thought that TolC and other efflux-related OMPs would have a similar structure to other bacterial outer membrane porins, such as OmpF of E. coli, in which the pore is formed from a multi-stranded P-barrel (Andersen et al, 2000; Andersen et al, 2001) and exists in the outer membrane as a homotrimer (Koebnik et al, 2000). Interestingly, the crystal structure of the trimeric TolC protein showed a single pore with an a-helical domain spanning the entire periplasm and a P-barrel domain in the outer membrane, whose entire structure was formed from three TolC monomers that each formed a third of the overall barrel structure (Koronakis et al, 2000). This new structure for TolC has provided some insight into the gating mechanism for these channel-forming OMPs. The periplasmic a-helical domain consists of three coiled-coil regions consisting of four helices each, an inner pair and an outer pair. The possible rotation of the inner pair of helices around the outer pair helices would serve to dilate and constrict the periplasmic end of the barrel (Koronakis et al, 2000). It is now believed that the direct interaction of inner and outer membrane components, most likely while the transporter is engaged by a specific substrate, triggers the movement of the helices and the dilation of the entrance of the barrel (Andersen et al, 2000; Koronakis et al, 2000). This new information has also called the role of the membrane fusion protein into question, since it is now apparent that a membrane fusion event is not necessary to facilitate one-step transport across two membranes. The current model suggests that the linker protein somehow promotes the interaction between the inner membrane transporter and the outer membrane pore but is not involved in any form of inner and outer membrane fusion thus making the term "membrane fusion protein" somewhat of a 10 misnomer (Andersen et al, 2000). The trimeric nature of the outer membrane components likely has a role in this interaction since it has been previously shown that the inner membrane translocases of these transport systems also act in groups of three (Thanabalu et al, 1998). 1.4 Aminoglycoside Uptake and Efflux Aminoglycosides are a class of polycationic, polysaccharide-based antibiotics that contain two or three amino sugars attached to a cyclitol ring (Davis, 1987). Aminoglycosides are routinely used to treat serious bacterial infections despite their toxicity (Davies and Wright, 1997). Since their discovery, there has been much controversy among researchers surrounding the mechanisms of uptake and action of aminoglycosides since they are incredibly pleiotropic in terms of their effects. Because aminoglycosides are cationic molecules, they compete with divalent cations such as magnesium for the polyanionic portions of the lipopolysaccharide (LPS) molecule on the outer leaflet of the Gram negative outer membrane (Mao et al, 2001), an observation supported by the well-characterized antagonism of aminoglycosides by salts (Hancock, 1981; Mingeot-LeClercq et al, 1999; Mao et al, 2001). Displacement of divalent cations by the much larger aminoglycoside molecule destabilizes and permeabilizes the outer membrane for aminoglycoside entry, a process known as self-promoted uptake (Mingeot-LeClercq et al, 1999; Mao et al, 2001). Aminoglycosides bypass the inner membrane in a two-step, energy-dependent manner characterized by an initial slow period of uptake (phase 1) followed by a rapid intracellular accumulation of the drug (phase 2) (Hancock, 1981; Mingeot-LeClercq et al, 1999). Early studies established that both functional electron transport and protein synthesis were absolutely 11 required for aminoglycoside uptake and action (Davies and Wright, 1997; Mingeot-LeClercq et al, 1999; Mao et al, 2001), which was supported by data that showed that energy uncouplers like carbonyl cyanide w-chlorophenyl hydrazone (CCCP) and protein synthesis inhibitors such as chloramphenicol were sufficient to prevent killing by aminoglycosides (Hancock, 1981; Davis, 1987). Since mutations to genes encoding ribosomal proteins such as rpsL result in high-level mutational resistance to some aminoglycosides, a ribosomal interaction most certainly plays a role (Moat and Foster, 1995; Davies and Wright, 1997; Mao et al, 2001), and there is strong evidence to suggest that membrane damage is also plays a role (Davies and Wright, 1997). Resistance to aminoglycosides takes many forms, many of which are various aminoglycoside-modifying enzymes. In fact, more than fifty enzymes have been shown to inactivate aminoglycosides according to three general modes of action: 1) ATP-dependent phosphorylation, 2) ATP-dependent adenylation, and 3) acetyl-CoA-dependent Af-acetylation (Davies and Wright, 1997; Mingeot-LeClercq et al, 1999). Although certain combinations of these enzymes can confer resistance to a variety of aminoglycosides, broad aminoglycoside resistance in the absence of these modifying enzymes has been noted in several clinical isolates (Westbrock-Wadman et al, 1999). The observation of broad-spectrum intrinsic aminoglycoside resistance in clinical isolates suggests the existence of an efflux pump capable of transporting aminoglycoside antibiotics. 1.5 MexX-MexY and Intrinsic Aminoglycoside Resistance Clinical isolates of P. aeruginosa are often resistant to aminoglycosides (Hancock and Speert, 2000). Curiously, despite empirical evidence of intrinsic aminoglycoside 12 resistance in this organism, none of the previously characterized efflux systems of P. aeruginosa, or of Gram negative bacteria at large, have been implicated in aminoglycoside efflux (Nikaido, 1996). Based on homology studies of the proposed aminoglycoside efflux pump and linker protein, AmrA and AmrB, identified in Burkholderia pseudomallei (Moore et al, 1999), an E. coli efflux pump protein, AcrD (Rosenberg, 2000), and a fourth multi-drug efflux system of P. aeruginosa, MexX-MexY, were recently identified and when overexpressed were shown to confer resistance to aminoglycosides (Aires et al, 1999; Mine et al, 1999; Westbrock-Wadman et al, 1999) as well as to many P-lactams, chloramphenicol, macrolides, quinolones, and tetracycline (Masuda et al, 2000b). The MexX-MexY system, like MexA-MexB-OprM, is expressed under normal laboratory conditions (Aires et al, 1999) and is responsible for conferring intrinsic aminoglycoside resistance in wildtype P. aeruginosa. Its expression is negatively regulated by the repressor MexZ, whose gene is encoded immediately upstream of mexXY. In addition, MexX-MexY was the first of the four characterized efflux systems of P. aeruginosa that has been shown to be induced by the presence of subinhibitory concentrations of some of its substrates, namely tetracycline, erythromycin, and gentamicin (Masuda et al, 2000a; Morita et al, 2001), although MexCD-OprJ was later identified as being inducible (Morita et al, 2001). Unlike the other three, previously-characterized, multi-drug efflux systems of P. aeruginosa, the three components that constitute the MexX-MexY pump are not encoded as a three-gene operon. Indeed, although mexX and mexY are co-transcribed, the outer membrane protein component is not contiguous with the two-gene mexXY operon, nor is there an outer membrane protein encoded immediately upstream or downstream of the 13 two genes (Westbrock-Wadman et al, 1999). This has led to some controversy as to the identity of the outer membrane component of MexX-MexY. While evidence showed that mexXY and oprM introduced on multicopy plasmids into E. coli (Mine et al, 1999) and P. aeruginosa (Aires et al, 1999; Masuda et al, 2000a) could constitute a working efflux pumps in both organisms, the observation that some broadly aminoglycoside resistant clinical isolates of P. aeruginosa have upregulated levels of MexX-MexY but downregulated levels of OprM (Westbrock-Wadman et al, 1999) suggests that the phenotype might result from overexpression of plasmid-borne genes and that the identity of the native OMP is still unknown. It is however not unlikely that OprM could function as the outer membrane component for more than one efflux system, as there is already a precedent for multi-functional outer membrane proteins. The well-studied outer membrane pore TolC of E. coli forms active transport complexes for Colicin V (CvaA-CvaB-TolC), hemolysin (HlyB-HlyD-TolC) (Postle et al, 2000) secretion, as well as with AcrA-AcrB for multi-drug efflux. 1.6 Outer Membrane Proteins of Pseudomonas aeruginosa A BLAST homology search of the recently completed Pseudomonas aeruginosa genome sequence revealed a total of seventeen homologues of OprM. A tree showing the eighteen members of the OprM family is shown in Figure 3. The proteins fall into two general clusters, those more closely similar to OprM and a second, looser cluster that comprises homologues of type I protein secretion and cation efflux pathways. The first cluster has eleven members and includes the three previously-characterized multi-drug efflux OMPs of P. aeruginosa, OprM, OprJ, and OprN. The second cluster contains seven members, including AprF, an OMP implicated in the transport of alkaline protease, 14 Figure 3. Phylogenetic analysis of the 18-member OprM family. The tree was constructed using the Neighbour-joining method from PHYLIP and Tree View. Uncharacterized OprM homologues are designated Opm, for probable outer membrane protein OprM family. (Drawn by Fiona Brinkman) OpmJ OpmK °PmL 15 OpmN, which is highly homologous to CzcC in Ralstonia eutropha, responsible for cadmium and zinc efflux, as well as the closest P. aeruginosa homologue of is. coli TolC, OpmH. Fourteen of these homologues, including OpmH and OpmN (CzcC), remain functionally uncharacterized. 1.7 Aims of this Study Despite the discovery of MexX-MexY in P. aeruginosa, the overall mechanism of intrinsic aminoglycoside resistance in this organism is still unclear. The identity of the outer membrane component for the MexX-MexY system is still under debate. Furthermore, it is not known whether outer membrane impermeability and active efflux by MexX-MexY are alone responsible for intrinsic resistance to aminoglycosides in P. aeruginosa (Mao et al, 2001). However, it is clear that impermeability-type resistance to aminoglycosides is a common feature in P. aeruginosa and active efflux is likely a major component (Mao et al, 2001). Therefore, in order to understand this phenomenon, it is essential that the identity of the OMP for MexX-MexY be determined. Given the large number of uncharacterized OprM homologues present in the genome sequence of P. aeruginosa, the goals of this thesis were (a) to screen knock-out mutants of all the uncharacterized OMP genes, using minimal inhibitory concentration (MIC) assays and identify candidate OMPs that may be responsible for mediating efflux of aminoglycosides; (b) to clone and express candidate OMPs and complement the aminoglycoside susceptibility of each knock-out mutant; and (c) to determine if compensatory upregulation of other OMPs in the OprM family in response to insertional inactivation of the OMP of interest played a role in determining the resistance phenotype in this organism. 16 METHODS AND MATERIALS 2.1 Strains, Plasmids, and Growth Conditions All strains used in this study are listed in Table I, and all plasmids used in this study are listed in Table II. Strains were grown at 37°C in Luria Broth (LB) medium {1.0% (w/v) tryptbne, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl for E. coli (LBNS) or 0.05% (w/v) NaCl for P. aeruginosa (LBLS)}, or on LB agar containing 2% (w/v) agar. All media components were obtained from Difco Laboratories (Detroit, MI, USA). Antibiotics were supplied at the following concentrations for plasmid maintenance in E. coli, ampicillin 100 p.g/ml; for plasmid maintenance in P. aeruginosa, carbenicillin 200 u.g/ml, tetracycline 100 ug/ml; for maintenance of insertion mutations in H956-H969, tetracycline 100 ug/ml; and for maintenance of the insertion mutation in K613, HgCl2 15 ug/ml. 2.2 General Techniques Protocols for general DNA techniques such as DNA isolation and agarose gel electrophoresis were found in Sambrook et al. (1989). DNA restriction and modifying enzymes were purchased from Invitrogen Life Technologies (Burlington, ON, Canada) or New England BioLabs (Mississauga, ON, Canada) and were used according to manufacturers' protocols. Plasmid DNA was isolated by alkaline lysis (Sambrook et al, 1987) or by using a QIAprep spin miniprep kit (Qiagen Inc., Chatsworth, California, USA), and PCR products were purified using a Qiaquick PCR purification kit (Qiagen Inc.). 17 Table I. Bacterial strains used in this study. Strain Description Reference/Source E. coli DH5a supEAA hsdRM recAl endAX gyrA96 thiA relAX Hanahan, 1983 A(/acZYA-argF)U 169 JeoR((|>80A/acZdM 15) P. aeruginosa H103 Wil dtypePAOl Nicas and Hancock, 1980 H911 Wil d type PAK Chiron (Pathogenesis Corp.) H956 H9 . 1 opmK::miniTn5-TcR Chiron (Pathogenesis Corp.) H957 H9 1 opmA::miniTn5-TcR Chiron (Pathogenesis Corp.) H958 H9 1 opmG::miniTn5-TcR Chiron (Pathogenesis Corp.) H959 H9 1 aprF::miniTn5-TcR Chiron (Pathogenesis Corp.) H960 H9 1 opmlv.miniTn 5'-TcR Chiron (Pathogenesis Corp.) H961 H9 1 opmE::miniTn5-TcR Chiron (Pathogenesis Corp.) H962 H9 1 opmL::miniTn5-TcR Chiron (Pathogenesis Corp.) H963 H9 1 opmD::miniTn5-TcR Chiron (Pathogenesis Corp.) H964 H9 1 opmJ::miniTn5-TcR Chiron (Pathogenesis Corp.) H965 H9 1 opmN::miniTn5-TcR Chiron (Pathogenesis Corp.) To be continued... 18 Table I. Bacterial strains used in this study. Strain Description Reference/Source P. aeruginosa H966 H911 opmH::miniTn5-TcR Chiron (Pathogenesis Corp.) H967 HI03 opmF::miniTn5-TcR Chiron (Pathogenesis Corp.) H968 H911 opmB::miniTn5-TcR Chiron (Pathogenesis Corp.) H969 H911 opmM::miniTn5-TcR Chiron (Pathogenesis Corp.) H730 (K372) PAO6609(me*9011 amiE200 rpsL pvd9) Pch", Poole etal., 1991 deficient in pyochelin production and ferripyochelin receptor production H743 (K613) K372 oprMy.QUg, OprM-deficient Poole etal., 1993 19 Table II. Plasmids used in this study. Plasmid Description Reference/Source pCR2.1 TA cloning vector; ApR Invitrogen pJJ104 pCR2AopmG; ApR This study pJJIOl pCR2.1opmH; ApR This study pUCP21 Escherichia - Pseudomonas shuttle vector; ApR Schweizer, 1991 pUCP27 Escherichia - Pseudomonas shuttle vector; TcR Schweizer, 1991 pJJ106 p\JCV27opmG; TcR This study pJJ105 pVmiopmH; TcR This study pJJ107 pUCPHopmG; ApR This study pJJ109 pUCP2\opmH; ApR This study 20 2.3 PCR All PCR was performed on an MJ Research Minicycler (MJ Research Inc., Waltham, MA, USA). PCR primers were synthesized by AlphaDNA (Montreal, PQ, Canada). PCR reactions were performed according to the manufacturer's protocol for Platinum Pfx DNA polymerase (Invitrogen Life Technologies). All PCR reactions, performed using PAOl genomic DNA, included 5% DMSO in the reaction mixtures. PCR primers used in this study are shown in Table III. 2.4 DNA Sequencing DNA plasmids for sequencing were isolated using a QIAprep spin miniprep kit (Qiagen Inc.). PCR primers were used for DNA sequencing. Sequencing reactions contained 3.2 pmol of primer, at least 200 ng of template DNA, and components from the BigDye Terminator Cycle Sequencing kit, according to manufacturer's protocols. Sequencing reactions were carried out in an MJ Research Minicycler (96°C for 30 sec, 50°C for 15 sec, 60°C for 4 min; 29 cycles), run on an Applied Biosystems 373 DNA Sequencer, and analysed using ABI 373 Data Collection and Analysis programs. PCR products to be sequenced were purified using a Qiaquick PCR purification kit (Qiagen Inc.). One-half volume sequencing reactions were carried out in the manner previously described and were run on the MJ Research Basestation 51 Automated DNA Fragment Analyzer. Sequence data was analyzed using the B.C.S. (Basestation Control Software) and Cartographer Analysis software. 2.5 Transfer of DNA into E. coli and P. aeruginosa Electrocompetent E. coli cells were prepared by growing cells to an OD55o of 0.4 -0.6 in LB with 0.5% w/v NaCl (LBNS). Cells were pelleted by centrifugation at 7000 21 Table III. Oligonucleotide primers used for the amplification of oprM homologues. Primer Sequence/Description opmG forward opmG reverse opmH forward opmH reverse oprM-left oprM-nght oprJAeft oprJ-nght oprN-left oprN-right uvrDAeft wvrTD-right opmA-left opmA-right opmBAeft opmB-nght opmDAeft opmD-rlght opmEAeft opmE-vight AAA GGA TCC ATG CCG TTC CCT CTT C AAA CTG CAG GTA GAA GAA CTC CCA CGC AAA GGA TCC CAC ATC GAT CCG GAC AAA CTG CAG CGA GCA GGA TGT ACC CAC CAT GAG CCG CCA ACT GTC C TGG GTC ACG GTC TGC TGG TTC C CGC AAC CTG CGG CAG AAA CAG C CTT GGC CAG CTT CAG GGC TTC G GCG CGA GAA GAT TGC CCT GAG C CTC CTG GCG CTT GCC ATA GTC G AAC GCC CTG ATC GCC AAC AAC C AGC GGC ATG TCC ATG ACC TTC G CAG TTG CGC GGC GAA CAG AAC C GTG CAT CGC GTC GTC GAC TTC C GAT CCG CCT GCT CAA CGA CAC G CCG CCG TCA AAC AGG GTC ATG G TTC GGT GCT CGC TGC CAG TTG C TCG ACC AGT TGC CGC AGG TCA C GAT GGT CGA ACG CCT GGT CAG C GCA GCG CCA TAG CCG TTG AAG G To be continued... 22 Primer Sequence/Description opmG-Mt TCG ACC AAC TGA TCG GCG AAG C opmG-nght ATC GCG CCG AGA TTG AGG TTG G opml-leR CGG CAC GCA TTT CGA GGT CAG C opml-nghX TTG CGC GGC GAA GTC CTT CTG G opmJ-Mt CGC TTG CCA GTC ATC TGC GTT G opmJ-nght TCG AGG CGG ACG GTG TGT TTC C opmQ-Mt GGC CTG GTG TTC GGC TTC ATG C opmQ-right CTC GCT GGC CTT GAG GCT TTC C aprF-lefl CAG CAC CGG CAA GTC CAA GTC C aprF-hght GAA GTT CGC CGC CAC CAG TTC C czcC-left AGG ACA CCC GCC AAG GCA ATC G czcC-right TCG TCG AGA TGG CGC AGC AAG G opmF-left CAA CGG ATC GCG CAG AAG TTC G opmF-right GCC TTG AGT TGC AGG CGA TTG G opmH-MX GCC AGC AAC TAC GCG GTC AAC G opmH-nght CTG CTT CAG GCG CAG GGT ATC G opmK-leR GCG ACC CTG CAG AAC ACC TTC G opmK-right CTA TGG CTG CGT GCC AGG TTG G opmL-\e& AGC GAT ATG GCG CGG GTG ATC C opmL-right AGT TGC CGC TCT GCC TCG AAC C opmM-Mt CAA CAA GGC GCG CAA CGA CTC C opmM-xight CCA GTT CGC GAT CCT CCA GTG C 23 RPM at 4°C for 10 min. The pellet was then resuspended in cold sterile dH20 and the process repeated. After resuspension and a second centrifugation, the pellet was resuspended in 10% v/v glycerol and pelleted by the same procedure. Two washes in 10% v/v glycerol were performed, and the pellet was finally resuspended in 3 ml 10% v/v glycerol and dispensed into sterile Eppendorf tubes in 100 \i\ aliquots. The aliquoted cells were then snap-frozen in a dry ice-EtOH bath and stored at -70°C. Electrocompetent P. aeruginosa cells were grown to a similar OD in LB with 0.05% w/v NaCl (LBLS) and washed using an identical preparation procedure as above, except that washes were performed in ice-cold magnesium electroporation buffer (MEB - ImM MgCb, ImM HEPES, [pH 7.0]). Cells were resuspended in ice-cold MEB, aliquoted, snap-frozen and stored at -70°C. Electroporation was performed by adding approximately 100 ng of DNA to 100 ul of cells in an ice-cold electroporation cuvette. The mixture was incubated on ice for 45 min, and then the cuvette was placed in the electroporator and subjected to 2.5V (25uFD, 200Q). 900 ml of SOC (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCh, 10 mM MgS04, and 20 mM glucose) was added to the cells and the mixture incubated at 37°C for 1 hour for ampicillin/carbenicillin selection or 3 hours for tetracycline selection. Following the incubation, cells were plated onto selective media and incubated at 37°C overnight. 2.6 Determining Minimal Inhibitory Concentration Minimal inhibitory concentration (MICs) of strains to antibiotics were determined according to the protocol for two-fold broth microdilution described by Amsterdam (1991). P. aeruginosa cells were grown in LB media with low salt (0.05% w/v NaCl). 24 After overnight incubation at 37°C, MICs were scored by noting the absence of bacteria in a dilution series of antibiotic, representing the well containing the lowest concentration of antibiotic needed to inhibit bacterial growth. Reported MICs were always the result of at least three independent trials, and changes in MIC of at least 4-fold are considered by convention to be significant. 2.7 Mini-microarrays 2.7.1 Microarray Construction Microarrays were constructed according to the protocol developed by Brazas and Hancock (unpublished data). Amplicons corresponding to 600 bp internal fragments of each of the OprM family homologues were amplified from PAOl genomic DNA using the gene-specific primers shown in table 4. The PCR reaction mixture (50 ul total volume) contained: 5 p.1 of 10 X amplification buffer, 1 mM MgS04, 50 uM of each dNTP, 200 uM of each of the forward and reverse primers, 5% DMSO, lOng DNA template, and 1.25 U of Platinum Pfx DNA polymerase (Invitrogen Life Technologies). The reactions were carried out for 30 cycles using a Minicycler (MJ Research Inc.) and each cycle included a denaturation step (94°C for 30 sec), followed by an annealing step (62-65°C for 45 sec), followed by an extension step (72°C for 1 min). The cycling was preceded by an initial denaturation step (94°C for 5 min) and followed by a final extension step (72°C for 5 min). Amplicons were purified using the Qiagen PCR Purification Kit and quantitated using an Ultrospec 2000 UV spectrophotometer (Amersham Pharmacia Biotech Piscataway, NJ, USA), and then resuspended in spotting solution (0.4 M NaOH, 10 mM EDTA [pH 8.2] in RNase free dH20) at a concentration of 20 ng/ul. The amplicons were denatured 25 at 100°C for 10 min and then immediately placed on ice and recollected by a brief centrifugation at 4°C. Samples were then transferred to a 96-well microtitre plate and spotted onto positively charged nylon membranes (Boehringer Mannheim Laval, PQ, Canada) in 0.5 ul spots using a 96-well groove-pin replicator (V & P Scientific San Diego, CA, USA). After air drying, the membranes were soaked in alkaline denaturing solution (1.5 M NaCl, 0.5 M NaOH in RNase free dH20) for 10 min and then transferred to neutralizing solution (1 M NaCl, 0.5 M Tris HC1 [pH 7.0] in RNase free dH20) for 5 min. Membranes were allowed to air dry and then were baked for 30 min at 80°C in a Tek Star Jr. hybridization oven (Bio/Can Scientific). Membranes were then wrapped in transparent plastic wrap and exposed to UV light for 30 seconds to crosslink the DNA to the membrane. The membranes were stored between filter papers at 4°C. 2.7.2 RNA Isolation Cultures of P. aeruginosa strains H911, H958, and H966 were grown overnight with appropriate selection and then subcultured to fresh media and allowed to grow to an OD of 0.5, corresponding to 5 x 108 cells/ml. All manipulations of RNA were carried out in designated RNase-free areas, and all solutions were treated overnight with 1% diethylpyrocarbonate (DEPC) and then autoclaved to inhibit RNases. RNA was isolated using the RNeasy mini RNA isolation kit (Qiagen): Pelleted cells were resuspended in 100 ul of TE buffer containing 400 ug/ml lysozyme and allowed to incubate at room temperature for 2.5 min. Buffer RLT (350 ul) containing P-mercaptoethanol was added and the sample vortexed. Ethanol (250 ul of 100%) was added and mixed by gentle pipetting. The mixture was applied to an RNeasy mini spin column. Following a 30 sec spin at 13000 rpm, 700 ul buffer RW1 was applied to the column and the mixture spun 26 again for 30 sec at 10000 rpm. The column was then washed twice with 500 ml RPE buffer containing ethanol and centrifuged once for 30 sec. Waste ethanol was removed from the collection tube, followed by a second 2 min centrifugation at 10,000 rpm to dry the column of ethanol. The column was then transferred to a new collection tube, spun at 13000 rpm for 1 min to clear all traces of RPE buffer and then transferred to another new collection tube. RNA was eluted in 40 jil of RNase free water (supplied) by a final spin at 13000 rpm. Contaminating genomic DNA was removed from the RNA sample using DNA-/ree kit (Ambion): 0.1 volume of 10 X DNase I buffer and 2 U of DNase were added to the RNA sample and the mixture incubate at 37°C of 30 min. After resuspending the DNase Inactivation Reagent by vortexing, 5 pi of the slurry was added to the tube and mixed by gentle pipetting. Following a 2 min incubation at room temperature, the sample was centrifuged at 13000 rpm for 1 min to pellet the DNase Inactivation Reagent. Purified RNA was quantitated in the Ultrospec 2000 UV spectrophotometer (Amersham Pharmacia Biotech) and stored at -20°C. 2.7.3 Reverse Transcription Reverse transcription reactions were performed according to the protocol for the use of Superscript II RNase FT reverse transcriptase (Invitrogen Life Technologies): 2 pg of RNA and 2 pi of 10 pM 5' reverse primer pool is added to the initial reaction mixture (12 pi), which is then heated to 70°C to denature the RNA and primers. Following a quick chill on ice and a brief centrifugation to collect the pellet, 4 pi of 5 X First Strand Buffer , 2 pi of 100 mM dithiothreitol, and 1 pi of 40 mM dNTP mix (10 mM each dNTP) were added to the reaction and heated to 42°C for 2 min. 200 U of Superscript II was added 27 and the mixture heated at 42°C for 50 min. The reaction was inactivated by heating for 15 min at 70°C. cDNA was stored at -20°C. 2.7.4 Radiolabelling DNA Probe and Hybridization a32P-dCTP was incorporated into DNA during quantitative PCR, carried out as follows: the PCR reaction mixture (50 ul total volume) contained: 5 ul of 10 X Amplification buffer, 1 mM MgS04, 50 uM of each dATP, dGTP, and dTTP, 50 uCi a32P-dCTP (Amersham Pharmacia Biotech), 200 uM of each of the forward (5') and reverse (3') primer pools (containing a mixture of the 19 primers used in the original amplification), 5% DMSO, 10 ng cDNA template, and 1.25 U of Platinum Pfx DNA Polymerase (Invitrogen Life Technologies). The reactions were carried out for 15 cycles using a Minicycler (MJ Research Inc.) and each cycle included a denaturation step (94°C for 30 sec), followed by an annealing step (63°C for 45 sec), followed by an extension step (72°C for 1 min). The cycling was preceded by an initial denaturation step (94°C for 5 min) and followed by a final extension step (72°C for 5 min). Unincorporated a32P-dCTP was purified from labeled PCR product using MicroSpin G-50 columns (Amersham Pharmacia Biotech). Incorporation of radioactive nucleotide was confirmed by adding 1 ul of PCR product to 5.5 ml scintillation fluid and measuring radioactivity on a Beckman 6000 IC Scintillation Counter. Prior to hybridization using labeled DNA probe, membranes were placed in glass hybridization tubes and incubated at 42°C for 3 hours with 5 ml of prehybridization buffer (5X SSC, 5X Denhardt's solution, 50% w/v formamide, 1% w/v sodium lauryl sulfate [SDS]), and 100 ug/ml denatured salmon sperm DNA to block nonspecific binding to the membranes. a32P-labeled DNA probe was denatured at 100°C for 5 min 28 and then chilled on ice. 14 pi of radioactive probe was added to the hybridization tube. Following an overnight incubation at 42°C, membranes were washed twice in 5 ml of each of 2X SSC/0.1% SDS and 0.2X SSC/0.1% SDS while rotating at room temperature and then twice in 0.2X SSC/0.1% SDS while rotating at 42°C. Membranes were subsequently blotted dry using filter paper and wrapped in transparent plastic wrap. 2.7.5 Autoradiographic Imaging Membranes were placed into MD Storage Phosphor Screen (Molecular Dynamics) for 72 hours. Autoradiographic imaging was performed on the Molecular Dynamics Phosphorlmager SI. Quantification of hybridization spots was performed using the ImageQuant version 1.1 (Molecular Dynamics) software. A circular box was drawn around the location of each spotted amplicon (regardless of hybridization signal), and local background (calculated as the intensity of the pixels surrounding each box) was subtracted from the intensity of the hybridization signal (calculated as the intensity of the pixels inside the box). The PCR-amplified uvrD gene, encoding a constitutively active DNA repair enzyme, was placed on the array as an internal control, since its expression had been previously shown to remain constant under a number of conditions (M. Brazas, personal communication). The calculated spot-density values were normalized by dividing by the density value for uvrD. Comparisons between conditions (wildtype and mutant strains) were performed by taking the ratio of spot density values of the wildtype over the mutant. Fold changes greater than 2-fold were determined to be significant. 29 RESULTS 3 OprM family outer membrane proteins from the P. aeruginosa genome 3.1 Introduction At the time of writing, the complete 6.3 megabase genome sequence of P. aeruginosa was among one of the largest known bacterial genomes, containing 5570 predicted ORFs (Stover et al, 2000). Members of the Pseudomonas genus are well-known for their ability to survive in several environments as well as their ability to catabolise almost every carbon source, so it is not surprising that almost 10% of these genes encode putative regulatory proteins. A search of the entire genome sequence also revealed the presence of a large number of outer membrane proteins (Hancock and Brinkman, 2002), 17 of which are related to OprM. These genes were given the designation opm, standing for probable outer membrane protein of the OprM family. Table IV gives summary data on the homology of these OMPs. The following chapter will explore the phylogenetic relationships of the members of this gene family. 3.2 Homology of Multi-Drug Efflux Pumps of Pseudomonas aeruginosa Following the completion of Pseudomonas aeruginosa genome sequence (Stover et al, 2000), three large families of outer membrane proteins were identified. One such group is the 18-member OprM family of OMPs, which includes a cluster of eleven OMPs closely related to OprM and most likely involved in the transport of small molecules, as well as seven members in a second more distantly related cluster that includes AprF, the outer membrane protein involved in alkaline protease secretion. Figure 3 demonstrates the phylogenetic relationship between the OprM and the 17 homologues. Further 30 a to o .e '5b u S bo | w o a o a,' £ o co _c 4) CO O o O o 0S-00 m a, O o l-l 03 oo in Q E ml a. OH OH OH OH O o o o o o o o o o o o » H—» •— t- 1— iH as o3 a3 cd 03 *—• —( £ E E | io co lo CO CO CO 0S- 6s- N° 0S- os v° Os cn O CN IT) m IT) in 3 bO _o "o E o .3 CO O e 'Bo s o 3 CO <D co O u co o e Sb s o 1-OH X 3 tD (D 3 03 3 O tD o C 03 co (D bO 2 -a CO 3 'o? 3 03 » 3 S S? 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While many of these operons share the same linker-pump-OMP gene organization as MexA-MexB-OprM, transposition of the genes within the operon is common. Interestingly, in three cases, the linker-pump-OMP gene organization is not preserved. The gene encoding opmH, which lies between a homologue of an E. coli thiamine biosynthesis gene and a gene bearing homology to a human NAD(P)H oxidoreductase, has no contiguous pump or linker proteins, while, in contrast, the genes encoding opmB and opmF are part of operons encoding two and three putative pump proteins respectively (Figure 4). This data is summarized in Table V. Table V also shows that the transport systems involved in efflux tend to encode inner membrane transporters of the RND- or the major facilitator superfamilies (the exceptions being opml and opmQ), while transport systems involved in secretion usually encode traffic ATPases (ABC transporter) proteins (the sole exception being czcC). Transport systems are usually categorized on the basis of their energy source, structure, and mode of transport (Saier, 2000). The four characterized efflux systems of P. aeruginosa are all members of the RND-family of efflux transporters. RND transporters, found only in Gram negative bacteria, are proton antiport systems known for their unusually broad substrate specificities. They contain twelve transmembrane domains (TMDs), with both N and C termini within the cytoplasm (Putman et al, 2000). The structural feature that distinguishes RND transporters from other transmembrane transporters is the presence of two large extracytoplasmic loops that project into the periplasm between TMDs 1 and 2, and TMDs 33 Figure 4. Gene organization of the OprM homologues. Most of the OprM homologues are encoded as three gene operons involving an inner membrane pump (of the RND-ABC- or MF- families), a periplasmic linker, as well as the outer membrane pore (OMP). Some operons include proximately-encoded putative regulators, though apart from the four characterized efflux systems of P. aeruginosa, the functions of the regulators have not yet been established. HP denotes a hypothetical protein. 34 £ 3 Q. a. H o a. O Q_ o c o a E Q. Q. E E 0> o 3 O) o S o s o Table V. Efflux genes of the MexA-MexB-OprM homologues. OMP gene Genes order3 PAID#b Strand Transporter family0 Cluster oprM ABM 0425-0426-0427 - RND Efflux oprJ ABM 4599-4598-4597 + RND Efflux oprN ABM 2493-2494-2495 - RND Efflux mexX-mexY'y AB 2019-2018 + RND Efflux opmA BAM 2835-2836-2837 - MFS Efflux opmB ABBM 2528-2527-2526-2525 + RND Efflux opmD ABM 4206-4207-4208 - RND Efflux opmE ABM 3523-3522-3521 + RND Efflux opmG MAB 5158-5159-5160 - MFS Efflux opml MBA 3894-3893-3892 + ABC Efflux opmJ MAB 1238-1237-1236 + MFS Efflux opmQ ABM 2389-2390-2391 - ABC Efflux aprF BAM 1246-1247-1248 - ABC Secretion czcC MAB 2522-2521-2520 + RND Secretion opmF BBBMA 4595-4594-4593-4592-4591 + ABC Secretion opmH M 4974 - N/A Secretion opmK ABM 4142-4143-4144 - ABC Secretion opmL MBA 1875-1876-1877 - ABC Secretion opmM BAM 3406-3405-3404 + ABC Secretion The order of the linker, pump, and OMP proteins in the operon with respect to the MexA-MexB-OprM system, where A denotes a linker, B denotes a pump, and M denotes an OMP. b The PAID# {Pseudomonas aeruginosa ID #) is the number of the ORF in the completed genome sequence. 0 The family to which the pump protein(s) belong, where RND = resistance/nodulation/cell division, MFS = major facilitator superfamily, and ABC = ATP binding cassette transporter. d MexX-MexY has no known cognate OMP to be named. 40 7 and 8 as shown in figure 2 (Gotoh et al, 1999; Guan et al, 1999). While their significance is not known, it is believed that these periplasmic extensions contact and interact with periplasmic domains of the OMPs, perhaps to initiate gating functions (Koronakis et al, 2000; Andersen et al, 2001). Transporters of the major facilitator superfamily (MFS) are found in a variety of organisms, including Gram positive bacteria and eukaryotes. They can have symport or antiport functions and are not dependent on periplasmic and outer membrane components for their function. Like RND pumps, their substrate specificities can be quite broad, including such diverse compounds as antibiotics, oligo- and polysaccharides, and intermediary metabolites (Paulsen et al, 1996). MFS are structurally divided into two groups, those with 12 transmembrane segments (TMS) and those with 14 TMS. They lack the large extracytoplasmic loops that distinguish RND transporters (Putman et al, 2000). Among the most well-known examples of MFS transporters are the E. coli TetB tetracycline efflux protein, part of the 12 TMS family (Paulsen et al, 1996) and the emrAB system of E. coli where EmrB is an MFS transporter of the 14 TMS family, while EmrA is a periplasmic protein that shares homology with other linker proteins (Paulsen et al, 1996; Nikaido, 1998). Traffic ATPases or ABC transporters, as their names imply, couple the energy of transport to the hydrolysis of ATP instead of the proton gradient. Present in many organisms including Gram negative bacteria, Gram positive bacteria, and eukaryotes, they are transmembrane proteins that usually have twelve transmembrane helices as well as a large cytoplasmic domain containing the Walker A and Walker B motifs as well as the nucleotide binding domain. Since ATP binding and hydrolysis occurs at the Walker 41 boxes and the nucleotide binding domain, these are the characteristic features of ABC transporters. ABC-based efflux systems are generally more specific than those of the RND- or MFS- families, though some broad spectrum ABC transporters have been identified (Putman et al, 2000). Perhaps the best known multi-drug transporter of this type is the human P-glycoprotein encoded by the mdr gene, which can export various cytotoxic drugs often used for cancer therapy (Paulsen et al, 1996; Mao et al, 2001), and bacterial homologues of P-glycoprotein exist, such as LmrA of Lactococcus lactis, an ABC-family multi-drug transporter that confers resistance to a wide range of structurally dissimilar amphiphilic compounds (van Veen et al, 1999; Margolles et al, 1999). Type I protein secretion involves a three-component secretion apparatus similar to the typical multi-drug efflux. Type I secretion systems, which are often responsible for the export of proteases, lipases, toxins, and other secreted protein factors, are comprised of inner membrane ABC transporters in conjunction with linker and outer membrane proteins (Thanassi and Hultgren, 2000). This structure facilitates secretion of whole proteins without a periplasmic intermediate, in much the same manner as efflux allows for one-step transport across both the inner and outer membranes. 3.3 The OprM Family of Outer Membrane Proteins The genome of Pseudomonas aeruginosa encodes a number of families of outer membrane proteins, including the 18-member OprM family. Table IV shows that the majority of these homologues, four from the efflux cluster and six from the secretion cluster, share their highest homology with outer membrane proteins from other bacteria, including PA4974, the OpmH protein which is 54% similar to the TolC protein of E. coli. 42 Figure 5. An alignment of the amino acid sequences of OprM and its seventeen homologues. The alignment was performed using the Clustal X (version 1.8) sequence alignment program and viewed using the GeneDoc software. Highly conserved residues are shaded in black with the single-letter amino acid code appearing in upper case above the alignment to denote those residues that are completely conserved among all eighteen members. This alignment was used to produce the unrooted tree shown in Figure 3. 43 2 0 4 0 60 MKR SF LSLAVAAWLSGCSLI PDYQR PEAPVAAAYPQGQAYGQNT GAAAVPAADIGWR : 58 MRKPAFGVSALLIALTLGACSMAPTYERPAAPVADSWSGAAAQRQ GAAID—TLDWK : 55 MIHAQSIRSGLASALGLF SLLALSACTVGPDYRT PDTAAAKIDATASKPYDRSRFE S LWW : 60 MKGTPLLLIASLALGACSLGPDFTRPDRPAPGEroSLQAAAGNPSHLAAAP-LAAQWM : 56 MKHT PSLLALALVAALGGCAIGPDYQRPDLAVPAEFKEAEGWRRAE PRDVFQRG—AWW : 57 — MKRSY PNLSRLALALAVGTGLAACSVGPDYQRPQS PPPRVASEHLGEF SGERREAP WW : 58 MK PYLRS SLSALILLGGCAAVGPDYAPPSASAPASFGAM PAGIDGS — G VEIEWW : 53 -MPF PLLHPWPQRLALASAILLAAGCVT SEGLE PNARLQPAGALQAGRSLDGVALS PAAWPRQDWW : 65 MVGSFVGFLWF SAISGCVSTGDIAPEAATLDANALATDHAIQAAAREAG-WPQAQWW : 57 M P LA S H LR CVALALGIS T ALGCAN RN Q PA P RAE S L D PGL S RVAGT R GDAL P AQWW : 55 MSMKNLSLISACLLLGACGST PAPLDSGLAAPSQWRYLAAGRSD ASDIR QWW : 52 MTMRRLMTWLFGAFLLLLREDAFALG : 2 6 MPILRPLASAGKRACWLLMGLCLGLPALANEAPVSFN G : 38 MNRWGLGVLWLVTALPVAASVNPALS PDVPSMAREQGRSVLLS : 43 MLRRLSLAAAVAAATGVAWAAQPT P LP : 27 HRALAGLLCGLLGLVPGAAAYEPDVF GTTGQVAGQAVYDLGGSGLP CRGG : 5 0 MRGRRQYARKGRRHGKGAIWLLSLGLP : 27 MNRLRACLLS SALLSAS SAQALG  23 100 EFFRDPQJjgQL GVAf|EN§RD|RVJiALNVEAFRAQYRIQRADLF SFIVDAE KQFDDPT TLFDDAQE ELYGDQT SFFDDPQ /RLjjD QAJ?AR jjH Dy R E |RAN LRSARALFDDRWLDQLJ RGFDE PA0E SL|QRA8AA1LD| TGLGDRQf KVYADPQl TLYQDPGj KAFGAPE TSIS EQVIDLS -TKTD— PPPTELS MFASAMPl 8 0 QQL GVA EM RRL DMA E'M MQL EQ: SG HAL QRVQRA: JLD|RSSAARLQQSRAIRRSLGGDAL JDLQMHLERSJMQTHAQSVAQFRQAEALVRGARAAFF LWGARLDEAKALLRENREEFLI DQL GEA QGTPD|QI|EARARQAAATAQAQDAARQ| DAWlJE KABDGSPGBAVUHARVRQAK SMAGLVE SIE S: 'HLKAAAgR H|JRDJlAAiDAH ARAL LGH L R GAQ GE RW D S LHQRAMLN S QD[jGA§VARVRQAQAS AVI AGA PLL LDGYHLAHEN1PQFQA§IQEHEAGRQYRALGRAALL EQA1ERA|RS1PEEJAAVGRETEIASGARQQAGLIPN SDAfflYLGfflRNlRGgRS|YLQRIAQKFDLRVAADAFN i SVYKEAJJDNJ EE SQADYLARKEWPQARAGLL PQTRLBWANAKAQAAQVGIGKSAYL DQA RAG AIHPE RS MAEADRAGTEVEMAKGGYY LDAYQLA RH PTFQA LHERRAGSENRAIGRAGLL A ERI CH  AlH 12 0 * RSGVDGSGTRQRLPGDLSTT- : 123 G?NAAATGNRQRQPADLSAG- : 12 0 T SRASADIGKGQQPG : 122 SffiDASGNYQRQRTTSAGLFDP : 122 SyTGNVGKTRSGQGGGDSTVL : 123 QJJTSQAGYSRSIEQQLDYDG- : 123 RGGPAF DYQARRRGEVET P— : 117 TfflDAKASYSGIRAPTSVAPAP : 131 QjjjEGKGSLVRHRWPDDYFYGP : 123 RTEVGYGYQYGRDGDDQTLAE : 121 EfJNATLGASRQKLLRDSGYSG : 118 RgVYSYNRGRSWSDVTQTTT- : 84 DgSWSVEDTRQGNRQTS : 97 KfflWRGDYRANRATE DRTRT S : 10 9 |QfflGAGARVGDTRIAFDE : 8 6 JR DGRLDASRGYSDMDYRDAP : 116 SfflTMSGGPQEFDFGEIVY : 90 S™RYDYNKARNDSTVSQ : 7 8 140 160 180 GS pAIS SQYGVTLGTTAWELDL NRSEVASSYQVGLALPEYELDLj" v TEDRVNSERYDLGLDSAWELDL;; s G KAGKGNYNHALAGF DASWELDF* LPGGSTVSS GGSGAIST SYSTNLSVSWEVDL| EPRRRLAESYRAGFDAQWEIDLi; AGQQRDIETYRGALDASWEIDLy LGGRY SAIKYL S LGFNYDFDL' G DLARTTSWNNSTEIGLNYKLDLAT DEDLHSQWKHTVRLDLSYQLDL, T DA TSDNDAVDSFSAGLSASYEVDF| R GDFKEDRDYDSYVSTLSLQQPL§DYEAFSRYRKGVAQA VSIAQPLELG£-KRGARVEVJJKRGS NVS PTATLLGEYGTRF SLAWVKQFRTADEAGRYRSDGLDLTWQPLLRDAGWDVTTAPLRLJ?RLSE R PATVKRN S QWQAT L S Q PL|RADRWFQWQA|KET S y LSGDGHRHRRGASLQLSWVLFDgg-RRSAALRN"QQLL g a -RLRSLRDQSLEQY -RVKSLTDA@LQQY -RIRRQLESSDALS -RVRRELEASDATV -KLRRQLEAHQASL -RLGRLSDAJLARA -RVRRSVEA0EAQA •GERAAWEA"LGQA j v-v -Li -3R VE - EVRARIAAIKADA -GRQAAYRS0LESL -DLTASQMLYDffi-RVTSKVDs|sATQ - GDARVERDYRSYASTLSLE QPL DYE AYARY R Q GE AQA 160 157 160 161 169 160 154 167 161 160 158 123 121 175 122 15 4 115 116 44 0 0 22C 240 260 LATEQAQRSAQTTjjVASVAT; LA S E E AAR AARIAMVAE V S Q i EAAEADLQQLQVsllAELVDJ EASENELRDVQVS§|LAEAARD| HASAADLAAVRLSQQSQLAQN \ EAADADLRLVRLSJJAADTARJ GSREALLRNVQASGAATVAMSJ NAARIDSQAARIGgSASIARi HMAAAEARQAQLE|EGNIVRJ EAAQAARDLLRVSIASQTTLJ KAS EYDRATVE LT jjL S GVAN s| LLSDERFRSQSQEHLVRVLE/ EIAWTQLEVRRAESRAQVRG; DANRLQLKASVSQTISQVIG; DQARLEFSATQQDSILRSAETJ LAANAS QDAT L QN T F ALAAQ; RKLSEAVLVARDDAALDIVETJ LFADEQFRGRSQEIAVRLF, LTLKADQAQLQ TKDTLGTYQKSFD TQRSYEA LSYDGALRRLA|TRQTLVSREYSFA||IDQRR; GQLRGAQLREK ALSHLENQKESRQ TEQLRD/ IQLRGEQNRAAMIRDNLETARRSLESTRTRLANI LQLRVMDEQIRILNDTVTAYERSLKIAENKYR/ FEIQGYQRRLDWARAQVRSWRDTLEPTRS SLQ FQLQGIEAELAIVHDIAGNQRDSLEIVERLVS. SDLAHAFTVRDgAEEELKRSQRMTEjSsQKRMS 'QLSLQYAEMD AKAMLQQQRDILA AQRRLR VRACALARRAE QRRSVGLLDASLA SERQLAA LQVLALREQQRHARLNLDNAEHVLRSVETRH_ _ TGALLAQDQIEIARAQKRSYREQFQ§NQRQFER| 'AALTAQERVRIAKT SLDLARRALQAADRRVK. RELLRAQEQAR§AREALARTQELLE|JNRAMIR. FTVLRAQDNLATSKAEEAAFKRQLDQANERFD' YDALAAQRSLAASRQVAELAAQNLEAADAKYRJ LDVLASERRVEAVREHIQRLDGIRE TQARGGDB SETLFAREQVVFJAEAQRRALETQLAFNQRAFEEJ VASALDLRQA 226 AATALDYQEA 223 VGAELDVLRA 226 VATDLEVAQA 227 IVTRADVAQA 235 SGLPEDVENA 226 SAHEFDRLRA 220 LDSKVQLQQT 233 IGTHFEVSQA 227 LSSELQRRRL 226 SATALEVAQQ 224 MGTRTDTLET 189 SISSVERVRA 187 RMAEFEIVQT 241 LSDKTDVLEA 188 AAALSDRLQA 220 YADRSELDRA 181 EGTRTDLLET 182 280 300 320 QTA E GAR AT L AQ Y T R L VAQ D QN A V L L SG 1 PANLPQGLGLDQTLLTEVPAGLPSDL QRR : LGL|EQARAEQERHLRQKQQAFNA^L|L|SDD--AAQAIPRSPGQRPKLLQDIAPGTPSELIERR DAR|AATAASVPQLQAEAERARHRMAT|L|QRP—EELTVDLSPRDLPAITKALPIGDPGELIRRR LAQSMSMEARLPEVEKNQAHLVNAFFLGYGVGAS PG-SLLAELGPARAIPRPPGSVPVGLPSELAQRR RTQFFLKSTQAQAIDLKYQRAQLEHAHAVWVGLP PAQFNLPPVASVPKLPDLPAWPSQLJJERR QANgLRSEAAI PPLTTALE SARYRjgDwgRgEAP—GSGAPILDGGAAAPLAKNLPLGDVDRLgjLQR EALFFLHNVEAAVPDLERRRAATRNA[JAVFFLLAEAP-QAF S PPVARASGERLTLRTLGVGDPAGL|JARR QTQJIATARQQLSAAEQDIASARIAPAVHLSF EVPIPETERRIEVIDEEIQLTRNLI; !L1KGP—DRGLELQRPQPLNPASLSLPSVLPAEL GRR -GEGRTIRRPSLNLAAQPSLPSALPAELGGRR LALRERTFLAALPMLEARRRAALYE AL S RS P-RQLDAPAATCAGIPQLRRALPTGDGWSL ARR SSLBASQRKQLPLLEQQAHEALIT|AT|I|EP—VQALQVAERPFDSLRWPETGAGLPSELSSRR QARFNLAQAQEIEARDSQDAALREfflER|V||APLEIADLAPLGERFQVRPLSPASYTAroRDLAfflAEN QVLADNAQLDLSQAELEQQRTYVQISSTWDEP QPGFARVGGALDAVPASITRGALLRHfflDES EAD^JASQELNVEE STNQVDSARLAfflLQfflLALD LSTQIRASDALAATPIEVDRQQAIRT AgQQQ QASYDTARANRLIAEQRVDDAFQAIIVTITNRDY—SAIEGMRHTLPWPPAPNDAKAWVDTAjJjQQN QTAJJsQASLAQVRDEGALSNALGVaALRMELAPDTPLRLSGELEAQPDTGFVKAIDEMLAEARREH NLEfflSRAQEQLSLEKGNLQDARNQYAlffVgQEP AD-LVE PE PMSLQRYLAASDMARV?JRE S RARisLTRAEEIAASDRAAAARRT|EAlL@QALEDRELAAPIERFPALRLQPATFEGWRQVA3QRS 340 DSLEAEHQ1MAANAS|GAA; DNLAAEHR|RARNAD|( D RAAERR AASTAD GVAT D RRAEARWHAATASSGVJ ~ D ASAERKIISANAQIGVJ D VSAERQ AASTEDIGAAT AD AAAERN AAATARGGVET AD VAARWR EAARRM DS D VARRWQ1AALAKG§DV D RAAERRgAAADAR RALAE 1ANAEAQ1AAAQAD|QV, " E|ASLRHA1DVARYE|EQN TBRLAAQEJ5ARGEAQ|DLE ^EYLQRLIGSRQADLNgVLA. LP. LAS NY A NAAEET P.QR BAILAAQARIKAAAAS .EES "LQRKALSDAHVAEAE REA' AE|GAQRHA|EAAAYE|ERN * 380 * sgTANAGTMSRQ LSGLFDAGSGSWLFQP SfflTGSFGTSSAE MSGLFDGGSRSWSFLSfflSGFLGFTAGR GSQIGSSAARAWSVGP jNGNFGFESLQ LSSLGDWDHRQFAIGSAAGGYRSGS LSNMI ST PNRFWSIGP JGGFIGFFALR SGDLGSAS-RAFELAIRGSIGLVAGN LDALDE SGT SFNVLNP GAM AGLAAL H T S DVLQAPS RFFQVAP [MASVGFSAVGGG MLEFFRSAKYTYSAGP 5 F AVGAE T S AAT LAGLGGS GAL AY AAG P THSASLSSGANR AADTFRN— PYYNLGA GgYASTGKSKSG SENTYNQRYETDSVGI |T JSIGSKYDQTAR DGRGE RVNLIGL SjjvGGASQIRDR YSEGGGDNSRSWDSYA DAVAQY QKGDN DAL GF AN S AAN PLVHYGKYVDE R SIGL flsANLARSHSD QAMAFNGDTRERDRSIGL flEASALRREIG GHPESDSWSsjfYASSSKTHSA SE STYEQKYDTDSVGL 45 400 420 440 460 I LP: LLP AFSLPi QFAMT TAGS LRASLDYAKIQKDINVAQYE KALQTAF Q E VAD GLAAR GTFTEQLQAQRDLVKA-S DGGRNRANLSLAEARKDSAVAAYEGTHQTAFREVADALAASDTLRREEKALRALANS-S DLGSVRARLRGAKADADAALASYEQQ|LLALEESANAFSDYGKRQERLVSLVRQSEA-S EGGRLRGRLELREAQQQEAAIDYQRT|LRAWQEVDDAMHDYAANQRRQERLGEAVAQ-N DGGLIGSQVDQAEATYDQTVATYRQTKLDGFREVEDYLVQLSVLDEESGVQREALES-A m mm m iH I mm. S|SWPAgRLGNVRARLRAVEAQSDAALARYQRS|LLAQEDVGNALNQLAEHQRRLVALFQSATH-G VyRWA^JLDRGRVWARIAASEARAQEALI LYDRTQLRALQET DDAFNGYGAAADRLRLRLLEATA-N AISLPMDGGRRRANLAERDADYDLAVGQYNKTIVQALGEVSDDLGKLRSLEQQVIDQRQARDI A TLP DGGRLRSQLGEAAAGYDAAVEQYNQT VDALKNISDQLIRLHSVDIQKDFAAQSVAS LMJSWRF PNRE SARGRLEISAAAERDAALARFDGAQLGALREVERALALYAGERQRRADLQRALDE NSLAP^JHGRLRAERDRSLARQEELLETYRKAMLTAFADTERSLNSIDGLDRQLHWQQQELEQ Q|SVPHSGGETLAATRQATHRMEKSHYDLDDK|RETLNQVRKMYNQSSSSAAKIRAYEMTVDS S?PLP»PD—RNQGNIYAAQSRADQARDLQRAT|LRLRSEAVQAYDQLRTSEQELALVRRDLLPGA G OVE PIGDLSRRQAEVRAQVDVENQKIL1EDARQTLEQNVIDAVRDLGTRWRQYQIAQRATALS E NIP SGGLT S SQVRE SYQRLNQSEQSREGQRRQWQDTRNLHRAVNTDVEQVQARRQAIIS-N 0 NIP EGFERTYQVRNALARREASEAELADTEQQVSLEVWNNYQSLSVETRSLARTRELVEQ-S RF RMDTJAQGLSNFRR PTAAQQRLE SAKWSADAMQRDI RP.QLQNLF DNGDT LRWREQSLTQQVTE- S R|JSLP3|EGGRVSAATRQAGDKYAQAQAELDAQS!5ASVINDLHSQFDLTASSLAKVRAYEMAVAA-A 410 408 411 413 418 410 406 418 414 412 406 376 366 426 383 409 357 369 480 500 5 2 0 DEYYQLADKR RTB NEALKLAKAP.SE sf RAAAQQAAIR|RE| RRALQSAREQRR, REALRLAEN'J K, ANALEIANEP. I REAARLARERS RSNFDLAMRP. GEJ QKTYDIATLASQRJ RHAYRLARSNRR; QRAFDLSDSRQQ. RTLVMATRKSI QSALDSMTRG|EM| RRKLEIEREKLRV QSSLEATEIGBQVI RQSLEWQGR RS| EQVGELYREQ|E-REQVTATRRSVA( VDNY T •DNHfflRY iTTDFjjvfl •.VDF|S| iTVDYTDl \.GSYJ!A| SDGEYHDI VGSYfflDA \.ETL1T| ^RVNfflDB SKFNF|D| SRSSN . 'TRNI|D| rcsMnEg 5RP.DV»JDJ# SERVNRDI DA RSLFTAQQQLITDRLNQLTSE|N|YKA| DA RSSFLNEIAFIDGSTQRQIALPDI DA REQLSAEDAQAQAEVELYRGL! DS RQLLDNQEQQVASDEAVSLTL TNGATALSNERTVLTLLGSRLTASQC E M R.ALY QIREELAQAETASF VN V EAHRSDYLSRRALSIARTEQRLA1 1QQLLVAERQLASLESQQIDLSJ5Q| ITRLFQQQLVQEQVQAARLAAHAS IR S LVAD RARLVDAEM RVAE R Q|j E jR T L Y AAQ D AAVQ L R L AR L Q A S §Gj a [ALQALY SAMNELSKAKY DY LT AWARGRFY/ IDAIRT LVGVRAQYVRALDAAAQAR|SI|E RL| ]SF1TDLRNVENTQLNALISFLNAQTQ|DLI| ]RQLYAAVRDYNNSRYDYILDT|R|KQ/ [ALTAYASAEDQHIRALGNWQT SRIJRBJAASIII JJVHRERFEAERQLINLRIERKRIEYRAA IDA QQFYGARRDLASARYAYLNAWJ?R3RQLA| GWNQQTVTQQQ : 47 6 GWDEGRSLWH : 474 GGWQPSA : 472 5GWS PT SDPASG : 479 5GWDSADIERTD : 484 IGSGDLAPGAGQ : 47 6 GGW E ACAGAR R C : 472 ,-GGFQPDSRSAAL : 484 gGGVGAGADTPAQ : 4 80 IGGWQAASSPSHQ : 47 8 iGGWQSDRQGLAR : 472 LDEADLELVAA : 4 42 EDIGHLGQ : 428 1TLDSKTEISLND : 492 TLS PADLEALSA : 44 9 RLGFWSLR : 471 LLGPLLENRLNH : 423 LEDRDLAVLAA : 435 540 560 TAKKEDPQA RGGRS ERLGRVEEGLPPSP LAAGETAGANR GVAT DDT S PGVARQR D SRS ATAKAPAE RKLAPENVPVRAVS SR ENGQ KD NFVSGET PARRRDCATTDCPAPLHTLS KTDTEENRSALN H YLKQDYDPDKDFLPPDLAKAAAEQLQSKPRQQY GS YFGAGEGRAQVT AAIR 485 479 498 487 491 492 496 482 474 481 493 482 425 451 46 Although there are few conserved residues among the family members, some regions have more conserved residues than others. For instance, those residues presumed present at the interfaces of the outer membrane are often aromatic amino acids. Figure 5 shows an alignment of the members of this family. 3.4 Conclusions To date, four RND efflux systems have been identified in the P. aeruginosa genome, which together have been shown to be capable of conferring resistance to almost every major class of antibiotics. However, since these genes have been present in the genome much longer than antibiotics have been in widespread use (Nikaido, 1998), the presence of at least eight other putative RND efflux operons alone in this family suggests that the ability to export toxic compounds en masse has played some important role in the evolution of this organism. Perhaps the number of systems capable of toxin export has contributed to the ubiquity of this organism in the environment. The observation that inactivation of one efflux systems can result in the upregulation of another (Li et al, 2000) suggests that the expression of the MDR systems is under a tight network of regulation that is not yet understood. 47 RESULTS 4 OpmG- and OpmH-mediated intrinsic aminoglycoside resistance 4.1 Introduction In patients with cystic fibrosis, the utility of aminoglycosides cannot be underrated. Although many aminoglycosides are highly toxic compounds with narrow therapeutic dose ranges, their synergistic effects when combined with some P-lactam antibiotics makes them ideal agents against recalcitrant Pseudomonas aeruginosa infection (Hancock and Speert, 2000). In the past, resistance to aminoglycosides has largely thought to be the result of aminoglycoside modifying enzymes; however, it is now believed that impermeability and efflux mechanisms are the predominant factors leading to broad-spectrum aminoglycoside resistance (Westbrock-Wadman et al, 1999) and are a major concern due to the already limited selection of available antibiotic therapies against Pseudomonas. The identification of the mexXY operon in P. aeruginosa has provided some insight into the specific mechanisms for broad aminoglycoside resistance, but, in the absence of a defined OMP component, our understanding is far from complete. From among the uncharacterized OprM homologues, three were found to play roles in aminoglycoside resistance. This chapter outlines the research into possible outer membrane components for this system. 4.2 Screening miniTn5 insertion mutants MiniTn5 insertion mutants (H956-H969) in each of the genes encoding the OprM homologues (except opmQ) were provided by PathoGenesis Corporation (Chiron). There are several derivatives of the miniTn5 transposon, largely differentiated on basis of the selectable marker encoded within the cassette. Mini transposons do not encode their own 48 transposases, which are instead supplied in trans from a donor suicide plasmid. Since the plasmid cannot be maintained inside cells, growth on the appropriate antibiotic selects for cells within which the transposon integrates into the chromosome (de Lorenzo et al, 1990;Herreroe/a/., 1990). The minimal inhibitory concentrations (MIC) of P. aeruginosa strains H956-H969 were determined for a number of antibiotics. Included in the screen were hydrophobic and hydrophilic antibiotics, dyes, detergents, the energy inhibitor CCCP, and ethidium bromide (Table VI), all of which are compounds that have been previously characterized as substrates for active export by tripartite multi-drug efflux systems in Gram negative bacteria. Only one set of mutants demonstrated a definite resistance pattern. These three mutants showed increased susceptibility to all four aminoglycoside antibiotics tested (Table VII). H958 (opmG) showed 8-fold decreases in MIC of kanamycin, gentamicin, and streptomycin, as well as a 4-fold decrease in the MIC of tobramycin, as compared to the wildtype H911 (PAK). H960 (opml) showed an 16-fold decrease in the MIC of streptomycin, an 8-fold decrease in the MIC of kanamycin, and 4-fold decreases in the MICs of gentamicin and tobramycin. H966 (opmH) showed 8-fold decreases in MICs of kanamycin and tobramycin, a 4-fold decrease in MIC streptomycin, and a 2-fold change in the MIC of gentamicin. Tetracycline resistance was observed, confirming maintenance of the transposon insertion. Carbenicillin is not a substrate for MexX-MexY (Masuda et al, 2000b) and all three mutants showed no change in the MIC of this p-lactam as compared to H911. For comparison, insertional inactivation of opmA, opmD, or opmL, all of which are relatively well expressed in H911 (see below), did not result in 49 aminoglycoside susceptibility. Indeed, none of the remaining eleven mutants showed altered susceptibility to any of the four aminoglycosides tested. Similarly, deletion of OprM, which some authors have proposed to be the cognate outer membrane pore of MexXY (Aires et al, 1999; Mine et al, 1999; Masuda et al, 2000a), only caused a 2-fold change in aminoglycoside susceptibility, which by convention is considered insignificant. Figure 3 shows that OpmG and Opml are highly related and are, in fact, more related to each other and outer membrane efflux pores from other organisms than to any P. aeruginosa genes. While OpmG and Opml are still part of the efflux cluster, OpmH is part of the secretion cluster (Figure 3), and its closest homologue both inside and outside of the P. aeruginosa genome, is the multifunctional E. coli OMP TolC. Interestingly, of the fifteen OprM homologues, OpmH is the sole member of the family that is not encoded as an operon (Figure 4), making it an attractive candidate for the role of the native OMP for mexXY. 4.3 Complementation of the opmG, opmH, and oprM mutants If opmG, opmH, and opml do play a role in aminoglycoside efflux, MICs values should be restored upon reintroduction of the genes into their respective mutants. However, since it is in fact inner membrane components that determine substrate specificity (Srikumar et al, 1997; Gotoh et al, 1998), if OpmG, OpmH, and Opml are indeed channel-forming OMPs, then each protein might also be able to complement an oprM~ defect. The opmG and opmH genes were PCR amplified from PAOl genomic DNA and cloned. The identities of opmG and opmH were confirmed by sequencing; however, since 50 Table VI. Compounds tested in initial MIC screen of miniTn5 insertion mutants. Compound Chloramphenicol Fusidic Acid Polymixin B Rifamycin Tetracycline Aminoglycosides Gentamycin Kanamycin Streptomycin Tobramycin Energy Inhibitors CCCP Macrolides Clindamycin Erythromycin Quinolones Nalidixic Acid Norfloxacin Dyes and Detergents Acriflavin Crystal Violet Ethidium Bromide SDS Dyes and Detergents P-Lactams Penicillins Carbenicillin Carbapenems Imipenem Meropenem Cephalosporins Cefpirome Cefsulodin Ceftazidime Ceftriaxone 51 Table VII. MICs to aminoglycosides, carbenicillin and tetracycline of P. aeruginosa mutants lacking selected outer membrane channel proteins. Strain Phenotype Km Gm MIC(pg/ml)a Sm Tm Cb Tc H911 Parent 100 0.8 8 0.25 50 1.3 H958 OpmG" 13 0.1 1 0.063 50 25 H966 OpmH" 13 0.4 2 0.031 50 25 H960 Opml" 13 0.2 0.5 0.063 100 13 H957 OpmA" 100 0.8 8 0.25 100 13 H963 OpmD" 100 0.8 4 0.25 100 25 H962 OpmL" 50 0.8 8 0.5 100 25 a Abbreviations: Km, kanamycin; Gm, gentamicin; Sm, streptomycin; Tm, tobramycin; Cb, carbenicillin; Tc, tetracycline. 52 this strategy was unsuccessful in cloning the opml gene, only the roles of OpmG and OpmH were explored in greater detail. 4.3.1 Complementation of an oprM mutant susceptibility phenotype H730 is a derivative of P. aeruginosa PAOl that has a number of mutations, including a mutation in the rpsL gene, the target of the aminoglycoside antibiotic, streptomycin. Consequently, H730 is highly resistant to the antibiotic streptomycin. H743 is an isogenic oprMy.Hg mutant of H730, created by inserting a mercury cassette into the oprM gene of strain H730. Mutants deficient in OprM are super-susceptible to many P-lactams (but not carbapenems or cephalosporins), chloramphenicol, macrolides, quinolones, and tetracycline. Table VIII shows that there were significant (4-fold or greater) decreases in MIC of nalidixic acid, carbenicillin, erythromycin, clindamycin, chloramphenicol, and tetracycline between strains H730 and H743, in agreement with previous studies (Li et al, 1995; Nikaido, 1996; Masuda et al, 2000b). The MICs of fusidic acid and acriflavin also decreased, indicating that these compounds were also substrates of MexA-MexB-OprM. As noted above, aminoglycoside susceptibility was not significantly changed. The PCR-amplified opmG and opmH genes were separately cloned behind the lac promoter on the pUCP27 E. coli-Pseudomonas shuttle vector to create pJJl 06 and pJJ 105, respectively. When multicopy plasmids bearing opmG (pJJ106) or opmH (pJJ105) were introduced into strain H743, antibiotic resistance was restored, indicated by the increased MICs of erythromycin, clindamycin, fusidic acid, chloramphenicol, nalidixic acid, and acriflavin. The MICs of erythromycin, chloramphenicol, and nalidixic acid were in fact higher in the complemented strains than those of the parent strain H730. 53 E 0, O -o c c3 o s OH O c3 OH o C a o c o e £ "OH E o o > H 60 u > 1H o < E CH <4-l cd X u N u ^ E u o H E u =3 PH u E 00 E o E CO CO *o 00 CO CO \D ,_, ro CO CO '—' O © © © ~ co O in CN O m CN in in in in >2. CN A CN A CN A fN CO O rN o © o © o © © m CO CO CO © O © © >5 m in in oo oo oo CN © © © in CO © o CN A o oo CO o o in © in CN © in CN >750 CO CN o in r-A o in A m CN oo o o in A o o in A >2500 o in CN © © in CN o o in CN _H 00 00 co © © © in in CN in CN o >> OH! m Although the MIC of tetracycline was also restored well above that in H730, it should be noted that this was probably due to the presence of a tetracycline resistance gene present in the vector pUCP27. 4.3.2 Complementation of opmG and opmH mutant susceptibility phenotypes Since both H958 and H966 contain the miniTn5 transposon encoding tetracycline resistance, plasmids pJJ106 and pJJ105, which also encode tetracycline resistance, could not be used to complement the original mutant strains. For this reason, the PCR-amplified opmG and opmH genes were separately cloned behind the lac promoter in the E. coli-Pseudomonas shuttle vector pUCP21, encoding ampicillin resistance, to create pJJ107 and pJJ109. Ampicillin-based selection in Pseudomonas is performed using the antibiotic carbenicillin, since the chromosomally-encoded P-lactamase gene of P. aeruginosa is ampicillin-inducible but not carbenicillin-inducible. Plasmid pJJ107 was introduced into strain H958, and pJJ109 was introduced into strain H966 by electroporation. Table IX summarizes the MIC data for the complementation experiments. Strain H958 (opmG::miniTn5-TcR) demonstrated an 8-fold decrease in the MICs of the three aminoglycosides kanamycin, gentamicin, and streptomycin, as well as a 4-fold decrease in the MIC of fusidic acid, as compared to the parent wildtype strain H911. There were no other significant changes in MIC as a result of insertional inactivation of the opmG gene. Introduction of pJJ107 (opmG) into strain H958 resulted in only partial complementation of the MIC of kanamycin but full complementation of the MICs of gentamicin, streptomycin, and fusidic acid. Therefore, OpmG plays a role in aminoglycoside and fusidic acid efflux, but other factors might be involved in kanamycin 55 U > u s-l o < PH HH CO X CO -4—» u T3 N u JO u U oo 3 PH £ £ 00 m in in CO CN CN CN ^H r—' CN CN CN co CO CO ' ' VO oo >—< '—1 o '—1 CO © © o © © IT) in in m in CN CN CN CN CN o © © © © CO CO CO CO CO CO CO CO CO CO vd OO oo oo oo oo o o o o o © © © © © CO CO CO in m '—1 >—1 o o © © © oo ^ o o o O o o in IT) O in o 00 OO CO o o o o CN CN CN CN A A A A in in in in o CN CN CN CN in ' 1 ' 1 CN CN oo O o in o llO in r- in CO co r-A A A m CO o CO m CN in CN CN OO 00 CN oo oo oo o o © © © o CO in CO in o *-H CN ^H CN '—' u + + box X ^ £ E E a E r=3 CH CH CH CH CH PH £ O OOO O + + ^H 00 O0 O vo ^H in m ^H ON ON ON G <3N ON X x X am X o CO O C*H O0" g 3 E PH ^3 ^ u .2 "2 'S ^ C N *J .£ 6 £ g o ex .„ o c E c " <+H o o H3 IH Ipq u pq •§ e :s | E ^ CH CH •fa 3 -£ " '3 | E c ^ oo « c X E g£ CO ^ SH g g C r, fj M-H E G o O ^ u £ H X g ^ C JH cO CO o o o m resistance, since resistance to this compound was not restored. The increase in carbenicillin resistance upon introduction of pJJ107 was due to the pMactamase marker on pUCP21. Strain H966 (op/n/-/"::miniTn5-TcR) showed less dramatic decreases in the MICs of the three aminoglycosides. There was an 8-fold reduction in the MIC of kanamycin, but only 2- and 4-fold reductions in the MICs of gentamicin and streptomycin, respectively. However, there were 8-fold reductions in the MICs of the dyes acriflavin and crystal violet. Reintroduction of the opmH gene in pJJ109 resulted in only 2-fold increases in the MICs to the three aminoglycosides, but also 4-fold (partial but still significant) restoration of the MICs to acriflavin and crystal violet. The ability to efflux the dye acriflavin is of particular interest since this compound has previously been noted to be an inducer of MexX-MexY (Morita et al., 2001). Control pUCP21 and pUCP27 plasmids lacking cloned OpmH or OpmG did not result in any significant alterations to antibiotic resistance except for that conferred by plasmid-borne resistance markers. 4.4 Use of DNA mini-microarrays to assess compensation It has been previously noted that the inactivation of one efflux pump can result in a compensatory increase in expression of another efflux pump (Li et al, 2000) and it would appear to be a reasonable extrapolation that compensatory decreases in expression might also occur. This phenomenon, if present as a result of insertional inactivation of any of the OprM homologues, would be problematic in masking the specific mutant phenotype. DNA mini-microarrays were used to assess expression levels of each of the OprM homologues in the opmG and opmH knockout strains, to determine if the antibiotic susceptibility phenotypes of H958 and H966 might be due to, or affected by, 57 compensatory alterations in expression of another outer membrane channel. Internal 600bp fragments of each OprM homologue and the uvrD gene were PCR amplified from PAOl genomic DNA. The DNA repair enzyme uvrD was previously determined to be constitutively expressed under all tested conditions (Brazas, unpublished data) and was used as an internal standard. RNA was isolated from H911, H958, and H966 and reverse transcribed to cDNA using a 5' primer pool consisting of a mixture of the 5' primers of each amplicon. cDNA was used as template for low-cycle PCR during which a32P-dCTP was incorporated. The resultant radioactive DNA, reflecting the amount of original mRNA, was used to probe the purified PCR amplicons that were spotted on positively charged nylon membranes using a replicator. Figure 6 shows the hybridization pattern for H911 compared with H958, while Figure 7 shows the hybridization pattern for H911 compared with H966. Each figure is a representative example of three independent trials. Table X compares the fold changes in expression of each OprM homologue between strains H911 and H958 (refer to figure 6), while Table XI compares strains H911 and H966 (refer to Figure 7). After background subtraction, fold changes were calculated by dividing the density of each hybridization spot by the density of the uvrD hybridization spot on that membrane. The ratio of the normalized values for each gene between the wildtype and mutant then represent the fold change. Greater than 2-fold changes in gene expression were considered significant. The spot densitometry results in Table X compares the quantified hybridization signals between the opmG mutant H958 (o/jwG::miniTn5-TcR) compared to the parent strain H911. When results were standardized to the level of expression of uvrD, the only significant changes were in the levels of opmG (as anticipated) and oprN message, which 58 were not observed in the mutant H958. Since it is known that OprN does not contribute to aminoglycoside resistance (Seiffer et al., 1993; Saier et al., 1994; Hancock and Speert, 2000), it is unlikely that the downregulation of OprN would be having any effect on the aminoglycoside resistance phenotype of strain H958. Similar expression studies were performed on the mutant H966 (opmH::mm\Tn5-TcR) compared to the parents strain H911, shown in Table XL As expected, opmH was not expressed in the mutant, but there were modest decreases in opmG (1.9-fold) and opml (2-fold) that may have contributed to the H966 resistance phenotype. No alteration in the expression of oprM was observed in either H958 or H966. 4.5 Conclusions By screening transposon insertion mutants of the fifteen uncharacterized homologues of OprM in the Pseudomonas aeruginosa genome, three were found to play a role in aminoglycoside resistance. When each of opmG, opmH, and opml were inactivated, the mutant strains were between 4- and 32-fold more susceptible to aminoglycosides. When opmG and opmH were each introduced into the antibiotic susceptible oprM~ strain H743, broad spectrum drug resistance was restored to H743, indicating that both OpmG and OpmH are channel-forming OMPs that are capable of functioning with RND pumps for drug efflux. In addition, OpmG was able to complement the aminoglycoside susceptiblility of H958. OpmH was able to fully restore resistance to the dyes acriflavin and crystal violet, but only partially restore resistance to aminoglycosides. Mini-microarray experiments were designed to monitor gene expression of each of the OprM homologues, and based on these experiments, compensatory upregulation of other oprM family OMPs is not a factor in masking the effects of knockouts in these 59 Figure 6. Comparison of expression of OprM homologue genes in wildtype PAK (H911; top panel) and the opmG mutant (H958; bottom panel) using mini-microarrays. RNA isolated from each strain was reverse transcribed to cDNA, radiolabeled with [a32P]dCTP, and hybridized to a nylon membrane containing 10 ng spots of 600 bp internal fragments corresponding to each of the OprM homologues. Spot densitometry revealed an absence of oprN expression as well as the expected absence of opmG expression. oprM oprJ oprN uvrD opmA opmB opmD opmE opmG opml * - i. ' _1 opmJ opmO aptF czcC opmF opmH opmK opmL opmM • H911 PAK wildly p.. oprM oprJ oprN uvrD m • • opmA opmB opmD opmE opmG opml m m opmJ opmQ aprF czcC opmF opmH opmK opmL opmM m H958 opmG mutant 60 Table X. Quantitated spot densitometry values for strains H911 and H958. Gene a) Spot density H911a b) Normalized Values H911b c) Spot Density H958a d) Normalized Values H958b e) Fold Change0 1 oprM 50357 1.3 50400 1.2 1.0 2 oprJ 25245 0.62 27874 0.66 0.93 3 oprN 12345 0.27 1364 0 N/A 4 uvrD 39803 1.0 41136 1.0 1.0 5 opmA 46138 1.2 39322 0.95 1.2 6 opmB 4682 0.071 6056 0.10 0.68 7 opmD 86457 2.21 51199 1.2 1.8 8 opmE 15846 0.37 16738 0.38 0.97 9 opmG 16352 0.38 1998 0 N/A 10 opml 16739 0.39 20060 0.46 0.84 11 opmJ 6516 0.12 5671 0.094 1.3 12 opmQ 7390 0.14 7980 0.15 0.93 13 aprF 21691 0.52 17683 0.40 1.3 14 czcC 17018 0.40 13722 0.30 1.3 15 opmF 18690 0.44 31050 0.74 0.59 16 opmH 10998 0.24 10224 0.21 1.1 17 opmK 7804 0.15 6665 0.12 1.3 18 opmL 28289 0.70 27582 0.65 1.1 19 opmM 7731 0.15 8740 0.17 0.88 a Spot density quantities are arbitrary values. b Normalized spot densities correspond to the spot density value for each gene divided by the spot density value of uvrD, i.e. an/a4forH911 and cn/c4for H958. c Fold change is calculated by taking the ratio of the normalized spot density values for the wildtype strain over the normalized spot density values of the mutant strain, i.e. bn/dn. Only differences greater than 2-fold were considered significant. 61 Figure 7. Comparison of expression of OprM homologue genes in wildtype PAK (H911; top panel) and the opmH mutant (H966; bottom panel) using mini-microarrays. RNA isolated from each strain was reverse transcribed to cDNA, radiolabelled with [a" PJdCTP, and hybridized to a nylon membrane containing 10 ng spots of 600 bp internal fragments corresponding to each of the OprM homologues. Spot densitometry revealed the expected absence of opmH expression as well as 1.9 and 2-fold downregulations of opmG and opml expression, respectively. oprM oprJ oprN uvrD opmA opmB opmD opmE opmG opml opmJ opmQ aprF czcC opmF opmH opmK opmL opmM H911 PAK wildtype oprM oprJ oprN uvrD opmA opmB opmD opmE opmG opml opmJ opmQ aprF czcC opmF opmH opmK opmL opmM H966 opmH mutant 62 Table XI. Quantitated spot densitometry values for strains H911 and H966. Gene a) Spot density H91 la b) Normalized Values H911b c) Spot Density H966a d) Normalized Values H966b e) Fold Change0 1 oprM 757 0.83 1034 0.88 0.95 2 oprJ 421 0.37 403 0.25 1.5 3 oprN 271 0.17 520 0.37 0.46 4 uvrD 878 1.0 1158 1.0 1.0 5 opmA 1018 1.2 1871 1.7 0.70 6 opmB 264 0.16 410 0.26 0.62 7 opmD 2143 2.7 2180 2.0 1.4 8 opmE 410 0.36 435 0.28 1.3 9 opmG 650 0.70 424 0.37 1.9 10 opml 517 0.51 401 0.25 2.0 11 opmJ 275 0.18 429 0.28 0.63 12 opmQ 317 0.23 435 0.28 0.82 13 aprF 667 0.71 645 0.49 1.4 14 czcC 574 0.58 485 0.33 1.7 15 opmF 714 0.78 716 0.56 1.4 16 opmH 259 0.15 147 0 N/A 17 opmK 286 0.19 413 0.26 0.72 18 opmL 565 0.57 840 0.69 0.83 19 opmM 206 0.13 435 0.25 0.52 a Spot density quantities are arbitrary values. b Normalized spot densities correspond to the spot density value for each gene divided by the spot density value of uvrD, i.e. an/a4for H911 and cn/c4for H966. c Fold change is calculated by taking the ratio of the normalized spot density values for the wildtype strain over the normalized spot density values of the mutant strain, i.e. bn/dn. Only differences greater than 2-fold were considered significant. 63 strains. However, this does not rule out other secondary mutations, such as mutations in drug targets, that might be having an affect on recorded MICs. While secondary mutations would not alter the role of these OMPs in determining aminoglycoside resistance, assessing the degree of the effect of the mutation or the ability to complement the mutant would be more complicated. It is clear, however, that OMPs other than OprM play a role in intrinsic aminoglycoside resistance, though it is not possible to conclude from these studies if their role also involves the MexX and MexY proteins. 64 DISCUSSION 5.1 Introduction At 6.3 Mb, Pseudomonas aeruginosa has one of the largest bacterial genomes. It is estimated that nearly 10% of the genes encode regulatory proteins, a testament to the environmental ubiquity of this organism (Stover et al, 2000). Several large families of outer membrane proteins have also been identified. The eighteen members of the OprM family have been implicated in forming the outer membrane repertoire of proteins required for efflux of toxic compounds and ions and secretion of extracellular proteins. Eleven of the OprM family members are encoded as part of an efflux operon, containing genes for an inner membrane RND or MFS efflux pump and periplasmic linker protein, and another seven are encoded in operons with genes for ABC-type transporters as well as those for periplasmic linker proteins. Of the 18 members of the OprM family, only opmH is encoded without genes encoding a cognate transporter and linker, the characteristics components of the tripartite efflux and type I secretion systems used to move compounds across the Gram negative double membrane. With seventeen homologous efflux/secretion systems (opmH is not part of an operon with any other components) present in the genome, it is clear that the ability to export harmful substances plays a large role in the ability of this organism to successfully colonize so many differing environments. The intrinsic resistance to antibiotic therapy that these efflux systems afford plays a large role in the success of P. aeruginosa at producing chronic infections in immunocompromised patients (Hancock and Speert, 2000). In particular, the aminoglycoside antibiotics are of particular interest since they are a common therapeutic drug for recalcitrant Pseudomonas infections (Davies and 65 Wright, 1997) and because it was believed until recently that hydrophilic molecules were not substrates for efflux (Nikaido, 1996). This project has examined two possible candidate outer membrane proteins that may function in conjunction with the MexY inner membrane RND efflux pump and the MexX linker in aminoglycoside efflux. Identifying the components of aminoglycoside efflux might provide leads to finding inhibitors of these systems, thus increasing the therapeutic value of currently available antibiotics. 5.2 Phylogenetic Analysis of OprM Homologues Table IV shows that the eleven uncharacterized members of the efflux cluster share between 45 and 55% sequence similarity with OprM or OprN. Likewise, the members of the secretion cluster are highly homologous (over 40% similarity at the amino acid level) with AprF, the outer membrane protein involved in secretion of alkaline protease. Interestingly, when the amino acid sequences of the eighteen OprM homologues are aligned against each other (Figure 5), there are few residues that are conserved among the entire family. A more extensive alignment of OprM-like outer membrane proteins from Gram negative bacteria also reveals few conserved residues, but the observation has been made that many of the conserved regions occur at critical points in the structure, such as the glycine residues present at tight turns and the aromatic amino acids at the membrane interfaces (Andersen et al, 2001). In addition, the phylogenetic analyses of these OMPs show a clustering of members according to the type of substrate exported: protein, cation, or antibiotic (Andersen et al, 2001). Since not all of the channel forming OMPs are encoded as units with inner membrane and periplasmic members (OpmH and E. coli TolC for example), it seems likely that these channel-forming OMPs are adaptable enough to function with more than one pump, a phenomenon noted with both TolC and 66 P. aeruginosa OprM. It would not be surprising to learn that more than one OMP can function with MexX and MexY to confer intrinsic aminoglycoside resistance. While several researchers (Aires et al, 1999; Masuda et al, 2000a; Masuda et al, 2000b) have shown that OprM can be co-expressed with MexX-MexY to confer aminoglycoside resistance, not all aminoglycoside-resistant isolates that highly express MexX and MexY also highly express OprM (Westbrock-Wadman et al, 1999). Determining the identity of the native OMP in particular is crucial to understanding the process of aminoglycoside efflux as well as the intricacies of the efflux of both hydrophilic and hydrophobic compounds by the same system. 5.3 OpmG and OpmH can complement an OprM" defect The genes encoding opmG and opmH were cloned behind the lac promoter of the pUCP27 vector to create the two recombinant vectors pJJ106 and pJJ105. Table VIII shows the ability of both the OpmG and OpmH proteins to complement the OprM" defect of strain H743. The ability to complement the antibiotic supersusceptibility associated with inactivation of oprM m strain H743, with the substrate specificity of MexA-MexB-OprM, demonstrates a number of important points: a) that both OpmG and OpmH are channel-forming outer membrane proteins capable of mediating efflux of antibiotics from the cell, b) that substrate specificity is indeed largely determined by the inner membrane components of the efflux pumps, as has been noted previously (Srikumar et al, 1997; Gotoh et al, 1998), c) that the ability of channel-forming OMPs to function with alternate pump and linker components (Gotoh et al, 1998; Yoneyama et al, 1998) is a common feature of the entire family, and d) that channel-forming OMPs, despite having 67 little sequence identity (Andersen et al, 2001), still share the same structure and mechanism of action of gating necessary to function as efflux OMPs. 5.4 OpmG and OpmH can complement opmG and opmH mutants OpmG was also able to complement the original mutant strains H958 when reintroduced on the pJJ107 vector (Table IX), a finding that supports the hypothesis that OpmG plays a role in aminoglycoside resistance. In contrast, complementation of aminoglycoside susceptibility by pJJ109 (opmH) in strain H966 (opm//::miniTn5-TcR) was only 2-fold. However, there was partial but significant complementation of the MICs to the two dyes acriflavin and crystal violet. The ability to complement an acriflavin susceptibility is a particularly interesting finding since the closest OpmH homologue (TolC of E. coli) was first implicated in the efflux of acriflavin (in conjunction with AcrA-AcrB), and because acriflavin was noted to be an inducer and a substrate for MexX-MexY (Morita et al, 2001). 5.5 Mini-microarray analysis MexC-MexD-OprJ and MexE-MexF-OprN have been shown to be upregulated in response to the loss of MexA-MexB-OprM by an unknown mechanism (10). The possibility that a miniTnJ insertion mutation in any one of the OprM family homologues might result in compensatory up or downregulation of other homologues would prove problematic to assessing the roles of OpmG and OpmH in aminoglycoside resistance. Mini-microarrays were constructed to monitor expression of all members of this family in the wildtype and mutant strains. It should be noted that microarray analysis measures the amount of transcript as opposed to the amount of protein. 68 Figure 2 shows one representative experiment comparing the expression of the 18 OprM homologues in the parent strain H911 (top panel) and the opmG mutant H958 (bottom panel). Quantification and normalization of the hybridization signals to the uvrD internal standard (Table X) showed the loss of opmG expression (as expected) as well as the loss of OprN expression. Since OprN plays no known role in aminoglycoside resistance (Seiffer et al, 1993; Saier et al, 1994; Hancock and Speert, 2000), it is unlikely that this change in OprN expression is influencing aminoglycoside resistance in strain H958. Thus, I conclude that OpmG is the major outer membrane channel responsible for aminoglycoside efflux. As it is the first gene in its efflux operon (in contrast to OprM, which is the third gene in the mexA-mexB-oprM) , it seems possible that OpmG is made at larger levels than its cognate linker and pump proteins, providing an excess of OMP to potentially form efflux complexes with MexX and MexY. Figure 3 shows one representative experiment comparing the expression of the 18 OprM homologues in the parent strain H911 (top panel) and the opmH mutant H966 (bottom panel). Similar quantification and normalization of the hybridization signals showed the expected loss of opmH expression. In addition there were modest decreases in opmG (1.9-fold) and opml (2-fold) expression. It is thus possible that the effect of opmH on aminoglycoside resistance is being exaggerated due to the downregulation of two other outer membrane proteins which also play a role in aminoglycoside resistance (Table VII). Nevertheless, it is clear that OpmH does function as a channel forming outer membrane protein since it can complement a mutant lacking OprM (Table 3), and its ability to partially restore MICs of aminoglycosides, macrolides, fusidic acid, acriflavin, 69 and crystal violet (Table 4), demonstrates that OpmH may play a minor role in resistance to these compounds. 5.6 Summary Despite the efforts of many separate research groups and the array of currently available pharmaceuticals, chronic infection by multi-drug resistant Pseudomonas aeruginosa remains a relevant clinical issue. The sheer number of proteins possessed by all bacteria that are dedicated to the modification, circumvention, and extrusion of toxic compounds, and the notorious ability of bacteria to horizontally transfer the genetic factors encoding many of these proteins, are major barriers to overcoming the problem of antibiotic resistance. Gram-negative tripartite multi-drug efflux systems are of particular concern since many RND- and MF- transporters have a broad spectrum of substrates that seems contrary to our traditional understanding of specific interactions between proteins and their substrates. The possibility exists that selective pressure applied to a population of bacteria in the form of a single antibiotic could result in the generation of a multi-drug resistant strain in a single step. While four RND efflux systems have been characterized in P. aeruginosa, little is known about the signals that regulate their expression. Since the discovery and widespread use of antibiotics did not occur until the 20th century, it is obvious that efflux systems predate their inception into general use. The ability of bacteria to adapt and utilize pre-existing transport systems for the purpose of exporting novel toxic compounds underscores the plasticity that allows them to survive the effects of antimicrobials, both natural and human-made. 70 The demonstration that wildtype P. aeruginosa strain PAK appears to express all eighteen OprM family outer membrane proteins, some almost as strongly as OprM, despite studies that have shown that at least two, OprJ and OprN, are not expressed under normal growth conditions in strain PAOl, emphasizes the limited understanding we have about the control of these efflux systems. Cross-hybridization of the probes to multiple membrane-bound amplicons could account for this, and this possibility needs to be examined, possibly by using the amplicons themselves as probes for the membrane. Nevertheless, it is known that the ability to export harmful substances plays a large role in the ability of this organism to successfully colonize so many differing environments, and the intrinsic resistance to antibiotic therapy that these efflux systems afford plays a significant role in the success of P. aeruginosa at producing chronic infections in immunocompromised patients. In particular, the efflux of aminoglycoside antibiotics are of particular interest since they are a common therapeutic drug for recalcitrant Pseudomonas infections (Davis, 1987) and because it was believed until recently that hydrophilic molecules were not substrates for efflux (Saier et al., 1994). This project has examined the roles of two candidate outer membrane proteins, OpmG and OpmH, and identified one, OpmG, that may function in conjunction with the MexY inner membrane RND efflux pump and the MexX linker in aminoglycoside efflux. Further studies into the regulation of these outer membrane proteins, and the role of a third protein Opml, will be required to evaluate their roles in determining resistance in P. aeruginosa. Identifying the components of aminoglycoside efflux might provide leads to finding ways to inhibit or circumvent these multi-drug efflux systems thus increasing the therapeutic lifespan and value of currently available antibiotics. 71 REFERENCES Aires, J. R., Kohler, T., Nikaido, H., and Plesiat, P. 1999. "Involvement of an Active Efflux System in the Natural Resistance of Pseudomonas aeruginosa to Aminoglycosides." Antimicrobial Agents and Chemotherapy 43(11): 2624-28. Amsterdam, D. 1991. Susceptibility testing of antimicrobials in liquid media, p72-78. In V. Lorian (ed.), Antibiotics in laboratory medicine, 3rd ed. Williams and Wilkins, Baltimore, Maryland. Andersen, C, Hughes, C, and Koronakis, V. 2000. "Chunnel Vision. Export and through bacterial channel-tunnels." EMBO Reports 1(4): 313-8. Andersen, C, Hughes, C, and Koronakis, V. 2001. "Protein export and drug efflux through bacterial channel-tunnels." Current Opinions in Cell Biology 13:412-16. Beinlich, K. L., Chuanchuen, R., and Schweizer, H. P. 2001. "Contribution of multidrug efflux pumps to multiple antibiotic resistance in veterinary clinical isolates of Pseudomonas aeruginosa^ FEMS Microbiology Letters 198: 129-34. Ciofu, O., Beveridge, T. J., Kadurgamuwa, J., Walther-Rasmussen, J., and Hoiby, N. 2000. "Chromosomal P-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa^ Journal of Antimicrobial Chemotherapy 45: 9-13. Davies, J. and Wright, G. D. 1997. "Bacterial resistance to aminoglycoside antibiotics." Trends in Microbiology 5(6): 234-40. Davis, B. 1987. "Mechanism of Bactericidal Action of Aminoglycosides." Microbiological Reviews (MMBR) 51(3): 341-50. de Lorenzo, V., Herrero, M., Jakubzik, U., and Timmis, K. N. 1990. "Mini-Tn5 Transposon Derivatives for Insertion Mutatgenesis, Promoter Probing, and Chromosomal Insertion of Cloned DNA in Gram-Negative Eubacteria." Journal of Bacteriology 172(11) 6568-72. Dinh, T., Paulsen, I. T., Saier, M. H. Jr. 1994. "A Family of Extracytoplasmic Proteins That Allow Transport of Large Molecules across the Outer Membranes of Gram-Negative Bacteria." Journal of Bacteriology 176(13): 3825-31. Gotoh, N., Tsujimoto, H., Nomura, A., Okamoto, K., Tsuda, M., and Nishino, T. 1998. "Functional replacement of OprJ by OprM in the MexCD-OprJ multidrug efflux system of Pseudomonas aeruginosa.'" FEMS Microbiology Letters 165: 21-7. 72 Gotoh, N., Kusumi, T., Tsujimoto, H., Wada, T., and Nishino, T. 1999. "Topological analysis of an RND family transporter, MexD of Pseudomonas aeruginosa." FEBS Letters 458: 32-36. Guan, L., Ehrmann, M., Yoneyama, H., and Nakae, T. 1999. "Membrane Topology of the Xenobiotic-exporting Subunit, MexB, of the MexA,B-OprM Extrusion Pump in Pseudomonas aeruginosa." The Journal of Biological Chemistry 274(15): 10517-22. Hanahan, D. 1983. "Studies on Transformation of Escherichia coli with Plasmids." Journal of Molecular Biology 166: 557-80. Hancock, R. E. W. 1981. "Aminoglycoside uptake and mode of action-with special reference to streptomycin and gentamicin." Journal of Antimicrobial Chemotherapy 8: 249-276, 429-445. Hancock, R. E. W., and Woodruff, W. A. 1988. "Roles of Porin and [3-Lactamase in P-Lactam Resistance of Pseudomonas aeruginosa." Reviews of Infectious Diseases 10(4): 770-5. Hancock, R. E. W. and Lam, J. S. 1998. "Pseudomonas aeruginosa: Infection and Immunity." In (P. J. Delves ed.) Encyclopedia of Immunology, 2nd Edition 4: 2042-45. Academic Press, London. Hancock, R. E. W., and Speert, D. 2000. "Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment." Drug Resistance Updates 3: 247-55. Hancock, R. E. W. and Brinkman, F. S. L. 2002. "Function of Pseudomonas Porins in Uptake and Efflux." Annual Review of Microbiology 56: 17-38. Handfield, M., and Levesque, R. C. 1999. "Strategies for isolation of in vivo expressed genes from bacteria." FEMS Microbiology Reviews 23: 69-91. Herrero, M., de Lorenzo, V., and Timmis, K. N. 1990. "Transposon Vectors Containing Non-Antibiotic Resistance Selection Markers for Cloning and Stable Chromosomal Insertion of Foreign Genes in Gram-Negative Bacteria." Journal of Bacteriology 172(11): 6557-67. Koebnik, R., Locher, K. P., and van Gelder, P. 2000. "Structure and function of bacterial outer membrane proteins: barrels in a nutshell." Microbiology 37(2): 239-53. Kohler, T., Michea-Hamzepour, M., Henze, U., Gotoh, N., Curty, L. K., and Pechere, J. C. 1997. "Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa." Molecular Microbiology 23(2): 345-54. 73 Koronakis, V., Sharff, A., Koronakis, E., Luisi, B., and Hughes, C. 2000. "Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export." Nature 405: 914-19. Levy, S. B. 1992. "Active Efflux Mechanisms for Antimicrobial Resistance." Antimicrobial Agents and Chemotherapy 36(4): 695-703. Li, X., Livermore, D. M., and Nikaido, H. 1994. "Role of Efflux Pump(s) in Intrinsic Resistance of Pseudomonas aeruginosa: Resistance to Tetracycline, Chloramphenicol, and Norfloxacin." Antimicrobial Agents and Chemotherapy 38(8): 1732-41. Li, X., Nikaido, H., and Poole, K. 1995. "Role of MexA-MexB-OprM in Antibiotic Efflux in Pseudomonas aeruginosa'' Antimicrobial Agents and Chemotherapy 39(9): 1948-53. Li, X., Barre, N., and Poole, K. 2000. "Influence of the MexA-MexB-OprM multidrug efflux system on expression of the MexC-MexD-OprJ and Mex-MexF-OprN multidrug efflux systems in Pseudomonas aeruginosa^ Journal of Antimicrobial Chemotherapy 46: 885-93. Mao, W., Warren, M. S., Lee, A., Mistry, A., and Lomovskaya, O. 2001. "MexXY-OprM Efflux Pump Is Required for Antagonism of Aminoglycosides by Divalent Cations in Pseudomonas aeruginosa^ Antimicrobial Agents and Chemotherapy 45(7): 2001-7. Margolles, A., Putman, M., van Veen, H. W., and Konings, W. N. 1999. "The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids." Biochemistry 38(49): 16298-306. Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Tsujimoto, T., and Nishino, T. 2000a. "Contribution of the MexX-MexY-OprM Efflux System to Intrinsic Resistance in Pseudomonas aeruginosa." Antimicrobial Agents and Chemotherapy 44(9): 2242-6. Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Tsujimoto, T., and Nishino, T. 2000b. "Substrate Specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM Efflux Pumps in Pseudomonas aeruginosa." Antimicrobial Agents and Chemotherapy 44(12): 3322-7. Mine, T., Morita, Y., Kataoka, A., Mizushima, T., Tsuchiya, T. 1999. "Expression in Escherichia coli of a New Multidrug Efflux Pump, MexXY from Pseudomonas aeruginosa." Antimicrobial Agents and Chemotherapy 43(2): 415-7. Mingeot-LeClercq, M. P., Glupczynski, Y., and Tulkens, P. M. 1999. "Aminoglycosides: Activity and Resistance." Antimicrobial Agents and Chemotherapy 43(4): 727-37. 74 Moat, A. G. and Foster, J. W. 1995. Microbial Physiology, 3r ed. Wiley-Liss Inc., New York, N.Y. Moore, R. A., DeShazer, D., Reckseidler, S., Weissman, A., and Woods D. E. 1999. "Efflux-Mediated Aminoglycoside Resistance in Burkholderia pseudomallei." Antimicrobial Agents and Chemotherapy 43(3): 465-47. Morita, Y., Komori, Y., Mima, T., Kuroda, T., Mizushima, T., and Tsuchiya, T. 2001. "Construction of a series of mutants lacking all of the four major mex operons for multidrug efflux pumps or possessing each one of the operons from Pseudomonas aeruginosa PAOl: MexCD-OprJ is an inducible pump." FEMS Microbiology Letters 202: 139-43. Nakae, T., Nakajima, A., Ono, T., Saito, K., and Yoneyama, H. 1999. "Resistance to |3-Lactam Antibiotics in Pseudomonas aeruginosa Due to Interplay between the MexAB-OprM Efflux Pump and P-Lactamase." Antimicrobial Agents and Chemotherapy 43(5): 1301-3. Nicas, T. I., and Hancock, R. E. W. 1980. "Outer Membrane Protein HI of Pseudomonas aeruginosa: Involvement in Adaptive and Mutational Resistance to Ethylenediaminetetraacetate, Polymixin B, and Gentamicin." Journal of Bacteriology 143(2): 872-8. Nikaido, H. 1996. "Multidrug Efflux Pumps of Gram-Negative Bacteria." Journal of Bacteriology 178(20): 5853-9. Nikaido, H. 1998. "Antibiotic Resistance Caused by Gram-Negative Multidrug Efflux Pumps." Clinical Infectious Diseases 27(Suppl 1): S32-41. Ochs, M. M., McCusker M. P., Bains. M., and Hancock R. E. 1999. "Negative regulation of the Pseudomonas aeruginosa outer membrane porin OprD selective for imipenem and basic amino acids." Antimicrobial Agents and Chemotherapy 43(5): 1085-90. Paulsen, I. T., Brown, M. H., and Skurray, R. A. 1996. "Proton-Dependent Multidrug Efflux Systems." Microbiological Reviews (MMBR) 60(4): 575-608. Poole, K., Neshat, S., and Heinrichs, D. 1991. "Pyoverdine-mediated iron transport in Pseudomonas aeruginosa: involvement of a high molecular mass outer membrane protein." FEMS Microbiology Letters 78: 1-5. Poole, K., Krebes, K., McNally, C, and Neshat, S. 1993. "Multiple Antibiotic Resistance in Pseudomonas aeruginosa: Evidence for Involvement of an Efflux Operon." Journal of Bacteriology 175(22): 7363-72. 75 Poole, K., Gotoh, N., Tsujimoto, H., Zhao, Q., Wada, A., Yamasaki, T., Neshat, S., Yamagachi, J., Li, X., and Nishino, T. 1996. "Overexpression of the mexC-mexD-oprJ efflux operon in rc/xfi-type multidrug-resistant strains of Pseudomonas aeruginosa." Molecular Microbiology 21(4): 713-24. Postle, K. and Vakharia, H. 2000. "TolC, a macromolecular periplasmic 'chunnel'." Nature Structural Biology 7(7): 527-30. Putman, M., van Veen, H. W., and Konings, W. N. 2000. "Molecular Properties of Bacterial Multidrug Transporters." Microbiology and Molecular Biology Reviews 64(4):672-93. Rosenberg, E. Y., Ma, D., and Nikaido, H. 2000. "AcrD of Escherichia coli Is an Aminoglycoside Efflux Pump." Journal of Bacteriology 182(6): 1754-6. Saier, M. H. Jr., Tarn, R., Reizer, A., and Reizer, J. 1994. "Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport." Molecular Microbiology 11(5): 841-7. Saier, M. H. Jr. 2000. "A Functional-Phylogenetic Classification System for Transmembrane Solute Transporters." Microbiology and Molecular Biology Reviews 64(2): 354-411. Saito, K., Yoneyama, H., and Nakae, T. 1999. "nalB-type mutations causing the overexpression of the MexAB-OprM efflux pump are located in the mexR gene of the Pseudomonas aeruginosa chromosome." FEMS Microbiology Letters 179: 67-72. Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Schweizer, H. P. 1991. "Escherichia-Pseudomonas shuttle vectors derived from pUC18/19." Gene 97(1): 109-21. Seiffer, D., Klein, J. R., and Plapp, R. 1993. "EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli." FEMS Microbiology Letters 107: 175-8. Srikumar, R., Li, X., and Poole, K. 1997. "Inner Membrane Efflux Components Are Responsible for P-lactam Specificity of Multidrug Efflux Pumps in Pseudomonas aeruginosa." Journal of Bacteriology 179(24): 7875-81. Srikumar, R., Paul, C. J., and Poole, K. 2000. "Influence of Mutations in the mexR Repressor Gene on Expression of the MexA-MexB-OprM Multidrug Efflux System of Pseudomonas aeruginosa." Journal of Bacteriology 182(5): 1410-4. 76 Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S. L., Hufnagle, W. O., Kowalik, D. J., Lagrou, J., Garber, R. L., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L. L., Coulter, S. N., Folger, K. R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G. K. -S., Wu, A., Paulsen, I. T., Reizer, J., Saier, M. H., Hancock, R. E. W., Lory, S., and Olson, M. V. 2000. "Complete genome sequence of Pseudomonas aeruginosa PAOl, an opportunistic pathogen." Nature 406: 954-60. Thanabalu, T., Koronakis, E., Hughes, C, and Koronakis, V. 1998. "Substrate-induced assembly of a contiguous channel for protein export from E.coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore." EMBO Journal 17: 6487-96. Thanassi, D. G., and Hultgren, S. J. 2000. "Multiple pathways allow protein secretion across the bacterial outer membrane." Current Opinion in Cell Biology 12: 420-30. van Veen, H. W., Putman, M., Margolles, A., Sakamoto, K., and Konings, W. N. 1999. "Structure-function analysis of multidrug transporters in Lactococcus lactis." Biochimica et Biophysica Acta 1461(2): 201-6. Westbrock-Wadman, S., Sherman, D. R., Hickey, M. J., Coulter, S. N., Zhu, Y. Q., Warrener, P., Nguyen, L. Y., Shawar, R. M., Folger, K. R., and Stover, C. K. 1999. "Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability." Antimicrobial Agents and Chemotherapy 43(12): 2975-83. Yoneyama, H., Ocaktan, A., Gotoh, N., Nishino, T., and Nakae, T. 1998. "Subunit Swapping in the Mex-Extrusion Pumps in Pseudomonas aeruginosa." Biochemical and Biophysical Research Communications 244: 898-902. Yoneyama, H., Maseda, H., Kamiguchi, H., and Nakae, T. 2000. "Function of the Membrane Fusion Protein, MexA, of the MexA, B-OprM Efflux Pump in Pseudomonas aeruginosa without an Anchoring Membrane." The Journal of Biological Chemistry 275(7): 4628-34. Zgurskaya, H. I., and Nikaido, H. 2000a. "Cross-Linked Complex between Oligomeric Periplasmic Lipoprotein AcrA and the Inner-Membrane-Associated Multidrug Efflux Pump AcrB from Escherichia coli." Journal of Bacteriology 182(15): 4264-7. Zgurskaya, H. I., and Nikaido, H. 2000b. "Multidrug resistance mechanisms: drug efflux across two membranes." Molecular Microbiology 37(2): 219-25. Ziha-Zarifi, I., Llanes, C, Kohler, T., Pechere, J. C, and Plesiat, P. 1999. "In Vivo Emergence of Multidrug-Resistant Mutants of Pseudomonas aeruginosa Overexpressing the Active Efflux System MexA-MexB-OprM." Antimicrobial Agents and Chemotherapy 43(2): 287-91. 77 


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