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

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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 S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR THE D E G R E E OF  M A S T E R OF SCIENCE In T H E F A C U L T Y OF G R A D U A T E STUDIES (MICROBIOLOGY P R O G R A M )  We accept this thesis as conforming to the required standard  U N I V E R S I T Y OF BRITISH C O L U M B I A June 2002 © James T. H. Jo, 2002  UBC  Rare Books and Special Collections - Thesis Authorisation Form  Page 1 of 1  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department o f  IAICTO\>(.OIQ^  T^iv^u-tno/c^  The U n i v e r s i t y o f B r i t i s h 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 recentlysequenced 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 OprMhomologues.  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  hi  LIST OF FIGURES  vi  LIST OF TABLES  vii  LIST OF ABBREVIATIONS  viii x  ACKNOWLEDGEMENTS  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  18  2.3. PCR  22  2.4. DNA Sequencing  22  2.5. 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  28  2.7.4. Radiolabelling DNA Probe and Hybridization  29  2.7.5. Autoradiographic Imaging  30  3. R E S U L T S : 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. R E S U L T S : OpmG- and OpmH-mediated intrinsic aminoglycoside resistance 4.1. Introduction  49  4.2. Screening miniTn5 insertion mutants  49  4.3. Complementation of the opmG, opmH, and oprM mutants 4.3.1. Complementation of an oprM mutant susceptibility phenotype  51 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  73  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  44  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 multidrug 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 M u l t i - D r u g E f f l u x Systems of G r a m Negative B a c t e r i a  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 drugfinallyexits 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 PH  G  CD CS  OH  o o  s 0) &  o  03  o 03 l-i 03 o 60  g  IS  '3  . 00 3  03  9 2S z  c -a o3 c SH HO  03  CN  S  o3  SH  — , I  C5  P  03  -a c o  o  S ^ 03 03 O 60  ^  ° o  E  O  c/)  2  CN pj  3  o  E  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 Cterminal 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 MexCMexD-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 MexCMexD-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 coiledcoil 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 selfpromoted 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; MingeotLeClercq 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). ribosomal proteins such as  rpsL  Since mutations to genes encoding  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) ATPdependent phosphorylation, 2) ATP-dependent adenylation, and 3) acetyl-CoAdependent 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.  efflux pump protein, AcrD  coli  (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.  efflux systems of P.  In addition, MexX-MexY was the first of the four characterized 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  P. aeruginosa  oprM  introduced on multicopy plasmids into E.  (Aires et  1999; Masuda et  al,  al,  coli  (Mine et  al,  1999) and  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. membrane pore TolC of E.  coli  The well-studied outer  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  °P  15  m L  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 A i m s 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-Tc  R  Chiron (Pathogenesis Corp.)  H957  H9 1 opmA::miniTn5-Tc  R  Chiron (Pathogenesis Corp.)  H958  H9 1 opmG::miniTn5-Tc  Chiron (Pathogenesis Corp.)  H959  H9 1 aprF::miniTn5-Tc  Chiron (Pathogenesis Corp.)  H960  H9 1 opmlv.miniTn 5'-Tc  R  Chiron (Pathogenesis Corp.)  H961  H9 1 opmE::miniTn5-Tc  R  Chiron (Pathogenesis Corp.)  H962  H9 1 opmL::miniTn5-Tc  R  Chiron (Pathogenesis Corp.)  H963  H9 1 opmD::miniTn5-Tc  Chiron (Pathogenesis Corp.)  H964  H9 1 opmJ::miniTn5-Tc  Chiron (Pathogenesis Corp.)  H965  H9 1 opmN::miniTn5-Tc  R  R  R  R  R  Chiron (Pathogenesis Corp.) To be continued...  18  Table I. Bacterial strains used in this study. Strain  Reference/Source  Description  P. aeruginosa  H966  H911 opmH::miniTn5-Tc  Chiron (Pathogenesis Corp.)  H967  HI03 opmF::miniTn5-Tc  Chiron (Pathogenesis Corp.)  H968  H911 opmB::miniTn5-Tc  H969  H911 opmM::miniTn5-Tc  H730 (K372)  PAO6609(me*9011 amiE200 rpsL pvd9) Pch", Poole etal., 1991  R  R  Chiron (Pathogenesis Corp.)  R  R  Chiron (Pathogenesis Corp.)  deficient in pyochelin production and ferripyochelin receptor production H743 (K613)  K372 oprMy.QUg, OprM-deficient  19  Poole etal., 1993  Table II. Plasmids used in this study. Reference/Source  Plasmid  Description  pCR2.1  TA cloning vector; Ap  pJJ104  pCR2AopmG; Ap  pJJIOl  pCR2.1opmH; Ap  pUCP21  Escherichia - Pseudomonas shuttle vector; Ap Schweizer, 1991  pUCP27  Escherichia - Pseudomonas shuttle vector; Tc  pJJ106  p\JCV27opmG; Tc  pJJ105  pVmiopmH; Tc  pJJ107  pUCPHopmG; Ap  R  This study  pJJ109  pUCP2\opmH; Ap  R  This study  Invitrogen  R  This study  R  This study  R  R  R  Schweizer, 1991 This study  R  This study  R  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 OD o of 0.4 55  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  AAA G G A T C C A T G C C G TTC C C T CTT C  opmG reverse  A A A C T G C A G G T A G A A G A A CTC C C A C G C  opmH forward  A A A G G A TCC C A C A T C G A T C C G G A C  opmH reverse  A A A C T G C A G C G A G C A G G A TGT A C C  oprM-left  C A C C A T G A G C C G C C A A C T GTC C  oprM-nght  TGG G T C A C G G T C TGC T G G TTC C  oprJAeft  CGC A A C CTG CGG CAG A A A C A G C  oprJ-nght  CTT G G C C A G CTT C A G G G C TTC G  oprN-left  G C G C G A G A A G A T T G C CCT G A G C  oprN-right  CTC C T G G C G CTT G C C A T A GTC G  uvrDAeft  A A C GCC CTG ATC GCC A A C A A C C  wvrTD-right  A G C G G C A T G T C C A T G A C C TTC G  opmA-left  C A G TTG CGC GGC G A A C A G A A C C  opmA-right  GTG C A T C G C GTC GTC G A C TTC C  opmBAeft  G A T C C G CCT GCT C A A C G A C A C G  opmB-nght  C C G C C G T C A A A C A G G GTC A T G G  opmDAeft  TTC GGT GCT C G C TGC C A G T T G C  opmD-rlght  T C G A C C A G T TGC C G C A G G T C A C  opmEAeft  G A T GGT C G A A C G CCT GGT C A G C  opmE-vight  GCA GCG CCA TAG CCG TTG A A G G To be continued...  22  Primer  Sequence/Description  opmG-Mt  TCG ACC A A C TGA TCG GCG A A G C  opmG-nght  A T C G C G C C G A G A TTG A G G TTG G  opml-leR  C G G C A C G C A TTT C G A GGT C A G C  opml-nghX  TTG C G C G G C G A A GTC CTT CTG G  opmJ-Mt  C G C T T G C C A G T C A T C T G C GTT G  opmJ-nght  T C G A G G C G G A C G G T G TGT TTC C  opmQ-Mt  G G C C T G G T G TTC G G C TTC A T G C  opmQ-right  CTC GCT G G C CTT G A G GCT TTC C  aprF-lefl  C A G C A C C G G C A A GTC C A A GTC C  aprF-hght  G A A GTT C G C C G C C A C C A G TTC C  czcC-left  A G G A C A CCC GCC A A G GCA ATC G  czcC-right  T C G T C G A G A TGG C G C A G C A A G G  opmF-left  C A A C G G A T C G C G C A G A A G TTC G  opmF-right  G C C TTG A G T T G C A G G C G A T T G G  opmH-MX  G C C A G C A A C T A C G C G GTC A A C G  opmH-nght  C T G CTT C A G G C G C A G GGT A T C G  opmK-leR  G C G A C C C T G C A G A A C A C C TTC G  opmK-right  C T A T G G C T G C G T G C C A G G TTG G  opmL-\e&  A G C G A T A T G G C G C G G GTG A T C C  opmL-right  A G T T G C C G C TCT G C C TCG A A C C  opmM-Mt  C A A C A A G G C G C G C A A C G A CTC C  opmM-xight  C C A GTT C G C G A T CCT C C A GTG C  23  RPM at 4°C for 10 min. The pellet was then resuspended in cold sterile dH 0 and the 2  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 D e t e r m i n i n g M i n i m a l 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 dH 0) at a concentration of 20 ng/ul. The amplicons were denatured 2  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 dH 0) for 10 min and then transferred 2  to neutralizing solution (1 M NaCl, 0.5 M Tris HC1 [pH 7.0] in RNase free dH 0) for 5 2  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 betweenfilterpapers 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 10 cells/ml. All manipulations of RNA were carried out in 8  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 T r a n s c r i p t i o n  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 D N A Probe and Hybridization a P-dCTP was incorporated into DNA during quantitative PCR, carried out as 32  follows: the PCR reaction mixture (50 ul total volume) contained: 5 ul of 10 X Amplification buffer, 1 mM MgS0 , 50 uM of each dATP, dGTP, and dTTP, 50 uCi 4  a P-dCTP (Amersham Pharmacia Biotech), 200 uM of each of the forward (5') and 32  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 a P-dCTP was purified from labeled PCR product using MicroSpin 32  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. a P-labeled DNA probe was denatured at 100°C for 5 min 32  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  O p r M 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.  30  Further  a  5 'b u  Q E a. O o  a, o  O O  S w obo | o a,' £ o co _ c  o  a  4) CO O  0 -  l-l 03  o  co  o t-  as *—•  o3 —(  io  co  £  0 S  oo in  3  s  3  3 03 3  03 »  E  S  3  tD (D  3  03  TD  TD  <D  <D  CO CO <D —  co CO  <D V-H  OH X  '5 o •—  tD  E  co  .3  CO O  u  bO  E  2  e 'Bo  -a  TD  <D O  3  3  S  TD  .3  MH  i  (D  <u <u 3 03 l-c  173  (D  3  <D  3  <H-H  E  o  u  s o 3  3 O  SU  <D  U  3 O  o 3 o3  DO 3  CO  <D co O  & H  tD O  u  CN  l-H  < c  '5  o  CO O K  'I  'o?  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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-pumpOMP 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 RNDfamily 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 RNDA B C - or M F - 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  £  O  o  3  Q.  a.  a. H Q_  o Q.  E  c a E Q.  0>  E  o  o 3 O)  o  S  o s  o  Table V. Efflux genes of the MexA-MexB-OprM homologues. OMP gene  Genes order  PAID#  Strand  Transporter family  Cluster  oprM  ABM  0425-0426-0427  -  RND  Efflux  oprJ  ABM  4599-4598-4597  +  RND  Efflux  oprN  ABM  2493-2494-2495  -  RND  Efflux  mexX-mexY'  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  3  y  b  0  d  b  0  The order of the linker, pump, and O M P 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. The PAID# {Pseudomonas aeruginosa ID #) is the number of the ORF in the completed genome sequence. The family to which the pump protein(s) belong, where R N D = resistance/nodulation/cell division, MFS = major facilitator superfamily, and A B C = A T P binding cassette transporter. M e x X - M e x Y has no known cognate O M P 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 T h e O p r M F a m i l y of O u t e r M e m b r a n e 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  20  40  60  MKR SF L S L A V A A W L S G C S L I PDYQR PEAPVAAAYPQGQAYGQNT GAAAVPAADIGWR MRKPAFGVSALLIALTLGACSMAPTYERPAAPVADSWSGAAAQRQ  :  58  :  55  LWW :  60  GAAID—TLDWK  MIHAQSIRSGLASALGLF SLLALSACTVGPDYRT PDTAAAKIDATASKPYDRSRFE S  MKGTPLLLIASLALGACSLGPDFTRPDRPAPGEroSLQAAAGNPSHLAAAP-LAAQWM  :  56  MKHT P S L L A L A L V A A L G G C A I G P D Y Q R P D L A V P A E F K E A E G W R R A E PRDVFQRG—AWW  :  57  WW :  58  — MKRSY P N L S R L A L A L A V G T G L A A C S V G P D Y Q R P Q S P P P R V A S E H L G E F S G E R R E A P MK PYLRS S L S A L I L L G G C A A V G P D Y A P P S A S A P A S F G A M PAGIDGS — G  VEIEWW  :  - M P F P L L H P W P Q R L A L A S A I L L A A G C V T S E G L E P N A R L Q P A G A L Q A G R S L D G V A L S PAAWPRQDWW  :  65  :  57  M V G S F V G F L W F SAISGCVSTGDIAPEAATLDANALATDHAIQAAAREAG-WPQAQWW M P L A S H LR C V A L A L G I S T A L G C A N RN Q PA P RAE S L D PGL S RVAGT R GDAL P M S M K N L S L I S A C L L L G A C G S T PAPLDSGLAAPSQWRYLAAGRSD  AQWW ASDIR  :  26  G :  38  MNRWGLGVLWLVTALPVAASVNPALS P D V P S M A R E Q G R S V L L S LP CRGG  MRGRRQYARKGRRHGKGAIWLLSLGLP MNRLRACLLS SALLSAS SAQALG 80 QLL E F F R D P Q J j gQQ SFIVDAE  RRL  KQFDDPT  MQL  TLFDDAQE H A L ELYGDQT  100  GG VV A A fEM |EN§RD|RVJiALNVEAFRAQYRIQRADLF DMA E'M EQ:  SG  12 0  :  43  :  27  :  50  :  27  :  23  *  RSGVDGSGTRQRLPGDLSTT-  :  123  G?NAAATGNRQRQPADLSAG-  :  12 0  T SRASADIGKGQQPG  Q R V Q R A :J L D | R S S A A R L Q Q S R A I R R S L G G D A L  55 52  MTMRRLMTWLFGAFLLLLREDAFALG  HRALAGLLCGLLGLVPGAAAYEPDVF GTTGQVAGQAVYDLGGSGLP  :  QWW :  MPILRPLASAGKRACWLLMGLCLGLPALANEAPVSFN MLRRLSLAAAVAAATGVAWAAQPT P  53  SffiDASGNYQRQRTTSAGLFDP  JDLQMHLERSJMQTHAQSVAQFRQAEALVRGARAAFF SyTGNVGKTRSGQGGGDSTVL /RLjjD QAJ?AR jjH Dy R E | R A N L R S A R A L F D D R W L D Q L J Q J J T S Q A G Y S R S I E Q Q L D Y D G -  :  122  :  122  :  123  :  123  R G F D E P A 0 E SL|QRA8AA1LD| L W G A R L D E A K A L L R E N R E E F L I RGGPAF DYQARRRGEVET P — TGLGDRQf DQL G E A Q G T P D | Q I | E A R A R Q A A A T A Q A Q D A A R Q | TfflDAKASYSGIRAPTSVAPAP KVYADPQl DAWlJE K A B D G S P G B A V U H A R V R Q A K S M A G L V E S I E S: QjjjEGKGSLVRHRWPDDYFYGP T L Y Q D P G j 'HLKAAAgR H|JRDJlAAiDAH A R A L L G H L R GAQ G E RW R T E V G Y G Y Q Y G R D G D D Q T L A E  :  117  :  121  KAFGAPE  :  118  SFFDDPQ  :  131  :  123  D S L H Q R A M L N S QD[jGA§VARVRQAQAS A V I AGA P L L E f J N A T L G A S R Q K L L R D S G Y S G L D G Y H L A H E N 1 P Q F Q A § I Q E H E A G R Q Y R A L G R A A L L RgVYSYNRGRSWSDVTQTTT-  :  84  EQA1ERA|RS1PEEJAAVGRETEIASGARQQAGLIPN  :  97  EQVIDLS  :  10 9  -TKTD—  :  86  :  116  TSIS  DgSWSVEDTRQGNRQTS  SDAfflYLGfflRNlRGgRS|YLQRIAQKFDLRVAADAFN KfflWRGDYRANRATE DRTRT S i SVYKEAJJDNJ S Q A D Y L A R K E W P Q A R A G L L |QfflGAGARVGDTRIAFDE P P P T E L S E E A E R I C H P Q T R L B W A N A K A Q A A Q V G I G K S A Y L JR DGRLDASRGYSDMDYRDAP MFASAMPl D Q AA R RA G A HP E RS M A E A D R A G T E V E M A K G G Y Y SfflTMSGGPQEFDFGEIVY AG A Il H LDAYQLA 140  RH  PTFQA  LHERRAGSENRAIGRAGLL 160  S™RYDYNKARNDSTVSQ  p  S  A I  S SQYGVTLGTTAWELDL  NRSEVASSYQVGLALPEYELDLj" v  TEDRVNSERYDLGLDSAWELDL;;  G  K A G K G N Y N H A L A G F DASWELDF*  LPGGSTVSS  GGSGAIST SYSTNLSVSWEVDL| EPRRRLAESYRAGFDAQWEIDLi; AGQQRDIETYRGALDASWEIDLy LGGRY S A I K Y L S L G F N Y D F D L '  G AT T  DA R  g  a -RLRSLRDQSLEQY -RVKSLTDA@LQQY -RIRRQLESSDALS -RVRRELEASDATV -KLRRQLEAHQASL -RLGRLSDAJLARA -RVRRSVEA0EAQA v-v" L -LiG- Q A • G E R A A W Ej A 3R V E  DLARTTSWNNSTEIGLNYKLDL' DEDLHSQWKHTVRLDLSYQLDL, - EVRARIAAIKADA TSDNDAVDSFSAGLSASYEVDF| -GRQAAYRS0LESL GDFKEDRDYDSYVSTLSLQQPL§DYEAFSRYRKGVAQA  VSIAQPLELG£-KRGARVEVJJKRGS NVS P T A T L L G E Y G T R F S L A W V K Q F R T A D E A G R Y R S D G L D L T W Q P L L R D A G W D V T T A P L R L J ? 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RMAEFEIVQT DANRLQLKASVSQTISQVIG; FTVL L RAQDNLATSKAEEAAFKRQLDQANERFD' L S D K T D V L E A DQARLEFSATQQDSILRSAETJ YDALA QRSLAASRQVAELAAQNLEAADAKYRJ AAALSDRLQA LAANAS QDAT L QN T F ALAL AD QV ;LAA S ERRVEAVREHIQRLDGIRE TQARGGDBYADRSELDRA RKLSEAVLVARDDAALDIVETJ SETLFAR EQVVFJAEAQRRALETQLAFNQRAFEEJ EGTRTDLLET LFADEQFRGRSQEIAVRLF, LATEQAQRSAQTTjjVASVAT;  LA S E E AAR AARIAMVAE V SLS QYiDGALRRLA|TRQTLVSREYSFA||IDQRR;  280  300  QTA E GAR AT L AQ Y T R L VAQ D QN A V L L SG  320  1 P A N L P Q G L G L D Q T L L T E V P A G L P S D L QRR  LGL|EQARAEQERHLRQKQQAFNA^L|L|SDD--AAQAIPRSPGQRPKLLQDIAPGTPSELIERR DAR|AATAASVPQLQAEAERARHRMATL |Q | RP—EELTVDLSPRDLPAITKALPIGDPGELIRRR :  LAQSMSMEARLPEVEKNQAHLVNAFFLGYGVGAS RTQFFLKSTQAQAIDLKYQRAQLEHAHAVWVGLP  PG-SLLAELGPARAIPRPPGSVPVGLPSELAQRR  PAQFNLPPVASVPKLPDLPAWPSQLJJERR  PPLTTALE SARYRjgDwgRgEAP—GSGAPILDGGAAAPLAKNLPLGDVDRLgjLQR E A L F F L H N V E A A V P D L E R R R A A T R N A [ J A V F F L L A E A P - Q A F S PPVARASGERLTLRTLGVGDPAGL|JARR !S LF 1KGP—DRGLELQRPQPLNPASLSLPSVLPAEL GRR QTQJIATARQQLSAAEQDIASARIAPAVHL -GEGRTIRRPSLNLAAQPSLPSALPAELGGRR EVPIPETERRIEVIDEEIQLTRNLI; LALRERTFLAALPMLEARRRAALYE AL S RS P-RQLDAPAATCAGIPQLRRALPTGDGWSL ARR SSLBASQRKQLPLLEQQAHEALIT|AT|I|EP—VQALQVAERPFDSLRWPETGAGLPSELSSRR QARFNLAQAQEIEARDSQDAALREfflER|V||APLEIADLAPLGERFQVRPLSPASYTAroRDLAfflAEN QVLADNAQLDLSQAELEQQRTYVQISSTWDEP QPGFARVGGALDAVPASITRGALLRHfflDES QANgLRSEAAI  E A D ^ J A S Q E L N V E E STNQVDSARLAfflLQfflLALD  L S T Q I R A S D A L A A T P I E V D R Q Q A I R T AgQQQ  QASYDTARANRLIAEQRVDDAFQAIIVTITNRDY—SAIEGMRHTLPWPPAPNDAKAWVDTAjJjQQN  QTAJJsQASLAQVRDEGALSNALGVaALRMELAPDTPLRLSGELEAQPDTGFVKAIDEMLAEARREH NLEfflSRAQEQLSLEKGNLQDARNQYAlffVgQEP A D - L V E PE PMSLQRYLAASDMARV?JRE S RARisLTRAEEIAASDRAAAARRT|EAlL@QALEDRELAAPIERFPALRLQPATFEGWRQVA3QRS 340  *  DSLEAEHQ1MAANAS|GAA; DNLAAEHR|RARNAD|(  D RAAERR AASTAD  GVAT D RRAEARWHAATASSGVJ ~  D ASAERKIISANAQIGVJ D VSAERQ A A S T E D I G A A T  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'  380  *  sgTANAGTMSRQ LSGLFDAGSGSWLFQP SfflTGSFGTSSAE MSGLFDGGSRSWSFLP SfflSGFLGFTAGR GSQIGSSAARAWSVGP jNGNFGFESLQ LSSLGDWDHRQFAIGP SAAGGYRSGS LSNMI ST PNRFWSIGP JGGFIGFFALR SGDLGSAS-RAFELAP IRGSIGLVAGN L D A L D E S G T SFNVLNP GAM A G L A A L H T S DVLQAPS RFFQVAP [MASVGFSAVGGG MLEFFRSAKYTYSAGP 5 FAVGAE T SAAT L A G L G G S G A L A Y AAG P THSASLSSGANR A A D T F R N — PYYNLGA GgYASTGKSKSG SENTYNQRYETDSVGI |T JSIGSKYDQTAR D G R G E RVNLIGL SjjvGGASQIRDR YSEGGGDNSRSWDSYA DAVAQY QKGDN D A L GF A N S A A N PLVHYGKYVDE R S I G L flsANLARSHSD QAMAFNGDTRERDRSIGL flEASALRREIG GHPESDSWSL sjfYASSSKTHSA SE STYEQKYDTDSVGL  AE|GAQRHA|EAAAYE|ERN  45  226 223 226 227 235 226 220 233 227 226 224 189 187 241 188 220 181 182  400  420  440  460  I LP:  T A G S L R A S L D Y A K I Q K D I N V A Q Y E K A L Q T A F QE V A D G L A A R G T F T E Q L Q A Q R D L V K A - S  410  LLP  DGGRNRANLSLAEARKDSAVAAYEGTHQTAFREVADALAASDTLRREEKALRALANS-S  408  DLGSVRARLRGAKADADAALASYEQQ|LLALEESANAFSDYGKRQERLVSLVRQSEA-S  411  EGGRLRGRLELREAQQQEAAIDYQRT|LRAWQEVDDAMHDYAANQRRQERLGEAVAQ-N  413  DGGLIGSQVDQAEATYDQTVATYRQTKLDGFREVEDYLVQLSVLDEESGVQREALES-A  418  AFSLPi QFAMT m  mm  iH I  m  410  mm.  S|SWPAgRLGNVRARLRAVEAQSDAALARYQRS|LLAQEDVGNALNQLAEHQRRLVALFQSATH-G  406  VyRWA^JLDRGRVWARIAASEARAQEALI LYDRTQLRALQET D D A F N G Y G A A A D R L R L R L L E A T A - N  418  AISLPMDGGRRRANLAERDADYDLAVGQYNKTIVQALGEVSDDLGKLRSLEQQVIDQRQARDI A  TLP  DGGRLRSQLGEAAAGYDAAVEQYNQT  414  VDALKNISDQLIRLHSVDIQKDFAAQSVAS  412  LMJSWRF P N R E S A R G R L E I S A A A E R D A A L A R F D G A Q L G A L R E V E R A L A L Y A G E R Q R R A D L Q R A L D E  406  NSLAP^JHGRLRAERDRSLARQEELLETYRKAMLTAFADTERSLNSIDGLDRQLHWQQQELEQ  376  Q|SVPHSGGETLAATRQATHRMEKSHYDLDDK|RETLNQVRKMYNQSSSSAAKIRAYEMTVDS  366  S?PLP»PD—RNQGNIYAAQSRADQARDLQRAT|LRLRSEAVQAYDQLRTSEQELALVRRDLLPGA G  OVE  E  NIP  0  NIP  426  PIGDLSRRQAEVRAQVDVENQKIL1EDARQTLEQNVIDAVRDLGTRWRQYQIAQRATALS SGGLT S SQVRE  383  SYQRLNQSEQSREGQRRQWQDTRNLHRAVNTDVEQVQARRQAIIS-N  409  EGFERTYQVRNALARREASEAELADTEQQVSLEVWNNYQSLSVETRSLARTRELVEQ-S  RF RMDTJAQGLSNFRR P T A A Q Q R L E SAKWSADAMQRDI  357  RP.QLQNLF DNGDT L R W R E Q S L T Q Q V T E - S  369  R|JSLP3|EGGRVSAATRQAGDKYAQAQAELDAQS!5ASVINDLHSQFDLTASSLAKVRAYEMAVAA-A  480 DEYYQLADKR  RTB V D N Y  T  GWNQQTVTQQQ  :  47 6  GWDEGRSLWH  :  474  :  472  5GWS PT SDPASG  :  479  TNGATALSNERTVLTLLGSRLTASQC  5GWDSADIERTD  :  484  EM  IGSGDLAPGAGQ  :  47 6 472  DA  RSLFTAQQQLITDRLNQLTSE|N|YKA|  •DNHfflRY D A  N E A L K L A K A P . S E sf  520  500  RSSFLNEIAFIDGSTQRQIALPDI  R A A A Q Q A A I R | R E | iTTDFjjvfl  DA  REQLSAEDAQAQAEVELYRGL!  RRALQSAREQRR,  DS  RQLLDNQEQQVASDEAVSLTL  REALRLAEN'J  K,  ANALEIANEP.  I  iTVDYTDl \.GSYJ!A|  SDGEYHDI  REAARLARERS RSNFDLAMRP.  •.VDF|S|  QKTYDIATLASQRJ RHAYRLARSNRR; QRAFDLSDSRQQ.  \.ETL1T|  RTLVMATRKSI  ^RVNfflDB  R.ALY Q I R E E L A Q A E T A S F VN V  GGW E ACAGAR R C  :  1QQLLVAERQLASLESQQIDLSJ5Q|  ,-GGFQPDSRSAAL  :  484  ITRLFQQQLVQEQVQAARLAAHAS  gGGVGAGADTPAQ  :  4 80  IR S LVAD RARLVDAEM RVAE R Q|j E  IGGWQAASSPSHQ  :  47 8  iGGWQSDRQGLAR  :  472  :  4 42  EAHRSDYLSRRALSIARTEQRLA  G E J VGSYfflDA  1  a  jR T L Y AAQ D AAVQ L R L AR L Q A S §Gj  [ALQALY S A M N E L S K A K Y DY L T A W A R G R F Y /  QSALDSMTRG|EM| SKFNF|D|  IDAIRT L V G V R A Q Y V R A L D A A A Q A R | S I | E R L |  R R K L E I E R E K L R V SRSSN  ]SF1TDLRNVENTQLNALISFLNAQTQ|DLI|  .  QSSLEATEIGBQVI ' T R N I | D | RQSLEWQGR R S | rcsMnEg EQVGELYREQ|E  -  REQVTATRRSVA(  GGWQPSA  ]RQLYAAVRDYNNSRYDYILDT|R|KQ/  540  EDIGHLGQ  :  428  1TLDSKTEISLND  :  492  T L S PADLEALSA  :  44 9  [ A L T A Y A S A E D Q H I R A L G N W Q T SRIJRBJAASIII RLGFWSLR  5RP.DV»JDJ# J J V H R E R F E A E R Q L I N L R I E R K R I E Y R A A SERVNRDI  LDEADLELVAA  IDA  QQFYGARRDLASARYAYLNAWJ?R3RQLA|  560  TAKKEDPQA RGGRS  485  ERLGRVEEGLPPSP  498  479  LAAGETAGANR  487  GVAT DDT S PGVARQR D SRS  491  ATAKAPAE  492  R K L A P E N V P V R A V S SR  496  ENGQ  482  KD  474  NFVSGET PARRRDCATTDCPAPLHTLS KTDTEENRSALN  481  H YLKQDYDPDKDFLPPDLAKAAAEQLQSKPRQQY  493 482  GS  425  YFGAGEGRAQVT A A I R  451  46  :  471  LLGPLLENRLNH  :  423  LEDRDLAVLAA  :  435  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  O p m G - 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 2fold 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  Macrolides  P-Lactams  Fusidic Acid  Clindamycin  Polymixin B  Erythromycin  Rifamycin  Penicillins Carbenicillin Carbapenems  Quinolones  Tetracycline  Nalidixic Acid  Imipenem  Aminoglycosides  Norfloxacin  Meropenem  Gentamycin  Dyes and Detergents  Cephalosporins  Kanamycin  Acriflavin  Cefpirome  Streptomycin  Crystal Violet  Cefsulodin  Tobramycin  Ethidium Bromide  Ceftazidime  SDS  Ceftriaxone  Energy Inhibitors CCCP  Dyes and Detergents  51  Table VII. MICs to aminoglycosides, carbenicillin and tetracycline of P. aeruginosa mutants lacking selected outer membrane channel proteins. MIC(pg/ml) Strain Phenotype  Sm 8  Tm 0.25  Cb 50  Tc 1.3  H911  Parent  Km 100  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  Gm 0.8  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  >  CO  CO  CO  *o 00  1H  CO \D  o  < E  CH  co  u  E  60  O  -o c  c3  o  s OH  O  CO  '—'  ©  ©  O  O  CN  CN  in in  in  CN  CN  CN  A  A  A  ©  o o  © ©  o ©  m  CO  CO  CO  ©  O  ©  ©  m oo  oo  in oo  ©  ©  ©  o  o  >2. fN  CO O  in CN  o H in  rN  CO  ©  A  E oo  CO  m  C  a o c o  e £ "OH  E o  o  > H  E  >2500  E  00  CO CN  oo  © in  o in r-  o in  A  A  o o in  o o in  © © in  o o in  00 ©  00  in  o  A  o in CN  CN  _H  o co E © in  © in CN  CN  OH  m >> O H !  o =3 o PH in  c3  o  CN  u  u  m  in  in  >750  0,  u  CO  ©  >5  cd  N  E  O  ~  u  u  ro  <4-l  X  ^  ,_,  in  CN  CN  CN  A  CN  ©  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-Tc ) demonstrated an 8-fold decrease in the MICs of R  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  >  m  in  in  CN  CN  u  r—  CO ^ H  CN  '  s-l  o CN co  <  ©  HH  CN o  CN  CO  CO  '—  1  ©  IT)  '  '  o  oo  VO  >—<  PH  CN  o o  ©  '—  CO  CO  1  O  ©  in  in  m  in  CN  CN  CN  CN  ©  ©  ©  ©  C*H  O0" 3  g  PH  E ^3  ^  u  CO  CO  CO  CO  X CO  -4—»  u T 3 N  u  CO  CO  CO  CO  "2 'S ^  vd  CO  CO  CO  OO  oo  oo  oo  oo  ©  ©  ©  ©  ©  C  o  o  o  CO  CO  o  o  CO  o  ©  o  '—  >—  U  o  JO oi n  O  o  O 0 0  IT)  u  o  E c  o  in  o  o  o  CN  CN  CN  A  in  in  in  in  CN  CN  CN  CN  '  '  1  A  o in CN  oo  3  PH  o in  O  llO CO  A m  o in  CO  CN  CN  oo  in r-  o in  co  A  u  pq  o OO  CN  1  <+H IH  o  o  A  I"H  H3  pq  A  .„  o c  ©  CO  U  ex  1  oo  ^  * J  og o  m  ©  N  .£ 6 £  in 1  .2  CO  •§ e :s |  E ^  CH  •fa 3 -£ " '3 | E c ^ oo « c  r-  A  m CN  CN  X  E g £ CO  SH  ^  g g r, fj M-H  0 0  OO  £ £  oo  oo  o  o  o o '—'  CO ^H  CN  00  ^ H ON  X  E  CH  E  CH  CH  X  O0  ON  ON  x  m  X  G  o  O  ^ £  u H  X  C  J H  g  ^  CN  box  0 0  in  in  + E  CH  O  o  CO  cO  o  +  + ^ H  ©  +  a OO O O  ^r=3 £C H £  ©  in  u  PH  oo  ©  CO *-H  E  CN  O vo ^H  G <3N a m  o ON  X  m  CH  C  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-Tc ) showed less dramatic decreases in the  MICs of the three aminoglycosides.  R  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 D N A 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 a P-dCTP was incorporated. 32  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-Tc ) compared to the parent R  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-  Tc ) compared to the parents strain H911, shown in Table XL As expected, R  not expressed in the mutant, but there were modest decreases in opml  opmG  opmH  was  (1.9-fold) and  (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 [a P]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. 32  oprM  oprJ  oprN  opmA  opmB  opmD  *  -  i .  opmJ  opmO  aptF  opmK  opmL  opmM  uvrD  opmE '  m  opmA  oprJ  opml  opmF  opmH  _1  czcC  • oprM  opmG  H 9 1 1 P A K wildly p.. oprN  uvrD  •  •  opmB  opmD  opmE  opmG  czcC  opmF  opml  m  m  opmJ  opmQ  aprF  opmK  opmL  opmM  m  H 9 5 8  60  opmG  opmH  mutant  Table X. Quantitated spot densitometry values for strains H911 and H958. c) Spot Density H958  d) Normalized Values H958  e) Fold Change  1.3  50400  1.2  1.0  25245  0.62  27874  0.66  0.93  oprN  12345  0.27  1364  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  10  opml  16739  0.39  20060  11  opmJ  6516  0.12  12  opmQ  7390  13  aprF  14  Gene  a) Spot density H911  1  oprM  50357  2  oprJ  3  a  b  a  b  0  0  0  N/A  N/A  0.46  0.84  5671  0.094  1.3  0.14  7980  0.15  0.93  21691  0.52  17683  0.40  1.3  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  b  Spot density quantities are arbitrary values. Normalized spot densities correspond to the spot density value for each gene divided by the spot density value of uvrD, i.e. a /a forH911 and c /c for H958. 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. b /d . Only differences greater than 2fold were considered significant. n  c  b) Normalized Values H911  4  n  4  n  61  n  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  opmA  opmJ  opmK  oprJ  oprN  uvrD  opmB  opmD  opmE  opmG  aprF  czcC  opmF  opmQ  opmL  opml  opmH  opmM H911  PAK  wildtype  oprM  oprJ  oprN  uvrD  opmA  opmB  opmD  opmE  opmG  opml  aprF  czcC  opmF  opmH  opmJ  opmK  opmQ  opmL  opmM  H966 opmH  62  mutant  Table XI. Quantitated spot densitometry values for strains H911 and H966.  Gene 1  oprM  2  oprJ  3  oprN  4  uvrD  5  opmA  6  opmB  7  opmD  8  opmE  9  opmG  10  opml  11  opmJ  12  opmQ  13  aprF  14  czcC  15  opmF  16  opmH  17  opmK  18  opmL  19  opmM  a  b  a) Spot density H91 l  b) Normalized Values H911 b  c) Spot Density H966 a  d) Normalized Values H966 b  e) Fold Change  0  757  0.83  1034  0.88  0.95  421  0.37  403  0.25  1.5  271  0.17  520  0.37  0.46  878  1.0  1158  1.0  1.0  1018  1.2  1871  1.7  0.70  264  0.16  410  0.26  0.62  2.7  2180  2.0  1.4  410  0.36  435  0.28  1.3  650  0.70  424  0.37  1.9  517  0.51  401  0.25  2.0  275  0.18  429  0.28  0.63  317  0.23  435  0.28  0.82  667  0.71  645  0.49  1.4  574  0.58  485  0.33  1.7  714  0.78  716  0.56  1.4  259  0.15  147  0  N/A  286  0.19  413  0.26  0.72  565  0.57  840  0.69  0.83  206  0.13  435  0.25  0.52  2143  Spot density quantities are arbitrary values. Normalized spot densities correspond to the spot density value for each gene divided by the spot density value of uvrD, i.e. a /a for H911 and c /c for H966. 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. b /d . Only differences greater than 2 fold were considered significant. n  c  a  4  n  4  n  63  n  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 O p m G and O p m H can complement an O p r M " 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-MexBOprM, 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 O p m G and O p m H 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-Tc ) R  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 S u m m a r y  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 20 century, it is obvious that efflux th  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). 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