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The role of OprH in the uptake of antibiotics across the outer membrane of Pseudomonas aeruginosa Young, Michele Louise 1992

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THE ROLE OF OprH IN THE UPTAKE OF ANTIBIOTICSACROSS THE OUTER MEMBRANE OF Pseudomonas aeruginosabyMICHELE LOUISE YOUNGB .Sc., McMaster University, 1990A THESIS SUBMI1TED IN PARTIAL FULFILLMENT OF THEREQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Microbiology)We accept this thesis as conformingto the required stapdardTHE UMVERSITY OF BRITISH COLUMBIAJuly1992© Michele Louise Young, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of (2,,The University of British ColumbiaVancouver, CanadaDate____________________DE-6 (2188)11ABSTRACTThe oprH gene of Pseudomonas aeruginosa was mutated byinserting a 1.4 kilobase pair fragment, encoding tetracyclineresistance, into the cloned gene followed by homologousrecombination into the P. aeruginosa chromosome. Growth of theresultant oprH::tet mutant in Mg2-deficient medium had little effecton susceptibility to polymyxin B, gentamicin or EDTA unlesscomplemented by the cloned oprH gene in plasmid pGB25. Incontrast growth of the parent strain on Mg2+deficient mediumresulted in high expression of OprH and resistance to all three agents.These data support the hypothesis that overexpression of OprHunder Mg2+-deficient growth conditions is obligately required forresistance to these 3 agents.OprH overexpression in P.aeruginosa also resulted insupersusceptibility to fluoroquinolones and chloramphenicol.Addition of the nucleosides deoxyadenosine or deoxycytosine did notreduce the MIC values for the quinolones or chioramphenicolrespectively as could be expected if OprH was a nucleoside-specificchannel. Addition of the base adenine as a potential competitor foruptake of either nalidixic acid or ciprofloxacin did not alter the MICvalues for the OprH-overexpressing mutant Hi 81. Overexpression ofOprH in the naiB strain PA06002 did not reverse the quinoloneresistance phenotype as it did for the parental strain PA0505. OprHoverexpression did not alter the susceptibility of E. coil to nalidixicacid, ciprofloxacin or chioramphenicol. Black lipid bilayer analysis ofFPLC-purified OprH did not result in an increase in membrane111conductance. These data suggest that OprH does not act as a porinprotein and that OprH mediates in a unique non-porin uptakepathway.ivTABLE OF CONTENTSPage1. ABSTRACT ii2. LIST OF TABLES v i3. LIST OF FIGURES viii4. ACKNOWLEDGEMENTS ix5. INTRODUCTION 16. MATERIALS AN]) METHODS 7A. Strains and media 7B. Plasmids 7C. Antibiotics 1 2D. Construction and characterization of the OprH-deficientmutant strain H703 1 2E. DNA techniques 1 4F. Southern blotting 1 4G. Biparental mating experiments 1 4H. Antibiotic susceptibility testing 1 4I. MIC determination 1 5J. Cell envelope isolation 1 6K. SDS-polyacrylamide gel electrophoresis 1 6VL. Selective solubilization of Opril.1 6M. FPLC purification of OprH 1 7N. Black lipid bilayer experiments 1 87. RESULTS 1 9A. Characterization of the OprH-deficient strain H703 1 9B. Antibiotic susceptibility testing to polymyxin B, gentamicinandEDTA 19C. Influence of OprH overexpression on susceptibility toquinolones and chioramphenicol 2 3D. Effect of excess monovalent and divalent cations onfluoroquinolone susceptibility to quinolones 28E. Is OprH a porin? 3 0F. OprH overexpression in E. coli 4 18. DISCUSSION 449. LITERATURE CITED 48viLIST OF TABLESTable Page1. P. aeruginosa strains.92. E. coli strains 1 03. Plasmids 114. Influence of the OprH phenotype on killing by polymyxin B,gentamicin and EDTA 235. Influence of OprH expression on MIC values of P. aeruginosa forvarious antimicrobial agents 2 66. Susceptibilities of various P. aeruginosa strains which vary inthe amount of OprH to polymyxin B and ciprofloxacin 277. The effect of excess monovalent or divalent cations on the MICvalue for ciprofloxacin 298. Nucleoside uptake assay in the wild-type strain; H103 3 29. Nucleoside uptake assay in the OprH-deficient strain; H703 3 310. Nucleoside uptake assay in the OprH-overexpressing strain;H181 3411. MIC assay using adenine as potential competitior forciprofloxacin and nalidixic acid uptake 3 512. Effect if OprH overexpression on the nalB4 strain PA06002 of P.aeruginosa 37vii13. Influence of OprH expression on fluoroquinolone susceptibility ofE.coli 43viiiLIST OF FIGURESFigure Page1. Diagram of plasmid pGB32B utilized for gene replacementmutagenisis 1 32. Comparison of 11103 and the OprH-deficient strain H703 bySouthern blotting 203. SDS-polyacrylamide gel electrophoresis of cell envelopepreparations demonstrating expression of Opril on Mg2+sufficient medium 2 14. Cell envelope preparation of P. aeruginosa nalB4 strain PA06002induced for OprH expression 3 65. Fast performance liquid chromatography profile of OprHpurification 3 86. FPLC-purified OprH 3 97. Black lipid bilayer analysis of FPLC-purified OprH 408. SDS-polyacrylamide gel analysis of OprH overexpression in E colistrainHBl0l 42ixACKNOWLEDGEMENTI wish to thank Manjeet Bains for all of her technical assistance ingetting me started in the laboratory. I also thank Francis forintroducing me to the wonderful world of antibiotics. I gratefullyacknowledge Dr. R.E.W. Hancock for his advice and directionthroughout my project. Especially, I thank my parents for their loveand support which has kept me smiling.1INTRODUCTIONPseudomonas aeruginosa is a gram-negative pathogen which is oneof the main causes of nosocomial infections in the hospitals of NorthAmerica. Individuals which are at risk from Pseudomonas infectionsinclude those who are compromised by ailments such as cancer,leukemia, cystic fibrosis or severe bodily infections. The most commonsites of infection include the lower respiratory tract and the urinarytract, as well as sites of a severe burn or wound. With the worldwideincrease of P. aeruginosa infections, chemotheraputic therapy has beenprominent.A major problem in the treatment of such infections is the highintrinsic resistance of P. aeruginosa to many of the conventially usedantibiotics. It has been well established that low outer membranepermeability contributes substantially to this resistance (15). Thecomposition of the outer membrane has been studied in detail and hasbeen shown to consist of an assymetric membrane, rich in proteins. Thebilayer may be strongly associated, either covalently or non-covalentlythrough interactions of its proteins with an underlying peptidoglycanlayer. The inner monolayer of the outer membrane comprisesphospholipid while the outer monolayer consists mainly of one or twospecies of lipopolysaccharide (LPS) molecules which are often categorizedinto three domains; the inner, hydrophobic lipid A, the coreoligosaccharide and the outer, hydrophilic, 0 side-chain polysaccharide.The P. aeruginosa outer membrane is highly negatively charged due tothe high amount of phosphate residues in the core- and 0-regions of theLPS (17). A contributing factor to the high stability of the P. aeruginosa2outer membrane is the presence of divalent cations such as Mg2+ andCa2 which non-covalently cross-bridge the LPS at negatively chargedsites.In recent years, there has been a significant increase in ourunderstanding of the mechanisms of uptake of antibiotics across theouter membrane of P. aeruginosa . Antibiotics cross the outer membraneboth by porin and non-porin routes. Hydrophilic antibiotics within acertain size limit, including B-lactams, are able to cross the outermembrane by diffusion through the water-filled channels of porinproteins. While, hydrophobic molecules such as rifampicin andtrimethoprim are generally restricted from penetration, however, severalhydrophobic drugs have been shown to diffuse through the LPSphospholipid bilayer of mutants (17). Conversely, polycationic antibioticssuch as polymyxin B and aminoglycosides access the self-promoteduptake pathway (17,15,28). This pathway involves the competitivedisplacement, by these bulky polycations, of the divalent cations whichnon-covalently crossbridge adj acent LPS molecules. Consequently, theouter membrane becomes distorted and more permeable, permittingincreased (self-promoted) uptake of the permeabilizing compounds. Theisolation of outer membrane mutants with altered susceptibility to theseagents argues powerfully that self-promoted uptake is relevant toeventual cell killing (17). The classical outer membrane permeabilizer,EDTA, a divalent cation chelator, functions by accessing the same divalentcation binding sites on LPS, as do these polycationic antibiotics (28).Nicas and Hancock (28) proposed that the P. aeruginosa outermembrane protein OprH (= Hi) blocked the self-promoted uptakepathway. Thus, overexpression of OprH by twenty-fold or more, either3as a result of adaptation to Mg2-deficient medium in the parent strainH103, or due to a mutation in strain 11181, was associated with resistanceto polymyxin B, gentamicin and EDTA (3,17,28). In contrast there was nochange in susceptibility of these cells to 8-lactams or tetracycline. It washypothesized that OprH acts by binding to LPS sites which are normallyoccupied by divalent cations, thereby preventing access of polymyxin,gentamicin and EDTA to these sites (17). Consistent with this theory,sequence analysis of the oprH gene indicated that OprH was a basicprotein (theoretical p1 = 8.6) permitting potential association withnegatively charged LPS molecules, whereas the OprH protein was shownto copurify with LPS (2). In addition, rough mutations altering P.aeruginosa LPS have been shown to abolish OprH mediated resistance topolymyxin B (2). Furthermore, evidence indicates that OprHoverexpression is associated with a decrease in the amount of divalentcations associated with the cell envelope (28).Antibiotic susceptibility testing has indicated that theoverexpression of OprH from the cloned gene in the wild type strainH103/pGB25 resulted in only partial resistance to EDTA, ambiguous datafor aminoglycosides and no resistance to polymyxin, compared to theOprH-overproducing mutant H181 which was resistant to all 3 agents(17). This led to the hypothesis that strain H181 (and strain H103adapted to Mg2-deficient medium which had an identical phenotype)contained a second alteration. Based on pseudorevertant studies, it wasproposed that this second alteration involved LPS (17). However, thesedata led us to reexamine whether OprH had any role in polymyxinresistance.4A second aspect of this research involves fluoroquinoloneantibiotics which have recently emerged as one of the most effectiveclasses of antibiotics against P. aeruginosa infections. Their broadspectrum of activity as well as their potential to be delivered orally topatients have been important in their clinical use as therapeutic agents.In P. aeruginosa, the outer membrane has been proposed to act as amajor permeability barrier to the antimicrobial activity offluoroquinolone antibiotics (1,7). Mutations which lead to decreaseddrug permeation through the disruption of uptake pathways areprominent. For example some studies indicated that alterations ordeficiencies in the outer membrane porin protein OprF of P. aeruginosa(7,30) are observed in mutants with decreased fluoroquinolonesusceptibility. Alternatively, the decreases in the amount of OprG (4) orthe lack of OprD (11,26) have been associated with quinolone resistance.Other studies have indicated that the acquisition of a 54 kDa outermembrane protein is associated with reduced fluoroquinolonepenetration (22). In contrast, Chamberland et al (5) could show nocorrelation between OprF deficiency or 54 kDa protein acquisition andresistance to quinolones. Yamano et al (34) proposed that outermembrane proteins C, D and E of P. aeruginosa played a role in quinoloneresistance (34). In addition it has been demonstrated that mutants withLPS modifications of the outer membranes of both E. coli and P.aerug inosa had altered fluoroquinolone permeation (5,6,19,23), whereasquinolones have been reported to penetrate the outer membrane of E.coli (18) and Salmonella typhimurium (19) through the lipid bilayer.Clearly these studies have provided conflicting data. Therefore, Iconsidered here the possibility that the various phenotypic alterations of5the above mutants were secondary manifestations of a primary mutationaffecting a non-porin pathway of uptake across the outer membrane.The self promoted (non-porin) uptake pathway (3,15,28) has alsobeen suggested as a contributing mechanism in fluoroquinolone uptake.Chapman and Georgopapadakou (6) have provided evidence that E. colicells treated with the fluoroquinolone fleroxacin release large amounts ofendotoxin (LPS) as well as display an increase in outer membranepermeability. In addition, excess magnesium was demonstrated toreduce fleroxacin uptake and increase the minimal inhibitoryconcentration for killing. Fluoroquinolones were also shown to act asdivalent cation chelating agents. Chapman et al (6) proposed thatfluoroquinolones might promote their own uptake through the chelationof divalent cations which cross-bridge adjacent LPS molecules, thusresulting in destabilizing of the outer membrane, thereby promotingtheir own uptake. Previously Nicas and Hancock (28,29) haddemonstrated that OprH overexpression in P. aeruginosa correlated withincreased resistance to the polycationic polymyxin B, gentamicin as wellas to the divalent cation chelator EDTA and it was proposed that OprHblocks the self-promoted uptake of these compounds.The initial aim of this project was to characterize a constructedOprH-deficient strain of P. aeruginosa. Through the use of the resultantOprH-deficient strain it was possible to demonstrate that OprH had anobligate role in polymyxin/gentamicin/EDTA resistance due to adaptationto Mg2-deficient growth conditions. I then proceeded to examine ifblocking the self-promoted uptake pathway through OprHoverexpression would lead to quinolone resistance. In contrast, I reporthere that OprH overproduction in P. aeruginosa rendered the cells6supersusceptible to the fluoroquinolones ciprofloxacin, norfioxacin andfleroxacin as well as to chioramphenicol. Therefore, I examined whetherthis supersusceptibility reflected a role for OprH as a porin forfluoroquinolone uptake across the outer membrane.7MATERIALS AND METHODSStrains and media. P. aeruginosa strains used are described in Table1. For short term maintenance of P. aeruginosa strains were maintainedon low-salt Luria Broth (LB) agar plates containing 1% Bactotryptone,0.5% Yeast extract, 0.1% NaC1 and 2 % agar. Exceptions included the OprHoverproducing strain H181 which was maintained on LB agar containing8 .tg of polymyxin B per ml and the OprH-deficient strain H703 whichwas maintained on LB agar containing 150 ig per ml of tetracycline-hydrochloride. The E. coli strains used (Table 2) were maintained short-term on normal salt LB agar as described in Maniatis et at (25). LBingredients were obtained from Difco Laboratories, Detroit, Michigan.Antibiotic susceptibility testing and MIC determination for P. aeruginosawas carried out in basal medium 2 (BM2) broth (9) or agar (2%)containing 0.4 % glucose as a carbon source and 10 iM FeSO4 . The P.aeruginosa strains PA0505 and PA06002 required 1 mM methionineand 1 mM leucine for growth in minimal medium. L-methionine and Lleucine were obtained from Sigma Chemical Co., St. Louis, Mo.Plasmids. Plasmids are been described in Table 3. The expressionvector pNM185 (27) and its derivative pGB25 containing the cloned oprHgene under control of the m -toluate regulatable tot promoter, wereinduced in P. aeruginosa by the addition of 5 mM meta-toluate. Inaddition, shuttle vector pRK767 and its oprH-containing derivativepGB23 under control of the lac promotor were induced for op rHexpression in E. coli. OprH expression was induced by the addition of81mM isopropylthio-13-D-galactoside (IPTO) obtained from BethesdaResearch Laboratories, Burlington, Ontario. Antibiotic concentrationsused to maintain the piasmids were tetracycline-hydrochloride at aconcentration of 150 jig/ml for P. aeruginosa and 12.5 jig/ml for E. coliand kanamycin acid sulfate at a concentration of 300 jig/mi for P.aeruginosa and 25 jig/mi for E. coli.Antibiotics. Tetracycline-hydrochloride, ampicillin, kanamycin acidsulfate, chioramphenicol, polymyxin B sulphate, nalidixic acid,ciprofloxacin and trimethoprim were obtained from Sigma Chemical Co.,St. Louis, Mo. Cefpirome was a gift from Hoechst-RousselPharmaceuticals Inc., Somerville, N. J. Fieroxacin was a gift fromHoffman-La Roche Inc., Nutley, N.J. Norfioxacin was a gift from Merck,Sharp and Dohme Research Lab, Rahway, N.J. Rifampicin was purchasedfrom Boehringer Mannheim, Laval, Quebec. Gentamicin sulphate waspurchased from ICN Biomedicais Inc., St. Laurent, Quebec.Construction and characterization of the OprH-deficient mutantstrain H703. Construction of the OprH deficient mutant was performedby Manjeet Bains and proceeded as follows: the 1.4 kb tetracycline genewas excised from pUC18T2 with Pstl and inserted between the tandemPstl sites placed 15 base pairs apart in the oprH gene of piasmid pGB32(2). Plasmid pGB32 was linearized using EcoRl and cloned into theEcoRl site of the gene replacement vector pRZ1O29Table 1. P. aeruginosa strainsStrain Relevant Characteristicsa Source or Reference11103 wild-type PAO1 prototroph This laboratory111 81 Pxr, OprH-overexpressing This laboratorymutant of H10311703 OprH::Tc mutant of H103 This studyPA0505 parental strain of PA06002 Rella et al, 1982methionine auxotrophPA06002 nalB4 mutant of PA0505 Rella et al, 1982methionine auxotropha Abbreviations: Pxr, polymyxin B-resistant; Tcr, tetracyclineresistant; Cipr, ciprofloxacin-resistant; Nalr, nalidixic acid-resistant10Table 2. E. coli strainsStrain Relevant Characteristics Source or ReferenceHB1O1 F, hsd S20 (r-b, rn-b), recAl3, This laboratoryproA2, rps L20(Smr), ara-14S 17-1 pro res- mod :: RP4 Simon et al, 1983Tc :: Mu Km Tn711Table 3. PlasmidsPlasmids Relevant Properties Source or ReferencepUC1 8T2 Co1E1 Tcr Ampr plac lacZ + S. LoryapGB32 pUC18 containingoprH on a A.Bell, Ph.D. thesis,19891.8 kB BamHl-Sall fragmentpRZ1O2 Co1E1 Tn5 Kmr Jorgensen, R.A. et al,1979pGB32A ColE 1 Ampr plac lacZ This studyoprH: :tetpGB32B EcoRl linearized pGB32A This studyinserted into pRZ1O2pGB25 2.8 kb EcoRl fragment containing Bell, et al, 1989oprH gene in pNM185pNM185 Kmr SmrJ2m (TOL) mob tra Mermod, N.,et al, 1986pGB23 2.8 kb EcoRl fragment (OprH) in Bell, Ph.D. thesis, 1989pRK767pRK767 Tcr plac lacZ + Ditta, G.S., et al, 198512(27) which contains a mobilization site necessary for transfer to P.aeruginosa however lacks a Pseudomonas origin of replication. Theresultant plasmid pGB32B (Fig. 1) was then transformed into themobilizing E. coli strain S17-1 [pro res- mod::RP4 (Tc::Mu Km::Tn7)](33), and transferred by conjugation into strain 11103. Since pGB32Bcould not replicate in P. aeruginosa, isolation of colonies resistant to 150.tg/ml tetracycline resulted in selection for mutants in which thechromosomal oprH gene was replaced by homologous recombination bythe interposon-mutated oprH::tet gene.DNA techniques. All DNA manipulations such as plasmid purification,restriction enzyme digestions, ligations, transformations and agarose gelelectrophoresis proceeded as previously described by Maniatis et al(25).Southern blotting. Alkaline southern blotting was carried out using aZeta-probe cationic nylon membrane (Bio-Rad Labotatories, Richmond,California). 32 random hexamer labelling of the oprH gene was carriedout as described previously (10). Restriction enzymes were obtainedfrom the Bethesda Research Laboratories (Burlington, Ontario) and usedas recommended.13Figure 1. Diagram of plasmid pGB32B utilized for gene replacementmutagenisis. The thick bar between the BamHl and Sail restrictionsites represents Pseudomonas DNA flanking (white bars) or encoding(black bars) the oprH gene whereas the stippled bar represents the Pstlfragment containing the tetracycline (Tc) resistance gene that was usedto interrupt oprH . The orientation of the oprH gene is given by N and C.Abbreviations: Km = kanamycin resistance gene, part of Tn5; Amp =ampicillin resistance gene; on = E. coli specific origin of replication, mob =mobilization sequence.30on EcoRlBamHl14Biparental mating experiments. Biparental mating was carried outas a modification of the triparental mating experiments previouslydescribed by Goldberg and Ohman (12). E. coli strain S17-1 cellscontained both mob and tra genes necessary for plasmid transfer to P.aeruginosa. Colonies containing the plasmid of interest were selected onthe basis of antibiotic resistance as previously described.Antibiotic Susceptibility testing. Antibiotic susceptibility wasexamined by cell killing assays using polymyxin B, gentamicin and EDTATris. The P. aeruginosa strains used were 11103, 11181 and 11703. Inaddition, H703 cells containing plasmids pGB25 and pNM185 were tested.Cells were grown to an O.D.600 of 0.6 in BM2-glucose medium using eitherMg2- sufficient (500 tM) or Mg2+ -deficient (20 jiM) conditions. Cellswere diluted 1000-fold into pre-warmed (37°C) 30 mM sodiumphosphate (pH 7.0) buffer or in 50 mM Tris-hydrochioride (pH 8.5). Thecells were then subject to killing for 5 mm at 37°C with the inclusion ofthe appropriate killing agent; polymyxin B (final concentration; 4 ug/ml),gentamicin (final concentration; 80 ug/mi) or 10 mM EDTA. Theinclusion of 0.4 % glucose was necessary for gentamicin killing. Cellswere then diluted and plated for viable counts on proteose peptone agarno. 2 (Difco Laboratories, Detroit, Michigan). Experiments were done 3times and statistically analyzed by the NCSS (Number Cruncher StatisticalSystem) computer program.MIC determination. To determine the minimal inhibitoryconcentration (MIC) value, each strain was grown overnight in BM2minimal medium containing 0.4% glucose as the carbon source for H103,15H181, H703, PA0505 and PA06002. MIC testing for E. coli was carriedout in LB medium. Agar plates of the appropriate growth mediacontaining serial two-fold dilutions of the appropriate antimicrobialagents were inoculated with approximately i0 cells in 10 pi volume.MICs were performed at least 3 times and were assessed after an 18 to32 hr incubation at 37°C. The MIC value was taken as the lowestantibiotic concentration at which cell growth was inhibited. MICcompetition assays were carried out in multiwell dishes in BM2 medium.Nucleosides used as potential competitors were 2’-deoxycytidine and 2’deoxyadenosine monohydrate which were obtained from ICNBiochemocals, St. Laurent, Quebec. The base adenine which was used incompetition assays with nalidixic acid and ciprofloxacin was obtainedfrom Sigma Chemical Co., St. Louis, Mo..Cell envelope isolation. Cells were grown to an O.D.600 of 0.6 to 0.8 inLB medium or in BM2 minimal medium with vigourous aeration at 37°Cwith appropriate antibiotic selection. Cells were then pelleted bycentrifugation in the Sorvall RC-5B rotor at 7,000 rpm for 10 minutes.The pellet was resuspended in 10 mM Na2PO4, 5 mM MgSO4, pH 7.4 withthe inclusion of pancreatic DNase 1 at a final concentration of 50 tg/ml.The suspension was then French pressed twice at 14,000 pounds persquare inch. Cell debris was removed by centrifugation at 3,000 rpm for10 minutes. The supernatant was centrifuged at 45,000 rpm for 1 hrand the cell envelope-containing pellet was resuspended in distilledwater.16SDS-polyacrylamide gel electrophoresis. Cell envelopepreparations were analyzed by SDS-PAGE using a 14% acrylamide gelpreviously described by Hancock and Carey (13). Solubilization prior toSDS-PAGE involved the dilution of the outer membrane protein sample ina 1:1 ratio with a reduction mix containing 4% (wt/vol) SDS, 20%(vol/vol) glycerol, 40 mM EDTA and 0.125 M Tris-hydrochioride (pH 6.8).As OprH is generally observed after SDS-PAGE in both its native 18 kDaform and its 21 kDa heat-modified form (2), to maximize the amount ofOprH in the 21 kDa band, samples were pretreated with 2% (vol/vol)trichioroacetic acid and heated at 100°C for 10 mm prior to loading.Selective solubilization of OprH. The OprH-overproducing strainH181 was used for the isolation of OprH. Cells from mid-logarithmicphase cultures (optical density at 600 nm of 0.6 - 0.8) were harvested at7,000 rpm in a Sorvall RC-5B rotor for 15 mm. The pellet wasresuspended in 20% (wt/vol) sucrose, 10 mM Tris-HC1 (pH 8.0) with theinclusion of 50 .tg of pancreatic DNase I (Sigma Chemical Co., St. Louis,Mo.) per ml. The cells were broken in a French Press at 14,000 psi andwhole cells removed by centrifugation at 3,000 rpm for 10 mm. A twostep sucrose gradient (50% and 70%) was set up onto which the samplewas applied. Overnight centrifugation at 100,000 x g in a Beckman 5W27rotor allowed separation of outer and inner membranes. The more denseouter membrane fraction was collected and diluted with distilled waterto a final sucrose concentration of approximately 5% and centrifuged at45,000 rpm for 1 hr. The pellet was resuspended in 10 mM Tris-HC1 (pH8.0), 0.5% octyl polyoxyethylene (octyl-POE; Bachem Bioscience Inc.,Philadelphia, PA) and sonicated 3 x 10 seconds using the Biosonik17sonicator (Bronwill Scientific, Inc., Fochester, N.Y.) and centrifuged at150,000 x g for 1 hr. The soluble fraction was retained for analysis andthe pellet was resuspended in 10 mM Tris-HC1 (pH 8.0), 3% octyl-POE andcentrifuged at 150,000 x g for 1 hr. The soluble fraction was retainedand the pellet subject to a second solubilization using 3% octyl-POE. Thefourth and fifth solubilization steps were carried out in 10 mM Tris-HC1(pH 8.0), 3% octyl-POE, 50 mM EDTA. All fractions were tested for thepresence of OprH by SDS-PAGE. OprH was primarily solubilized in 10mM Tris-HC1 (pH 8.0), 3% octyl-POE, 50 mM EDTA.FPLC purification of OprH. Fast Performance Liquid Chromatography(FPLC) was carried out in order to further purify OprH from othercontaminating proteins within the fraction obtained by selectivesolubilization. The column used was a Mono Q anion exchanger(Pharmacia, Laboratory Separation Division, Uppsala, Sweden). Thestarting buffer contained 10 mM Tris-hydrochioride (pH 8.0), 10 mMEDTA and 1% octyl-POE. A sample containing 6-8 mg of protein wasloaded onto the column and the proteins were eluted gradually as thesalt concentration increased from 0 to 1 M. Fractions were collected in 1ml aliquots throughout the FPLC run and tested for the presence of OprHby SDS-polyacrylamide gel electrophoresis.Black lipid bilayer experiments. The method has been previouslydescribed in detail by Hancock and Benz (16). The apparatus consisted ofa Teflon chamber containing two compartments. Conductance wasmeasured across a lipid bilayer in the presence of 10-12 M of FPLCpurified OprH in 1 M KC1. The lipid membrane was formed using 1%18oxidized cholesterol in n-decane. The applied voltage was 10 mV.Purified OprP (16) provided by Dr. C. Egli from our laboratory, was usedas a control.19RESULTSCharacterization of the OprH-deficient mutant strain H703.In order to access whether the chromosomal oprH gene of H103had been replaced by the oprH::tet cartridge of 11703, Southernhybridizations of EcoRl digested chromosomal DNA from both the wild-type strain H103 (Figure 2, lane A) and the OprH-deficient strain H703(Figure 2, lane B) with a 32P-labelled oprH gene probe was carried out.The results confirmed that the 2.8 kb oprH chromosomal gene wasinterrupted by a 1.4 kb tet gene. The observation that the gene probedid not hybridize to other areas of the blot confirmed the lack of asecond cross-hybridizing gene. SDS-PAGE of cell envelopes indicated inFigure 3 confirmed the lack of OprH protein in strain H703 (lane A). Aslight band in the 21 kDa region in cell envelope preparations of H703was consistently present. However, comparisons of heated and unheatedsamples indicated that the band was not heat-modifiable suggesting thatthis protein likely represents one of the minor proteins in the outermembrane and is not OprH. Expression of protein Hi from theexpression plasmid pGB25 in strain H703 resulted in OprH levelscomparable to that seen in OprH-overexpressing strain 11181 (Figure 3;B,C).Antibiotic susceptibility testing to polymyxin B, gentamicinand EDTA. To assess the extent of OprH involvement in resistance topolymyxin B, gentamicin and EDTA, susceptibility testing was carried outusing the OprH-deficient mutant strain H703. In contrast20kb4.073.052.04 -1.64-ABFigure 2. Comparison of H103 and the OprH-deficient strain H703 bysouthern blotting. Autoradiogram of southern hybridization of 32 -labelled oprH gene to a gel containing EcoRl digested chromosomal DNAfrom P. aeruginosa wild-type strain H103 (Lane A) and OprH-deficientstrain 11703 (Lane B). Bacteriophage lambda Hindill fragments wereused as molecular size markers, which are identified in kilobase pairs onthe left.21OprFOprGOprHFigure 3. SDS-polyacrylamide gel electrophoresis of cell envelopepreparations demonstrating expression of OprH on Mg2+sufficientmedium. Lane A - Strain H703 oprH ::tet ; Lane B- H703/pGB25 inducedwith 5 mM m-toluate; Lane C - H181; Lane D - H103. The runningpositions of OprH, OprG and the major porin OprF are indicated. Thesmall amount of protein observed just above the running position ofOprH in strain H703 was not heat-modifiable and was not inducible bygrowth in Mg2-deficient medium like OprH and thus was concluded tobe another protein.22to the situation with parent strain H103, the OprH-deficient, oprH: :tetmutant strain H703 remained susceptible to all 3 agents regardless of theMg2 concentration during growth (Table 4). These data supported thehypothesis that overproduction of OprH during growth on Mg2 deficientmedium was obligately required to permit expression of thepolymyxin/gentamicin/EDTA resistance phenotype.To confirm that this result reflected an inability to produce andinduce OprH, two controls were performed. First, it was demonstratedthat H703 had the same MIC (1 ig/ml) to the j3-lactam cefpirome, as theparent strain H103, in both Mg2-sufficient and -deficient medium,arguing against a non-specific change in permeability. Second, plasmidpGB25, which contains the oprH gene behind a m-toluate-induciblepromoter, was introduced into strain H703. The resultant strainH703/pGB25, when grown on Mg2+-deficient medium was resistant toboth polymyxin B and EDTA compared to the vector plasmid controlstrain H703/pNM185 and the parent strain H703 (Table 4, N.B.gentamicin resistance could not be tested in the presence of theseplasmids; 3).Two sets of results confirmed previous suggestions (17) that asecond cellular alteration was required to give the full resistancephenotype observed in strain H103 grown in Mg2-deficient medium.First, as previously observed for strain H103/pGB25 (17), overexpressionof OprH in strain H703/pGB25 was not sufficient to give the fullresistance phenotype when strains were grown in Mg2+sufficientmedium. Indeed both H703/pGB25 (Table 4) and H1O3IpGB25 (17)when grown on Mg2+sufficient medium demonstrated only partialresistance to EDTA and full susceptibility to polymyxin B, despite23Table 4. Influence of the OprH phenotype on killing by polymyxin B,gentamicin and EDTA.Strain Mg2 OprH Survivors (%)b(tM) levelaPolymyxin Gentamicin EDTAH103 20 90±3d 78±3d 89±3eH103 500 + 4±3 2±2 2±3H181 20 95±3d 85±3d 87±7dH181 500 81 ± 5d 78 ± 5d 85 ± 18dH703 20- 8±4 12±5 7±1H703 500 - 2±1 4±3 5±2H703/pGB25 20 79 ± 5d -c 77 ± 9dH703/pGB25 500 3 ± 1 -c 30 ± ld,eH703/pNM185 20- 6 ± 5 -c 8 ± 4H703/pNM185 500- 3 ± 2 -c 2 ± 224Table 4 (continued)a As judged by SDS-PAGE experiments such as those shown in Fig. 2. +++= overexpression of OprH such that this was the major cell envelopeprotein (Figure 2,), + = small amount of OprH produced, - = noobservable expression of OprH.b Percent survivors after 5 mm of treatment with 4 ji.g/ml polymyxin B,80 .tg/m1 gentamicin or 10 mM EDTA. Results are the means ±standard deviations of 3 experiments, all numbers are rounded off tothe nearest integer.C Due to the aminoglycoside modifying enzyme genes on the plasmidspGB25 and pNM185, gentamicin susceptibility could not be tested.d Significantly different (p<O.05) by Student’s t test to the result forH103 grown on Mg2- sufficient mediumSignificantly different (p<O.05) to the result for H703/pGB25 grown onMg2-deficient medium25overexpression of OprH. Second, growth of strain H703 (andH703/pNM185) on Mg2-deficient medium consistently gave increasedresistance to these agents, although the increase was far less than thatobserved for OprH-overexpressing strains.Influence of OprH overexpression on susceptibility toquinolones and chioramphenicol. To test whether quinolonesaccessed the self-promoted uptake pathway to cross the outer membraneof P. aeruginosa, the isogenic strains; H103, H703 and H181, containingnormal or altered expression of OprH were utilized. As demonstratedpreviously OprH expression in wild type strain H103 was influenced bythe level of Mg2 in the medium. In low Mg2 (20 riM) medium, OprHwas overexpressed at least 24-fold (28,29; confirmed in this study) andcells were 4-fold more resistant to polymyxin and gentamicin comparedto strain H103 cells grown in Mg2-sufficient (500 jiM) medium (27;Table 5). In strain H703 cells in which the oprH gene was mutated byinsertion of a tet gene, OprH was not expressed regardless of themedium Mg2+ concentration, and polymyxin and gentamicin MICs wereidentical to those levels observed for H103 expressing low levels of OprH,in Mg2-sufficient medium (Table 5). In strain 11181, whichconstitutively overexpresses OprH (28), cells were resistant to polymyxinand gentamicin at levels similar to those observed for strain H103 grownon Mg2-deficient medium (Table 5).These strains were utilized to see if OprH could also block uptake ofquinolones. In contrast, cells overexpressing OprH (i.e. strain H181grown in Mg2-sufficient or Mg2-deficient medium or strain H10326Table 5. Influence of OprH expression on MIC values P. aeruginosafor various antimicrobial agents.MIC(ig/ml)aStrain Mg2 OprH(mM) levelPX GM NAL NOR CIP FLX CAP RIF TMP CFPH103 0.5 + 1 1 256 2 0.5 1 128 8 1024 10.02 4 4 8 0.25 0.06 0.13 6 8 64 1H703 0.5 - 1 1 256 2 0.5 1 128 8 1024 10.02 - 1 1 256 2 0.5 1 128 8 1024 111181 0.5 --++ 8 8 16 0.13 0.03 0.06 6 8 64 10.02 -1±’- 8 8 16 0.13 0.03 0.06 6 8 64 1a Abbreviations: PX, polymyxin B; GM, gentamicin; NAL, nalidixic acid;NOR, norfioxacin; CIP, ciprofloxacin; FLX, fleroxacin; CAP,chioramphenicol; CFP, cefpirome; RIF, rifampicin; TMP, trimethoprim27grown in Mg2+deficient medium) were supersusceptible to thequinolones nalidixic acid, norfioxacin, ciprofloxacin and fleroxacin andto chioramphenicol when compared to cells with normal (strain H103grown in Mg2+sufficient medium) or no (strain 11703) expression ofOprH. The increase in susceptibility varied from 8 to 32 fold in OprHoverexpressing cells. No alteration in cefpirome susceptibility wasobserved in any of the above cells.To test if OprH overexpression was solely responsible for thesupersusceptibility phenotype, the quinolone susceptibility of strainH103 carrying the control plasmid pNM185 was compared with H103carrying the OprH expression plasmid pGB25 after induction with 5 mMm -toluate. Again OprH overexpression was correlated withsupersusceptibility to ciprofloxacin (Table 6), nalidixic acid, norfioxacinand chioramphenicol. In addition, MIC values indicated that polymyxinresistance was observed for the constitutively OprH-overproducing strainH181 however, this resistance was not observed in the wild-type H103overexpressing OprH from expression plasmid pGB25.To address the possibility that OprH overexpression leads to ageneral increase in hydrophobic antibiotic uptake, MIC values werecalculated for the antibiotics; rifampicin and trimethoprim. OprHoverexpression in H181 or in H103 grown in Mg2-deficient conditionscorrelated with trimethoprim supersusceptibility, however, susceptibilityof P. aeruginosa to rifampicin remained constant, irrespective of the levelof OprH expressed (Table 5).Effect of excess monovalent or divalent cations onfluoroquinolone susceptibility. The antagonism of excess divalent28Table 6. Susceptibilities of various P. aeruginosa strains which vary inthe amount of OprH to polymyxin B and ciprofloxacinStrain Plasmidsa OprH MIC(jlg/ml)CphenotypebPX NAL NOR FLX CIPH103 none + 1 256 2 1 0.5H103 pNM185 + 1 256 2 1 0.5H103 pGB25 1 8 0.25 0.13 0.03H703 none- 1 256 2 1 0.5H181 none -H-I- 4 8 0.25 0.13 0.03a plasmids were induced for gene expression with 5 mM m-toluateb OprH expression based on SDS-polyacrylamide gel analysisC Abbreviations: PX, polymyxin B; NAL, nalidixic acid; NOR, norfioxacin;FLX, fleroxacin; CIP, ciprofloxacin29cation, Mg2+ as previously noted by Chapman et at (6) was confirmed asseen in Table 7. A twenty fold excess of monovalent Na+ ions did notalter the MIC values for any of the strains tested. In contrast, 5 mMMg2+ increased the MIC values for all of the strains tested. A substantialincrease in the MIC value (4-8 fold) was however limited to strainsH181, H103/pGB25 and H703/ pGB25, all of which overexpressed OprH.The MIC values calculated for the B-lactam cefpirome were not altered inthe presence of excess Mg2+ or Na+ for any of the strains tested.Is OprH a porin? There were two possible explanations for quinoloneand chioramphenicol supersusceptibility due to OprH overexpression.Either OprH is a porin or OprH mediates in a non-porin uptake pathway.I reasoned that if Opril was a porin it should be specific for thequinolones and chloramphenicol since OprH overexpression did not resultin supersusceptibility to other antibiotics. Therefore the possibility thatthese antibiotics were pyrimidine and purine analogs respectively andthat OprH was a nucleoside-specific channel like E. coli protein Tsx (24)was considered. As seen in Tables 8,9 and 10, the addition of 1 mM or0.1 mM deoxyadenosine as a potential competitor for quinolone uptakedid not influence susceptibility in strains H103, H703 and H181.Similarly, addition of deoxycytidine as a competitor for chloramphenicoluptake did not alter the susceptibility of the strains tested. Thus it wasconcluded that these compounds were unable to bind to a specificbinding site and block quinolone uptake. Additional experiments werecarried out to access the possibility that adenine could compete foruptake with ciprofloxacin and nalidixic acid30Table 7. The effect of excess monovalent cation or divalent cations onthe MIC value for ciprofloxacin.Strain Opril MIClevelsa (jig/ml)bno addition 5mM MgC12 1 00mMNaC1H103 + 2 4 1H181 -i-i-i- 0.13 1 0.1311703- 2 4 1H103/pGB25 -‘-f+ 0.13 0.5 0.13H103/pNM185 + 1 2 1H703/pGB25 -i-f-i- 0.13 0.5 0.13H703/pNM185- 1 2 1a as judged by SDS-polyacrylamide gel electrophoresisb The MIC value calculated for the 8-lactam cefpirome was consistently1.0 jig/ml.31Table 8. Nucleoside uptake assay in the wild-type strain; H103Antibiotic Mg2 deoxyadenosine(jiM) concentration (jiM)0 0.1 1 10nalidixic acid20 8 8 NGa500 128 128 128 NGciprofloxacin20 0.06 0.06 NG NG500 0.25 0.25 0.25 NGcefp ir o me20 1 1 NG NG500 0.5 0.5 0.5 NOchloramphenic olb20 6.3 6.3 6.3 NO500 128 128 256 NOa NO; no growthb deoxycytidine was used as a competitor for chioramphenicol uptake32Table 9. Nucleoside uptake assay in the OprH-deficient strain; H703.Antibiotic Mg2 deoxyadenosine(jiM) concentration (jiM)0 0.1 1 10nalidixic acid20 128 128 NG NG500 128 128 64 NGciprofloxacin20 0.25 0.25 NG500 0.25 0.5 0.25 NGcefpirome20 0.5 1 M3 I’U500 0.5 0.5 0.5 I’Uchioramphenicoib20 128 128 128 128500 256 256 256 256a NG; no growthb deoxycytidine was used as a competitor for chioramphenicol33Table 10. Nucleoside uptake assay in the OprH-overexpressing strain;H181.Antibiotic Mg2 deoxyadenosine(.tM) concentration (jiM)0 0.1 1 10nalidixic acid20 8 8 NGa J..fl500 16 16 16 NGciprofloxacin20 0.03 0.03 NG NG500 0.03 0.03 0.03 NGc e fp i rome20 1 1 NG NG500 1 1 1 NGchioramphenicoib20 3.2 3.2 NG NG500 3.2 3.2 3.2 3.2a NG; no growthb deoxycytidine was used as a competitor for chioramphenicol uptake34in the OprH-overexpressing strain H181. No change in the MIC valueswere seen in the presence of 0.1 mM or 1.0 mM adenine (table 11).Adenine and deoxyadenosine at a concentration of 10 mM inhibited cellgrowth.A second test involved an attempt to examine the ability of OprH toreverse the quinolone resistance phenotype of several mutant strains.The nalB4 mutant strain PA06002 and its parent strain PA0505 (31)were transformed with plasmids pGB25 and pNM185. Figure 4 indicatesthat OprH overexpression was attained in both the naiB mutant strain aswell as the parental strain; PA0505. MIC values calculated indicate thatquinolone supersusceptibility was attained in the wild-type PA0505when OprH was overexpressed. However OprH overexpression did notalter the susceptibility of PA06002 to either nalidixic acid orciprofloxacin (Table 12).To further confirm that OprH was not acting as a porin forquinolone uptake, OprH was purified by selective solubilization followedby FPLC in the presence of the detergent octyl-POE for analysis in blacklipid bilayer experiments. In the selective solubilization, OprH wasprimarily solubilized in 3% octyl-POE with 50 mM EDTA. In the FPLCprofile (Figure 5) a peak which was visible prior to the addition of NaC1was protein Hi. As expected, since OprH is a basic protein, it eluted inthe void volume. SDS-PAGE analysis (Figure 6, lane A) confirmed thatOprH eluted in a pure form. The FPLC-purified sample was diluted in 1%octyl-POE and 0.1% Triton-XiOO for analysis in black lipid bilayerexperiments. Addition of purified OprH to a final concentration of 8 pgor 80 pg per ml of bathing solution, resulted in no increase in membraneconductance (Figure 7; A,B). Stepwise increases in membrane35Table 11. MIC analysis using adenine as potential competitor forciprofloxacin and nalidixic acid uptake.Antibiotic Mg2 adenine(iiM) concentration (riM)0 0.1 1 10nalidixic acid20 16 16 8 NGa500 16 16 16ciprofloxacin20 0.03 0.03 0.03500 0.03 0.03 0.03 I’Uce fp i rome20 1 1 1500 0.5 1 1a NG; no growth36OprFoprL OprHFigure 4. Cell envelope preparations of P. aeruginosa naiB strainPA06002 induced for OprH expression. SDS-polyacrylamide gelelectrophoresis was carried out. Samples were pretreated with 2%trichloroacetic acid and heated at 1000C for 10 minutes prior to loading.Analysis was on a 14% acrylamide gel. Running positions of OprH andthe major outer membrane porin protein OprF have been denoted. LaneA- PA0505, lane B- PA0505/pGB25 induced with 5 mM m-toluate, laneC- PA06002, lane D- PA06002/pGB25 induced with 5 mM m-toluate.ABCD37Table 12. Effect of OprH overexpression on the nalB4 mutant strainPA06002 of P. aeruginosa.Straina Plasmidb OprHC MICd(ig/ml)NAL CIP CFPH103 none + 256 0.5 1H181 none -i-i-i- 16 0.03 1PA0505 none + 32 0.5 1PA0505 pNM185 + 32 0.5 1PA0505 pGB25 ±1-f- 4 0.03 1PA06002 none + 100 0.25 1PA06002 pNM185 + 100 0.25 1PA06002 pGB25 +-H- 100 0.25 1a Strain PA0505 is parental to naiB strain PA06002b Plasmid induction with 5 mM m-toluateC As examined by SDS-PAGEd Abbreviations: NAL, nalidixic acid; CIP, ciprofloxacin; CFP, cefpirome38Figure 5. Fast performance liquid chromatography profile of OprHpurification. Proteins were eluted as the salt concentration increasedgradually from 0 to 1M. Ultra-violet light at 280 nm was used forprotein absorption. The fractions in peaks 1, 2, 3, and 4 were analyzedfor Opril. Peaks 2, 3 and 4 contained unidentified proteins in minuteamounts. OprH elutes in the void volume as peak 1. The remainder ofthe proteins in the sample were eluted in 1M NaC1.380.0.3 1 5004000.2 “,0300 Eoco—C)Cu• z‘20001• 4“100,0.0 rrrrrrrrrrrrrrf ‘‘‘‘i’’’’I’’’’I’’’’I’’’’ •00 5 10 15 20 25 30 35 40Fraction no.39OprH LLAOprF‘OprLFigure 6. FPLC-purified OprH. Purification of outer membrane proteinOprH from P. aeruginosa strain H181 by selective solubilization of OprHfrom cell envelope preparations of 11181 grown in BM2-glucoseproceeded as indicated in the text. Samples were run on a 14%polyacrylamide gel. Lane A- FPLC-purified OprH; Lane B- H181 cellenvelope preparation.B40I I I--I5OOSA B4C5mm 2OsecFigure 7. Black lipid bilayer analysis of FPLC-purified OprH. Themembrane was made from 1% cholesterol in n-decane. The appliedvoltage was 10 mV. Three separate sections from a chart recording areshown: arrow A, 8 pg/ml purified OprH was added to the chamber;arrow B, a further 80 pg/ml purified OprH was added to the chamber;arrow C, 8 pg/mI purified OprP was added to the chamber leading tostepwise increases in conductance. The time scale on the left refers tothe chart sections between arrows A and C, the time scale on the rightrefers to the section of chart recording after arrow C.41conductance of the appropriate size(s) were however observed for thecontrol porin OprP when added at a concentration of 8 pg per ml to thec h amber.OprH overexpression in E. coli. E. coli strain HB1O1 expressing OprHfrom the IPTG-inducable expression plasmid pGB23 resulted in OprH asthe major outer membrane protein as seen in figure 8. The MIC valuescalculated (Table 13) indicated that OprH overexpression did not alterthe susceptibility of E. coli to either nalidixic acid, ciprofloxacin,chioramphenicol or cefpirome.42OprHFigure 8. SDS-polyacrylamide gel analysis of OprH overexpression in E.coli strain HB1O1. Acrylamide concentration is 14%. Cell envelopepreparations were treated with 2% trichioroacetic acid and heated to1000C for 10 mm prior to loading. Expression vector plasmid pRK767and its OprH-expressing derivative pGB23 were induced with 1 mMIPTG. Lane A- HB1O1; Lane B- HB1O1/pGB23; Lane C- HB1O1/pRK767.A BC43Table 13. Influence of OprH expression on fluoroquinolonesusceptibility of E. coliStrain Plasmida OprH MIC (.tg/m1)cleveibNAL CIP CAP CFPHB1O1 none - 4 0.01 8 0.25HB1O1 pGB23 -i-H- 4 0.01 8 0.25HB1O1 pRK767 - 4 0.01 8 0.25a Plasmids were induced for expression with 1 mM IPTGb As estimated by SDS-polyacrylamide gel electrophoresisC Abbreviations: NAL, nalidixic acid; CIP, ciprofloxacin; CAP,chioramphenicol; CFP, cefpirome44DISCUSSIONIt has been previously demonstrated that overexpression of OprH in theparent strain H103 grown on Mg2-deficient (20 pM Mg2+) BM2 glucoseminimal medium, in the polymyxin resistant mutant H181, wasaccompanied by resistance to killing by polymyxin B, gentamicin andEDTA, when compared to results for strain H103 grown on Mg2+..sufficient (500 jiM Mg2+) BM2-glucose minimal medium (3,27). Thisobservation was confirmed here (Table 4) in killing assay experiments.In addition, construction of an OprH-deficient mutant allowedconfirmation that the resistance phenotype observed in the OprHoverexpressing strains was due to the ability to express OprH as a majorouter membrane protein. Furthermore, this data suggests that althoughOprH overexpression is required for the polymyxin/gentamicin/EDTAresistance phenotype, it is not by itself sufficient to explain thisphenotype. Previous pseudorevertant studies suggested that LPS mightalso play a role. However methods for examining the rather complex LPSof P. aeruginosa are difficult to perform and beyond the scope of thisthesis.Utilizing the strains from which the above conclusions arose, Itested the hypothesis of Chapman and Georgopapadakou (6) thatfluoroquinolones could penetrate the outer membrane via the selfpromoted uptake pathway. I confirmed their observation (6) thatexogenous Mg2+ increased the MIC for fluoroquinolones, as shownpreviously for other agents that access the self-promoted uptakepathway, antagonizes the effects of polymyxin and gentamicin (27). Itwas evident that this antagonizing effect of Mg2 on fluoroquinolone45uptake was more extreme in cells which overexpress OprH. This seemsto indicate a direct involvement of high levels of OprH and the Mg2 +antagonism with the quinolone resistance/susceptibility phenotype of P.aeruginosa. Researchers have been unable to demonstrate thatfluoroquinolones were capable of increasing the permeability of theouter membrane to the hydrophobic fluorophor, NPN, the 13-lactamnitrocefin or the protein lysozyme (our laboratory, data not shown), incontrast to results obtained for polymyxin, gentamicin and otherpolycations that access self-promoted uptake (28). Furthermoreoverexpression of OprH correlated with supersusceptibility to quinolones(and chloramphenicol) not resistance as observed for gentamicin andpolymyxin (Table 5). Thus we can conclude that the quinolones andchloramphenicol are not taken up by self-promoted uptake in P.aeruginosa. In addition, it is evident that OprH overexpression does notresult in a general increase in the uptake of hydrophobic antibiotics asseen by the unaltered susceptibility of OprH overexpressing cells torifampicin.Results obtained using cells in which OprH was overexpressed fromthe cloned gene confirmed that the supersusceptibility phenotype wasentirely due to the presence of OprH in the outer membrane. In addition,three lines of evidence argued against OprH forming a channel for porins.Thus I hypothesize that Opril increases the rate of non-porin uptake ofquinolones and chloramphenicol across the outer membrane of P.aeruginosa. The observed supersusceptibility to trimethoprim, ahydrophobic compound structurally unrelated to either the quinolones orchloramphenicol, is consistent with the hypothesis that OprH may bemediating a general non-porin pathway. It must be emphasized46however that quinolone antibiotics are water soluble and thus notconsidered hydrophobic. Therefore it seems unlikely that this postulatednon-porin pathway is equivalent to the previously described (17)hydrophobic uptake pathway. Although not currently in a position todefine the actual mechanism of uptake of fluoroquinolones, it is possiblethat OprH, a basic protein might act by partly neutralizing the highlynegative surface charge of P. aeruginosa, as indicated by phasepartitioning experiments with strains 11103 and H181 (A. Bell, 1989,Ph.D. thesis, U.B.C.). This might then permit quinolones to better accessspecific’sites involved in uptake via a non-porin pathway. One potentialclass of uptake sites might involve LPS aggregates in the outermembrane. Alternatively uptake may occur at the interface betweenOprH and LPS in the outer membrane. Nevertheless uptake at such siteswould not seem to account for the quinolone susceptibility of wild type P.aeruginosa. Consistent with this, MICs for quinolones andchloramphenicol were not different for strain H103 grown in 500 jiMMg2 (which expresses low levels of OprH) and the oprH::tet mutant(which cannot produce OprH).Information regarding the route of uptake of quinolones across theouter membrane of P. aeruginosa appears contradictory. Thus OprC, D, E,F and G as well as LPS (3,15,28,29) have been variously suggested to beinvolved in fluoroquinolone uptake across the outer membrane of P.aeruginosa based on crude compositional analyses of quinolone resistantmutants. Thus it is possible that the variety of phenotypic alterations inresistant mutants reflect a chemically more subtle alteration to a nonporin pathway of quinolone uptake. It is known that LPS alterations caninfluence outer membrane protein composition (17) and LPS chemistry is47so complex that changes to LPS can evade definition by simple chemicalanalyses. Several authors have suggested that LPS may be involved insome way in fluoroquinolone uptake in P. aeruginosa (34) and E. coil(6,23). Thus, unless a thorough analysis of LPS is performed, it wouldseem inappropriate to conclude solely on the basis of the lack ofexpression of specific outer membrane proteins in a quinolone-resistantmutant, that these proteins are porins which mediate fluoroquinoloneuptake.The results indicate that OprH-overexpression alone renders strainssupersensitive to quinolones and chloramphenicol. I have also providedevidence that OprH is not a porin. I therefore propose a model ofquinolone uptake which accounts for these findings in which quinolonespenetrate the outer membrane of OprH-overexpressing strains via a nonporin pathway, and further propose that this general mode of uptakemay also be utilized in wild type P. aeruginosa cells.48LITERATURE CITED1. Bedard, J., S. Chamberland, S. Wong, T. Schollaardt, and L.E. Bryan.1989. Contribution of permeability and sensitivity to inhibition ofDNA synthesis in determining susceptibilities of Escherichia coli,Pseudomonas aeruginosa, and Alcaligenes faecalis to ciprofloxacin.Antimicrob. Agents Chemother. 33: 1457-1464.2. Bell, A., and R.E.W. Hancock. 1989. Outer membrane protein Hi ofPseudomonas aeruginosa: Purification, gene cloning and nucleotidesequence. J. Bacteriol. 17 1:321 1-3217.3. Bell, A., Bains, M., and R.E.W. Hancock. 1991. Pseudomonasaeruginosa outer membrane protein OprH: Expression from thecloned gene and function in EDTA and gentamicin resistance. 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