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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 ANTIBIOTICS  ACROSS THE OUTER MEMBRANE OF Pseudomonas aeruginosa by MICHELE LOUISE YOUNG  B .Sc., McMaster University, 1990  A THESIS SUBMI1TED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology)  We accept this thesis as conforming to the required stapdard  THE UMVERSITY OF BRITISH COLUMBIA  July1992 © Michele Louise Young, 1992  In presenting this thesis in  partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  (2,,  The University of British Columbia Vancouver, Canada  Date  DE-6 (2188)  11  ABSTRACT  The oprH inserting  1.4  a  resistance,  gene of Pseudomonas  into  kilobase the  pair  cloned  recombination into the P.  was mutated by  aeruginosa  fragment, gene  encoding  followed  by  tetracycline homologous  Growth of the  aeruginosa chromosome.  resultant oprH::tet mutant in Mg -deficient medium had little effect 2 on  susceptibility  complemented  by  to  polymyxin  the cloned  B,  gentamicin  or  EDTA  unless  oprH gene in plasmid pGB25.  +deficient 2 contrast growth of the parent strain on Mg  In  medium  resulted in high expression of OprH and resistance to all three agents. These data  support the hypothesis  that  overexpression  of OprH  +-deficient growth conditions is obligately required for 2 under Mg resistance to these 3 agents. OprH overexpression in P.aeruginosa also resulted in supersusceptibility  to  fluoroquinolones  and  chloramphenicol.  Addition of the nucleosides deoxyadenosine or deoxycytosine did not reduce  the  MIC  values  for  the  quinolones  or  chioramphenicol  respectively as could be expected if OprH was a nucleoside-specific channel.  Addition of the base adenine as a potential competitor for  uptake of either nalidixic acid or ciprofloxacin did not alter the MIC values for the OprH-overexpres sing mutant Hi 81. OprH in the naiB  Overexpression of  strain PA06002 did not reverse the quinolone  resistance phenotype as it did for the parental strain PA0505.  OprH  overexpression did not alter the susceptibility of E. coil to nalidixic acid, ciprofloxacin or chioramphenicol. FPLC-purified  Black lipid bilayer analysis of  OprH did not result in  an increase in membrane  111  conductance. protein  and  pathway.  These data suggest that OprH does not act as a porin that  OprH  mediates  in  a  unique  non-porin  uptake  iv  TABLE OF CONTENTS  Page 1. ABSTRACT  ii  2. LIST OF TABLES  vi  3. LIST OF FIGURES  viii  4. ACKNOWLEDGEMENTS  ix  5. INTRODUCTION  1  6. MATERIALS AN]) METHODS  7  A. Strains and media  7  B. Plasmids  7  C. Antibiotics  12  D. Construction and characterization of the OprH-deficient mutant strain H703  1 2  E. DNA techniques  14  F. Southern blotting  14  G. Biparental mating experiments  14  H. Antibiotic susceptibility testing  14  I. MIC determination  15  J. Cell envelope isolation  16  K. SDS-polyacrylamide gel electrophoresis  16  V  L. Selective solubilization of Opril  .  1 6  M. FPLC purification of OprH  17  N. Black lipid bilayer experiments  1 8  7. RESULTS  1 9  A.  Characterization of the OprH-deficient strain H703  B.  Antibiotic susceptibility testing to polymyxin B, gentamicin andEDTA  C.  19  Influence of OprH overexpression on susceptibility to quinolones and chioramphenicol  D.  1 9  23  Effect of excess monovalent and divalent cations on fluoroquinolone susceptibility to quinolones  28  E.  Is OprH a porin?  30  F.  OprH overexpression in E. coli  41  8. DISCUSSION  44  9. LITERATURE CITED  48  vi  LIST OF TABLES  Table  Page  1.  P. aeruginosa strains  2.  E. coli strains  10  3.  Plasmids  11  4.  Influence of the OprH phenotype on killing by polymyxin B, gentamicin and EDTA  5.  23  Influence of OprH expression on MIC values of P. aeruginosa  for  various antimicrobial agents 6.  Susceptibilities of various  26 P. aeruginosa  strains which vary in  the amount of OprH to polymyxin B and ciprofloxacin 7.  9  .  27  The effect of excess monovalent or divalent cations on the MIC value for ciprofloxacin  29  8.  Nucleoside uptake assay in the wild-type strain; H103  32  9.  Nucleoside uptake assay in the OprH-deficient strain; H703  33  10. Nucleoside uptake assay in the OprH-overexpressing strain; H181  34  11. MIC assay using adenine as potential competitior for ciprofloxacin and nalidixic acid uptake 12. Effect if OprH overexpression on the nalB4 aeruginosa  35 strain PA06002 of P. 37  vii 13.  Influence of OprH expression on fluoroquinolone susceptibility of  E.coli  43  viii  LIST OF FIGURES  Figure 1.  Page  Diagram of plasmid pGB32B utilized for gene replacement  mutagenisis 2.  Comparison of 11103 and the OprH-deficient strain H703 by Southern blotting  3.  13  20  SDS-polyacrylamide gel electrophoresis of cell envelope + 2 preparations demonstrating expression of Opril on Mg sufficient medium  4.  Cell envelope preparation of P. aeruginosa nalB4 strain induced for OprH expression  5.  6.  PA06002 36  Fast performance liquid chromatography profile of OprH purification  38  FPLC-purified OprH  39  7. Black lipid bilayer analysis of FPLC-purified OprH 8.  21  40  SDS-polyacrylamide gel analysis of OprH overexpression in E coli strainHBl0l  42  ix ACKNOWLEDGEMENT  I wish to thank Manjeet Bains for all of her technical assistance in getting me started in the laboratory.  I also thank Francis for  introducing me to the wonderful world of antibiotics.  I gratefully  acknowledge Dr. R.E.W. Hancock for his advice and direction throughout my project.  Especially, I thank my parents for their love  and support which has kept me smiling.  1 INTRODUCTION  Pseudomonas  aeruginosa  is a gram-negative pathogen which is one  of the main causes of nosocomial infections in the hospitals of North America. include  Individuals which are at risk from Pseudomonas those  who  are  compromised  ailments  by  leukemia, cystic fibrosis or severe bodily infections.  such  infections as  cancer,  The most common  sites of infection include the lower respiratory tract and the urinary tract, as well as sites of a severe burn or wound. increase of P.  With the worldwide  aeruginosa infections, chemotheraputic therapy has been  prominent. A major problem in the treatment of such infections is the high intrinsic resistance of P. aeruginosa antibiotics.  It has  permeability  contributes  been well  to many of the conventially used  established that low  substantially to this  outer membrane  resistance (15).  The  composition of the outer membrane has been studied in detail and has been shown to consist of an  assymetric membrane,  rich in proteins.  The  bilayer may be strongly associated, either covalently or non-covalently through interactions of its proteins with an underlying peptidoglycan layer.  The  inner  monolayer  of  the  outer  membrane  comprises  phospholipid while the outer monolayer consists mainly of one or two species of lipopolysaccharide (LPS) molecules which are often categorized into  three  domains;  the  inner,  hydrophobic  lipid  A,  the  core  oligosaccharide and the outer, hydrophilic, 0 side-chain polysaccharide. The P.  aeruginosa  outer membrane is highly negatively charged due to  the high amount of phosphate residues in the core- and 0-regions of the LPS (17).  A contributing factor to the high stability of the P.  aeruginosa  2 Mg and + outer membrane is the presence of divalent cations such as 2 2 which non-covalently cross-bridge the LPS at negatively charged Ca sites. In recent years,  there  has  been  significant increase in  a  our  understanding of the mechanisms of uptake of antibiotics across the outer membrane of P. aeruginosa  Antibiotics cross the outer membrane  .  both by porin and non-porin routes. certain  size  membrane  limit,  by  proteins.  including  diffusion  While,  Hydrophilic antibiotics within a  B-lactams,  through  the  hydrophobic  are able  to cross  the outer  water-filled  channels  of porin  molecules  such  rifampicin  as  and  trimethoprim are generally restricted from penetration, however, several hydrophobic  drugs  have  been  shown  phospholipid bilayer of mutants (17). such  as polymyxin  uptake  pathway  B  and  diffuse  to  This  the  LPS  Conversely, polycationic antibiotics  aminoglycosides  (17,15,28).  through  access the  pathway  involves  self-promoted  the  competitive  displacement, by these bulky polycations, of the divalent cations which non-covalently crossbridge adj acent LPS molecules. outer  membrane  becomes  distorted  and  more  Consequently, the  permeable,  permitting  increased (self-promoted) uptake of the permeabilizing compounds.  The  isolation of outer membrane mutants with altered susceptibility to these agents  argues  powerfully  eventual cell killing (17).  that  self-promoted  uptake  is  relevant  to  The classical outer membrane permeabilizer,  EDTA, a divalent cation chelator, functions by accessing the same divalent cation binding sites on LPS, as do these polycationic antibiotics (28). Nicas and Hancock (28) proposed that the P. membrane pathway.  protein  OprH  (=  Hi)  blocked  the  aeruginosa outer  self-promoted  uptake  Thus, overexpression of OprH by twenty-fold or more, either  3 -deficient medium in the parent strain 2 as a result of adaptation to Mg H103, or due to a mutation in strain 11181, was associated with resistance to polymyxin B, gentamicin and EDTA (3,17,28).  In contrast there was no  change in susceptibility of these cells to 8-lactams or tetracycline. It was hypothesized that OprH acts by binding to LPS sites which are normally occupied 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 basic  protein  permitting  (theoretical  p1  =  8.6)  potential  association  with  negatively charged LPS molecules, whereas the OprH protein was shown to copurify with LPS (2).  In addition, rough mutations altering P.  aeruginosa  LPS have been shown to abolish OprH mediated resistance to  polymyxin  B  (2).  Furthermore,  evidence  indicates  that  overexpression is associated with a decrease in the amount of  OprH divalent  cations associated with the cell envelope (28). Antibiotic  susceptibility  testing  has  indicated  that  the  overexpression of OprH from the cloned gene in the wild type strain H103/pGB25 resulted in only partial resistance to EDTA, ambiguous data for aminoglycosides and no resistance to polymyxin, compared to the OprH-overproducing mutant H181 which was resistant to all 3 agents (17).  This led to the hypothesis that strain H181  (and strain H103  adapted to Mg -deficient medium which had an identical phenotype) 2 contained a second alteration.  Based on pseudorevertant studies, it was  proposed that this second alteration involved LPS (17).  However, these  data led us to reexamine whether OprH had any role in polymyxin resistance.  4 second  A  of  aspect  this  involves  research  fluoroquinolone  antibiotics which have recently emerged as one of the most effective classes of antibiotics against P.  aeruginosa infections.  Their broad  spectrum of activity as well as their potential to be delivered orally to patients have been important in their clinical use as therapeutic agents. In P.  aeruginosa, the outer membrane has been proposed to act as a permeability  major  barrier  fluoroquinolone antibiotics (1,7). drug  permeation  prominent.  For  through  the  example  some  the  to  antimicrobial  activity  Mutations which lead to decreased disruption studies  of  uptake  pathways  indicated that  alterations  deficiencies in the outer membrane porin protein OprF of P. are  (7,30)  observed  susceptibility.  in  of  mutants  with  decreased  are or  aeruginosa  fluoroquinolone  Alternatively, the decreases in the amount of OprG (4) or  the lack of OprD (11,26) have been associated with quinolone resistance. Other studies have indicated that the acquisition of a 54 kDa outer membrane  protein  penetration (22).  is  associated  with  reduced  In contrast, Chamberland et al  fluoroquinolone  (5) could show no  correlation between OprF deficiency or 54 kDa protein acquisition and resistance  to  quinolones.  Yamano  et al  (34)  proposed  that outer  membrane proteins C, D and E of P. aeruginosa played a role in quinolone resistance (34). LPS  In addition it has been demonstrated that mutants with  modifications of the outer membranes  of both E.  coli  and P.  aerug inosa had altered fluoroquinolone permeation (5,6,19,23), whereas quinolones have been reported to penetrate the outer membrane of E. coli (18) and Salmonella Clearly  these  studies  typhimurium (19) through the lipid bilayer.  have  provided  conflicting  data.  Therefore, I  considered here the possibility that the various phenotypic alterations of  5 the above mutants were secondary manifestations of a primary mutation affecting a non-porin pathway of uptake across the outer membrane. The self promoted (non-porin) uptake pathway (3,15,28) has also been suggested as a contributing mechanism in fluoroquinolone uptake. Chapman and Georgopapadakou (6) have provided evidence that E. coli cells treated with the fluoroquinolone fleroxacin release large amounts of endotoxin  (LPS) as  permeability. reduce  In  fleroxacin  well as display an increase in addition, uptake  magnesium  excess and  increase  outer membrane  was  the  demonstrated  minimal  to  inhibitory  concentration for killing.  Fluoroquinolones were also shown to act as  divalent  agents.  cation  chelating  Chapman  (6) proposed that  et al  fluoroquinolones might promote their own uptake through the chelation of divalent cations which resulting their  in  own  destabilizing uptake.  cross-bridge adjacent LPS of the  Previously  outer membrane, Nicas  and  molecules,  thus  thereby promoting  Hancock  demonstrated that OprH overexpression in P. aeruginosa  (28,29)  had  correlated with  increased resistance to the polycationic polymyxin B, gentamicin as well as to the divalent cation chelator EDTA and it was proposed that OprH blocks the self-promoted uptake of these compounds. The initial aim of this project was to characterize a constructed OprH-deficient strain of P.  aeruginosa.  Through the use of the resultant  OprH-deficient strain it was possible to demonstrate that OprH had an obligate role in polymyxin/gentamicin/EDTA resistance due to adaptation to Mg -deficient growth conditions. 2 blocking  the  self-promoted  I then proceeded to examine if  uptake  pathway  overexpression would lead to quinolone resistance. here that OprH  overproduction in P.  through  OprH  In contrast, I report  aeruginosa rendered the cells  6 supersusceptible to the fluoroquinolones ciprofloxacin, norfioxacin and fleroxacin as well as to chioramphenicol. this  supersusceptibility  reflected  a  role  Therefore, I examined whether for  OprH  fluoroquinolone uptake across the outer membrane.  as  a  porin  for  7  MATERIALS AND METHODS  Strains and media. P.  1.  aeruginosa strains used are described in Table  For short term maintenance of P. aeruginosa  strains were maintained  on low-salt Luria Broth (LB) agar plates containing 1% Bactotryptone, 0.5% Yeast extract, 0.1% NaC1 and 2 % agar. Exceptions included the OprH overproducing strain H181 which was maintained on LB agar containing 8 .tg of polymyxin B per ml and the OprH-deficient strain H703 which was maintained on LB agar containing 150 ig per ml of tetracyclineThe E. coli  hydrochloride.  strains used (Table 2) were maintained short-  term on normal salt LB agar as described in Maniatis et at  (25). LB  ingredients were obtained from Difco Laboratories, Detroit, Michigan. Antibiotic susceptibility testing and MIC determination for P. aeruginosa was carried out in basal medium 2  (BM2) broth (9) or agar (2%)  containing 0.4 % glucose as a carbon source and 10 iM FeSO4 aeruginosa  strains PA0505 and PA06002 required 1  and 1 mM leucine for growth in minimal medium.  .  The P.  mM methionine  L-methionine and L  leucine were obtained from Sigma Chemical Co., St. Louis, Mo.  Plasmids.  Plasmids are been described in Table 3.  The expression  vector pNM185 (27) and its derivative pGB25 containing the cloned oprH gene under control of the m -toluate induced in P.  aeruginosa  regulatable  tot  promoter, were  by the addition of 5 mM meta-toluate.  addition, shuttle vector pRK767 and its oprH-containing pGB23  In  derivative  under control of the lac promotor were induced for op rH  expression in E. coli.  OprH expression was induced by the addition of  8 1mM  Research Laboratories, used  (IPTO)  isopropylthio-13-D-galactoside  to  maintain  the  obtained  Burlington, Ontario. piasmids  were  from  Bethesda  Antibiotic concentrations  tetracycline-hydrochloride at a  concentration of 150 jig/ml for P. aeruginosa  and 12.5 jig/ml for E. coli  and kanamycin acid sulfate at a concentration of 300 jig/mi for P. aeruginosa  and 25 jig/mi for E. coli.  Antibiotics. sulfate,  Tetracycline-hydrochloride,  chioramphenicol,  polymyxin  ampicillin,  kanamycin  acid  sulphate,  nalidixic  acid,  B  ciprofloxacin and trimethoprim were obtained from Sigma Chemical Co., St.  Louis,  Mo.  Cefpirome  was  a  Pharmaceuticals Inc., Somerville, N. J. Hoffman-La Roche Inc., Nutley, N.J.  gift  from  Hoechst-Roussel  Fieroxacin was a gift from  Norfioxacin was a gift from Merck,  Sharp and Dohme Research Lab, Rahway, N.J.  Rifampicin was purchased  from Boehringer Mannheim, Laval, Quebec.  Gentamicin sulphate was  purchased from ICN Biomedicais Inc., St. Laurent, Quebec.  Construction strain H703.  and  characterization  of the  OprH-deficient  mutant  Construction of the OprH deficient mutant was performed  by Manjeet Bains and proceeded as follows:  the 1.4 kb tetracycline gene  was excised from pUC18T2 with Pstl and inserted between the tandem Pstl sites placed 15 base pairs apart in the oprH (2).  gene of piasmid pGB32  Plasmid pGB32 was linearized using EcoRl and cloned into the  EcoRl site of the gene replacement vector pRZ1O2  9  Table 1.  P. aeruginosa  Strain  Relevant Characteristicsa  Source or Reference  11103  wild-type PAO1 prototroph  This laboratory  111 81  Pxr, OprH-overexpressing  This laboratory  strains  mutant of H103  11703  OprH::Tc mutant of H103  This study  PA0505  parental strain of PA06002  Rella et al, 1982  methionine  PA06002  nalB4  auxotroph  mutant of PA0505  methionine  Rella et al, 1982  auxotroph  a Abbreviations: Pxr, polymyxin B-resistant; Tcr, tetracycline resistant; Cipr, ciprofloxacin-resistant; Nalr, nalidixic acid-resistant  10  Table 2.  E. coli  strains  Strain  Relevant Characteristics  Source or Reference  HB1O1  F, hsd S20 (r-b, rn-b), recAl3, proA2, rps L20(Smr), ara-14  This laboratory  S 17-1  pro res- mod :: RP4 Tc :: Mu Km Tn7  Simon et al, 1983  11  Table  3.  Plasmids  Plasmids  Relevant Properties  pUC1 8T2  Co1E1 Tcr Ampr plac lacZ  pGB32  pUC18 containingoprH on a  Source or Reference  +  S. Lorya  A.Bell, Ph.D. thesis,1989  1.8 kB BamHl-Sall fragment  pRZ1O2  Co1E1 Tn5 Kmr  Jorgensen, R.A. et al, 1979  pGB32A  ColE 1 Ampr plac lacZ  This study  oprH: :tet  pGB32B  EcoRl linearized pGB32A  This study  inserted into pRZ1O2  pGB25  2.8 kb EcoRl fragment containing Bell, et al, 1989 oprH  gene in pNM185  pNM185  Kmr SmrJ2m (TOL) mob tra  Mermod, N.,et al, 1986  pGB23  2.8 kb EcoRl fragment (OprH) in  Bell, Ph.D. thesis, 1989  pRK767  pRK767  Tcr plac lacZ  +  Ditta, G.S., et al, 1985  12 (27) which contains a mobilization site necessary for transfer to P.  resultant  origin of replication.  however lacks a Pseudomonas  aeruginosa  plasmid  pGB32B  mobilizing E. coli  (Fig.  1)  was  then  transformed  strain S17-1 [pro res- mod::RP4  into  The the  (Tc::Mu Km::Tn7)]  (33), and transferred by conjugation into strain 11103.  Since pGB32B  could not replicate in P. aeruginosa, isolation of colonies resistant to 150 .tg/ml  tetracycline  chromosomal  resulted  in  for  mutants  in  which  the  oprH gene was replaced by homologous recombination by  the interposon-mutated oprH::tet  DNA  selection  techniques.  gene.  All DNA manipulations such as plasmid purification,  restriction enzyme digestions, ligations, transformations and agarose gel electrophoresis  proceeded  as  previously described  by  Maniatis  et al  (25).  Southern  blotting.  Alkaline southern blotting was carried out using a  Zeta-probe cationic nylon membrane (Bio-Rad Labotatories, Richmond, California).  32  random hexamer labelling of the oprH  out as described previously (10).  gene was carried  Restriction enzymes were obtained  from the Bethesda Research Laboratories (Burlington, Ontario) and used as recommended.  13  Figure 1. Diagram of plasmid pGB32B utilized for gene replacement mutagenisis. The thick bar between the BamHl and Sail restriction sites represents Pseudomonas DNA flanking (white bars) or encoding (black bars) the oprH gene whereas the stippled bar represents the Pstl fragment containing the tetracycline (Tc) resistance gene that was used to 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. .  30  on  EcoRl BamHl  14 Biparental mating as  a modification  experiments.  Biparental mating was carried out  of the triparental mating experiments previously  described by Goldberg and Ohman (12).  E. coli strain S17-1 cells  contained both mob and tra genes necessary for plasmid transfer to P. aeruginosa.  Colonies containing the plasmid of interest were selected on  the basis of antibiotic resistance as previously described.  Antibiotic  Susceptibility  testing.  Antibiotic  susceptibility  was  examined by cell killing assays using polymyxin B, gentamicin and EDTA Tris.  The P.  aeruginosa strains used were 11103, 11181 and 11703.  In  addition, 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 either - sufficient (500 tM) or Mg 2 Mg + -deficient (20 jiM) conditions. 2 were  diluted  1000-fold  into  pre-warmed  (37°C)  30  mM  Cells sodium  phosphate (pH 7.0) buffer or in 50 mM Tris-hydrochioride (pH 8.5). cells were then subject to killing for 5 mm  The  at 37°C with the inclusion of  the appropriate killing agent; polymyxin B (final concentration; 4 ug/ml), gentamicin  (final concentration;  80 ug/mi) or  10 mM EDTA.  The  inclusion of 0.4 % glucose was necessary for gentamicin killing.  Cells  were then diluted and plated for viable counts on proteose peptone agar no. 2 (Difco Laboratories, Detroit, Michigan).  Experiments were done 3  times and statistically analyzed by the NCSS (Number Cruncher Statistical System) computer program.  MIC  determination.  To  determine  the  minimal  inhibitory  concentration (MIC) value, each strain was grown overnight in BM2 minimal medium containing 0.4% glucose as the carbon source for H103,  15 H181, H703, PA0505 and PA06002.  MIC testing for E. coli was carried  out in LB  of the  medium.  containing  serial  Agar plates  two-fold  dilutions  of  appropriate growth media  the  agents were inoculated with approximately  appropriate  antimicrobial  i0 cells in 10 pi volume.  MICs were performed at least 3 times and were assessed after an 18 to 32 hr incubation at 37°C. antibiotic  concentration  at  The MIC value was taken as the lowest which  cell  growth  was  inhibited.  MIC  competition assays were carried out in multiwell dishes in BM2 medium. Nucleosides used as potential competitors were 2’-deoxycytidine and 2’ deoxyadenosine  monohydrate  Biochemocals, St. Laurent, Quebec.  which  were  obtained  from  ICN  The base adenine which was used in  competition assays with nalidixic acid and ciprofloxacin was obtained from Sigma Chemical Co., St. Louis, Mo..  Cell envelope isolation.  Cells were grown to an O.D. 600 of 0.6 to 0.8 in  LB medium or in BM2 minimal medium with vigourous aeration at 37°C with  appropriate  antibiotic  selection.  Cells  were  then  pelleted  by  centrifugation in the Sorvall RC-5B rotor at 7,000 rpm for 10 minutes. The pellet was resuspended in 10 mM , 4 P 2 Na , pH 7.4 with 4 O 5 mM MgSO the inclusion of pancreatic DNase 1 at a final concentration of 50 tg/ml. The suspension was then French pressed twice at 14,000 pounds per square inch.  Cell debris was removed by centrifugation at 3,000 rpm for  10 minutes.  The supernatant was centrifuged at 45,000 rpm for 1 hr  and  envelope-containing pellet was resuspended  the cell  water.  in distilled  16 SDS-polyacrylamide  gel  Cell  electrophoresis.  envelope  preparations were analyzed by SDS-PAGE using a 14% acrylamide gel previously described by Hancock and Carey (13).  Solubilization prior to  SDS-PAGE involved the dilution of the outer membrane protein sample in a 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 kDa form and its 21 kDa heat-modified form (2),  to maximize the amount of  OprH in the 21 kDa band, samples were pretreated with 2% (vol/vol) trichioroacetic acid  Selective  and heated at 100°C for 10 mm  solubilization  of  prior to loading.  The OprH-overproducing strain  OprH.  H181 was used for the isolation of OprH.  Cells from mid-logarithmic  phase cultures (optical density at 600 nm of 0.6 7,000 rpm in a Sorvall RC-5B rotor for  15  -  0.8) were harvested at mm.  The pellet was  resuspended in 20% (wt/vol) sucrose, 10 mM Tris-HC1 (pH 8.0) with the inclusion 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 and  whole cells removed by centrifugation at 3,000 rpm for 10 mm.  A two  step sucrose gradient (50% and 70%) was set up onto which the sample was applied.  Overnight centrifugation at 100,000 x g in a Beckman 5W27  rotor allowed separation of outer and inner membranes.  The more dense  outer membrane fraction was collected and diluted with distilled water to a final sucrose concentration of approximately 5% and centrifuged at 45,000 rpm for 1 hr.  The pellet was resuspended in 10 mM Tris-HC1 (pH  8.0), 0.5% octyl polyoxyethylene (octyl-POE; Bachem Bioscience Inc., Philadelphia, PA) and sonicated 3  x  10 seconds using the Biosonik  17 sonicator (Bronwill Scientific, Inc., Fochester, N.Y.) and centrifuged at 150,000 x g for 1 hr.  The soluble fraction was retained for analysis and  the pellet was resuspended in 10 mM Tris-HC1 (pH 8.0), 3% octyl-POE and centrifuged at 150,000 x g for 1 hr.  The soluble fraction was retained  and the pellet subject to a second solubilization using 3% octyl-POE.  The  fourth and fifth solubilization steps were carried out in 10 mM Tris-HC1 (pH 8.0), 3% octyl-POE, 50 mM EDTA. presence of OprH by SDS-PAGE.  All fractions were tested for the  OprH was primarily solubilized in 10  mM 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 other contaminating solubilization.  proteins The  within  column  (Pharmacia, Laboratory  the  used  fraction was  a Mono  Separation Division,  starting buffer contained  obtained  Q  Uppsala,  by  anion  selective exchanger  Sweden).  10 mM Tris-hydrochioride (pH 8.0),  EDTA and 1% octyl-POE.  The 10 mM  A sample containing 6-8 mg of protein was  loaded onto the column and the proteins were eluted gradually as the salt concentration increased from 0 to 1 M.  Fractions were collected in 1  ml aliquots throughout the FPLC run and tested for the presence of OprH by SDS-polyacrylamide gel electrophoresis.  Black  lipid  bilayer  experiments.  The method has been previously  described in detail by Hancock and Benz (16).  The apparatus consisted of  a Teflon chamber containing two compartments.  Conductance was  measured across a lipid bilayer in the presence of 10-12 M of FPLC purified OprH in 1 M KC1.  The lipid membrane was formed using 1%  18 oxidized cholesterol in  n-decane.  The applied voltage was 10 mV.  Purified OprP (16) provided by Dr. C. Egli from our laboratory, was used as a control.  19  RESULTS  Characterization  of the  OprH-deficient mutant  strain  H703.  In order to access whether the chromosomal oprH gene of H103 had been replaced by the oprH::tet  cartridge  of  11703,  Southern  hybridizations of EcoRl digested chromosomal DNA from both the wildtype strain H103 (Figure 2, lane A) and the OprH-deficient strain H703 (Figure 2, lane B) with a 32 P-labelled oprH  gene probe was carried out.  The results confirmed that the 2.8 kb oprH interrupted by a 1.4 kb tet  chromosomal gene was  The observation that the gene probe  gene.  did not hybridize to other areas of the blot confirmed the lack of a second cross-hybridizing gene.  SDS-PAGE of cell envelopes indicated in  Figure 3 confirmed the lack of OprH protein in strain H703 (lane A). A slight band in the 21 kDa region in cell envelope preparations of H703 was consistently present.  However, comparisons of heated and unheated  samples indicated that the band was not heat-modifiable suggesting that this protein likely represents one of the minor proteins in the outer membrane  and is not OprH.  expression  plasmid  pGB25  in  Expression strain  H703  of protein Hi resulted  in  from the  OprH levels  comparable to that seen in OprH-overexpressing strain 11181 (Figure 3; B,C).  Antibiotic and  EDTA.  susceptibility  testing  to  polymyxin  B,  gentamicin  To assess the extent of OprH involvement in resistance to  polymyxin B, gentamicin and EDTA, susceptibility testing was carried out using  the  OprH-deficient  mutant  strain  H703.  In  contrast  20  kb  4.07 3.05  2.04  -  1.64-  AB Figure 2. Comparison of H103 and the OprH-deficient strain H703 by southern blotting. Autoradiogram of southern hybridization of 32 labelled oprH gene to a gel containing EcoRl digested chromosomal DNA from P. aeruginosa wild-type strain H103 (Lane A) and OprH-deficient Bacteriophage lambda Hindill fragments were strain 11703 (Lane B). used as molecular size markers, which are identified in kilobase pairs on the left. -  21  OprF OprG OprH  Figure 3. SDS-polyacrylamide gel electrophoresis of cell envelope +sufficient 2 preparations demonstrating expression of OprH on Mg Strain H703 oprH ::tet ; Lane B- H703/pGB25 induced medium. Lane A The running H103. H181; Lane D with 5 mM m-toluate; Lane C The indicated. are porin OprF positions of OprH, OprG and the major small amount of protein observed just above the running position of OprH in strain H703 was not heat-modifiable and was not inducible by -deficient medium like OprH and thus was concluded to 2 growth in Mg be another protein. -  -  -  22 to the situation with parent strain H103, the OprH-deficient, oprH: :tet mutant strain H703 remained susceptible to all 3 agents regardless of the 2 concentration during growth (Table 4). Mg  These data supported the  hypothesis that overproduction of OprH during growth on Mg 2 deficient was  medium  obligately  required  to  permit  expression  of  the  polymyxin/gentamicin/EDTA resistance phenotype. To confirm that this result reflected an inability to produce and induce OprH, two controls were performed.  First, it was demonstrated  that H703 had the same MIC (1 ig/ml) to the j3-lactam cefpirome, as the parent strain H103, in both 2 Mg sufficient  and  -deficient  arguing against a non-specific change in permeability.  medium,  Second, plasmid  pGB25, which contains the oprH gene behind a m-toluate-inducible promoter,  was  introduced  into  strain  H703.  The  resultant  strain  H703/pGB25, when grown on Mg +-deficient medium was resistant to 2 both polymyxin B and EDTA compared to the vector plasmid control strain  H703/pNM185  and  the  parent  gentamicin resistance could not be  strain  H703  in  tested  (Table  4,  N.B.  the presence of these  plasmids; 3). Two sets of results confirmed previous suggestions (17) that a second  cellular  alteration  was  required  to  give  the  full  resistance  phenotype observed in strain H103 grown in 2 Mg deficient  medium.  First, as previously observed for strain H103/pGB25 (17), overexpression of OprH in  strain H703/pGB25  was not sufficient to give the full Mg + 2 sufficient  resistance  phenotype  medium.  Indeed both H703/pGB25 (Table 4) and H1O3IpGB25 (17)  when grown on  when  strains  Mg + 2 sufficient  were  medium  grown  in  demonstrated  only  resistance to EDTA and full susceptibility to polymyxin B, despite  partial  23 Table  4. Influence of the OprH phenotype on killing by polymyxin B, gentamicin and EDTA.  Strain  2 Mg  OprH  (tM)  levela  Survivors (%)b  Polymyxin  Gentamicin  EDTA  90±3d  78±3d  89±3e  4±3  2±2  2±3  H103  20  H103  500  H181  20  95±3d  85±3d  87±7d  H181  500  81 ± 5d  78 ± 5d  85 ± 18d  H703  20  8±4  12±5  7±1  H703  500  2±1  4±3  5±2  H703/pGB25  20  79 ± 5d  -c  77 ± 9d  H703/pGB25  500  3 ± 1  -c  30 ± ld,e  H703/pNM185  20  6 ± 5  -c  8 ± 4  H703/pNM185  500  3 ± 2  -c  2 ± 2  +  -  -  -  -  24 Table 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 envelope  protein (Figure 2,),  +  =  small amount of OprH produced,  -  =  no  observable 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 to the nearest integer. C  Due to the aminoglycoside modifying enzyme genes on the plasmids pGB25 and pNM185, gentamicin susceptibility could not be tested.  d Significantly different (p<O.05) by Student’s  t  test to the result for  H103 grown on Mg - sufficient medium 2 Significantly different (p<O.05) to the result for H703/pGB25 grown on -deficient medium 2 Mg  25 OprH.  overexpression  of  H703/pNM185)  on Mg -deficient medium consistently gave increased 2  Second,  growth  of  strain  H703  (and  resistance to these agents, although the increase was far less than that observed for OprH-overexpressing strains.  Influence  of  quinolones  OprH  and  overexpression  chioramphenicol.  To  on test  susceptibility  to  whether quinolones  accessed the self-promoted uptake pathway to cross the outer membrane of P. aeruginosa, the isogenic strains; H103, H703 and H181, containing normal or altered expression of OprH were utilized.  As demonstrated  previously OprH expression in wild type strain H103 was influenced by the level of Mg 2 in the medium.  In low Mg 2 (20 riM) medium, OprH  was overexpressed at least 24-fold (28,29; confirmed in this study) and cells were 4-fold more resistant to polymyxin and gentamicin compared to strain H103 cells grown in Mg -sufficient (500 jiM) medium (27; 2 Table 5).  In strain H703 cells in which the oprH  insertion of a tet  gene was mutated by  gene, OprH was not expressed regardless of the  + concentration, and polymyxin and gentamicin MICs were 2 medium Mg identical to those levels observed for H103 expressing low levels of OprH, in  -sufficient 2 Mg  medium  (Table  5).  In  11181,  strain  which  constitutively overexpresses OprH (28), cells were resistant to polymyxin and gentamicin at levels similar to those observed for strain H103 grown on Mg -deficient medium (Table 5). 2 These strains were utilized to see if OprH could also block uptake of quinolones.  In contrast, cells overexpressing OprH (i.e. strain H181  grown in Mg -sufficient or Mg 2 -deficient medium 2  or  strain H103  26  Table  5.  Influence of OprH expression on MIC values P. aeruginosa for various antimicrobial agents.  MIC(ig/ml)a  Strain  2 Mg  OprH  (mM) level PX GM NAL  H103  0.5  +  0.02  H703  0.5 0.02  11181  -  -  NOR  CIP  FLX  CAP  RIF  TMP  CFP  1  1  256  2  0.5  1  128  8  1024  1  4  4  8  0.25  0.06  0.13  6  8  64  1  1  1  256  2  0.5  1  128  8  1024  1  1  1  256  2  0.5  1  128  8  1024  1  0.5  --++  8  8  16  0.13  0.03  0.06  6  8  64  1  0.02  -1±’-  8  8  16  0.13  0.03  0.06  6  8  64  1  a Abbreviations:  PX, polymyxin B; GM, gentamicin; NAL, nalidixic acid;  NOR, norfioxacin; CIP, ciprofloxacin; FLX, fleroxacin; CAP, chioramphenicol; CFP, cefpirome; RIF, rifampicin; TMP, trimethoprim  27 grown  +deficient 2 Mg  in  medium)  supersusceptible  were  the  to  quinolones nalidixic acid, norfioxacin, ciprofloxacin and fleroxacin and to chioramphenicol when compared to cells with normal (strain H103 +sufficient medium) or no (strain 11703) expression of 2 grown in Mg OprH.  The increase in susceptibility varied from 8 to 32 fold in OprH  overexpressing  cells.  No  alteration  in cefpirome  susceptibility was  observed in any of the above cells. To test if OprH overexpression was supersusceptibility  phenotype,  the  solely responsible for the  quinolone  susceptibility  of  strain  H103 carrying the control plasmid pNM185 was compared with H103 carrying the OprH expression plasmid pGB25 after induction with 5 mM  m -toluate.  Again  OprH  overexpression  was  correlated  with  supersusceptibility to ciprofloxacin (Table 6), nalidixic acid, norfioxacin In addition, MIC values indicated that polymyxin  and chioramphenicol.  resistance was observed for the constitutively OprH-overproducing strain H181 however, this resistance was not observed in the wild-type H103 overexpressing OprH from expression plasmid pGB25. To address the possibility that OprH overexpression leads to a general  increase  calculated  for  in hydrophobic the  antibiotics;  antibiotic rifampicin  uptake, and  MIC  values  trimethoprim.  overexpression in H181 or in H103 grown in Mg -deficient 2  were OprH  conditions  correlated with trimethoprim supersusceptibility, however, susceptibility of P. aeruginosa to rifampicin remained constant, irrespective of the level of OprH expressed (Table 5).  Effect  of  excess  fluoroquinolone  monovalent  susceptibility.  or  divalent  cations  on  The antagonism of excess divalent  28  Table 6.  Susceptibilities of various P. aeruginosa strains which vary in the amount of OprH to polymyxin B and ciprofloxacin  Strain  Plasmidsa  MIC(jlg/ml)C  OprH phenotypeb  PX  NAL  NOR  FLX  CIP  H103  none  +  1  256  2  1  0.5  H103  pNM185  +  1  256  2  1  0.5  H103  pGB25  1  8  0.25  0.13  0.03  H703  none  1  256  2  1  0.5  H181  none  4  8  0.25  0.13  0.03  -  -H-I-  a plasmids were induced for gene expression with 5 mM m-toluate b OprH expression based on SDS-polyacrylamide gel analysis C  Abbreviations:  PX, polymyxin B; NAL, nalidixic acid; NOR, norfioxacin;  FLX, fleroxacin; CIP, ciprofloxacin  29 Mg as previously noted by Chapman et at + cation, 2  (6) was confirmed as  A twenty fold excess of monovalent Na+ ions did not  seen in Table 7.  alter the MIC values for any of the strains tested.  In contrast, 5 mM  + increased the MIC values for all of the strains tested. 2 Mg  A substantial  increase in the MIC value (4-8 fold) was however limited to strains H181, H103/pGB25 and H703/ pGB25, all of which overexpressed OprH. The MIC values calculated for the B-lactam cefpirome were not altered in + or Na+ for any of the strains tested. 2 the presence of excess Mg  Is OprH a porin? and  There were two possible explanations for quinolone  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 the quinolones and chloramphenicol since OprH overexpression did not result in supersusceptibility to other antibiotics.  Therefore the possibility that  these antibiotics were pyrimidine and purine analogs respectively and that 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 or  0.1 mM deoxyadenosine as a potential competitor for quinolone uptake did  not  influence  susceptibility  in  strains  H103,  H703  and  H181.  Similarly, addition of deoxycytidine as a competitor for chloramphenicol uptake did not alter the susceptibility of the strains tested. concluded  that these compounds  were unable  binding site and block quinolone uptake.  to  bind  Thus it was to  a  specific  Additional experiments were  carried out to access the possibility that adenine could compete for uptake  with  ciprofloxacin  and  nalidixic  acid  30 Table 7.  The effect of excess monovalent cation or divalent cations on the MIC value for ciprofloxacin.  Strain  Opril  MIC  levelsa  (jig/ml)b  no addition  5mM MgC12  1 00mM NaC1  H103  +  2  4  1  H181  -i-i-i-  0.13  1  0.13  2  4  1  11703  -  H103/pGB25  -‘-f+  0.13  0.5  0.13  H103/pNM185  +  1  2  1  H703/pGB25  -i-f-i-  0.13  0.5  0.13  1  2  1  H703/pNM185  -  a as judged by SDS-polyacrylamide gel electrophoresis b  The MIC value calculated for the 8-lactam cefpirome was consistently  1.0  jig/ml.  31  Table 8.  Nucleoside uptake assay in the wild-type strain; H103  Antibiotic  2 Mg (jiM)  deoxyadenosine concentration (jiM) 0  0.1  1  20  8  8  NGa  500  128  128  128  NG  20  0.06  0.06  NG  NG  500  0.25  0.25  0.25  NG  20  1  1  NG  NG  500  0.5  0.5  0.5  NO  20  6.3  6.3  6.3  NO  500  128  128  256  NO  10  nalidixic acid  ciprofloxacin  ce fp ir o me  chloramphenic olb  a NO; no growth b deoxycytidine was used as a competitor for chioramphenicol uptake  32  Table 9.  Nucleoside uptake assay in the OprH-deficient strain; H703.  Antibiotic  2 Mg (jiM)  deoxyadenosine concentration (jiM) 0  0.1  1  10  20  128  128  NG  NG  500  128  128  64  NG  20  0.25  0.25  NG  500  0.25  0.5  0.25  NG  20  0.5  1  M3  I’U  500  0.5  0.5  0.5  I’U  20  128  128  128  128  500  256  256  256  256  nalidixic acid  ciprofloxacin  ce fpir o me  chioramphenicoib  a NG; no growth  b deoxycytidine was used as a competitor for chioramphenicol  33  Table 10.  Nucleoside uptake assay in the OprH-overexpressing strain; H181.  Antibiotic  Mg 2 (.tM)  deoxyadenosine concentration (jiM) 0  0.1  1  10  20  8  8  NGa  J..fl  500  16  16  16  NG  20  0.03  0.03  NG  NG  500  0.03  0.03  0.03  NG  20  1  1  NG  NG  500  1  1  1  NG  20  3.2  3.2  NG  NG  500  3.2  3.2  3.2  3.2  nalidixic acid  ciprofloxacin  c e fp i rome  chioramphenicoib  a NG; no growth b deoxycytidine was used as a competitor for chioramphenicol uptake  34 in the OprH-overexpressing strain H181.  No change in the MIC values  were 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 cell  growth. A second test involved an attempt to examine the ability of OprH to reverse 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 indicates  that OprH overexpression was attained in both the naiB well as the parental strain; PA0505. quinolone  supersusceptibility  was  when OprH was overexpressed. alter  the  susceptibility  of  mutant strain as  MIC values calculated indicate that attained  in  the  wild-type  PA0505  However OprH overexpression did not  PA06002  to  either  nalidixic  acid  or  ciprofloxacin (Table 12). To further confirm that OprH was not acting as a porin for quinolone uptake, OprH was purified by selective solubilization followed by FPLC in the presence of the detergent octyl-POE for analysis in black lipid bilayer experiments.  In the selective solubilization, OprH was  primarily solubilized in 3% octyl-POE with 50 mM EDTA.  In the FPLC  profile (Figure 5) a peak which was visible prior to the addition of NaC1 was protein Hi. the void volume.  As expected, since OprH is a basic protein, it eluted in SDS-PAGE analysis (Figure 6, lane A) confirmed that  OprH eluted in a pure form.  The FPLC-purified sample was diluted in 1%  octyl-POE and 0.1% Triton-XiOO for experiments.  analysis in black lipid bilayer  Addition of purified OprH to a final concentration of 8 pg  or 80 pg per ml of bathing solution, resulted in no increase in membrane conductance  (Figure  7;  A,B).  Stepwise  increases  in  membrane  35  Table 11.  MIC analysis using adenine as potential competitor for ciprofloxacin and nalidixic acid uptake.  Antibiotic  Mg 2 (iiM)  adenine concentration (riM) 0  0.1  1  10  20  16  16  8  NGa  500  16  16  16  20  0.03  0.03  0.03  500  0.03  0.03  0.03  20  1  1  1  500  0.5  1  1  nalidixic acid  ciprofloxacin  ce fp i rome  a NG; no growth  I’U  36  OprF  OprH  oprL  ABCD  Figure 4. Cell envelope preparations of P. aeruginosa naiB strain SDS-polyacrylamide gel PA06002 induced for OprH expression. Samples were pretreated with 2% electrophoresis was carried out. trichloroacetic acid and heated at 1000C for 10 minutes prior to loading. Running positions of OprH and Analysis was on a 14% acrylamide gel. Lane the major outer membrane porin protein OprF have been denoted. A- PA0505, lane B- PA0505/pGB25 induced with 5 mM m-toluate, lane C- PA06002, lane D- PA06002/pGB25 induced with 5 mM m-toluate.  37  Table 12.  Effect of OprH overexpression on the nalB4  mutant strain  PA06002 of P. aeruginosa.  Straina  Plasmidb  OprHC  MICd (ig/ml) NAL  CIP  CFP  H103  none  +  256  0.5  1  H181  none  -i-i-i-  16  0.03  1  PA0505  none  +  32  0.5  1  PA0505  pNM185  +  32  0.5  1  PA0505  pGB25  ±1-f-  4  0.03  1  PA06002  none  +  100  0.25  1  PA06002  pNM185  +  100  0.25  1  PA06002  pGB25  +-H-  100  0.25  1  a Strain PA0505 is parental to naiB  strain PA06002  b Plasmid induction with 5 mM m-toluate C  d  As examined by SDS-PAGE Abbreviations:  NAL, nalidixic acid; CIP, ciprofloxacin; CFP, cefpirome  38  Figure 5. Fast performance liquid chromatography profile of OprH purification. Proteins were eluted as the salt concentration increased gradually from 0 to 1M. Ultra-violet light at 280 nm was used for protein absorption. The fractions in peaks 1, 2, 3, and 4 were analyzed for Opril. Peaks 2, 3 and 4 contained unidentified proteins in minute amounts. OprH elutes in the void volume as peak 1. The remainder of the proteins in the sample were eluted in 1M NaC1.  380.  0.3  1  500  400  0.2  ,0  “ 300  o co  E —  C)  Cu  •  ‘200  4  01•  “100  , 0.0  rrrrrrrrrrrrrrf 15 10 5 0  ‘‘‘‘i’’’’I’’’’I’’’’I’’’’  20  25  Fraction no.  30  35  •0 40  z  39  OprF  OprH  LL  A  ‘OprL  B  Figure 6. FPLC-purified OprH. Purification of outer membrane protein OprH from P. aeruginosa strain H181 by selective solubilization of OprH from cell envelope preparations of 11181 grown in BM2-glucose Samples were run on a 14% proceeded as indicated in the text. gel. Lane A- FPLC-purified OprH; Lane B- H181 cell polyacrylamide envelope preparation.  40  B  A  C  5OOS  4 sec 2 O  5mm I  I  I-  -I  The Black lipid bilayer analysis of FPLC-purified OprH. Figure 7. The applied membrane was made from 1% cholesterol in n-decane. are recording chart from a sections mV. Three separate voltage was 10 arrow A, 8 pg/ml purified OprH was added to the chamber; shown: 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 to The time scale on the left refers to stepwise increases in conductance. the chart sections between arrows A and C, the time scale on the right refers to the section of chart recording after arrow C.  41 conductance of the appropriate size(s) were however observed for the control porin OprP when added at a concentration of 8 pg per ml to the c h amber.  OprH overexpression in E. coli.  E. coli strain HB1O1 expressing OprH  from the IPTG-inducable expression plasmid pGB23 resulted in OprH as the major outer membrane protein as seen in figure 8.  The MIC values  calculated (Table 13) indicated that OprH overexpression did not alter the  susceptibility  of E.  coli  chioramphenicol or cefpirome.  to  either  nalidixic  acid,  ciprofloxacin,  42  OprH  A  BC  Figure 8. SDS-polyacrylamide gel analysis of OprH overexpression in E. Cell envelope Acrylamide concentration is 14%. coli strain HB1O1. preparations were treated with 2% trichioroacetic acid and heated to 1000C for 10 mm prior to loading. Expression vector plasmid pRK767 and its OprH-expressing derivative pGB23 were induced with 1 mM IPTG. Lane A- HB1O1; Lane B- HB1O1/pGB23; Lane C- HB1O1/pRK767.  43 Table 13.  Influence of OprH expression on fluoroquinolone susceptibility of E. coli  Strain  Plasmida  MIC  OprH  (.tg/m1)c  leveib  HB1O1  none  HB1O1  pGB23  HB1O1  pRK767  NAL  CIP  CAP  CFP  4  0.01  8  0.25  4  0.01  8  0.25  4  0.01  8  0.25  -  -i-H-  -  a Plasmids were induced for expression with 1 mM IPTG  b As estimated by SDS-polyacrylamide gel electrophoresis C  Abbreviations:  NAL,  nalidixic  chioramphenicol; CFP, cefpirome  acid;  CIP,  ciprofloxacin;  CAP,  44 DISCUSSION  It has been previously demonstrated that overexpression of OprH in the parent strain H103 grown on Mg -deficient (20 pM Mg 2 +) BM2 glucose 2 minimal  medium,  in  the  polymyxin  resistant  mutant  H181,  was  accompanied by resistance to killing by polymyxin B, gentamicin and +.. 2 grown on Mg  EDTA, when compared to results for strain H103  sufficient (500 jiM Mg +) BM2-glucose minimal medium (3,27). 2  This  observation was confirmed here (Table 4) in killing assay experiments. In  addition,  confirmation  construction that  the  of  an  resistance  OprH-deficient  phenotype  mutant  observed  in  allowed  the  OprH  overexpressing strains was due to the ability to express OprH as a major outer membrane protein. OprH  Furthermore, this data suggests that although  overexpression is required  resistance phenotype.  phenotype,  it  is  not  for the polymyxin/gentamicin/EDTA sufficient  itself  by  to  explain  this  Previous pseudorevertant studies suggested that LPS might  also play a role.  However methods for examining the rather complex LPS  aeruginosa are difficult to perform and beyond the scope of this  of P. thesis.  Utilizing the strains from which the above conclusions arose, I tested  the  hypothesis  fluoroquinolones promoted  uptake  could  of  Chapman  penetrate  pathway.  I  and  the  Georgopapadakou  outer  confirmed  membrane their  via  + increased the MIC for fluoroquinolones, 2 Mg  previously  for  agents  that  access  the  that  the  self  observation  exogenous  other  (6)  (6) as  self-promoted  shown uptake  pathway, antagonizes the effects of polymyxin and gentamicin (27). was evident that this antagonizing effect of Mg 2  on  that  It  fluoroquinolone  45 uptake was more extreme in cells which overexpress OprH.  This seems  to indicate a direct involvement of high levels of OprH and the Mg 2  +  antagonism with the quinolone resistance/susceptibility phenotype of P.  aeruginosa.  Researchers  fluoroquinolones  were capable of increasing the permeability  have  been  outer membrane to the hydrophobic  unable  to  fluorophor,  demonstrate  NPN,  the  that  of the  13-lactam  nitrocefin or the protein lysozyme (our laboratory, data not shown), in contrast  to  results that  polycations  obtained  access  for  polymyxin,  self-promoted  gentamicin  uptake  (28).  and  other  Furthermore  overexpression of OprH correlated with supersusceptibility to quinolones (and chloramphenicol) not resistance as observed for gentamicin and polymyxin (Table 5). chloramphenicol  aeruginosa.  are  Thus we can conclude that the quinolones and not  taken  up  by  self-promoted  uptake  in  P.  In addition, it is evident that OprH overexpression does not  result in a general increase in the uptake of hydrophobic antibiotics as seen by the unaltered susceptibility of OprH overexpressing cells to rifampicin. Results obtained using cells in which OprH was overexpressed from the cloned gene confirmed that the supersusceptibility phenotype was entirely 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 of quinolones  and  aeruginosa.  chloramphenicol The observed  across  the  outer  supersusceptibility  membrane to  of P.  trimethoprim,  a  hydrophobic compound structurally unrelated to either the quinolones or chloramphenicol, is consistent with the hypothesis that OprH may be mediating  a  general  non-porin  pathway.  It  must  be  emphasized  46 however  that  quinolone  considered hydrophobic. non-porin  pathway  are  antibiotics  water  soluble  and  thus  not  Therefore it seems unlikely that this postulated  is  equivalent  hydrophobic uptake pathway.  to  the  previously  described  (17)  Although not currently in a position to  define the actual mechanism of uptake of fluoroquinolones, it is possible that OprH, a basic protein might act by partly neutralizing the highly negative  surface  charge  of P.  aeruginosa,  as  indicated by phase  partitioning experiments with strains 11103 and H181 Ph.D. thesis, U.B.C.).  (A. Bell,  This might then permit quinolones to better access  specific’sites involved in uptake via a non-porin pathway. class  of  membrane.  uptake  sites  1989,  might  involve  LPS  aggregates  One potential in  the  outer  Alternatively uptake may occur at the interface between  OprH and LPS in the outer membrane.  Nevertheless uptake at such sites  would not seem to account for the quinolone susceptibility of wild type P. aeruginosa.  Consistent  with  this,  MICs  for  quinolones  and  chloramphenicol were not different for strain H103 grown in 500 jiM 2 (which expresses low levels of OprH) and the oprH::tet mutant Mg (which cannot produce OprH). Information regarding the route of uptake of quinolones across the outer 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 be involved in fluoroquinolone uptake across the outer membrane of P. aeruginosa based on crude compositional analyses of quinolone resistant mutants.  Thus it is possible that the variety of phenotypic alterations in  resistant mutants reflect a chemically more subtle alteration to a non porin pathway of quinolone uptake.  It is known that LPS alterations can  influence outer membrane protein composition (17) and LPS chemistry is  47 so complex that changes to LPS can evade definition by simple chemical analyses.  Several authors have suggested that LPS may be involved in  some way in fluoroquinolone uptake in P. (6,23).  aeruginosa (34) and E. coil  Thus, unless a thorough analysis of LPS is performed, it would  seem inappropriate to conclude  solely on  the basis  of the lack of  expression of specific outer membrane proteins in a quinolone-resistant mutant,  that these proteins are porins which mediate fluoroquinolone  uptake. The results indicate that OprH-overexpression alone renders strains supersensitive to quinolones and chloramphenicol. evidence that OprH is not a porin.  I have also provided  I therefore propose a model of  quinolone uptake which accounts for these findings in which quinolones penetrate the outer membrane of OprH-overexpressing strains via a non porin pathway, and further propose that this general mode of uptake may also be utilized in wild type P. aeruginosa cells.  48  LITERATURE CITED  1.  Bedard, J., S. Chamberland, S. Wong, T. Schollaardt, and L.E. Bryan. 1989.  Contribution of permeability and sensitivity to inhibition of  DNA 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. Pseudomonas aeruginosa:  Outer membrane protein Hi of  Purification, gene cloning and nucleotide  J. Bacteriol. 17 1:321 1-3217.  sequence.  3.  1989.  Bell, A., Bains, M., and R.E.W. Hancock. aeruginosa  1991. Pseudomonas  outer membrane protein OprH:  Expression from the  cloned gene and function in EDTA and gentamicin resistance.  J.  Bacteriol. 173:6657-6664.  4.  Chamberland, S., Bayer, A.S., Schollaardt, T., Wong, S.A., and L.A. Bryan.  1989.  Characterization of mechanisms of quinolone  resistance in Pseudomonas  aeruginosa strains isolated in vitro and in  vivo during experimental endocarditis.  Antimicrob. Agents  Chemother. 33:624-634.  5.  Chamberland, S., F. Malouin, H.R. Rabin, T. Schollaardt, T.R. Parr, and L.E. Bryan.  1990.  Persistence of Pseudomonas aeruginosa during  ciprofloxacin therapy of a cystic fibrosis patient: transient resistance  49 to quinolones and protein F-deficiency.  J. Antimicrob. Chemother..  25:995-1010.  6.  Chapman, J.S., and N.H. Georgopapadakou. permeation in Escherichia coli.  1988.  Routes of quinolone  Antimicrob. Agents Chemother.  3..2.:43 8-442. 7.  Diakos, D.L., V.T. Lolands, and G.G.Jackson. outer membrane proteins of Pseudomonas  1988.  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Hirai, K., H. Aoyama, T. Irikura, S. lyobe, and S. Mitsuhashi.  1986.  Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli. Antimicrob. Agents Chemother. 2.9: 535-538.  20. Hirai, K., S. Suzue, T. Irikura, S. lyobe, and S. Mitsuhashi.  1987.  Mutations producing resistance to norfioxacin in Pseudomonas aeruginosa.  Antimicrob. Agents Chemother. 3j:5 82-586.  21. Ishimoto, K.S., and S. Lory.  1989.  Formation of pilin in Pseudomonas  aeruginosa requires an alternative a factor (RpoN) subunit of RNA polymerase.  Proc. Nati. Acad. Sic. USA :1954-1957.  22. Jorgensen, R.A., S.J. Rothstein, and W.S. Reznikoff.  1979.  A  restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance.  Molec. Gen. Genet. 121:65-72.  23. Legakis, N.J., L.S. Tzouvelekis, A. Makris, and H. Kotsifaki.  1989.  Outer membrane alterations in multiresistant mutants of Pseudomonas aerug inosa selected by ciprofloxacin.  Antimicrob.  Agents Chemother. 33:124-127.  24. Maier, C., E. Bremer, A. Schmid and R. Benz.  1988.  Pore-forming  activity of the Tsx protein from the outer membrane of Escherichia coli. J. Biol. Chem. 23:2493-2499.  52  25. Maniatis, T., E.F. Fritsch, and J. Sambrook. A laboratory manual.  1982.  Molecular cloning:  Cold Spring Harbor Laboratory.  Cold Spring  Harbour, New York.  26. Michéa-Hamzehpour, M., R. Auckenthaler, P. Regamy, and J.-C. Pechère.  1987.  Resistance occurring after fluoroquinolone therapy  of experimental Pse udomonas aerug inosa peritonitis.  Antimicrob.  Agents Chemother. j:l8O3-18O8.  27. Mermod, N., J.L. Ramos, P.R. Lehrbach, and K.N. Timmis.  1986.  Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria.  J. Bacteriol. 1i: 447-454.  28. Nicas, T.I., and R.E.W. Hancock. Pseudomonas  aeruginosa:  1980.  Outer membrane protein Hi of  involvement in adaptive and mutational  resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin.  I. Bacteriol. 143: 872-878.  29. Nicas, T.I. and R.E.W. Hancock.  1983.  Alteration of susceptibility to  EDTA, polymyxin B and gentamicin in Pseudomonas aeruginosa by divalent cation regulation of outer membrane protein Hi 129:509517.  30. Piddock, L.J.V. and R. Wise.  1989.  quinolones and clinical perspectives. : 475-483.  Mechanisms of resistance to J. Antimicrob. Chemother.  53 3 1. Rella, M. and D. Haas.  1982.  Resistance of Pseudomonas aeruginosa  PAO to nalidixic acid and low levels of B-lactam antibiotics: mapping of chromosomal genes.  Antimicrob. Agents Chemother.  32. Robillard, N.L., and A.L. Scarpa.  1988.  2:242-249.  Genetic and physiological  characterization of ciprofloxacin resistance in Pseudo mona s aeruginosa PAO.  Antimicrob. Agents Chemother. 3.2:535-539.  33. Simon, R., U. Preifer, and A. Puhier.  1983.  A broad host range  mobilization system for in vivo genetic engineering: mutagenesis in Gram negative bacteria.  Transposon  Biotechnol. 1:784-790.  34. Yamano, Y., T. Nishikawa, and Y. Komatsu.  1990.  Outer membrane  proteins responsible for the penetration of 13-lactams and quinolones  in Pseudomonas aeruginosa.  J. Antimicrob. Chemother. :175-184.  


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