Correlation between lipopolysaccharide structure and permeability resistance in beta-lactam-resistant Pseudomonas aeruginosa

Godfrey, A J; Hatlelid, L; Bryan, L. E.

doi:10.1128/aac.26.2.181

Cited by 56 publications

(29 citation statements)

References 28 publications

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“…In addition, we previously suggested that the LPS of P. aeruginosa may alter porin function (1). Godfrey et al also presented data suggesting that LPS influenced porin function in P. aeruginosa (6). In light of the data presented in this paper, it may be useful to reexamine the conclusions drawn from these studies.…”

Section: Discussionmentioning

confidence: 57%

Lipopolysaccharide-free Escherichia coli OmpF and Pseudomonas aeruginosa protein P porins are functionally active in lipid bilayer membranes

Parr¹,

Poole²,

Crockford³

et al. 1986

J Bacteriol

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Escherichia coli porin OmpF and Pseudomonas aeruginosa porin protein P were eluted from sodium dodecyl sulfate-polyacrylamide gels. The resultant porin preparations were found to be devoid of detectable lipopolysaccharide (LPS) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining for LPS, direct enzyme-linked immunosorbent assays with LPS-specific monoclonal antibodies, and 2-keto-3-deoxyoctulosonic acid assays. The average conductances, ionic selectivities and incorporation rates of the electroeluted porins were identical to those of their conventionally purified counterparts. These data suggest that LPS is not required per se for porin function.The outer membranes of gram-negative bacteria are known to contain water-filled channels formed by a class of proteins termed porins, which allow the diffusion across these membranes of hydrophilic compounds up to specific molecular size cutoffs (14). Study of these proteins often involves the detergent isolation and purification of the porins, followed by examination of their functional properties under a variety of experimental conditions in liposome or black lipid bilayer systems (14). The information gathered with such techniques has considerably expanded our understanding of porins.Many porin molecules isolated from outer membranes are tightly associated with lipopolysaccharide (LPS) (5,14,16,18). This association has been suggested as being necessary for the reconstitution of functional pores in planar membrane systems (12, 18). However, we feel that the requirement for LPS in the formation of functional porin molecules has not yet been fully defined.In an attempt to resolve this problem and to obtain porins in their native trimeric configuration for use as immunogens in monoclonal antibody production, we developed a technique which allows for the purification of functional porin trimers devoid of detectable LPS. MATERIALS AND METHODSStrains, growth conditions, and protein isolations. Escherichia coli CGSC 6047 (JF733; proC-24 ompA-252 his-53 ompC-262 purE41 ilv-277 met-65 lacY-29 xyl-14 rpsL-97 cycA-1 cycB? tsx-63 X-) (4) and Pseudomonas aeruginosa H287 (ATCC 8689; prototroph) (9) were grown as previously described to obtain outer membranes containing protein OmpF and protein P, respectively (9, 11). Cells were harvested, outer membranes were obtained, and proteins were detergent extracted and purified as previously described (9, 11). The purified protein preparations were loaded onto sodium dodecyl sulfate(SDS)-polyacrylamide gels (1.5 mm thickness) containing 14% acrylamide and electrophoresed by a methodology previously described (7). Solubilization of the proteins in gel sample buffer was performed at 20°C in a solubilization mixture without 2-mercaptoethanol (7). A portion of the gel was stained with Coomassie blue to locate * Corresponding author. the appropriate trimeric bands, and a corresponding region was cut from the unstained, unfixed portion of the gel. The porin-containing gel was crushed with a glass rod to increase its ...

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Section: Discussionmentioning

confidence: 57%

Lipopolysaccharide-free Escherichia coli OmpF and Pseudomonas aeruginosa protein P porins are functionally active in lipid bilayer membranes

Parr¹,

Poole²,

Crockford³

et al. 1986

J Bacteriol

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“…It has been reported that mechanisms of resistance to nalidixic acid in P. aeruginosa involve alterations in replicative DNA synthesis in permeabilized cells and alterations in cell permeability as a result of mutations in the nalA and nalB loci, respectively (32 (1,3,6,7,19,22,27,29,37). No (27).…”

Section: Discussionmentioning

confidence: 99%

Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa

Hirai¹,

Suzue²,

Irikura³

et al. 1987

Antimicrob Agents Chemother

187

164

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Two genetically distinct classes of norfloxacin-resistant Pseudomonas aeruginosa PA04009 mutants were isolated spontaneously. Two norfloxacin resistance genes, nfxA and nfxB, were mapped between hex-9001 and leu-9005 and between pro-9031 and ilv-9023, respectively, on the P. aeruginosa PAO chromosome. The nfxA gene was shown to be an allele of nalA by transductional analysis with bacteriophage F116L. The nfxB mutant showed a 16-fold increase in resistance to norfloxacin and a slight increase in resistance to nalidixic acid. The nfxB mutant was unique in that it showed hypersusceptibility to beta-lactam and aminoglycoside antibiotics. This mutant had about a threefold-lower rate of norfloxacin uptake than that of the wild-type strain or nfxA mutant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins demonstrated the appearance of a 54,000-dalton protein in the nfxB mutant. These findings suggested that the norfloxacin resistance mechanism in the nfxB mutant might be an alteration in outer membrane permeability to norfloxacin.Norfloxacin and other new 4-quinolones show potent antibacterial activity against gram-negative and grampositive bacteria (11,14,20). They also have high in vitro and in vivo antibacterial activity against Pseudomonas aeruginosa strains that show a strong intrinsic resistance to various antimicrobial agents including older quinolones such as nalidixic acid (11,20,42). The high antibacterial activity of new quinolones might be due to their strong inhibitory action against DNA gyrase, a target enzyme of quinolones, which has been isolated from various bacteria including Pseudomonas aeruginosa (5,16,26,28,42).Drug resistance mediated by plasmids or transposons is a serious clinical problem. However, resistance to nalidixic acid and other quinolones in bacteria is due to chromosomal mutations (10,13,(16)(17)(18)32). Plasmids or transposons that carry quinolone resistance genes have not been found in bacteria (4). Several chromosomal mutations, gyrA, nalB, nalC (gyrB), and nalD, conferring nalidixic acid resistance were identified and mapped on the Escherichia coli K-12 chromosome (10,17,18). Recently, we (13) and Hooper et al. (16) identified norfloxacin resistance mutations (norA, norB, norC, nfxA, and nfxB) in E. coli K-12. norA and nfxA were alleles of gyrA encoding the A subunit of DNA gyrase, while norB, norC, and nfxB determined outer membrane permeability resistance to norfloxacin, were associated with a decrease in OmpF porin protein, and were mapped at 34 min, near 8 min, and at 20 to 22 min, respectively (13, 16). Two loci coding for resistance to nalidixic acid, nalA and nalB, have also been mapped on the P. aeruginosa PAO chromosome (32). It has been reported that DNA replication is resistant to nalidixic acid in permeabilized cells of nalA mutants and that nalB mutants cause a decrease of cell permeability to nalidixic acid and carbenicillin (32).To gain information on the resistance mechanisms to norfloxacin in P. aeruginosa, we isolated spontaneous...

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“…OMPs were isolated by the method of Hancock and Nikaido,lg as modified by Godfrey et a1., 20 . Protein concentration was determined by the method of Lowry21 with bovine serum albumin as a standard.…”

Section: Isolation and Characterisation Of Outer-membrane Proteins (Omentioning

confidence: 99%

Iron sources for Haemophilus ducreyi

Lee¹

1991

Journal of Medical Microbiology

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Summary. The ability of various haem-and non-haem-iron-containing compounds to support the growth of iron-limited cultures of Haemophilus ducreyi was assessed in a plate bioassay. Only haemin or the haem-containing proteins, bovine haemoglobin, human haemoglobin and bovine catalase, but not equine cytochrome C1 1, were capable of serving as the sole exogenous iron source. Complexes of haptoglobin-haemoglobin and haem-serum albumin retained the ability to function as iron substrates. In contrast, no growth was observed with FeCl,, human lactoferrin and human transferrin. Siderophore production was not detected with a universal chemical assay. Outer-membrane-protein profiles derived from iron-starved cultures revealed four iron-regulated polypeptides of 65,50,45.5 and 40.5 Kda. These results indicate that haem can supply the requisite iron for growth of H. ducreyi.

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Correlation between lipopolysaccharide structure and permeability resistance in beta-lactam-resistant Pseudomonas aeruginosa

Cited by 56 publications

References 28 publications

Lipopolysaccharide-free Escherichia coli OmpF and Pseudomonas aeruginosa protein P porins are functionally active in lipid bilayer membranes

Lipopolysaccharide-free Escherichia coli OmpF and Pseudomonas aeruginosa protein P porins are functionally active in lipid bilayer membranes

Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa

Iron sources for Haemophilus ducreyi

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