Pseudomonas aeruginosa outer membrane protein OprH: expression from the cloned gene and function in EDTA and gentamicin resistance.

Overexpression of major outer membrane protein OprH of Pseudomonas aeruginosa as a result of mutation (in strain H181) or adaptation to low Mg2+ concentrations (in parent strain H103) is accompanied by increased resistance to polymyxin B, gentamicin, and EDTA. A 2.8-kb EcoRI fragment containing the oprH gene was subcloned into several different expression plasmids in Escherichia coli. These experiments showed that significant levels of OprH could be produced from a promoter on the EcoRI fragment; that the cloned oprH gene was not regulated by Mg2+ deficiency; that there were no differences in the expression of OprH in any construction, regardless of whether the gene from strain H103 or its OprH-overexpressing, polymyxin B-resistant derivative, strain H181, was used; and that overexpression of OprH in E. coli to the level observed in P. aeruginosa H181 did not result in a resistance phenotype. These results favored the conclusion that the mutation in strain H181 was a regulatory rather than a promoter mutation. The oprH gene was cloned behind the benzoate-inducible pm promoter in plasmid pGB25 and transferred to P. aeruginosa H103. Overexpression of OprH from the cloned gene in H103/pGB25 resulted in EDTA resistance but not polymyxin B resistance. This result suggested that another factor, possibly lipopolysaccharide, was affected by the mutation in strain H181. Consistent with this suggestion was the demonstration that mutants of strain H181 with alterations in lipopolysaccharide had reverted to wild-type polymyxin B susceptibility but had unaltered gentamicin and EDTA resistance. These data were consistent with the hypothesis that OprH replaces outer membrane-stabilizing divalent cations.     

Complete genome sequences of three Pseudomonas aeruginosa isolates with phenotypes of polymyxin B adaptation and inducible resistance

Clinical "superbug" isolates of Pseudomonas aeruginosa were previously observed to be resistant to several antibiotics, including polymyxin B, and/or to have a distinct, reproducible adaptive polymyxin resistance phenotype, identified by observing "skipped" wells (appearance of extra turbid wells) during broth microdilution testing. Here we report the complete assembled draft genome sequences of three such polymyxin resistant P. aeruginosa strains (9BR, 19BR, and 213BR).     

Preparation of Pseudomonas aeruginosa recombinant outer-membrane protein F and the study of its antigenic properties

In Escherichia coli M15, the gene of P. aeruginosa recombinant outer-membrane protein F (OprF) was cloned. OprF, chromatographically purified on Ni-agarose and containing an additional sequence of 6 histidines on the N-end, was obtained. The purified OprF specifically reacted with rabbit serum, hyperimmune to P. aeruginosa, and in the mice injected with this protein specific IgG antibodies were synthesized. The optimum concentrations of P. aeruginosa OprF were selected for further tests of its protective properties from infection induced by P. aeruginosa.     

Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin.

It is well established that Pseudomonas aeruginosa cells grown in Mg2+-deficient medium acquire nonmutational resistance to the chelator ethylenediaminetetraacetate and to the cationic antibiotic polymyxin B; this type of resistance can be reversed by transferring the cells to Mg2+-sufficient medium for a few generations. Stable mutants resistant to polymyxin B were isolated and shown to have also gained ethylenediaminetetraacetate resistance. Both the mutants and strains grown on Mg2+-deficient medium had greatly enhanced levels of outer membrane protein H1 when compared with the wild-type strain or with revertants grown in Mg2+-sufficient medium. It was determined that in all strains and at all medium Mg2+ concentrations, the cell envelope Mg2+ concentration varied inversely with the amount of protein H1. In addition, the increase in protein H1 in the mutants was associated with an increase in resistance to another group of cationic antibiotics, the aminoglycosides, e.g., gentamicin. We propose that protein H1 acts by replacing Mg2+ at a site on the lipopolysaccharide which can otherwise be attacked by the cationic antibiotics or ethylenediaminetetraacetate.     

Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major nosocomial pathogen that infects cystic fibrosis and immunocompromised patients. The impermeability of the P. aeruginosa outer membrane contributes substantially to the notorious antibiotic resistance of this human pathogen. This impermeability is partially imparted by the outer membrane protein H (OprH). Here we have solved the structure of OprH in a lipid environment by solution NMR. The structure reveals an eight-stranded β-barrel protein with four extracellular loops of unequal size. Fast time-scale dynamics measurements show that the extracellular loops are disordered and unstructured. It was previously suggested that the function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with lipopolysaccharide (LPS) molecules. Using in vivo and in vitro biochemical assays, we show that OprH indeed interacts with LPS in P. aeruginosa outer membranes. Based upon NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, we conclude that the interaction is predominantly electrostatic and localized to charged regions near both rims of the barrel, but also through two conspicuous tyrosines in the middle of the bilayer. These results provide the first molecular structure of OprH and offer evidence for multiple interactions between OprH and LPS that likely contribute to the antibiotic resistance of P. aeruginosa.     

Membrane topology of the outer membrane protein OprH from Pseudomonas aeruginosa: PCR-mediated site-directed insertion and deletion mutagenesis.

The 21-kDa outer membrane protein OprH from Pseudomonas aeruginosa is overexpressed under Mg2+ starvation conditions and when overproduced causes resistance to polymyxin B, gentamicin, and EDTA. By circular dichroism analysis, OprH revealed a calculated beta-sheet structure content of 47.3%. PCR-based site-directed deletion and epitope insertion mutagenesis was used to test a topological model of OprH as an eight-stranded beta-barrel. Three permissive and seven nonpermissive malarial epitope insertion mutants and four permissive and four nonpermissive deletion mutants confirmed the general accuracy of this model. Thus, OprH is the smallest outer membrane protein to date to be confirmed as a beta-stranded protein.     

Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism

The isolated outer membrane from cells of a Pseudomonas aeruginosa strain exhibiting adaptive resistance to polymyxin was not affected by polymyxin treatment, as monitored by electron microscopy of negatively stained preparations. This was in sharp contrast with extensive disruption by polymyxin of the outer membranes of the parent polymyxin-sensitive strain and the resistant strain following reversion to greater polymyxin sensitivity. The isolated cytoplasmic membrane of the polymyxin-resistant strain, on the other hand, remained sensitive to the disruptive effects of polymyxin treatment. The permeability characteristics of the resistant strains appear to be altered, as indicated by differences in minimal inhibitory concentrations for a variety of antibiotics between the polymyxin-sensitive and polymyxin-resistant strains. No evidence was found for a polymyxin-inactivating enzyme in osmotic shock fluid from the polymyxin-resistant strain. No evidence for a cytoplasmic membrane repair mechanism was found in the polymyxin-resistant strain. These observations suggest that the mechanism of adaptive polymyxin resistance in this model system is the alteration of the outer membrane so that it excludes polymyxin from reaching the still sensitive cytoplasmic membrane.     

Growth characteristics and expression of iron-regulated outer-membrane proteins of chemostat-grown biofilm cells of Pseudomonas aeruginosa.

An in vitro chemostat system was used to study the growth and the expression of iron-regulated outer-membrane proteins (IROMPs) by biofilm cells of Pseudomonas aeruginosa cultivated under conditions of iron limitation. The population of the planktonic cells decreased when the dilution rate was increased. At a dilution rate of 0.05 h-1, the populations of planktonic cells of both mucoid and nonmucoid P. aeruginosa were 3 x 10(9) cells/mL. This value dropped to 5 x 10(6) cells/mL when the dilution rate was increased to 1.0 h-1. The reverse was observed for the biofilm cells. The number of biofilm cells colonising the silicone tubing increased when the dilution rate was increased. The number of biofilm cells of the mucoid strain at steady state was 2 x 10(8) cells/cm (length) when the dilution rate was fixed at 0.05 h-1. The figure increased to 8 x 10(9) cells/cm when the dilution rate was increased to 1.0 h-1. The population of biofilm cells of the nonmucoid strain was 9 x 10(7) cells/cm (length) when the dilution rate was 0.05 h-1. It increased to 2 x 10(9) cells/cm when the dilution rate was set at 1.0 h-1. The expression of IROMPs was induced in the biofilm cells of both mucoid and nonmucoid strains when the dilution rates were 0.05 and 0.2 h-1. IROMPs were reduced but still detectable at the dilution rate of 0.5 h-1. However, the expression of IROMPs was repressed when the dilution rate was increased to 1.0 h-1. The data suggest that the biofilm cells of P. aeruginosa switch on the expression of IROMPs to assist iron acquisition when the dilution rate used for the chemostat run is below 0.5 h-1. The high affinity iron uptake system is not required by the biofilm cells when the dilution rate is increased because the trace amount of iron present in the chemostat is sufficient for the growth of adherent biofilm cells.     

Effective antibiotics in combination against extreme drug-resistant Pseudomonas aeruginosa with decreased susceptibility to polymyxin B

Objective

Extreme drug-resistant Pseudomonas aeruginosa (XDR-PA) with decreased susceptibility to polymyxin B (PB) has emerged in Singapore, causing infections in immunocompromised hosts. Combination therapy may be the only viable therapeutic option until new antibiotics become available. The objective of this study is to assess the in vitro activity of various antibiotics against local XDR-PA isolates.

Methods

PA isolates from all public hospitals in Singapore were systematically collected between 2006 and 2007. MICs were determined according to CLSI guidelines. All XDR-PA isolates identified were genotyped using a PCR-based method. Time-kill studies (TKS) were performed with approximately 10⁵ CFU/ml at baseline using clinically achievable unbound concentrations of amikacin (A), levofloxacin (L), meropenem (M), rifampicin (R) and PB alone and in combination. Bactericidal activity (primary endpoint) was defined as a ≥3 log₁₀ CFU/ml decrease in the colony count from the initial inoculum at 24 hours.

Results

22 clinical XDR-PA isolates with PB MIC 2–16 µg/ml were collected. From clonal typing, 5 clonal groups were identified and nine isolates exhibited clonal diversity. In TKS, meropenem plus PB, amikacin plus meropenem, amikacin plus rifampicin, amikacin plus PB exhibited bactericidal activity in 8/22, 3/22, 1/22 and 6/22 isolates at 24 hours respectively. Against the remaining ten isolates where none of the dual-drug combination achieved bactericidal activity against, only the triple-antibiotic combinations of ARP and AMP achieved bactericidal activity against 7/10 and 6/10 isolates respectively.

Conclusion

Bactericidal activity with sustained killing effect of ≥99.9% is critical for eradicating XDR-PA infections, especially in immunocompromised hosts. These findings underscore the difficulty of developing combination therapeutic options against XDR-PA, demonstrating that at least 3 antibiotics are required in combination and that efficacy is strain dependant.     

Gene cloning and expression of the Pseudomonas aeruginosa periplasmic phosphate-binding protein

Abstract The pstS gene, encoding the Pseudomonas aeruginosa phosphate-binding protein, was cloned onto a cosmid vector into Escherichia coli , and localized by subcloning, mapping the insertion site of Tn 501 in a P. aeruginosa pstS ::Tn 501 mutant, and hybridization to an oligonucleotide pool synthesized according to the aminoterminal amino acid sequence of the purified protein. The cloned pstS gene was transferred to P. aeruginosa pstS mutants. The P. aeruoginosa phosphate-binding protein was also expressed and secreted into the periplasm of E. coli pstS mutants.     

Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa

The two-component regulatory system PhoP-PhoQ of Pseudomonas aeruginosa regulates resistance to cationic antimicrobial peptides, polymyxin B and aminoglycosides in response to low Mg2+ conditions. We have identified a second two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides. This system responds to limiting Mg2+, and is affected by a phoQ, but not a phoP mutation. Inactivation of the pmrB sensor kinase and pmrA response regulator greatly decreased the expression of the operon encoding pmrA-pmrB while expression of the response regulator pmrA in trans resulted in increased activation suggesting that the pmrA-pmrB operon is autoregulated. Interposon mutants in pmrB, pmrA, or in an intergenic region upstream of pmrA-pmrB exhibited two to 16-fold increased susceptibility to polymyxin B and cationic antimicrobial peptides. The pmrA-pmrB operon was also found to be activated by a number of cationic peptides including polymyxins B and E, cattle indolicidin and synthetic variants as well as LL-37, a component of human innate immunity, whereas peptides with the lowest minimum inhibitory concentrations tended to be the weakest inducers. Additionally, we showed that the putative LPS modification operon, PA3552-PA3559, was also induced by cationic peptides, but its expression was only partially dependent on the PmrA-PmrB system. The discovery that the PmrA-PmrB two-component system regulates resistance to cationic peptides and that both it and the putative LPS modification system are induced by cationic antimicrobial peptides has major implications for the development of these antibiotics as a therapy for P. aeruginosa infections.