Biochemical Characterization of MsbA from Pseudomonas aeruginosa

Ghanei, Hamed; Abeyrathne, Priyanka D.; Lam, Joseph S.

doi:10.1074/jbc.m702952200

Cited by 26 publications

(25 citation statements)

References 47 publications

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“…In addition, MsbA increases resistance to erythromycin by 86-fold when it is expressed in L. lactis [22]. In contrast, expression of MsbA in Pseudomonas aeruginosa did not confer resistance to erythromycin, but introducing E. coli msbA into P. aeruginosa decreased the susceptibility of this bacterium to erythromycin by 4-fold [46]. Hence, we investigated whether the isogenic msbA mutant was more sensitive to erythromycin than wild-type H. pylori .…”

Section: Resultsmentioning

confidence: 99%

Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori

et al. 2009

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BackgroundContamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism.ResultsThe minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4–10 μg/ml was higher than that in strains with the MICs of 1–3 μg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs.ConclusionThe expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.

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Section: Resultsmentioning

confidence: 99%

Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori

et al. 2009

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“…The truncated LPS was isolated primarily in the IM fraction, and the apparent lengthening of the IM is more consistent with specific IM localization. P. aeruginosa lipid A-core chemically stripped of all phosphates (including the lipid A phosphates) failed to stimulate the ATPase activity of isolated P. aeruginosa MsbA in vitro (16), but this could not be attributed to a particular phosphate(s). In our electron micrographs, there was no obvious blebbing on the inner leaflet of the IM previously seen with MsbA temperature-sensitive mutants of E. coli (20).…”

Section: Discussionmentioning

confidence: 99%

“…The process emerging has several interesting features; lipid A-core, synthesized on the inner leaflet of the inner membrane (IM), is flipped across the IM by the ATP-binding cassette (ABC) transporter MsbA (16, 17). On the periplasmic side, LPS is first extracted from the outer leaflet of the IM by LptC in coordination with the LptBFG transporter complex and then conveyed by the periplasmic protein LptA to the LptDE complex, which inserts fully assembled LPS to the outer leaflet of the OM through an as-yet-uncharacterized process (13, 18).…”

Section: Introductionmentioning

confidence: 99%

Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in Pseudomonas aeruginosa PAO1

DeLucia¹,

Six²,

Caughlan³

et al. 2011

mBio

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Gram-negative outer membrane (OM) integrity is maintained in part by Mg2+ cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria, waaP, encoding an inner-core kinase, could not be inactivated in Pseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential in P. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), two l-glycero-d-manno-heptoses (Hep), and one hexose but completely devoid of phosphates, indicating that phosphorylation by WaaP is necessary for subsequent core phosphorylations. MS analysis of lipid A from WaaP-depleted cells revealed extensive 4-amino-4-deoxy-l-arabinose modification. OM prepared from these cells by Sarkosyl extraction of total membranes or by sucrose density gradient centrifugation lacked truncated LPS. Instead, truncated LPS was detected in the inner membrane fractions, consistent with impaired transport/assembly of this species into the OM.

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“…Attempted mutant studies determined that msbA is essential for in E. coli (Zhou et al, 1998), and this was independently confirmed in Pseudomonas aeruginosa (Ghanei et al, 2007). Similar studies were conducted in N. meningitidis , with somewhat differing results: total LPS levels for the mutant msbA strain were shown to be lower in the parental strain, indicating the potential presence of a feedback inhibition pathway not seen in E. coli (Tefsen et al, 2005).…”

Section: Lipopolysaccharidementioning

confidence: 97%

“…A growing field of knowledge regarding the trafficking and insertion of bacterial membrane components has succeeded in characterizing the key factors involved in membrane biogenesis in a variety of model systems. The eventual targeting of these essential barriers by molecular inhibitors or using them as vaccine candidates has the potential for inducing either bactericidal effects outright, or increased susceptibility to more established antibiotics (Chiu et al, 2007, 2009; Ghanei et al, 2007). Enzymes/proteins found in the cytoplasm, periplasmic space, and outside the bacterial membrane have all evolved under the confines and conditions of each of these unique microenvironments.…”

Section: Introductionmentioning

confidence: 99%

Outer membrane biogenesis in Escherichia coli, Neisseria meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori

Liechti¹,

Goldberg²

2012

Front. Cell. Inf. Microbio.

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The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM). Lipopolysaccharide (LPS) and numerous OM proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its OM profile limits the effectiveness of vaccines or therapeutics that target any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins (OMPs) are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ε proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ε proteobacteria, while the inner and OM associated apparatus of LPS, lipoprotein, and OMP transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to their E. coli counterparts. Eventual targeting of these pathways would have the net effect of severely limiting the delivery/transport of components to the OM and preventing the bacterium's ability to infect its human host.

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Biochemical Characterization of MsbA from Pseudomonas aeruginosa

Abstract: Lipopolysaccharide of

Cited by 26 publications

References 47 publications

Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori

Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori

Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in Pseudomonas aeruginosa PAO1

Outer membrane biogenesis in Escherichia coli, Neisseria meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori

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