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

Chiu HC, Lin TL, Yang JC, Wang JT - BMC Microbiol. (2009)

Bottom Line: 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.Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei City 10051, Taiwan, Republic of China. gaki98@yahoo.com.tw

ABSTRACT

Background: Contamination 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.

Results: The 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 microg/ml was higher than that in strains with the MICs of 1-3 microg/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.

Conclusion: The 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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Silver-stained of proteinase-K digested whole cell lysate from H. pylori wild-type and isogenic mutants. (A) Lanes 1–7 were all loaded with 2.5 × 108 proteinase K-digested bacteria (~130 μg total protein). Lane 1, 26695; lane 2, wild-type; lane 3, imp/ostA single mutant strain; lane 4, imp/ostA complementation strain; lane 5, msbA single mutant strain; lane 6, msbA complementation strain; lane 7,imp/ostA and msbA double mutant strain. Molecular weights of the prestained markers are indicated. (B-C) Immunoblots of LPS from H. pylori with anti-Lea or anti-Leb monoclonal antibodies. (B) anti-Lea (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody, or (C) anti-Leb (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody.
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Figure 7: Silver-stained of proteinase-K digested whole cell lysate from H. pylori wild-type and isogenic mutants. (A) Lanes 1–7 were all loaded with 2.5 × 108 proteinase K-digested bacteria (~130 μg total protein). Lane 1, 26695; lane 2, wild-type; lane 3, imp/ostA single mutant strain; lane 4, imp/ostA complementation strain; lane 5, msbA single mutant strain; lane 6, msbA complementation strain; lane 7,imp/ostA and msbA double mutant strain. Molecular weights of the prestained markers are indicated. (B-C) Immunoblots of LPS from H. pylori with anti-Lea or anti-Leb monoclonal antibodies. (B) anti-Lea (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody, or (C) anti-Leb (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody.

Mentions: To investigate whether imp/ostA and msbA participated in LPS biogenesis, the equivalent amounts of proteinase K-digested whole cells were analyzed by silver staining (Fig. 7A). The total amount of LPS was drastically reduced in the imp/ostA single mutant compared with that in the wild-type strain (Fig. 7A, lane 3). This result indicated that imp/ostA participated in LPS biogenesis and is consistent with a similar finding in N. meningitidis [20]. Mutation of msbA decreased LPS production, although small amounts of LPS could be detected (Fig. 7A, lane 5). Furthermore, deletion of both imp/ostA and msbA severely reduced LPS production. The LPS in H. pylori was detected by using anti-Lea (Fig. 7B) or anti-Leb antibody (Fig. 7C). H. pylori 26695 strain expressing Lex and Ley but not Lea or Leb antigens was used as a negative control to ensure the specificity of the anti-Lea or anti-Leb antibody (Fig. 7B and 7C, lane 1). The data indicated that the NTUH-S1 strain expressed both the Lea and Leb antigens. In addition, the amounts of O-antigen (~34 kDa) in the imp/ostA or msbA single mutant were reduced, and it was especially reduced in the imp/ostA and msbA double mutant. The growth curves of the wild-type and mutant strains were also examined, and the growth rates of these mutants did not differ from that of the wild-type strain (data not shown). This result demonstrated that both imp/ostA and msbA were involved in the production of LPS.


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

Chiu HC, Lin TL, Yang JC, Wang JT - BMC Microbiol. (2009)

Silver-stained of proteinase-K digested whole cell lysate from H. pylori wild-type and isogenic mutants. (A) Lanes 1–7 were all loaded with 2.5 × 108 proteinase K-digested bacteria (~130 μg total protein). Lane 1, 26695; lane 2, wild-type; lane 3, imp/ostA single mutant strain; lane 4, imp/ostA complementation strain; lane 5, msbA single mutant strain; lane 6, msbA complementation strain; lane 7,imp/ostA and msbA double mutant strain. Molecular weights of the prestained markers are indicated. (B-C) Immunoblots of LPS from H. pylori with anti-Lea or anti-Leb monoclonal antibodies. (B) anti-Lea (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody, or (C) anti-Leb (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719649&req=5

Figure 7: Silver-stained of proteinase-K digested whole cell lysate from H. pylori wild-type and isogenic mutants. (A) Lanes 1–7 were all loaded with 2.5 × 108 proteinase K-digested bacteria (~130 μg total protein). Lane 1, 26695; lane 2, wild-type; lane 3, imp/ostA single mutant strain; lane 4, imp/ostA complementation strain; lane 5, msbA single mutant strain; lane 6, msbA complementation strain; lane 7,imp/ostA and msbA double mutant strain. Molecular weights of the prestained markers are indicated. (B-C) Immunoblots of LPS from H. pylori with anti-Lea or anti-Leb monoclonal antibodies. (B) anti-Lea (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody, or (C) anti-Leb (1:3000) as the primary antibody and anti-mouse IgG (1:5000) as the secondary antibody.
Mentions: To investigate whether imp/ostA and msbA participated in LPS biogenesis, the equivalent amounts of proteinase K-digested whole cells were analyzed by silver staining (Fig. 7A). The total amount of LPS was drastically reduced in the imp/ostA single mutant compared with that in the wild-type strain (Fig. 7A, lane 3). This result indicated that imp/ostA participated in LPS biogenesis and is consistent with a similar finding in N. meningitidis [20]. Mutation of msbA decreased LPS production, although small amounts of LPS could be detected (Fig. 7A, lane 5). Furthermore, deletion of both imp/ostA and msbA severely reduced LPS production. The LPS in H. pylori was detected by using anti-Lea (Fig. 7B) or anti-Leb antibody (Fig. 7C). H. pylori 26695 strain expressing Lex and Ley but not Lea or Leb antigens was used as a negative control to ensure the specificity of the anti-Lea or anti-Leb antibody (Fig. 7B and 7C, lane 1). The data indicated that the NTUH-S1 strain expressed both the Lea and Leb antigens. In addition, the amounts of O-antigen (~34 kDa) in the imp/ostA or msbA single mutant were reduced, and it was especially reduced in the imp/ostA and msbA double mutant. The growth curves of the wild-type and mutant strains were also examined, and the growth rates of these mutants did not differ from that of the wild-type strain (data not shown). This result demonstrated that both imp/ostA and msbA were involved in the production of LPS.

Bottom Line: 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.Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei City 10051, Taiwan, Republic of China. gaki98@yahoo.com.tw

ABSTRACT

Background: Contamination 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.

Results: The 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 microg/ml was higher than that in strains with the MICs of 1-3 microg/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.

Conclusion: The 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.

Show MeSH
Related in: MedlinePlus