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Moonlighting of Helicobacter pylori catalase protects against complement-mediated killing by utilising the host molecule vitronectin.

Richter C, Mukherjee O, Ermert D, Singh B, Su YC, Agarwal V, Blom AM, Riesbeck K - Sci Rep (2016)

Bottom Line: Surprisingly, by using proteomics, we found that the hydrogen peroxide-neutralizing enzyme catalase KatA is a major Vn-binding protein.Deletion of the katA gene in three different strains resulted in impaired binding of Vn.Taken together, the virulence factor KatA is a Vn-binding protein that moonlights on the surface of H. pylori to promote bacterial evasion of host innate immunity.

View Article: PubMed Central - PubMed

Affiliation: Clinical Microbiology, Department of Translational Medicine, Lund University, SE-205 02 Malmö, Sweden.

ABSTRACT
Helicobacter pylori is an important human pathogen and a common cause of peptic ulcers and gastric cancer. Despite H. pylori provoking strong innate and adaptive immune responses, the bacterium is able to successfully establish long-term infections. Vitronectin (Vn), a component of both the extracellular matrix and plasma, is involved in many physiological processes, including regulation of the complement system. The aim of this study was to define a receptor in H. pylori that binds Vn and determine the significance of the interaction for virulence. Surprisingly, by using proteomics, we found that the hydrogen peroxide-neutralizing enzyme catalase KatA is a major Vn-binding protein. Deletion of the katA gene in three different strains resulted in impaired binding of Vn. Recombinant KatA was generated and shown to bind with high affinity to a region between heparin-binding domain 2 and 3 of Vn that differs from previously characterised bacterial binding sites on the molecule. In terms of function, KatA protected H. pylori from complement-mediated killing in a Vn-dependent manner. Taken together, the virulence factor KatA is a Vn-binding protein that moonlights on the surface of H. pylori to promote bacterial evasion of host innate immunity.

No MeSH data available.


Related in: MedlinePlus

Deposition of MAC is increased in H. pylori strains lacking katA.Deposition of C9 on CCUG18943, KR697, and KR497 wt and ΔkatA strains was analysed by ELISA. Bacteria were coated onto 96-well Maxisorp plates and incubated with 5% NHS negative for anti-Hp IgG. C9 deposition was detected using specific antibodies. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (*) equals p < 0.05 and (**) equals p < 0.01. Data presented are the mean and SE of three independent biological replicates performed in duplicates.
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f8: Deposition of MAC is increased in H. pylori strains lacking katA.Deposition of C9 on CCUG18943, KR697, and KR497 wt and ΔkatA strains was analysed by ELISA. Bacteria were coated onto 96-well Maxisorp plates and incubated with 5% NHS negative for anti-Hp IgG. C9 deposition was detected using specific antibodies. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (*) equals p < 0.05 and (**) equals p < 0.01. Data presented are the mean and SE of three independent biological replicates performed in duplicates.

Mentions: As mentioned above, Vn interferes with formation of the MAC. Consequently, if H. pylori used KatA to bind Vn, MAC deposition should be decreased on H. pylori wt strains compared to those in which katA has been deleted. We therefore conducted an ELISA, in which we determined C9 deposition on wt and ΔkatA of CCUG18943, KR697, and KR497 after incubation with 5% anti-Hp IgG negative NHS (Fig. 8). Indeed, we observed a statistically significant increased MAC deposition on all three ΔkatA strains further confirming a role of KatA in complement resistance. Surprisingly, this finding also included strain KR497, where no significant difference was seen between wt and ΔkatA in the complement killing assays. This discrepancy is best explained with the different nature of the two assays. While the complement killing assay, which is based upon CFU count, gives information on differences in the number of culturable bacteria, the ELISA does not discriminate between viable but not culturable (VBNC) and culturable forms. It is currently impossible to revive coccoid VBNC H. pylori under laboratory conditions, but there are hints that this form is still relevant for pathogenesis in the host40. Hence we cannot exclude a possible effect of a KatA-dependent complement resistance related to Vn for strain KR497, even though it might not be visible in a CFU based assay. Taken together, our data on complement resistance clearly demonstrate the importance of KatA in high and medium Vn-binding strains and even indicate involvement of KatA in Vn-mediated complement resistance in low Vn-binding strains.


Moonlighting of Helicobacter pylori catalase protects against complement-mediated killing by utilising the host molecule vitronectin.

Richter C, Mukherjee O, Ermert D, Singh B, Su YC, Agarwal V, Blom AM, Riesbeck K - Sci Rep (2016)

Deposition of MAC is increased in H. pylori strains lacking katA.Deposition of C9 on CCUG18943, KR697, and KR497 wt and ΔkatA strains was analysed by ELISA. Bacteria were coated onto 96-well Maxisorp plates and incubated with 5% NHS negative for anti-Hp IgG. C9 deposition was detected using specific antibodies. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (*) equals p < 0.05 and (**) equals p < 0.01. Data presented are the mean and SE of three independent biological replicates performed in duplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834553&req=5

f8: Deposition of MAC is increased in H. pylori strains lacking katA.Deposition of C9 on CCUG18943, KR697, and KR497 wt and ΔkatA strains was analysed by ELISA. Bacteria were coated onto 96-well Maxisorp plates and incubated with 5% NHS negative for anti-Hp IgG. C9 deposition was detected using specific antibodies. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (*) equals p < 0.05 and (**) equals p < 0.01. Data presented are the mean and SE of three independent biological replicates performed in duplicates.
Mentions: As mentioned above, Vn interferes with formation of the MAC. Consequently, if H. pylori used KatA to bind Vn, MAC deposition should be decreased on H. pylori wt strains compared to those in which katA has been deleted. We therefore conducted an ELISA, in which we determined C9 deposition on wt and ΔkatA of CCUG18943, KR697, and KR497 after incubation with 5% anti-Hp IgG negative NHS (Fig. 8). Indeed, we observed a statistically significant increased MAC deposition on all three ΔkatA strains further confirming a role of KatA in complement resistance. Surprisingly, this finding also included strain KR497, where no significant difference was seen between wt and ΔkatA in the complement killing assays. This discrepancy is best explained with the different nature of the two assays. While the complement killing assay, which is based upon CFU count, gives information on differences in the number of culturable bacteria, the ELISA does not discriminate between viable but not culturable (VBNC) and culturable forms. It is currently impossible to revive coccoid VBNC H. pylori under laboratory conditions, but there are hints that this form is still relevant for pathogenesis in the host40. Hence we cannot exclude a possible effect of a KatA-dependent complement resistance related to Vn for strain KR497, even though it might not be visible in a CFU based assay. Taken together, our data on complement resistance clearly demonstrate the importance of KatA in high and medium Vn-binding strains and even indicate involvement of KatA in Vn-mediated complement resistance in low Vn-binding strains.

Bottom Line: Surprisingly, by using proteomics, we found that the hydrogen peroxide-neutralizing enzyme catalase KatA is a major Vn-binding protein.Deletion of the katA gene in three different strains resulted in impaired binding of Vn.Taken together, the virulence factor KatA is a Vn-binding protein that moonlights on the surface of H. pylori to promote bacterial evasion of host innate immunity.

View Article: PubMed Central - PubMed

Affiliation: Clinical Microbiology, Department of Translational Medicine, Lund University, SE-205 02 Malmö, Sweden.

ABSTRACT
Helicobacter pylori is an important human pathogen and a common cause of peptic ulcers and gastric cancer. Despite H. pylori provoking strong innate and adaptive immune responses, the bacterium is able to successfully establish long-term infections. Vitronectin (Vn), a component of both the extracellular matrix and plasma, is involved in many physiological processes, including regulation of the complement system. The aim of this study was to define a receptor in H. pylori that binds Vn and determine the significance of the interaction for virulence. Surprisingly, by using proteomics, we found that the hydrogen peroxide-neutralizing enzyme catalase KatA is a major Vn-binding protein. Deletion of the katA gene in three different strains resulted in impaired binding of Vn. Recombinant KatA was generated and shown to bind with high affinity to a region between heparin-binding domain 2 and 3 of Vn that differs from previously characterised bacterial binding sites on the molecule. In terms of function, KatA protected H. pylori from complement-mediated killing in a Vn-dependent manner. Taken together, the virulence factor KatA is a Vn-binding protein that moonlights on the surface of H. pylori to promote bacterial evasion of host innate immunity.

No MeSH data available.


Related in: MedlinePlus