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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

KatA interacts with Vn via an unusual binding site.(a) Inhibition of the KatA-Vn interaction by heparin was tested by ELISA. KatA (100 nM) was immobilised on plates and Vn80–396 was added after being pre-incubated with different concentrations of heparin. (b) The ability of Vn-fragments to bind KatA was tested by ELISA. KatA (100 nM) was immobilised and incubated with different Vn-fragments at a concentration of 20 nM. Data shown are the mean and SD of three independent experiments performed in technical triplicate. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (*) equals p < 0.05 and (***) equals p < 0.001.
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f4: KatA interacts with Vn via an unusual binding site.(a) Inhibition of the KatA-Vn interaction by heparin was tested by ELISA. KatA (100 nM) was immobilised on plates and Vn80–396 was added after being pre-incubated with different concentrations of heparin. (b) The ability of Vn-fragments to bind KatA was tested by ELISA. KatA (100 nM) was immobilised and incubated with different Vn-fragments at a concentration of 20 nM. Data shown are the mean and SD of three independent experiments performed in technical triplicate. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (*) equals p < 0.05 and (***) equals p < 0.001.

Mentions: There are two major bacterial binding regions within the Vn molecule. Most pathogens bind to the C-terminal HBD-325. The second binding region, used by N. meningitidis Vn-binding proteins Msf and Opc is located at the N-terminal end of the Vn molecule and the importance of sulphated tyrosine residues Y56 and Y59 was demonstrated for Opc binding to Vn2627. Since KatA bound to Vn80–396, we suspected an interaction with HBD-3 without involvement of the N-terminus. When bacterial Vn-binding proteins utilise the C-terminal HBD-3, binding can be effectively blocked by heparin. Thus, we tested the influence of heparin on the Vn-KatA interaction in an ELISA (Fig. 4a), and pre-incubated Vn with increasing concentrations of heparin (0.1–100 μg/ml). Heparin inhibited binding of KatA to Vn by only 80%, and higher heparin concentrations had no stronger inhibitory effect (data not shown). This result suggests that, in contrast to most other known Vn-binding proteins, KatA does not use HBD-3 as a major binding site but may have additional binding region(s). To narrow down the binding site, binding of KatA to a range of truncated Vn molecules was tested by ELISA (Fig. 4b). KatA was immobilised in 96 well plates and incubated with different Vn-fragments, which were detected using anti-Vn antibodies (Abs). Removal of the C-terminus distal of HBD-3 (Vn80–373) had no significant effect on binding when compared to Vn80–396. Similarly, Vn80–339, which lacks the whole HBD-3, showed only a minor decrease in binding KatA, which is in agreement with our observation that heparin had only a partial inhibitory effect. Strikingly, binding was substantially impaired with fragment Vn80–229 (Fig. 4b). Reciprocal experiments, i.e., when Vn-fragments were immobilised, incubated with KatA and binding was detected using anti-KatA Abs, gave similar results (data not shown). We observed some residual binding of Vn80–229 and cannot fully exclude involvement of residues located further upstream. However, the primary binding site for KatA is located within amino acids 229 and 339 of the Vn-molecule, a region not involved in interactions with any bacterial Vn-binding proteins described to date. The fact that heparin has an inhibitory effect on the interaction, even though HBD-3 is not the primary binding site, can be explained with the model structures of Vn prepared by two independent research groups2829. Both models proposed a close proximity of the central domain and the C-terminal HBD resulting in a large inter-domain contact surface, which contains the putative heparin-binding groove. Given the spatial proximity between HBD-3 and our predicted KatA binding region within the central domain, it is feasible that bound heparin sterically disrupts the interaction between KatA and Vn rather than occupying the binding site itself. Docking of heparin to Vn28 supports this hypothesis. The use of an alternative binding site also explains why our kinetic data differed from data obtained for UspA2 and protein F, which both use HBD-3 as binding site2224. In summary, H. pylori KatA uses a not previously characterised bacterial binding site on the Vn-molecule, which allows a high affinity interaction.


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)

KatA interacts with Vn via an unusual binding site.(a) Inhibition of the KatA-Vn interaction by heparin was tested by ELISA. KatA (100 nM) was immobilised on plates and Vn80–396 was added after being pre-incubated with different concentrations of heparin. (b) The ability of Vn-fragments to bind KatA was tested by ELISA. KatA (100 nM) was immobilised and incubated with different Vn-fragments at a concentration of 20 nM. Data shown are the mean and SD of three independent experiments performed in technical triplicate. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (*) equals p < 0.05 and (***) equals p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: KatA interacts with Vn via an unusual binding site.(a) Inhibition of the KatA-Vn interaction by heparin was tested by ELISA. KatA (100 nM) was immobilised on plates and Vn80–396 was added after being pre-incubated with different concentrations of heparin. (b) The ability of Vn-fragments to bind KatA was tested by ELISA. KatA (100 nM) was immobilised and incubated with different Vn-fragments at a concentration of 20 nM. Data shown are the mean and SD of three independent experiments performed in technical triplicate. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (*) equals p < 0.05 and (***) equals p < 0.001.
Mentions: There are two major bacterial binding regions within the Vn molecule. Most pathogens bind to the C-terminal HBD-325. The second binding region, used by N. meningitidis Vn-binding proteins Msf and Opc is located at the N-terminal end of the Vn molecule and the importance of sulphated tyrosine residues Y56 and Y59 was demonstrated for Opc binding to Vn2627. Since KatA bound to Vn80–396, we suspected an interaction with HBD-3 without involvement of the N-terminus. When bacterial Vn-binding proteins utilise the C-terminal HBD-3, binding can be effectively blocked by heparin. Thus, we tested the influence of heparin on the Vn-KatA interaction in an ELISA (Fig. 4a), and pre-incubated Vn with increasing concentrations of heparin (0.1–100 μg/ml). Heparin inhibited binding of KatA to Vn by only 80%, and higher heparin concentrations had no stronger inhibitory effect (data not shown). This result suggests that, in contrast to most other known Vn-binding proteins, KatA does not use HBD-3 as a major binding site but may have additional binding region(s). To narrow down the binding site, binding of KatA to a range of truncated Vn molecules was tested by ELISA (Fig. 4b). KatA was immobilised in 96 well plates and incubated with different Vn-fragments, which were detected using anti-Vn antibodies (Abs). Removal of the C-terminus distal of HBD-3 (Vn80–373) had no significant effect on binding when compared to Vn80–396. Similarly, Vn80–339, which lacks the whole HBD-3, showed only a minor decrease in binding KatA, which is in agreement with our observation that heparin had only a partial inhibitory effect. Strikingly, binding was substantially impaired with fragment Vn80–229 (Fig. 4b). Reciprocal experiments, i.e., when Vn-fragments were immobilised, incubated with KatA and binding was detected using anti-KatA Abs, gave similar results (data not shown). We observed some residual binding of Vn80–229 and cannot fully exclude involvement of residues located further upstream. However, the primary binding site for KatA is located within amino acids 229 and 339 of the Vn-molecule, a region not involved in interactions with any bacterial Vn-binding proteins described to date. The fact that heparin has an inhibitory effect on the interaction, even though HBD-3 is not the primary binding site, can be explained with the model structures of Vn prepared by two independent research groups2829. Both models proposed a close proximity of the central domain and the C-terminal HBD resulting in a large inter-domain contact surface, which contains the putative heparin-binding groove. Given the spatial proximity between HBD-3 and our predicted KatA binding region within the central domain, it is feasible that bound heparin sterically disrupts the interaction between KatA and Vn rather than occupying the binding site itself. Docking of heparin to Vn28 supports this hypothesis. The use of an alternative binding site also explains why our kinetic data differed from data obtained for UspA2 and protein F, which both use HBD-3 as binding site2224. In summary, H. pylori KatA uses a not previously characterised bacterial binding site on the Vn-molecule, which allows a high affinity interaction.

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