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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 binds vitronectin with its extended wrapping loop.Dimer of H. pylori KatA. Each monomer consists of an N-terminal arm (green), a central β-barrel domain (orange), the wrapping-loop (magenta), and the helical domain (blue). The C-terminal amino acids 492–505 are missing in the structure. (b) KatA-fragments were designed based on structural features shown in (a) and the Vn-binding capacity was compared to full length KatA using 20 nM Vn in an ELISA. Data shown are the mean and SD of at three independent experiments performed in technical triplicate. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (***) equals p < 0.001. (c) Surface structure-model of KatA dimer-surface depicting the area of the Vn-binding site (magenta).
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f5: KatA binds vitronectin with its extended wrapping loop.Dimer of H. pylori KatA. Each monomer consists of an N-terminal arm (green), a central β-barrel domain (orange), the wrapping-loop (magenta), and the helical domain (blue). The C-terminal amino acids 492–505 are missing in the structure. (b) KatA-fragments were designed based on structural features shown in (a) and the Vn-binding capacity was compared to full length KatA using 20 nM Vn in an ELISA. Data shown are the mean and SD of at three independent experiments performed in technical triplicate. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (***) equals p < 0.001. (c) Surface structure-model of KatA dimer-surface depicting the area of the Vn-binding site (magenta).

Mentions: H. pylori KatA, like other catalases, forms tetramers30. Each KatA monomer consists of 505 amino acids and exhibits the topology typical for small subunit, clade 3 catalases (Fig. 5a): an N-terminal protruding arm (aa 1–55), involved in formation of the homo-multimer, a central β-barrel domain (aa 56–315), and a C-terminal helical domain (aa 429–500), which is linked to the β-barrel domain by an extended ‘wrapping’ loop (aa 316–428)30. The unique feature of H. pylori catalase is a four lysine-motif at the C-terminus (aa 501–505), the function of which is at present unknown. Based on this topology a range of KatA fragments were designed, and their ability to interact with recombinant Vn was investigated by ELISA (Fig. 5b). KatA1–49 did not show any binding to Vn and, accordingly, KatA51–505 was not impaired in binding Vn when compared to the full-length protein (Fig. 5b). These results indicate that the N-terminal arm of KatA is dispensable for the interaction with Vn. This was expected, since the N-terminus is not exposed in the tetramer. As mentioned above, H. pylori catalases comprise a tetra-lysine motif as part of an unstructured C-terminus. It was proposed that this motif could be involved in the transport of KatA to the surface or in anchoring KatA to the membrane30; however, neither of these theories has to date been proven. We wanted to know, whether this C-terminal motif mediates the interaction with Vn and tested a fragment comprising amino acids 51–488 (Fig. 5b). Indeed, there was a slight reduction in binding to Vn, albeit not statistically significant. Therefore, the C-terminal tetra-lysine motif is, if at all, only marginally involved in Vn binding. Removal of the central β-barrel and the N-terminal part of the wrapping loop resulted in a small, but not statistically significant, reduction in binding (Fig. 5b). Finally, we tested KatA400–488, which additionally lacked most of the wrapping loop. When this KatA fragment was included in our analysis a strong reduction of Vn binding was observed when compared to the full length KatA. Therefore, the region, which binds Vn, most likely is located within the central part of the extended wrapping loop of KatA (Fig. 5b,c). Disordered regions of proteins, which include loops, have a high propensity for involvement in protein-protein interactions31, a recent example being the fibronectin protein FNE in Streptococcus equi32.


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 binds vitronectin with its extended wrapping loop.Dimer of H. pylori KatA. Each monomer consists of an N-terminal arm (green), a central β-barrel domain (orange), the wrapping-loop (magenta), and the helical domain (blue). The C-terminal amino acids 492–505 are missing in the structure. (b) KatA-fragments were designed based on structural features shown in (a) and the Vn-binding capacity was compared to full length KatA using 20 nM Vn in an ELISA. Data shown are the mean and SD of at three independent experiments performed in technical triplicate. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (***) equals p < 0.001. (c) Surface structure-model of KatA dimer-surface depicting the area of the Vn-binding site (magenta).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: KatA binds vitronectin with its extended wrapping loop.Dimer of H. pylori KatA. Each monomer consists of an N-terminal arm (green), a central β-barrel domain (orange), the wrapping-loop (magenta), and the helical domain (blue). The C-terminal amino acids 492–505 are missing in the structure. (b) KatA-fragments were designed based on structural features shown in (a) and the Vn-binding capacity was compared to full length KatA using 20 nM Vn in an ELISA. Data shown are the mean and SD of at three independent experiments performed in technical triplicate. Statistically significant differences were determined using one-way ANOVA and Bonferroni’s post-test where (***) equals p < 0.001. (c) Surface structure-model of KatA dimer-surface depicting the area of the Vn-binding site (magenta).
Mentions: H. pylori KatA, like other catalases, forms tetramers30. Each KatA monomer consists of 505 amino acids and exhibits the topology typical for small subunit, clade 3 catalases (Fig. 5a): an N-terminal protruding arm (aa 1–55), involved in formation of the homo-multimer, a central β-barrel domain (aa 56–315), and a C-terminal helical domain (aa 429–500), which is linked to the β-barrel domain by an extended ‘wrapping’ loop (aa 316–428)30. The unique feature of H. pylori catalase is a four lysine-motif at the C-terminus (aa 501–505), the function of which is at present unknown. Based on this topology a range of KatA fragments were designed, and their ability to interact with recombinant Vn was investigated by ELISA (Fig. 5b). KatA1–49 did not show any binding to Vn and, accordingly, KatA51–505 was not impaired in binding Vn when compared to the full-length protein (Fig. 5b). These results indicate that the N-terminal arm of KatA is dispensable for the interaction with Vn. This was expected, since the N-terminus is not exposed in the tetramer. As mentioned above, H. pylori catalases comprise a tetra-lysine motif as part of an unstructured C-terminus. It was proposed that this motif could be involved in the transport of KatA to the surface or in anchoring KatA to the membrane30; however, neither of these theories has to date been proven. We wanted to know, whether this C-terminal motif mediates the interaction with Vn and tested a fragment comprising amino acids 51–488 (Fig. 5b). Indeed, there was a slight reduction in binding to Vn, albeit not statistically significant. Therefore, the C-terminal tetra-lysine motif is, if at all, only marginally involved in Vn binding. Removal of the central β-barrel and the N-terminal part of the wrapping loop resulted in a small, but not statistically significant, reduction in binding (Fig. 5b). Finally, we tested KatA400–488, which additionally lacked most of the wrapping loop. When this KatA fragment was included in our analysis a strong reduction of Vn binding was observed when compared to the full length KatA. Therefore, the region, which binds Vn, most likely is located within the central part of the extended wrapping loop of KatA (Fig. 5b,c). Disordered regions of proteins, which include loops, have a high propensity for involvement in protein-protein interactions31, a recent example being the fibronectin protein FNE in Streptococcus equi32.

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