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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

Identification of H. pylori Vn-binding proteins.Outer-membrane fractions of strains CCUG18943 (a) and KR697 (b) were subjected to 2D-analysis. Left panels show Coomassie stained gels, panels to the right show detection of Vn-binding proteins by far-Western blotting. Vn-binding spots were identified by MALDI-TOF as KatA (1) and UreA (2 and 3). Spot 4 corresponds to UreB.
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f1: Identification of H. pylori Vn-binding proteins.Outer-membrane fractions of strains CCUG18943 (a) and KR697 (b) were subjected to 2D-analysis. Left panels show Coomassie stained gels, panels to the right show detection of Vn-binding proteins by far-Western blotting. Vn-binding spots were identified by MALDI-TOF as KatA (1) and UreA (2 and 3). Spot 4 corresponds to UreB.

Mentions: The first aim of this study was to identify the major Vn-binding protein in H. pylori since the bacterium has previously been shown to bind Vn with varying binding capacity for different isolates12. We therefore tested binding of Vn, which was purified from sera obtained from healthy human volunteers, to 13 clinical strains from our collection (Supplementary Fig. S1 and Supplementary Table 1). For further analysis we chose three H. pylori with strong (CCUG18943; 92% binding), intermediate (KR697; 69%), and weak Vn binding (KR497; 15%). Outer membrane fractions of these three strains were prepared and subjected to 2D-gel electrophoresis followed by far-Western blotting against human serum Vn (Fig. 1a and b). Three Vn-binding protein spots were detected in both H. pylori CCUG19843 (Fig. 1a) and KR697 (Fig. 1b). A fourth intense spot corresponded to urease subunit B (UreB). No Vn-binding proteins were detected in the weak Vn-binding strain KR497 (data not shown). Interestingly, subsequent MALDI-TOF MS analysis of proteins in KR697 and CCUG18943 identified spots 1 and 2 as two isoforms of H. pylori urease subunit A (UreA) and spot 3 as catalase KatA. To further verify our findings, we performed a pull-down experiment, in which the outer membrane fraction of H. pylori CCUG18943 was subjected to a Vn-coupled Sepharose column (data not shown). MALDI-TOF MS identified KatA, but neither UreA nor UreB could be detected in this assay, despite the fact that mild conditions (pH 7.4, 140 mM NaCl) were used during the pull-down. It is possible, however, that the conditions chosen in the pull-down experiment were not optimal for urease and we therefore, at this point, cannot exclude that urease binds Vn. However, the fact that KatA could successfully be identified as a Vn-binding protein in two different experimental setups suggested a significant interaction between the two proteins and prompted us to focus our attention on the characterisation of KatA in the sequel of this study.


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)

Identification of H. pylori Vn-binding proteins.Outer-membrane fractions of strains CCUG18943 (a) and KR697 (b) were subjected to 2D-analysis. Left panels show Coomassie stained gels, panels to the right show detection of Vn-binding proteins by far-Western blotting. Vn-binding spots were identified by MALDI-TOF as KatA (1) and UreA (2 and 3). Spot 4 corresponds to UreB.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Identification of H. pylori Vn-binding proteins.Outer-membrane fractions of strains CCUG18943 (a) and KR697 (b) were subjected to 2D-analysis. Left panels show Coomassie stained gels, panels to the right show detection of Vn-binding proteins by far-Western blotting. Vn-binding spots were identified by MALDI-TOF as KatA (1) and UreA (2 and 3). Spot 4 corresponds to UreB.
Mentions: The first aim of this study was to identify the major Vn-binding protein in H. pylori since the bacterium has previously been shown to bind Vn with varying binding capacity for different isolates12. We therefore tested binding of Vn, which was purified from sera obtained from healthy human volunteers, to 13 clinical strains from our collection (Supplementary Fig. S1 and Supplementary Table 1). For further analysis we chose three H. pylori with strong (CCUG18943; 92% binding), intermediate (KR697; 69%), and weak Vn binding (KR497; 15%). Outer membrane fractions of these three strains were prepared and subjected to 2D-gel electrophoresis followed by far-Western blotting against human serum Vn (Fig. 1a and b). Three Vn-binding protein spots were detected in both H. pylori CCUG19843 (Fig. 1a) and KR697 (Fig. 1b). A fourth intense spot corresponded to urease subunit B (UreB). No Vn-binding proteins were detected in the weak Vn-binding strain KR497 (data not shown). Interestingly, subsequent MALDI-TOF MS analysis of proteins in KR697 and CCUG18943 identified spots 1 and 2 as two isoforms of H. pylori urease subunit A (UreA) and spot 3 as catalase KatA. To further verify our findings, we performed a pull-down experiment, in which the outer membrane fraction of H. pylori CCUG18943 was subjected to a Vn-coupled Sepharose column (data not shown). MALDI-TOF MS identified KatA, but neither UreA nor UreB could be detected in this assay, despite the fact that mild conditions (pH 7.4, 140 mM NaCl) were used during the pull-down. It is possible, however, that the conditions chosen in the pull-down experiment were not optimal for urease and we therefore, at this point, cannot exclude that urease binds Vn. However, the fact that KatA could successfully be identified as a Vn-binding protein in two different experimental setups suggested a significant interaction between the two proteins and prompted us to focus our attention on the characterisation of KatA in the sequel of this study.

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