Limits...
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 is a Vn-binder with high affinity.(a) Binding of recombinant KatA to Vn80–396 was determined by ELISA. Plates coated with 100 nM KatA were incubated with increasing concentrations (2.5–80 nM) of Vn. Protein P09011 from Haemophilus influenzae incubated with 20 nM Vn served as negative control (inset). Statistical significance was determined using Student’s t-test, where (***) equals p ≤ 0.001. Data presented are the mean and SD of at three independent experiments performed in technical triplicates. (b) Binding of KatA to monomeric (native) or polymeric (activated) Vn. Plates coated with 50 nM Vn were incubated with increasing concentrations (2.5–80 nM) of KatA. (c) Vn80–396 was immobilised on amine reactive AR2G sensors and biolayer-interferometry was performed with KatA as analyte using a 1:3 dilution series (0.055–50 nM). (d) The affinity constant (KD) was calculated from steady state analysis based on R equilibrium values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4834553&req=5

f3: KatA is a Vn-binder with high affinity.(a) Binding of recombinant KatA to Vn80–396 was determined by ELISA. Plates coated with 100 nM KatA were incubated with increasing concentrations (2.5–80 nM) of Vn. Protein P09011 from Haemophilus influenzae incubated with 20 nM Vn served as negative control (inset). Statistical significance was determined using Student’s t-test, where (***) equals p ≤ 0.001. Data presented are the mean and SD of at three independent experiments performed in technical triplicates. (b) Binding of KatA to monomeric (native) or polymeric (activated) Vn. Plates coated with 50 nM Vn were incubated with increasing concentrations (2.5–80 nM) of KatA. (c) Vn80–396 was immobilised on amine reactive AR2G sensors and biolayer-interferometry was performed with KatA as analyte using a 1:3 dilution series (0.055–50 nM). (d) The affinity constant (KD) was calculated from steady state analysis based on R equilibrium values.

Mentions: Next, we wanted to characterise the KatA-Vn interaction at the protein level. We performed an ELISA using full length recombinant KatA and a dilution series (2.5–80 nM) of Vn80–396, which spans most known bacterial binding sites. Binding of KatA and Vn was dose-dependent and saturable, confirming a specific interaction (Fig 3a). To exclude any effects resulting from trace amounts of Escherichia coli proteins, P09011 from H. influenzae22, which was purified using the same protein expression and purification protocol as for KatA, served as negative control for Vn binding (Fig. 3a, inset). Furthermore, we wanted to reveal whether KatA preferentially targets native (monomeric) Vn or the activated polymeric form. Binding of KatA to either of the two forms of Vn was investigated by ELISA and showed that KatA bound both monomeric and polymeric Vn (Fig 3b). However, binding to the activated, polymeric Vn was nearly twice as strong. Given that H. pylori does not typically enter the bloodstream and Vn occurs predominantly in the activated form in tissues and the ECM, targeting mainly polymeric Vn is certainly an advantage for the bacterium. A similar preference for activated Vn has been described for other bacteria including Streptococcus pneumoniae and N. meningitidis2023. It should be noted that the activated form of Vn undergoes conformational changes and partial unfolding, and thereby exposes potential binding sites10, which is likely a reason why the activated form is predominantly targeted by bacteria.


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 is a Vn-binder with high affinity.(a) Binding of recombinant KatA to Vn80–396 was determined by ELISA. Plates coated with 100 nM KatA were incubated with increasing concentrations (2.5–80 nM) of Vn. Protein P09011 from Haemophilus influenzae incubated with 20 nM Vn served as negative control (inset). Statistical significance was determined using Student’s t-test, where (***) equals p ≤ 0.001. Data presented are the mean and SD of at three independent experiments performed in technical triplicates. (b) Binding of KatA to monomeric (native) or polymeric (activated) Vn. Plates coated with 50 nM Vn were incubated with increasing concentrations (2.5–80 nM) of KatA. (c) Vn80–396 was immobilised on amine reactive AR2G sensors and biolayer-interferometry was performed with KatA as analyte using a 1:3 dilution series (0.055–50 nM). (d) The affinity constant (KD) was calculated from steady state analysis based on R equilibrium values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: KatA is a Vn-binder with high affinity.(a) Binding of recombinant KatA to Vn80–396 was determined by ELISA. Plates coated with 100 nM KatA were incubated with increasing concentrations (2.5–80 nM) of Vn. Protein P09011 from Haemophilus influenzae incubated with 20 nM Vn served as negative control (inset). Statistical significance was determined using Student’s t-test, where (***) equals p ≤ 0.001. Data presented are the mean and SD of at three independent experiments performed in technical triplicates. (b) Binding of KatA to monomeric (native) or polymeric (activated) Vn. Plates coated with 50 nM Vn were incubated with increasing concentrations (2.5–80 nM) of KatA. (c) Vn80–396 was immobilised on amine reactive AR2G sensors and biolayer-interferometry was performed with KatA as analyte using a 1:3 dilution series (0.055–50 nM). (d) The affinity constant (KD) was calculated from steady state analysis based on R equilibrium values.
Mentions: Next, we wanted to characterise the KatA-Vn interaction at the protein level. We performed an ELISA using full length recombinant KatA and a dilution series (2.5–80 nM) of Vn80–396, which spans most known bacterial binding sites. Binding of KatA and Vn was dose-dependent and saturable, confirming a specific interaction (Fig 3a). To exclude any effects resulting from trace amounts of Escherichia coli proteins, P09011 from H. influenzae22, which was purified using the same protein expression and purification protocol as for KatA, served as negative control for Vn binding (Fig. 3a, inset). Furthermore, we wanted to reveal whether KatA preferentially targets native (monomeric) Vn or the activated polymeric form. Binding of KatA to either of the two forms of Vn was investigated by ELISA and showed that KatA bound both monomeric and polymeric Vn (Fig 3b). However, binding to the activated, polymeric Vn was nearly twice as strong. Given that H. pylori does not typically enter the bloodstream and Vn occurs predominantly in the activated form in tissues and the ECM, targeting mainly polymeric Vn is certainly an advantage for the bacterium. A similar preference for activated Vn has been described for other bacteria including Streptococcus pneumoniae and N. meningitidis2023. It should be noted that the activated form of Vn undergoes conformational changes and partial unfolding, and thereby exposes potential binding sites10, which is likely a reason why the activated form is predominantly targeted by bacteria.

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