Importance of the Cys124-Cys128 intermolecular disulfide bonding for oligomeric assembly and hemolytic activity of the Helicobacter pylori TlyA hemolysin

Nitchakan Samainukul; Aung Khine Linn; Mohammad Bagher Javadi; Somsri Sakdee; Chanan Angsuthanasombat; Gerd Katzenmeier

doi:10.1016/j.bbrc.2019.04.096

Importance of the Cys¹²⁴-Cys¹²⁸ intermolecular disulfide bonding for oligomeric assembly and hemolytic activity of the Helicobacter pylori TlyA hemolysin

Biochem Biophys Res Commun. 2019 Jun 25;514(2):365-371. doi: 10.1016/j.bbrc.2019.04.096. Epub 2019 Apr 27.

Authors

Nitchakan Samainukul¹, Aung Khine Linn¹, Mohammad Bagher Javadi², Somsri Sakdee², Chanan Angsuthanasombat³, Gerd Katzenmeier⁴

Affiliations

¹ Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; Bacterial Toxin Reseach Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand.
² Bacterial Toxin Reseach Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand.
³ Bacterial Toxin Reseach Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand; Division of Biochemistry and Biochemical Technology, Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. Electronic address: chanan.ang@mahidol.ac.th.
⁴ Bacterial Toxin Reseach Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand. Electronic address: katzenmeier.ger@mahidol.ac.th.

PMID: 31040022
DOI: 10.1016/j.bbrc.2019.04.096

Abstract

Although the TlyA hemolysin from Helicobacter pylori has been implicated as a potential virulence factor involved in mediating host cell colonization and hence disease progression, its structural determinants underlying the biological activity are still largely uncertain. In this study, an important role of the formation of a particular disulfide bond for functional oligomeric assembly of the H. pylori TlyA toxin was evidently elucidated. The 27-kDa TlyA recombinant protein was overexpressed in Escherichia coli, subsequently purified to near homogeneity by cation exchange chromatography, and proven to be hemolytically active against sheep erythrocytes. Additionally, TlyA-induced hemolytic activity was significantly diminished under conditions of disulfide bond reduction with a thiol-reducing agent, dithiothreitol. When the purified TlyA protein was subjected to modified SDS-PAGE under non-reducing conditions, the presence of an oligomeric state of this protein was clearly revealed by its apparent molecular mass of ∼48 kDa. Recombinant E. coli cells expressing TlyA also displayed contact-dependent hemolysis of erythrocytes, suggesting TlyA localization at the bacterial outer membrane and thus supporting the formation of disulfide-bonded TlyA. Homology-based modeling and in silico structural assembly analysis of TlyA signified potential intermolecular, rather than intramolecular, disulfide bonding through Cys¹²⁴ and Cys¹²⁸. Subsequently, single substitution of either of these Cys residues with Ser severely affected the oligomeric assembly of both TlyA mutants and hence abolished their hemolytic activity. Altogether, our present data provide pivotal evidence that the formation of intermolecular disulfide bonding between Cys¹²⁴ and Cys¹²⁸ plays a critical role in structural assembly of a biologically active-TlyA oligomer.

Keywords: Helicobacter pylori; Homology-based modeling; Intermolecular disulfide bonding; Modified SDS-PAGE; Oligomerization; TlyA-induced hemolysis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Bacterial Proteins / analysis
Bacterial Proteins / chemistry*
Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
Computer Simulation
Cysteine / chemistry
Cysteine / metabolism*
Disulfides / chemistry
Disulfides / metabolism*
Electrophoresis, Polyacrylamide Gel
Erythrocytes / pathology
Escherichia coli
Helicobacter pylori* / chemistry
Helicobacter pylori* / metabolism
Hemolysis*
Mutation
Oxidation-Reduction
Sheep
Virulence Factors / analysis
Virulence Factors / chemistry*
Virulence Factors / genetics
Virulence Factors / metabolism*

Substances

Bacterial Proteins
Disulfides
TlyA protein, Helicobacter pylori
Virulence Factors
Cysteine