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

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.

The Forgotten Virulence Factor: The 'non-conventional' Hemolysin TlyA And Its Role in Helicobacter pylori Infection.

Helicobacter pylori is a human-specific Gram-negative pathogenic bacterium which colonizes the gastric mucosal layer in the stomach causing diseases such as peptic ulcer, adenocarcinoma, and gastric lymphoma. It is estimated that approximately half of the world's population is infected with H. pylori making it the most intensively characterized microbial pathogen up to now. Hemolysis has been suggested to significantly contribute to colonization of the stomach and disease progression by H. pylori. In a number of earlier studies, TlyA was characterized as a putative pore-forming cytolysin. Although a few observations in the literature suggest a role for TlyA as significant virulence factor of H. pylori, the molecular and structural characterization of this protein is much curtailed at present. Given the intensive characterization of numerous H. pylori virulence factors over the past decade, surprisingly little information exists for the TlyA toxin and its significance for pathogenesis. This review provides a brief overview on microbial hemolysis and its role for pathogenesis and discusses recent research efforts aimed at an improved understanding of the role of the 'non-conventional' hemolysin and its associated RNA methyltransferase TlyA from H. pylori.

Vacuolating cytotoxin A (VacA) - A multi-talented pore-forming toxin from Helicobacter pylori.

Helicobacter pylori is associated with severe and chronic diseases of the stomach and duodenum such as peptic ulcer, non-cardial adenocarcinoma and gastric lymphoma, making Helicobacter pylori the only bacterial pathogen which is known to cause cancer. The worldwide rate of incidence for these diseases is extremely high and it is estimated that about half of the world's population is infected with H. pylori. Among the bacterial virulence factors is the vacuolating cytotoxin A (VacA), which represents an important determinant of pathogenicity. Intensive characterization of VacA over the past years has provided insight into an ample variety of mechanisms contributing to host-pathogen interactions. The toxin is considered as an important target for ongoing research for several reasons: i) VacA displays unique features and structural properties and its mechanism of action is unrelated to any other known bacterial toxin; ii) the toxin is involved in disease progress and colonization by H. pylori of the stomach; iii) VacA is a potential and promising candidate for the inclusion as antigen in a vaccine directed against H. pylori and iv) the vacA gene is characterized by a high allelic diversity, and allelic variants contribute differently to the pathogenicity of H. pylori. Despite the accumulation of substantial data related to VacA over the past years, several aspects of VacA-related activity have been characterized only to a limited extent. The biologically most significant effect of VacA activity on host cells is the formation of membrane pores and the induction of vacuole formation. This review discusses recent findings and advances on structure-function relations of the H. pylori VacA toxin, in particular with a view to membrane channel formation, oligomerization, receptor binding and apoptosis.