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

https://doi.org/10.1016/j.bbrc.2019.04.096Get rights and content

Highlights

  • Hemolysis induced by H. pylori TlyA was drastically decreased under reducing conditions.

  • 27-kDa TlyA formed ∼48-kDa oligomers as analyzed by non-reducing semi-native SDS-PAGE.

  • Cys124−Cys128 intermolecular disulfide bonding was revealed through TlyA-dimers by in silico analysis.

  • TlyA-induced hemolysis and oligomeric assembly were affected by C124S or C128S single-mutation.

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.

Introduction

Helicobacter pylori infects approximately half of the world's population and it is estimated that 90% of duodenal ulcers and up to 80% of gastric ulcers are caused by this microorganism. H. pylori infection significantly increases gastric cancer risk and about 3% of infected patients developed gastric cancer, making H. pylori the only bacterial pathogen classified as a class I carcinogen [1]. H. pylori contains a wide range of virulence factors which are essential for continual infection and disease development such as urease, catalase, lipopolysaccharides, adhesins, the cytotoxin-associated gene A and vacuolating cytotoxin A [2].

TlyA is a hemolysin/cytolysin encoded by HP1086 gene (Gene ID: 899622) within H. pylori strain 26695 (NC_000915.1) [3]. Although hemolysis is the only reported biological activity for the TlyA hemolysin from H. pylori, its homologues from other pathogenic bacteria display S-adenosyl-l-methionine-dependent RNA 2′-O-methyltransferase activity which specifically methylates cytosine residues in the 16S rRNA and 23S rRNA [4]. While wild-type H. pylori causes hemolysis of human and animal erythrocytes, H. pylori TlyA-negative mutants showed reduced hemolytic activity, decreased adhesion to human gastric adenocarcinoma cells in vitro and failure to colonize the gastric mucosa of mice in vivo [5]. When produced as a recombinant protein, TlyA displayed hemolytic activity although the physiological significance of such hemolytic activity in vivo is still unclear [6]. It was speculated that TlyA has a role in the acquisition of nutrients such as iron [7]. TlyA was shown to trigger liposome fusion leading to disruption of lipid membranes [8] and its hemolytic activity is inhibited in the presence of osmoprotective saccharides, thus suggesting the potential formation of TlyA-induced transmembrane pores [6].

TlyA is synthesized as a 27-kDa hemolytic protein which lacks a signal sequence and currently no crystallographic structure is available. Therefore, fundamental correlations linking TlyA structural assembly, biological activity and functionally critical residue/s still remain unclear at present. Here we showed that (i) TlyA-induced hemolytic activity was significantly decreased under reducing conditions, (ii) TlyA could form oligomers of approximately 48 kDa under non-reducing conditions, (iii) E. coli cells expressing soluble TlyA display hemolytic activity thus suggesting location of TlyA at the outer membrane and (iv) TlyA-Cys mutants (i.e., C124S and C128S) displayed drastically reduced hemolytic activity and oligomeric assembly compared to wild-type TlyA, signifying that the Cys124−Cys128 intermolecular disulfide bonding plays a critical role in structural assembly and hemolytic activity of TlyA.

Section snippets

Recombinant plasmid construction

DNA sequence encoding TlyA from H. pylori 26695 (NC_000915.1) was retrieved (NCBI Gene ID: 899622) and custom synthesized (DNA 2.0, USA). The FLAG tag was fused to the N-terminus of TlyA for downstream applications. After the fusion gene segment was sub-cloned into the pD441 vector under control of the T5 promoter, the resulting plasmid designated as pFLAG/TlyA235 (see Supplementary Fig. S1a) was analyzed by restriction digestion and DNA sequencing prior to transformation into E. coli strain

Biochemical and structural characteristics, and hemolytic activity of purified TlyA

TlyA overexpressed as a soluble FLAG-tagged protein with high yields (∼5–7 mg/L bacterial culture) was obtained at 6-h IPTG induction at 28 °C (Fig. 1a, left panel). SDS-PAGE analysis revealed that TlyA was produced as a 27-kDa protein which corresponds to the molecular mass calculated from its deduced amino acid sequence. Additionally, the expressed protein was immunoreactive with anti-FLAG antiserum in Western blotting, verifying the presence of the FLAG affinity tag (Fig. 1a, right panel).

Acknowledgements

This work was supported by grant RSA5580047 from the Thailand Research Fund (TRF, to GK). A TRF-Royal Golden Jubilee Ph.D. scholarship (to AKL) is gratefully acknowledged.

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