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