Abstract

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.

Highlights

  • Can cause diverse gastric diseases such as peptic ulcers, chronic gastritis, mucosa-associated lymphoid tissue lymphoma, and gastric cancer [2,3,4]

  • We expected that if HP0894 is a TA toxin, it would bind to HP0895, a hypothetical protein and putative TA antitoxin of H. pylori

  • HP0894 was expressed as a C-terminal His tag fusion (HP0894-His) and HP0895 expressed without a purification tag

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Summary

Introduction

Can cause diverse gastric diseases such as peptic ulcers, chronic gastritis, mucosa-associated lymphoid tissue lymphoma, and gastric cancer [2,3,4]. SDS-PAGE analysis of the elution fraction from the Ni2ϩ-affinity column containing co-expressed HP0316 and HP0894 (amounts of both expressed proteins were nearly the same) did not show a band corresponding to the HP0316, unlike the result with the HP0894-His/HP0895 mixture (Fig. 2B).

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