Identification of chromosomal HP0892-HP0893 toxin-antitoxin proteins in Helicobacter pylori and structural elucidation of their protein-protein interaction.
ABSTRACT Bacterial chromosomal toxin-antitoxin (TA) systems have been proposed not only to play an important role in the stress response, but also to be associated with antibiotic resistance. Here, we identified the chromosomal HP0892-HP0893 TA proteins in the gastric pathogen, Helicobacter pylori (H. pylori), and structurally characterized their protein-protein interaction. Previously, HP0892 protein was suggested to be a putative TA toxin based on its structural similarity to other RelE family TA toxins. In this study, we demonstrated that HP0892 binds to HP0893 strongly with a stoichiometry of 1:1 and HP0892-HP0893 interaction occurs mainly between the N-terminal secondary structure elements of HP0892 and the C-terminal region of HP0893. HP0892 cleaved mRNA in vitro, preferentially at 5 end of A or G, and the RNase activity of HP0892 was inhibited by HP0893. In addition, heterologous expression of HP0892 in E. coli cells led to cell growth arrest, and the cell toxicity of HP0892 was neutralized by co-expression with HP0893. From these results and a structural comparison with other TA toxins, it is concluded that HP0892 is a toxin with intrinsic RNase activity and HP0893 is an antitoxin against HP0892 from a TA system of H. pylori. It has been known that hp0893 gene and another TA antitoxin gene, hp0895, of H. pylori, are both genomic open reading frames that correspond to genes that are potentially expressed in response to interactions with the human gastric mucosa. Therefore, it is highly probable that TA systems of H. pylori are involved in virulence of H. pylori.
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ABSTRACT: The Vibrio cholerae type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.PLoS Pathogens 12/2013; 9(12):e1003752. · 8.06 Impact Factor
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ABSTRACT: Due to the widespread and increasing appearance of antibiotic resistance, a new strategy is needed for developing novel antibiotics. Especially, there are no specific antibiotics for Helicobacter pylori (H. pylori). H. pylori are bacteria that live in the stomach and are related to many serious gastric problems such as peptic ulcers, chronic gastritis, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Because of its importance as a human pathogen, it's worth studying the structure and function of the proteins from H. pylori. After the sequencing of the H. pylori strain 26695 in 1997, more than 1,600 genes were identified from H. pylori. Until now, the structures of 334 proteins from H. pylori have been determined. Among them, 309 structures were determined by X-ray crystallography and 25 structures by Nuclear Magnetic Resonance (NMR), respectively. Overall, the structures of large proteins were determined by X-ray crystallography and those of small proteins by NMR. In our lab, we have studied the structural and functional characteristics of small proteins from H. pylori. In this review, 25 NMR structures of H. pylori proteins will be introduced and their structure-function relationships will be discussed.Molecules 11/2013; 18(11):13410-24. · 2.10 Impact Factor
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ABSTRACT: Antibiotic resistance and microorganism virulence have been consistently exhibited by bacteria and archaea, which survive in conditions of environmental stress through toxin-antitoxin (TA) systems. The HP0892-HP0893 toxin-antitoxin system is one of the two known TA systems belonging to Helicobacter pylori. The antitoxin, HP0893, binds and inhibits the HP0892 toxin and regulates the transcription of the TA operon. Here, we present the crystal structure of the zinc-bound HP0892 toxin at 1.8 Å resolution. Reorientation of residues at the mRNase active site was shown. The involved residues, namely E58A, H86A and H58A/ H60A, were mutated and the binding affinity was monitored by ITC studies. Through the structural difference between the apo and the metal-bound state, and by using a homology modeling tool, the involvement of the metal ion in mRNase active site could be identified. The most catalytically important residue, His86, reorients itself to exhibit RNase activity. His47, Glu58 and His60 are involved in metal binding where Glu58 acts as a general base and His47 and His60 may also act as a general acid in enzymatic activity. Glu58 and Asp64 are involved in substrate binding and specific sequence recognition. Arg83 is involved in phosphate binding and stabilization of the transition state, and Phe90 is involved in base packing and substrate orientation.Protein Science 03/2014; · 2.86 Impact Factor