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Functional and structural analysis of HicA3-HicB3, a novel toxin-antitoxin system of Yersinia pestis.

ABSTRACT: The mechanisms involved in the virulence of Yersinia pestis, the plague pathogen, are not fully understood. In previous research, we found that a Y. pestis mutant lacking the HicB3 (YPO3369) putative orphan antitoxin was attenuated for virulence in a murine model of bubonic plague. Toxin-antitoxin systems (TASs) are widespread in prokaryotes. Most bacterial species possess many TASs of several types. In type II TASs, the toxin protein is bound and neutralized by its cognate antitoxin protein in the cytoplasm. Here we identify the hicA3 gene encoding the toxin neutralized by HicB3 and show that HicA3-HicB3 constitutes a new functional type II TAS in Y. pestis. Using biochemical and mutagenesis-based approaches, we demonstrate that the HicA3 toxin is an RNase with a catalytic histidine residue. HicB3 has two functions: it sequesters and neutralizes HicA3 by blocking its active site, and it represses transcription of the hicA3B3 operon. Gel shift assays and reporter fusion experiments indicate that the HicB3 antitoxin binds to two operators in the hicA3B3 promoter region. We solved the X-ray structures of HicB3 and the HicA3-HicB3 complex; thus, we present the first crystal structure of a TA complex from the HicAB family. HicB3 forms a tetramer that can bind two HicA3 toxin molecules. HicA3 is monomeric and folds as a double-stranded-RNA-binding domain. The HicB3 N-terminal domain occludes the HicA3 active site, whereas its C-terminal domain folds as a ribbon-helix-helix DNA-binding motif.

SUBMITTER: Bibi-Triki S

PROVIDER: S-EPMC4248797 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Functional and structural analysis of HicA3-HicB3, a novel toxin-antitoxin system of Yersinia pestis.

Bibi-Triki Sabrina S Li de la Sierra-Gallay Inès I Lazar Noureddine N Leroy Arnaud A Van Tilbeurgh Herman H Sebbane Florent F Pradel Elizabeth E

Journal of bacteriology 20140811 21

The mechanisms involved in the virulence of Yersinia pestis, the plague pathogen, are not fully understood. In previous research, we found that a Y. pestis mutant lacking the HicB3 (YPO3369) putative orphan antitoxin was attenuated for virulence in a murine model of bubonic plague. Toxin-antitoxin systems (TASs) are widespread in prokaryotes. Most bacterial species possess many TASs of several types. In type II TASs, the toxin protein is bound and neutralized by its cognate antitoxin protein in ...[more]

PMID: 25112480

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Project description:Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed, as a consequence of horizontal exchange, and the breadth of their functionality in bacterial physiology remains unknown. Here, we characterize a TA system in Legionella pneumophila that is highly conserved across Legionella species. This system is distantly related to Escherichia coli HipBST, and we demonstrate that it is a functional tripartite TA system (denoted HipBSTLp). We identify HipBSTLp homologs in diverse taxa, yet in the Gammaproteobacteria, these are almost exclusively found in Legionella species. Notably, the toxin HipTLp was previously reported to be a pathogenic effector protein that is translocated by L. pneumophila into its eukaryotic hosts. Contrary to this, we find no signal of HipTLp translocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBSTLp, and identify key variations between this system and HipBSTEc. Finally, we show that the target of HipTLp is likely not conserved with any characterized HipA or HipT toxin. This work serves as a useful comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family. IMPORTANCE Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen Legionella pneumophila that is highly conserved across Legionella species genomes. This system (denoted HipBSTLp) is a distant homolog of the recently discovered split-HipA system in Escherichia coli (HipBSTEc). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipTLp) possesses bifunctionality as an L. pneumophila virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements.

Dataset Information

Functional and structural analysis of HicA3-HicB3, a novel toxin-antitoxin system of Yersinia pestis.

Publications

Functional and structural analysis of HicA3-HicB3, a novel toxin-antitoxin system of Yersinia pestis.

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