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 DNAbinding motif.

HicA toxin of Escherichia coli derepresses hicAB transcription to selectively produce HicB antitoxin

“…In contrast, high toxin:antitoxin ratios (>1) leads to destabilization of the TA complex bound to the promoter region in vitro and strong induction of TA transcription in vivo (Page and Peti, ). There are also examples in which an antitoxin represses TA operon transcription independently of the toxins, that is, the toxin does not function as a co‐repressor (Brown et al ., ; Ruangprasert et al ., ; Bibi‐Triki et al ., ) and in one of these cases (mqsRA of E. coli K‐12) toxin excess derepresses TA operon transcription (Brown et al ., ).…”

Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σ E in Escherichia coli

“…To examine whether the mRNA interferase activity is required for suppression of the E essentiality by HicA, we constructed HicA mutants lacking mRNA interferase activity by mutating one of the two evolutionally conserved His residues (His-23 or His-38) to Ala. The importance of His-23 for mRNA interferase activity was recently suggested based on structural and functional analyses of the H24A and H28A mutant proteins of the HicA homologues from Burkholderia pseudomallei and Y. pestis, respectively (30,31). His-38 is located in the ␤3-␣2 loop (30), in which basic residues are implicated in substrate binding of the double-stranded RNA-binding domain proteins (44).…”

“…HicA adopts an ␣1␤1␤2␤3␣2 fold characteristic of a double-stranded RNA-binding domain (29,30), indicating that HicA may cleave mRNAs and tmRNA around their double-stranded regions. Recent biochemical and structural analyses of the HicA3-HicB3 complex, a HicAB family TA system of Yersinia pestis, revealed that the tetrameric HicB3 antitoxin binds two HicA3 toxin monomers, occluding the catalytic His28 residue of HicA3 (31). Both the HicB3 tetramer and the HicA3-HicB3 complex act as transcriptional autorepressors of the operon (31).…”

“…Recent biochemical and structural analyses of the HicA3-HicB3 complex, a HicAB family TA system of Yersinia pestis, revealed that the tetrameric HicB3 antitoxin binds two HicA3 toxin monomers, occluding the catalytic His28 residue of HicA3 (31). Both the HicB3 tetramer and the HicA3-HicB3 complex act as transcriptional autorepressors of the operon (31). The physiological function of the HicAB TA system is not known; its involvement in cell death or dormancy appears unlikely, as cells lacking HicB show an apparently normal growth phenotype (14).…”

Plausible role of bacterial toxin–antitoxin system in persister cell formation and elimination

“…For instance, in the mqsRA TA system of E. coli , the binding of toxin (MqsR) to antitoxin (MqsA) results in falling off DNA by the TA complex as the binding sites of MqsRA to DNA and to MqsR overlap with one another indicating that this TA system is never regulated by this auto inhibition strategy (Brown, Lord, Grigorius, Peti, & Pages, ). Others, including YafQ/DinJ TA system in E. coli (Ruangprasert et al, ), HicA3/HicB3 TA system in Yersinia pestis (Bibi‐Triki et al, ) and the HigB‐HigA TA system of Proteus vulgaris (Schureck et al,) are also not governed by conditional cooperativity indicating that some other phenomenon could be operating for the elimination of persisters in these bacterial species.…”