Dataset Information

Structural and functional analysis of the Klebsiella pneumoniae MazEF toxin-antitoxin system.

ABSTRACT: Bacterial toxin-antitoxin (TA) systems correlate strongly with physiological processes in bacteria, such as growth arrest, survival and apoptosis. Here, the first crystal structure of a type II TA complex structure of Klebsiella pneumoniae at 2.3 Å resolution is presented. The K. pneumoniae MazEF complex consists of two MazEs and four MazFs in a heterohexameric assembly. It was estimated that MazEF forms a dodecamer with two heterohexameric MazEF complexes in solution, and a truncated complex exists in heterohexameric form. The MazE antitoxin interacts with the MazF toxin via two binding modes, namely, hydro-phobic and hydro-philic interactions. Compared with structural homologs, K. pneumoniae MazF shows distinct features in loops β1-β2, β3-β4 and β4-β5. It can be inferred that these three loops have the potential to represent the unique characteristics of MazF, especially various substrate recognition sites. In addition, K. pneumoniae MazF shows ribonuclease activity and the catalytic core of MazF lies in an RNA-binding pocket. Mutation experiments and cell-growth assays confirm Arg28 and Thr51 as critical residues for MazF ribonuclease activity. The findings shown here may contribute to the understanding of the bacterial MazEF TA system and the exploration of antimicrobial candidates to treat drug-resistant K. pneumoniae.

SUBMITTER: Jin C

PROVIDER: S-EPMC8086154 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Structural and functional analysis of the Klebsiella pneumoniae MazEF toxin-antitoxin system.

Jin Chenglong C Kang Sung-Min SM Kim Do-Hee DH Lee Bong-Jin BJ

IUCrJ 20210305 Pt 3

Bacterial toxin-antitoxin (TA) systems correlate strongly with physiological processes in bacteria, such as growth arrest, survival and apoptosis. Here, the first crystal structure of a type II TA complex structure of Klebsiella pneumoniae at 2.3 Å resolution is presented. The K. pneumoniae MazEF complex consists of two MazEs and four MazFs in a heterohexameric assembly. It was estimated that MazEF forms a dodecamer with two heterohexameric MazEF complexes in solution, and a trunca ...[more]

PMID: 33953923

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Project description:Toxin-antitoxin (TA) modules containing a Gcn5-related N-acetyltransferase (GNAT) toxin domain regulate bacterial physiology under adverse environmental stresses. Multiple GNAT-ribbon-helix-helix domain (RHH) TA loci have been identified in single bacterial genomes. However, their diversity and interactions are still obscure. Our previous analysis showed that the GNAT toxin of Klebsiella pneumoniae, KacT, introduces antibiotic tolerance and the toxicity of GNAT is neutralized by KacA, an RHH antitoxin. We here present a phylogenetic analysis of GNAT toxins of more than 1,000 GNAT-RHH pairs detected in completely sequenced K. pneumoniae genomes, revealing that the GNAT toxins are diverse and grouped into four distinct clades. Overexpression of GNAT toxins representative of each of the four clades halts the cell growth of K. pneumoniae, while the coexpression of the cognate RHH antitoxin neutralizes GNAT toxicity. We also identify point mutations that inactivate the GNAT toxins. Moreover, we observe a cross-interaction between GNAT-RHH pairs encoded by different replicons, where a chromosomal toxin (KacT2) can be neutralized by its cognate RHH antitoxin (KacA2 on a chromosome) and another antitoxin (KacA3 on a plasmid). Finally, statistical analysis of the distribution of GNAT-RHH loci in K. pneumoniae strains shows pronounced deviation from random distribution within the same clades. Moreover, we also obtain statistically significant correlations between different clades, which we discuss in terms of the experimental results. IMPORTANCE Elucidating the roles of multifaceted GNAT-RHH TA loci is essential for understanding how these TAs interact among themselves. Recently, the reaction mechanisms and structures of several GNAT-RHH pairs have been reported. While bacterial strains can carry multiple GNAT-RHH loci with diverse origins, studies on the possible cross-interactions of these TA pairs are still limited. Here, we find that 1,000 predicted GNAT toxins of K. pneumoniae can be grouped into four distinct clades. The distributions of TA loci within these clades in K. pneumoniae strains are highly nonrandom, with the presence of a single locus of each clade per strain being highly overrepresented. Moreover, the toxicity of a GNAT toxin encoded by a chromosome was alleviated by a noncognate RHH antitoxin on a plasmid. These results might yield a profound understanding of the widespread GNAT-RHH TA pairs and the cross-interactions between noncognate TA pairs located on different replicons.

Project description:Bacterial cells respond to environmental stresses by modulating their gene expression and adjusting their proteome. In Staphylococcus aureus, selective degradation by ClpP protease eliminates damaged proteins and regulates the abundance of functional proteins such as many important stress-induced transcriptional regulators. Degradation by ClpP requires the unfolding activity of partner Clp ATPases, such as ClpX and ClpC, and assistance of substrate-specific adaptor proteins such as YjbH and TrfA. Herein, we demonstrated that YjbH is aggregated in response to growth stress stimuli, such as oxidative and antibiotic stresses. In consequence, its function as an adaptor protein is compromised. YjbH controls the degradation of the stress-induced transcriptional regulator, Spx. Aggregated YjbH cannot assist Spx degradation, which results in Spx accumulation. We discovered that depending on the stress stimulus, Spx can be soluble or insoluble, and, consequently, transcriptionally active or inactive. Therefore, Spx accumulation and solubility are key components governing activation of Spx-dependent genes. Spx positively regulates expression of a ClpCP adaptor protein TrfA. TrfA in turn is required for degradation of MazE antitoxin, the unstable component of the MazEF toxin-antitoxin system, that neutralizes the endoribonuclease activity of MazF toxin. Bacterial toxin-antitoxin systems are associated with dormancy and tolerance to antibiotics that are related to chronic and relapsing infections, and it is at present a key unresolved problem in medicine. MazF activity was linked to growth stasis, yet the precise environmental signals that trigger MazE degradation and MazF activation are poorly understood. Here we propose a model where YjbH serves as a sensor of environmental stresses for downstream regulation of MazEF activity. YjbH aggregation, soluble Spx, and TrfA, coordinately control MazE antitoxin levels and consequently MazF toxin activity. This model implies that certain stress conditions culminate in modulation of MazF activity resulting in growth stasis during in vivo infections.

Dataset Information

Structural and functional analysis of the Klebsiella pneumoniae MazEF toxin-antitoxin system.

Publications

Structural and functional analysis of the <i>Klebsiella pneumoniae</i> MazEF toxin-antitoxin system.

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