Identification and activity assay of the SezAT toxin-antitoxin system of highly pathogenic Streptococcus suis serotype 2

Abstract:

[Objective] Bioinformatics analysis revealed that the 89K pathogenicity island (PAI) of highly pathogenic Streptococcus suis serotype 2 (SS2) in China encodes a putative type II toxin-antitoxin (TA) system named SezAT, which is homologous to the epsilon-zeta system from S. pyogenes. SezAT is presumed to be requisite for the stability of the 89K PAI in SS2. To confirm the SezAT system is a functional TA system. [Methods] The sequence characteristics of SezAT were subjected to further bioinformatics analysis. RT-PCR was performed to analyze the transcriptions of the sezAT locus and the flanking genes. The SezT toxin and SezA antitoxin proteins were selectively overexpressed in Escherichia coli. Deletion mutagenesis was carried out to obtain a SezAT-deficient mutant. [Results] Bioinformatics analysis and RT-PCR results suggest that sezAT are in the same operon. Overexpression of SezT led to severe growth inhibition of the host bacteria, while this toxicity was counteracted by the expression of SezA. Finally, the toxin-encoding gene sezT was successfully knockout by allelic replacement. [Conclusion] All of these results suggest that SezAT is an activated toxin-antitoxin (TA) system. Moreover, our results provide foundations for investigating the potential stabilized effect of SezAT in the 89K PAI, and screening an 89K-negative mutant to better understand the pathopoiesis of 89 K in highly pathogenic SS2.