The Phd-Doc locus of the Gram-positive pathogen Streptococcus pneumoniae encodes a functional toxin-antitoxin system

Chieng, Yeo Chew and Chan, Wai Ting and Espinosa, Manuel (2014) The Phd-Doc locus of the Gram-positive pathogen Streptococcus pneumoniae encodes a functional toxin-antitoxin system. In: 21st Malaysian Society for Molecular Biology & Biotechnology (MSMBB) Annual Scientific Meeting, 01-03 October 2014, Monash University Malaysia.

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Abstract

Type II toxins-antitoxins (TAs) are found abundantly in prokaryotes and archaea, but not in eukaryotes. Bacterial toxin-antitoxin loci usually consist of two genes organised as an operon, where their products are bound together and are inert under normal conditions. However, under stressful circumstances, the antitoxin which is more labile will be degraded more rapidly, thereby unleashing its cognate toxin to act on the cell. This, in turn, causes cell stasis or cell death, depending on the type of TAs and/or time of toxin exposure. These circumstances have led to the proposal that the toxins of the TA pair could be used as potential antimicrobials. An in depth in silico study showed that Streptococcus pneumoniae, a human pathogen that causes 2 million deaths per year, harbours between 4 – 10 putative TA loci. In addition to the three well characterised TAs, namely RelBE2, YefM-YoeB and PezAT, here we present proof of the existence of a fourth functional pneumococcal TA, Phd-Doc. Overproduction of the Doc toxin in its natural host S. pneumoniae suppressed cell growth although growth was slowly resumed after a few hours. Co-expression of its cognate Phd antitoxin in cis was able to neutralise the toxicity of Doc, signifying Phd-Doc as a bona fide TA. The gene encoding the Doc toxin could not be cloned into the heterologous host Escherichia coli, most likely because of its high toxicity. Bioinformatics analyses demonstrated that the phd-doc locus was present in 43 out of 48 pneumococcal strains studied. Transcriptional fusions with Green Fluorescent Protein showed that the phd-doc operon was negatively autoregulated by its own proteins with the Phd antitoxin serving as a weak repressor, and Doc toxin as a corepressor to further repress transcription to nearly basal levels. The genetic organisation of phd-doc was also analysed and discussed in this study.

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