VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.

The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the sp...

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Autores principales:	Alexandre P Y Lopes, Luana M Lopes, Tatiana R Fraga, Rosa M Chura-Chambi, André L Sanson, Elisabeth Cheng, Erika Nakajima, Ligia Morganti, Elizabeth A L Martins
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9d3561fb310e4fcebb82a2243db521a2
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spelling	oai:doaj.org-article:9d3561fb310e4fcebb82a2243db521a22021-11-25T06:07:49ZVapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.1932-620310.1371/journal.pone.0101678https://doaj.org/article/9d3561fb310e4fcebb82a2243db521a22014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25047537/?tool=EBIhttps://doaj.org/toc/1932-6203The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation.Alexandre P Y LopesLuana M LopesTatiana R FragaRosa M Chura-ChambiAndré L SansonElisabeth ChengErika NakajimaLigia MorgantiElizabeth A L MartinsPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 7, p e101678 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Alexandre P Y Lopes Luana M Lopes Tatiana R Fraga Rosa M Chura-Chambi André L Sanson Elisabeth Cheng Erika Nakajima Ligia Morganti Elizabeth A L Martins VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.
description	The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation.
format	article
author	Alexandre P Y Lopes Luana M Lopes Tatiana R Fraga Rosa M Chura-Chambi André L Sanson Elisabeth Cheng Erika Nakajima Ligia Morganti Elizabeth A L Martins
author_facet	Alexandre P Y Lopes Luana M Lopes Tatiana R Fraga Rosa M Chura-Chambi André L Sanson Elisabeth Cheng Erika Nakajima Ligia Morganti Elizabeth A L Martins
author_sort	Alexandre P Y Lopes
title	VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.
title_short	VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.
title_full	VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.
title_fullStr	VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.
title_full_unstemmed	VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.
title_sort	vapc from the leptospiral vapbc toxin-antitoxin module displays ribonuclease activity on the initiator trna.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/9d3561fb310e4fcebb82a2243db521a2
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VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.

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