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...

Descripción completa

Guardado en:
Detalles Bibliográficos
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:
R
Q
Acceso en línea:https://doaj.org/article/9d3561fb310e4fcebb82a2243db521a2
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:doaj.org-article:9d3561fb310e4fcebb82a2243db521a2
record_format dspace
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
work_keys_str_mv AT alexandrepylopes vapcfromtheleptospiralvapbctoxinantitoxinmoduledisplaysribonucleaseactivityontheinitiatortrna
AT luanamlopes vapcfromtheleptospiralvapbctoxinantitoxinmoduledisplaysribonucleaseactivityontheinitiatortrna
AT tatianarfraga vapcfromtheleptospiralvapbctoxinantitoxinmoduledisplaysribonucleaseactivityontheinitiatortrna
AT rosamchurachambi vapcfromtheleptospiralvapbctoxinantitoxinmoduledisplaysribonucleaseactivityontheinitiatortrna
AT andrelsanson vapcfromtheleptospiralvapbctoxinantitoxinmoduledisplaysribonucleaseactivityontheinitiatortrna
AT elisabethcheng vapcfromtheleptospiralvapbctoxinantitoxinmoduledisplaysribonucleaseactivityontheinitiatortrna
AT erikanakajima vapcfromtheleptospiralvapbctoxinantitoxinmoduledisplaysribonucleaseactivityontheinitiatortrna
AT ligiamorganti vapcfromtheleptospiralvapbctoxinantitoxinmoduledisplaysribonucleaseactivityontheinitiatortrna
AT elizabethalmartins vapcfromtheleptospiralvapbctoxinantitoxinmoduledisplaysribonucleaseactivityontheinitiatortrna
_version_ 1718414178253799424