title: VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.
abstract: 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.
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.
authors.forename: Alexandre P Y
authors.initials: AP
authors.lastname: Lopes
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.
authors.forename: Luana M
authors.initials: LM
authors.lastname: Lopes
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.
authors.forename: Tatiana R
authors.initials: TR
authors.lastname: Fraga
authors.affiliation: Centro de Biotecnologia, Instituto de Pesquisas Energéticas e Nucleares, Comissão Nacional de Energia Nuclear, São Paulo, São Paulo, Brazil.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Chura-Chambi
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.
authors.forename: André L
authors.initials: AL
authors.lastname: Sanson
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Cheng
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.
authors.forename: Erika
authors.initials: E
authors.lastname: Nakajima
authors.affiliation: Centro de Biotecnologia, Instituto de Pesquisas Energéticas e Nucleares, Comissão Nacional de Energia Nuclear, São Paulo, São Paulo, Brazil.
authors.forename: Ligia
authors.initials: L
authors.lastname: Morganti
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, São Paulo, Brazil.
authors.forename: Elizabeth A L
authors.initials: EA
authors.lastname: Martins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000992:Antitoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D007921:Leptospira interrogans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009876:Operon; D058849:Protein Refolding; D012358:RNA, Transfer, Met / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*
chemical_list: D000992:Antitoxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D012358:RNA, Transfer, Met; D011994:Recombinant Proteins; C077517:VapB protein, Bacteria; D012260:Ribonucleases