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Study of toxin-antitoxin systems of Leptospira interrogans: biochemical and functional characterization and role in the pathogenesis of bacteria


Leptospira interrogans is one of the causative agents of human leptospirosis. Toxin-Antitoxin (TA) operons are widely distributed among bacteria and encode a stable toxin and an unstable antitoxin. Its general function remains controversial, but the physiological action of the TA system is the reversible cessation of cell growth under stress conditions. Members of these systems have been related to the virulence of several bacteria during infection, including L. interrogans (Komi et al., 2015). TA systems are classified into types and families. Virulence Associated Proteins B and C (VapBC) constitute the main TA family, grouped due to the homology of a PIN domain of the toxin which acts as a ribonuclease. The TA Data Base (TADB) shows that L. interrogans serovar Copenhageni has four vapBC loci, all on chromosome I. We have previously published the biochemical and functional characterization of the first member of the VapBC family described in leptospira that corresponds to the locus vapBC-3 (Lopes et al., 2014). This project will provide the characterization of the other VapBC modules with the cloning and expression of vapBC operons 1, 2 and 4 in E. coli; purification of recombinant proteins; characterization of operon functionality by determination of toxic and antitoxic activity on E.coli growth; recognition tests (Western blotting) on different leptospira strains; evaluation of cross-reactivity between toxins and antitoxins; toxin and antitoxin in vitro affinity tests (ligand affinity blotting) and differential detection (Western blotting) of Vap B and C by Western blotting in leptospires under stress conditions. Ultimately, this work aims to elucidate if the VapBC modules are functional and contribute to the study of their role in the physiology of bacterial stress, leading to future studies of their role in the pathogenicity of bacteria. (AU)