RESEARCH ARTICLE Identification of Critical Amino Acids Conferring Lethality in VopK, a Type III Effector Protein of Vibrio cholerae: Lessons from Yeast Model System

VopK, a type III effector protein, has been implicated in the pathogenesis of Vibrio cholerae strains belonging to diverse serogroups. Ectopic expression of this protein exhibits strong toxicity in yeast model system. In order to map critical residues in VopK, we scanned the pri-mary sequence guided by available data on various toxins and effector proteins. Our in sil-ico analysis of VopK indicated the presence of predicted MCF1-SHE (SHxxxE) serine peptidase domain at the C-terminus region of the protein. Substitution of each of the pre-dicted catalytic triad residues namely Ser314, His353 and Glu357 with alanine resulted in recombinant VopK proteins varying in lethality as evaluated in yeast model system. We observed that replacement of glutamate357 to alanine causes complete loss in toxicity while substitutions of serine314 and histidine353 with alanine exhibited partial loss in toxicity with-out affecting the stability of variants. In addition, replacement of another conserved serine residue at position 354 (S354) within predicted S314H353E357 did not affect toxicity of VopK. In essence, combined in silico and site directed mutagenesis, we have identified critical amino acids contributing to the lethal activity of VopK in yeast model system