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 primary sequence guided by available data on various toxins and effector proteins. Our in silico 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 predicted 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 without 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.
Institute of Microbial Technology, Council of Scientific and Industrial Research, Molecular Biology Division, Chandigarh, India.
Leela Krishna
LK
Bankapalli
Institute of Microbial Technology, Council of Scientific and Industrial Research, Molecular Biology Division, Chandigarh, India.
Rahul Chandra
RC
Mishra
Institute of Microbial Technology, Council of Scientific and Industrial Research, Molecular Biology Division, Chandigarh, India.
Balvinder
B
Singh
Institute of Microbial Technology, Council of Scientific and Industrial Research, Molecular Biology Division, Chandigarh, India.
Saumya
S
Raychaudhuri
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D014734:Vibrio cholerae / Q000378:metabolism*; D015003:Yeasts / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012694:Serine; D000409:Alanine