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

Abstract

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.

VopE, a Vibrio cholerae Type III Effector, Attenuates the Activation of CWI-MAPK Pathway in Yeast Model System

“…Previously, yeast has been used as a model organism to study the activity of several other V. cholerae T3SS effector proteins (Tripathi et al, 2010, 2013; Bankapalli et al, 2015; Seward et al, 2015). …”

“…Yeast growth assays were performed as described earlier (Tripathi et al, 2010 ; Bankapalli et al, 2015 ). Briefly, overnight cultures of all recombinant strains in selective SC Raf medium were diluted and grown again in same media at 30°C until exponential phase (OD 600 ~ 0.8–1.0).…”

“…Growth was monitored after 60–70 h at 30°C and photographed accordingly. Liquid growth assay was done as described earlier (Bankapalli et al, 2015 ). For yeast viability plating assay (cfu), recombinant stains carrying VopE or its variants were grown overnight in selective SC Raf media.…”

Vibrio variations on a type three theme

“…In AM-19226, no 3' encoded effector is required for infant mouse colonization, but in an infant rabbit model of infection, moderate reductions in the incidence and severity of diarrhea as well as a slight decrease in colonization is observed during infection with VopK or VopY deletion strains [18]. In yeast, VopK toxicity is dependent on residues in the C-terminal domain postulated to comprise an MCF1-SHE serine peptidase domain, although peptidase activity was not detected and motif conservation is imprecise [80].…”

Regulation by ToxR-Like Proteins Converges on vttR B Expression To Control Type 3 Secretion System-Dependent Caco2-BBE Cytotoxicity in Vibrio cholerae

“…Previous studies using O39 serogroup strain AM-19226, which lacks the TCP and CT, have shown that T3SS activity is required for colonization and disease in murine and rabbit animal models of infection (24,25). T3SS genomic island-encoded, translocated proteins have been identified and named Vops, and several Vops have been assigned functions (25)(26)(27)(28)(29)(30).…”