Alanine-scanning mutagenesis of WH2 domains of VopF reveals residues important for conferring lethality in a Saccharomyces cerevisiae model

Tripathi, Ratnakar; Kaithwas, Vikas; Dureja, Chetna; Raychaudhuri, Saumya

doi:10.1016/j.gene.2013.04.071

Cited by 8 publications

(13 citation statements)

References 21 publications

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“…The data clearly demonstrated significant loss in lethality of VopE R125A variant, thereby underscoring the importance of arginine residue in the functionality of VopE in yeast system (Figure 1B left panel). To investigate in vivo protein stability, wild type and VopE R125A were C-terminally tagged with FLAG epitope and western blot analysis using anti-FLAG antibody was carried out (Zhang et al, 2011; Tripathi et al, 2013). The results indicated that VopE and VopE R125A were found to be stable (Figure 2C).…”

Section: Resultsmentioning

confidence: 99%

“…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). …”

Section: Discussionmentioning

confidence: 99%

“…The immunoblot analysis was carried out based on previous work (Tripathi et al, 2013; Bankapalli et al, 2015). All the recombinant yeast strains carrying wild type VopE and its congeners were grown in selective SC Raf medium at 30°C until mid-log phase.…”

Section: Methodsmentioning

confidence: 99%

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

Bankapalli

Mishra

Raychaudhuri

2017

Front. Cell. Infect. Microbiol.

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VopE, a mitochondrial targeting T3SS effector protein of Vibrio cholerae, perturbs innate immunity by modulating mitochondrial dynamics. In the current study, ectopic expression of VopE was found to be toxic in a yeast model system and toxicity was further aggravated in the presence of various stressors. Interestingly, a VopE variant lacking predicted mitochondrial targeting sequence (MTS) also exhibited partial lethality in the yeast system. With the aid of yeast genetic tools and different stressors, we have demonstrated that VopE and its derivative VopEΔMTS modulate cell wall integrity (CWI-MAPK) signaling pathway and have identified several critical residues contributing to the lethality of VopE. Furthermore, co-expression of two effectors VopEΔMTS and VopX, interfering with the CWI-MAPK cellular pathway can partially suppress the VopX mediated yeast growth inhibition. Taken together, these results suggest that VopE alters signaling through the CWI-MAPK pathway, and demonstrates the usefulness of yeast model system to gain additional insights on the functionality of VopE.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Bankapalli

Mishra

Raychaudhuri

2017

Front. Cell. Infect. Microbiol.

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“…Growth was monitored after 60-70h at 30°C and photographed accordingly. Liquid growth assay was done as described earlier [ 7 , 15 ]. Briefly, overnight cultures of recombinant yeast strains harboring wild type and alanine variants of VopK grown in raffinose were diluted back to an OD 600 of 0.05 in 25 ml of indicated media with appropriate amino acids supplementation.…”

Section: Methodsmentioning

confidence: 99%

“…Of these, VopF has received much of the attention. By employing yeast, tissue culture and animal model systems, many crucial facts on the structural and functional aspects of VopF have been garnered [ 2 – 7 ]. In addition to VopF, the existence of additional 11 effector proteins has been revealed in the T3SS island of Vibrio cholerae strain AM-19226 [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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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.

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Substitution of leucine by glutamate perturbs VopE localization to mitochondria: Lessons from yeast model system

Sharma,

Heer,

Raychaudhuri

2025

Mitochondrion

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Alanine-scanning mutagenesis of WH2 domains of VopF reveals residues important for conferring lethality in a Saccharomyces cerevisiae model

Cited by 8 publications

References 21 publications

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

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

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

Substitution of leucine by glutamate perturbs VopE localization to mitochondria: Lessons from yeast model system

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