Using S. cerevisiae as a Model System to Investigate V. cholerae VopX-Host Cell Protein Interactions and Phenotypes

Abstract

Most pathogenic, non-O1/non-O139 serogroup Vibrio cholerae strains cause diarrheal disease in the absence of cholera toxin. Instead, many use Type 3 Secretion System (T3SS) mediated mechanisms to disrupt host cell homeostasis. We identified a T3SS effector protein, VopX, which is translocated into mammalian cells during in vitro co-culture. In a S. cerevisiae model system, we found that expression of VopX resulted in a severe growth defect that was partially suppressed by a deletion of RLM1, encoding the terminal transcriptional regulator of the Cell Wall Integrity MAP kinase (CWI) regulated pathway. Growth of yeast cells in the presence of sorbitol also suppressed the defect, supporting a role for VopX in destabilizing the cell wall. Expression of VopX activated expression of β-galactosidase from an RLM1-reponsive element reporter fusion, but failed to do so in cells lacking MAP kinases upstream of Rlm1. The results suggest that VopX inhibits cell growth by stimulating the CWI pathway through Rlm1. Rlm1 is an ortholog of mammalian MEF2 transcription factors that are proposed to regulate cell differentiation, proliferation, and apoptosis. The collective findings suggest that VopX contributes to disease by activating MAP kinase cascades that elicit changes in cellular transcriptional programs.

Keywords: S. cerevisiae CWI pathway; Type 3 Secretion System; VopX; cholera.

Figure 1

Deletion analysis identifies sequences important for function. (A) VopX deletions beyond the N-terminal 50 amino acids attenuate yeast inhibition. Strains were grown overnight at 30 °C in Synthetic Complete Dextrose (SCD) media to repress VopX expression, then 10-fold serially diluted (10⁻¹ to 10⁻⁴, represented by the triangle) and spotted on media containing either dextrose to repress VopX expression, or galactose to induce VopX expression. VopX FL represents the predicted full length VopX protein, consisting of 252 amino acids. The doublet likely represents degradation or cleavage of a C-terminal protein fusion tag that is part of the expression construct; (B) Western analysis identified full length and deletion mutant VopX proteins, using an anti-HA antibody to detect a C-terminal tag present in the parent vector pBG1805-VopX and derived constructs [13]. Anti-Pgk1 antibody was used as a loading control.

Figure 2

The VopX growth defect is temperature sensitive and can be partially relieved by sorbitol at 20 °C. 10-fold serial dilutions of cultures grown at 30 °C were spotted on plates containing either dextrose (represses GAL promoter) or galactose (induces GAL promoter) to control VopX and Yal expression, and with or without 1M sorbitol. Plates were incubated for 72 h at the temperatures indicated.

Figure 3

VopX expression activates Rlm1 responsive promoter element activity. A plasmid based PRM5-lacZ reporter fusion was introduced into S. cerevisiae strains possessing an intact CWI pathway or deleted for different MAPK components. Strains expressing VopX or Yal control sequences were grown at 30 °C under conditions promoting VopX or Yal expression, then processed for the assay. Three colonies were used for each strain, and the assay was repeated with similar results. Not shown: WT strains grown under non-inducing conditions expressed 13 Miller units of activity when carrying Yal sequences and 15 Miller units of activity when carrying VopX.

Figure 4

VopX expression influences the expression of Rlm1 responsive genes. Semi-quantitative RT-PCR was performed on RNA extracted from cultures grown in the presence of galactose. Primers were designed to amplify genes that are targets of Rlm1 transcriptional activation, encoding cell wall proteins, an Rlm1 associated protein, or stress response proteins.

Figure 5

VopX expression alters yeast cell cycle progression. S. cerevisiae strains carrying pBG1805-VopX or pBG1805-Yal were grown to log phase in repressing medium (2% SCD-URA), normalized by OD₆₀₀, washed, and resuspended in inducing medium (2% SCG-URA). Samples of each strain were removed every hour for eight hours and imaged by light microscopy. For each strain, ~200 randomly selected yeast cells were counted at each time point and growth phases recorded.

Figure 6

Expression of VopX depolarizes actin cytoskeleton. Wild-type and rlm1Δ strains carrying empty vector (pBG1805) or plasmid containing VopX were grown in the presence of galactose for 5 h. Cells were fixed, processed and stained with Alexa Fluor 546 phalloidin to visualize the actin cytoskeleton.