A Novel Connection between the Yeast Cdc42 GTPase and the Slt2-mediated Cell Integrity Pathway Identified through the Effect of Secreted Salmonella GTPase Modulators

Abstract

Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential for Salmonella typhimurium to enter into non-phagocytic cells. Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells. Furthermore, it promotes the activation of the cell integrity MAPK Slt2. Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs. These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway. Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway. Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S. cerevisiae, down-regulating MAPK-mediated signaling. Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways. Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes.

Impaired PRPP-synthesizing capacity compromises cell integrity signalling in Saccharomyces cerevisiae

“…In the wild-type, incubation at 37 u C leads to an increase in the signal for phosphorylated Slt2 from that obtained at 28 uC in agreement with published data (Lagorce et al, 2003;Martin et al, 2000;Rodriguez-Pachon et al, 2002). Western blot analysis shows that, in contrast to the wildtype, in a prs1D strain the signal corresponding to phosphoSlt2 is stronger at 28 u C and is maintained over a period of 4 h during incubation at 37 u C (Fig.…”

PtdIns(3)P accumulation in triple lipid-phosphatase-deletion mutants triggers lethal hyperactivation of the Rho1p/Pkc1p cell-integrity MAP kinase pathway

“…Of the known PtdIns(3)P effector-domaincontain proteins in yeast, the PX-domain-containing Bem1p [a positive regulator of Cdc24p guanine-nucleotide exchange factor activity towards Cdc42p (Zheng et al, 1995)] and Bem3p [a GTPase-activating protein for Cdc42p (Zheng et al, 1994)] proteins could be attractive candidates for performing such a function. In line with this, it is noteworthy that a recent report from Molina and coworkers indicated that Cdc42p signaling can influence Slt2p activity (Rodriguez-Pachon et al, 2002). Therefore, it is intriguing that overexpression of BEM3 in ymr1⌬ sjl2 ts sjl3⌬ cells prevented growth at the normally permissive temperature of 34°C (our unpublished observations).…”

Development of a Novel Functional High-Throughput Screening System for Pathogen Effectors in the YeastSaccharomyces cerevisiae

“…Yersinia enterocolitica effectors, YopE, YopM, and YpkA, and Salmonella typhimurium effectors, SopE2, SptP, and SigD/SopB, were amplified by PCR using plasmids pCFL110, pCFL140, and pCFL150 (gifts of Dr. Samuel Miller), 9) and pKG-SopE2, pKG-SptP, and YCpLG-SigD (gifts of Dr. Maria Molina) 10,11) respectively. Oligonucleotides were designed so that their 3 0 ends were specific to the various pathogen effector genes and their 5 0 ends partially overlapped the sequences of the attB-adaptor primers, as described in the GatewayÔ technology instruction manual (Invitrogen) ( Table 3).…”

“…Numerous effectors from various bacterial pathogens such as Yersinia, 8,9) Salmonella, 10,11) Pseudomonas, 12,13) Vibrio, 14) Legionella, 15,16) Campylobacter, 17) and Chlamydia 18,19) have been expressed in yeast and have been found to interfere with cellular functions related to their proposed targets within the host cell, such as Rho-type small GTPase, mitogen-activated protein kinase (MAPK) cascades, vesicle trafficking, DNA, the actin cytoskeleton, and microtubules. These reports indicate that the yeast system is powerful tool in the identification and characterization of pathogen effectors.…”

“…[9][10][11] Yersinia enterocolitica effectors (YopE, Rho-GAP; YopM, a leucine-rich repeat containing protein, unknown function; YpkA, protein kinase and RhoGDI) and Salmonella typhimurium effectors (SopE2, Rho-GEF; SptP, RhoGAP; SigD/SopB, Phosphoinositide phosphatase) were cloned into yeast-inducible expression plasmids, pMT751 and pMT830. Yeast cells carrying GFP-tagged pathogen effector expression plasmids were spotted in 10-fold serial dilution onto the induction (ÀDox for the Tet-Off promoter vectors and galactose for the GAL1 promoter vectors) or noninduction (þDox for the Tet-Off promoter vectors and fructose for the GAL1 promoter vectors) plates.…”