Inhibition of Cdc42-dependent signalling in Saccharomyces cerevisiae by phosphatase-dead SigD/SopB from Salmonella typhimurium

Abstract

Heterologous expression of bacterial virulence factors in Saccharomyces cerevisiae is a feasible approach to study their molecular function. The authors have previously reported that the Salmonella typhimurium SigD protein, a phosphatidylinositol phosphatase involved in invasion of the host cell, inhibits yeast growth, presumably by depleting an essential pool of phosphatidylinositol 4,5-bisphosphate, and also that a catalytically inactive version, SigDR468A, was able to arrest growth by a different mechanism that involved disruption of the actin cytoskeleton. This paper describes marked differences between the phenotypes elicited by expression of SigD and SigDR468A in yeast. First, expression of SigDR468A caused accumulation of large unbudded cells and loss of septin organization, while SigD expression caused none of these effects. Second, growth inhibition by SigDR468A was mediated by a cell cycle arrest in G2 dependent on the Swe1 morphogenetic checkpoint, but SigD-induced growth inhibition was cell cycle independent. And third, SigD caused strong activation of the yeast MAP kinase Slt2, whereas SigDR468A rather inactivated another MAP kinase, Kss1. In a screen for suppressors of SigDR468A-induced growth arrest by overexpression of a yeast cDNA library, the Cdc42 GTPase was isolated. Furthermore, SigDR468A was co-purified with Cdc42 from yeast lysates. It is concluded that the Salmonella SigD protein deprived of its phosphatase activity is able to disrupt yeast morphogenesis by interfering with Cdc42 function, opening the possibility that the SigD N-terminal region might directly modulate small GTPases from the host during infection.

Impact ofSalmonella entericaType III Secretion System Effectors on the Eukaryotic Host Cell

“…Both, a catalytically inactive SopB and a truncated SopB devoid of the phosphatase domain are able to complex with yeast Cdc42 and to inhibit Cdc42-dependent pathways when expressed in the eukaryotic cell model S. cerevisiae [153]. SopB also interacts with mammalian Cdc42 [268] through its N-terminal region independently of its catalytic activity and regardless of the activation state of the GTPase [269].…”

“…Studying the effects of the expression of individual effectors in host cells, although nonphysiological, is a powerful tool that can help in the discovery of their activities. This can be carried out not only in mammalian cells but also in more simple models, like the yeast, that are more amenable to genetic analysis [152,153]. Defining host substrates for the catalytic activities of effectors is another relevant subject of research.…”

The Phosphoinositides: Key Regulators of Salmonella Containing Vacuole (SCV) Trafficking and Identity

“…This interaction occurs independent of the activation state of CDC42. Interestingly, Salmonella strains harbouring SopB-mutations that render it unable to bind CDC42 presented similar invasion efficiencies when compared to the wild-type but reduced intracellular replication (Rodriguez-Escudero, Ferrer et al, 2011;Rodriguez-Escudero, Rotger et al, 2006). This indicates that the SopB-CDC42 association is pertinent to the intracellular adaptation of the pathogen rather than the actinremodeling that occurs upon invasion as one might expect.…”

Global impact of Salmonella type III secretion effector SteA on host cells

“…Expression of individual effectors, like AvrA or SopB, in the budding yeast Saccharomyces cerevisiae, that serves as a simplified heterologous model [14], has been a productive approach to study the effects of these proteins on host cells [15,16,17], thereby helping in the discovery of their activities. Here, we use mammalian cells as a relevant model to specifically analyze the effect on the host transcriptome of the effector SteA.…”