Yeast Functional Genomic Screens Lead to Identification of a Role for a Bacterial Effector in Innate Immunity Regulation

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Abstract
Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins. Many bacterial pathogens use specialized secretion systems to deliver effector proteins directly into host cells. The effector proteins mediate the subversion or inhibition of host cell processes to promote survival of the pathogens. Although these proteins are critical elements of pathogenesis, relatively few are well characterized. They often lack significant homology to proteins of known function, and they present special challenges, biological and practical, to study in vivo. For example, their functions often appear to be redundant or synergistic, and the organisms that produce them can be dangerous or difficult to culture, requiring special facilities. The yeast Saccharomyces cerevisiae has recently emerged as a model system to both identify and functionally characterize effector proteins. This work describes how genome-wide phenotypic screens and mRNA profiling of yeast expressing the Shigella effector OspF led to the discovery that OspF inhibits mitogen-activated protein kinase signaling in both yeast and mammalian cells. This inhibition of mitogen-activated protein kinase signaling is associated with attenuation of the host innate immune response. This study demonstrates how yeast functional genomic studies can contribute to the understanding of pathogenic effector proteins.