Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor ∼1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to culture.
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.
Here we describe the protein interaction platform assay, a method for identifying interacting proteins in Saccharomyces cerevisiae. This assay relies on the reovirus scaffolding protein μNS, which forms large focal inclusions in living cells. When a query protein is fused to μNS and potential interaction partners are fused to a fluorescent reporter, interactors can be identified by screening for yeast that display fluorescent foci.A variety of methods have been developed to screen for interacting proteins in living cells, including yeast two-hybrid (Y2H) and protein-fragment complementation assays1 , 2. We recently demon-strated a complementary approach: the protein interaction plat-form (PIP) assay, in which we generate inclusions that serve as platforms for assembling interacting proteins in living mammalian cells. Here, we investigated whether this assay could be adapted to the yeast S. cerevisiae, in which (unlike in mammalian systems) plasmids for expressing proteins are readily maintained.PIP relies on the reovirus scaffolding protein μNS to form distinctively large focal cytoplasmic inclusions in mammalian cells3 ,4 . The C-terminal one-third of μNS (μNS residues 471-721) formed inclusions when expressed in S. cerevisiae (Fig. 1a). These inclusions did not perturb yeast growth, and mutations in the putative zinc-hook motif of μNS that disrupt the formation of platforms in mammalian cells 5 also disrupted their formation in yeast (Fig. 1b).The basic design of PIP as adapted to yeast involved fusing one protein (A) to μNS and a second protein (B) to GFP, so that if A and B interact, fluorescent green inclusions are visible in individual yeast cells (Fig. 1c,d). We created a series of recombination-based destination vectors using Gateway technology (Invitrogen) to facilitate the transfer of open reading frames such that they are fused to μNS at their N termini and expressed from either a constitutive (GPD) or an inducible (GAL1) yeast promoter. Many Gram-negative bacterial pathogens, including Shigella spp. and Salmonella spp., directly inject virulence proteins, called effectors, into host cells via specialized secretion systems. Some effectors require chaperones for delivery to the bacterial secretion apparatus. Shigella flexneri encodes four chaperones and at least 30
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.