Microbial pathogens often co-opt host cellular processes to promote their own survival, replication and spread. For example, many Gram-negative bacterial pathogens utilize specialized type 3 secretion systems to directly inject tens of proteins, referred to as effectors, directly into host cells. Studies devoted to determining the roles of these proteins in pathogenesis as well as their molecular modes of action have been instrumental in advancing our understanding of the means by which pathogens establish an infection and cause disease. Nevertheless, a function for only a minority of effectors is current known, as it is often challenging to determine their roles in pathogenesis. Effectors can be difficult to study given that their sequence uniqueness in general provides no clues as to their function and loss of expression of individual effectors often does not result in a detectable phenotype, as they often act in a functional redundant manner. Furthermore, genetic approaches to identify roles of effectors are similarly complicated given that the bacteria deliver tens to hundreds of proteins into host cells. For these reasons, to gain insights regarding the roles of effectors in pathogenesis, many groups initially focus their efforts on biochemical approaches aimed at identifying mammalian proteins that directly bind the effectors, an approach that also has it's caveats. For example, the relatively low-levels of effectors injected into host cells coupled with their tendency to act catalytic thus presumably only transiently with target proteins complicates co-immunoprecipitation based approaches, particularly in the context of an infection. Similarly, even targeted assays directed at following up candidate interacting proteins can be difficult, as the heterologous expression of effectors in yeast and mammalian cells is often toxic. We recently developed the Protein Interaction Platform assay or PIP, a powerful and innovative means to study binary protein interactions. PIP consistently outperforms Y2H in detecting interactions between effectors and their interacting proteins. Our long-term goal is to develop a large comprehensive set of yeast strains, each of which express a mammalian protein fused to ?NS, that can be used in small or large-scale studies to identify new effector target proteins. However, such an endeavor is a huge undertaking and requires additional extensive supportive preliminary data. Here, towards this goal, we propose to compare the ability of PIP and Y2H to detect additional known effector-target interactions (Aim 1), conduct a small-scale screen to identify new candidate interactions between effectors and mammalian proteins involved in innate immune regulation (Aim 2) and convert PIP to a genetic selection (Aim 3).

Public Health Relevance

In order to establish an infection many Gram-negative bacterial pathogens usurp host cell proteins by the direct delivery of virulence proteins referred to as effectors into the host cell cytosol. An understanding of the means by which these effectors act to mediate pathogenesis is essential, as it can provide new insights regarding targets for the development of novel therapeutic agents. Here, we propose to establish the yeast Protein Interaction Platform as a novel high throughput system to accurately and efficiently identify host cell proteins targeted by the secreted effectors.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-IDM-B (80))
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Alexander, William A
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Massachusetts General Hospital
United States
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