- Central to the pathogenesis of many bacteria are sophisticated secretion systems that translocateproteins from the cytosol of bacteria into target eukaryotic cells. Despite a great deal of interest, there remainsa fundamental gap in our understanding of the molecular function of most bacterial effectors. This gaprepresents an important problem because the virulence of intracellular bacterial pathogens relies on theseproteins; therefore, understanding how individual effectors contribute to bacterial replication and survival is keyto understanding mechanisms of pathogenesis and developing effective therapeutics. The long-term goal ofthis project is to elucidate how Salmonella enterica serovar Typhimurium (STm) effectors modulate endocytictrafficking through manipulation of the host retromer complex. The retromer is a key component of theendosomal protein sorting machinery and mostly functions to recognize and sort cargo from the canonicalendocytic pathway to either the plasma membrane or the Golgi network. Subversion of retrograde transport isemerging as an important adaptation for intracellular pathogen survival, with the retromer being a favoritetarget of translocated bacterial effectors. The central hypothesis of this proposal is that direct engagement ofthe retromer by the STm effector SseC is required for maintaining the Salmonella-containing vacuole andestablishing of a replicative niche in host cells. An innovative high-throughput yeast genetic screen identifiedthe retromer as a potential target of SseC. Although strong follow-up experiments clearly show that SseCdirectly interacts with components of the human retromer complex (VPS35 and VPS26A) with nanomolaraffinity, why this interaction is important for STm survival remains unclear. As Î”sseC Salmonella arecompletely avirulent in a mouse model of infection, modulation of the retromer may be a crucial component ofSalmonella pathogenesis. This proposal pursues two specific aims: 1) Demonstrate a role for the retromer indetermining the outcome of STm infection; and 2) Determine the structure of the SseC:retromer complex andtranslate knowledge of the SseC-retromer interface to structure-function analysis during STm infection. Thefirst aim combines live-cell imaging and the generation of retromer knockout human cell lines with STminfection to determine how modulation of the retromer affects STm trafficking and survival in infection-relevantcell types. The second aim leverages the already solved crystal structure of the retromer complex to probe theSseC:retromer interface at the atomic level and uses this structure to inform structure-function experiments incells. The approach is innovative in its application of our novel yeast screen to characterize SseC mutantsand in that no one has implicated the retromer complex in STm infection to date. The proposed research issignificant because it will illuminate mechanisms that bacteria have evolved to subvert or co-opt the retromer,thus contributing to the design of host-directed therapeutics that may be effective against phylogeneticallydiverse bacterial pathogens.!