Molecular mechanisms of bacterial type III protein translocation across membranes

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Type III secretion systems (T3SS) deliver bacterial proteins into host cells, enabling diverse lifestyles such as mutualism, parasitism, and commensalism. T3SS facilitated the expansion of E. coli into warm-blooded mammals where they eventually evolved commensal relationships in ruminant hosts. Many of these E. coli strains are now widely distributed in commercial livestock where they imperil food and water safety as specific adulterants and environmental contaminants. T3SS inhibitors have potential applications in emerging husbandry practices where the goal is to de-colonize animals at the source. This would benefit the agricultural and food sectors as a way to mitigate the billion-dollar losses suffered due to E. coli contamination. However, such applications need to be informed by a meticulous understanding of T3SS function and this provides our focus. This proposal examines the enigmatic process of secretion hierarchy', which describes how T3SS cargo is selected and prioritized for release in a stepwise fashion. Our focus has been on T3SS-associated chaperone proteins that impose regulatory control on cargo selection through unknown mechanisms. Our short-term objectives are two-fold. We will first determine the molecular requirements for secretion hierarchy in E. coli using the multi-cargo chaperone CesT and the effector Tir that regulates T3SS cargo selection. We solved the crystal structure of a Tir-CesT complex that revealed a previously unknown CesT binding domain in Tir. Guided by our structure, we will use Tir mutants to probe how the Tir-CesT complex interacts with the T3SS gatekeeper protein and whether this interaction is the regulatory step between middle to late effector release. In the second objective, we seek to establish how reversible post-translational modifications in chaperones regulate their functions in cargo selection. Our preliminary data shows that reversible phosphorylation of the CesT chaperone controls secretion hierarchy through interaction with the mRNA regulator CsrA. We have identified the phosphatase involved and will characterize when and how it acts on phosphorylated CesT substrate. We designed a genetic screen that takes advantage of CsrA antagonism by phosphorylated CesT to identify the CesT kinase and will determine the structural basis of CesT-CsrA interaction by crystallography. This research program will deliver a new molecular understanding of how T3SS cargo selection is regulated, with implications for agriculture and food sectors where T3SS-containing bacteria are common contaminants. Our long-term objective is to understand how bacteria use type III secretion for parasitic, commensal and mutualistic associations with hosts so that we may perhaps harness its widespread role as a colonization factor in the bacterial world.