Modulation of host actin cytoskeleton and membrane transport systems during Salmonella invasion and intracellular survival

Shipan Dai, Purdue University

Abstract

Salmonella typhimurium is a facultative intracellular pathogen. Through the co-evolution with its vertebrate hosts, it has acquired sophisticated strategies to manipulate specific and highly regulated host cell responses, inducing actin cytoskeleton rearrangement that facilitates its entry and subsequent survival within a membrane bound compartment, the Salmonella containing vacuole (SCV). Two questions we want to address during Salmonella invasion are: how actin depolymerization and turnover contribute to Salmonella entry; and whether Salmonella uses membrane sources other than the plasma membrane for membrane ruffling. The role of host actin depolymerizing factor, ADF/Cofilin, during Salmonella invasion was determined. Efficient Salmonella internalization involved an initial dephosphorylation of ADF/Cofilin followed by phosphorylation, suggesting ADF/cofilin activities were increased briefly and had to be reversed to facilitate efficient bacterial entry. SopE was responsible for ADF/Cofilin phosphorylation and SipA bound actin filaments were resistant to the depolymerizing activity of ADF/Cofilin. The molecular mechanism for membrane supplementation during Salmonella invasion was also explored. VAMP8, a v-SNARE in endocytic pathway contributed to efficient Salmonella entry. VAMP8-containing vesicles were recruited and focally exocytosed at the site of bacterial entry. We found that SopB generated PtdIns(3)P was responsible for VAMP8 recruitment by direct interaction with VAMP8. Our results demonstrated that host membrane transport system was explored by bacterial pathogen for efficient entry. After Salmonella gets into the host cell, it stays in SCV where it replicates and survives. The role of SseF in this process was studied. We identified its chaperone, SscB, which was important for SseF stability and function. SseF localized to SCV and Sifs after translocation into host cell and targeted to Golgi complex after exogenous expression. Golgi-targeting domain of SseF was identified, and was important for SseF function in both Salmonella intracellular survival and Sif formation. In addition, four SNAREs in late endocytic pathway were found to be associated with SCV at different times during Salmonella infection and all had elevated level upon Salmonella infection. VAMP8 supported Salmonella intracellular survival in a SPI-2 independent manner, while VAMP7 inhibited the bacterial replication in a SPI-2 dependent, specifically SseF-mediated way.

Degree

Ph.D.

Advisors

Zhou, Purdue University.

Subject Area

Cellular biology|Microbiology

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