Characterization of translocation inhibition on needle/translocon complex assembly in Yersinia pseudotuberculosis.
Harmon, Dana.
2012
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Abstract: The
pathogenic Yersiniae express a type three secretion system (TTSS) for the translocation
of effector proteins into eukaryotic cells during infection. The process of
translocation is essential for virulence, but the mechanisms that underlie this process
are poorly understood. Several steps are required for translocation, including synthesis
of Yops, assembly of the TTSS, adherence ... read moreto cells, pore-formation and triggering of
effector release. The interactions between the bacterial cell and the host cell that
lead to successful polarized transfer of effectors are complex. It has been the goal of
this thesis to identify and understand necessary steps in this process. To dissect the
mechanisms underlying effector translocation in Y. pseudotuberculosis we have made of
use inhibition techniques, both chemical and genetic to characterize the TTSS and
translocon complex under these conditions. To identify chemical inhibitors of
translocation we carried out a high throughput screen for small molecule inhibitors that
blocked translocation. In this screen we identified 6 molecules that effectively
inhibited the translocation of YopE into HEp-2 cells without interfering with Yop
synthesis or secretion. One of these molecules, C20, caused reduced adherence of Y.
pseudotuberculosis to HEp-2 cells in a YadA- and invasin- independent manner
demonstrating the importance of generalized adherence mechanisms in the process of
translocation. To probe pore-formation dynamics, we devised an assay to recover
membranes from infected HEp-2 cells and for the first time demonstrated an LcrV
association with membranes during infection. We also established a chemical crosslinking
assay that we could use to assess LcrV on both needle tips, and at the membrane of
eukaryotic cells. Using this technique we were able to demonstrate that we could
crosslink LcrV tip-complexes on needles in vitro and show that LcrV can oligomerize in
response to lipid. We also found that a mutant, yscF D28A_D46A, is unable to form a
stable tip-complex. Examining this mutant further we found that stable tip-complex
formation is not required for triggering of
secretion.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Joan Mecsas.
Committee: Ralph Isberg, Michael Malamy, and Andrew Camilli.
Keyword: Microbiology.read less - ID:
- n8710337b
- Component ID:
- tufts:20357
- To Cite:
- TARC Citation Guide EndNote
