Characterization of the activity of the cloned SPI-2 type III secretion system in Salmonella Typhimurium

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Description: Salmonella Typhimurium encodes two distinct type III secretion systems (T3SS) at genomic loci termed SPI-1 and SPI-2. While the SPI-1 T3SS secretes proteins to allow S. Typhimurium host cell invasion, the SPI-2 T3SS targets proteins to allow phagosome remodeling and intracellular survival. To study the activity of these systems outside of the context of their S. Typhimurium chromosomal locations, both the SPI-1 and SPI-2 T3SS have been separately cloned onto the plasmid vector R995. My work focuses on the characterization of the cloned SPI-2 T3SS. We do not know the extent of substrates secreted by the R995 + SPI-2 T3SS clone. In addition, we do not know the role of the SPI-2-encoded SsrA/B regulation system in controlling SPI-2 gene expression from the R995 + SPI-2 construct. Moreover, we do not know how key translocation proteins encoded by the sseB, sseC, and sseD genes contribute to the phenotypes displayed by R995 + SPI-2. My specific aims were to: (1) identify the substrates secreted by cloned R995 + SPI-2 system, (2) determine the role of the SsrA/SsrB regulatory system in the R995 + SPI-2 clone and achieve inducible control of SPI-2 gene expression, and (3) determine the role of SseB, SseC, and SseD in R995 + SPI-2 function. During my thesis work, I was able to: (1) modify a secretion preparation protocol to improve our ability to detect secreted proteins from R995 + SPI-2 strains, (2) design and construct an IncQ pJAK16 and a pBR322 pBAD18Tp plasmid expressing SseF-HA or SifB-FLAG tagged proteins and observe its expression, (3) delete the SsrA/SsrB regulatory system from R995 + SPI-2 and assay subsequent effects on cloned SPI-2 expression, (4) place the sseA operon under control of the arabinose inducible pBAD promoter, and (5) delete the sseB, sseC and sseD translocon genes from R995 + SPI-2. These studies will impact our ability to understand how we can use R995 + SPI-2 to engineer bacteria for beneficial purposes related to protein secretion, bacterial-host cell interactions, and vaccine development.
Language: English
Format: Degree Work