Proteomics Analysis Of Salmonella Typhimurium Under The Regulation Of RstA/RstB And PhoP/PhoQ Systems

The facultative intracellular gram-negative bacteria Salmonella enterica serovar Typhimurium (S. Typhimurium) is the causative agent of gastroenteritis and septicemia in human. To survive and develop in various host tissues, bacteria require the capability to sense host environmental signals and adapt accordingly by regulating various gene expression. This sensing and adaptation process often involves a bacterial specific signal transduction system, the so-called two-component regulatory system (TCS). In S. Typhimurium, PhoP/PhoQ is a master TCS system which controls the expression of regulates the transcription of more than 100 genes including some other TCSs. It has been reported recently that in Salmonella, RstB, a sensor histidine kinase of RstA/RstB TCS, enhances the activity of PhoQ by increasing the transcription of PhoP-activated genes, and RstA, the cognate response regulator for RstB, is activated by the PhoP/PhoQ under conditions of either low Mg2+ or acidic pH. These studies suggest that there may be cross regulation between the PhoP/PhoQ and the RstA/RstB systems. In this study, to study the RstA/RstB regulatory function as well as the cross regulation between the RstA/RstB and PhoP/PhoQ, we performed a comparative proteomics investigation.In Chapter 2, we analyzed the proteome landscape change introduced by ΔrstB mutation of S. Typhimurium growing under a low Mg2+(10 μM) condition. Three biological replicates were performed with ～69%(1587 out of 2297 proteins) protein data overlap. We found that a total of 6.92% of 2297 proteins identified from the ΔrstB strain was significantly changed. Bioinformatics analysis revealed that the 80 metabolic pathways from identified proteins. A’statistical enrichment test’ (which evaluates if certain protein groups are nonrandomly distributed with respect to the entire list of identified proteins) showed that 6 biological processes (BP),6 molecular functions (MF) and 1 cellular component (CC) were overrepresented. Three molecular functions were underrepresented. A’statistical overrepresentation test’(which compares the input protein list with a reference protein source by calculating the variance between the expected and actually observed protein numbers) indicated that 6 BPs,3 MFs, and 8 CCs were overrepresented while only one category was underrepresented for BP, MF and CC. Among the highly regulated proteins, there are 2 proteins involved in pyrimidine metabolism and 7 proteins involved in iron acquisition (5 proteins required for Fe3+ acquisition and 2 proteins required for Fe2+ transport) indicating RstA/RstB may have functions in these metabolic processes. Based on regulated proteins and their physiological implications, we used functional assays to demonstrate that RstA/RstB regulated bacterial cellular motility and host cell invasion.In Chapter 3, we analyzed the proteome landscape change introduced by △phoPQ mutation. Three biological replicates were performed with ～82%(1884 out of 2288 proteins) protein data overlap. We found that a total of 14.9% of 2288 proteins were significantly changed. The overlaps in three biological replicates were about 80%(1884 proteins). Bioinformatics analysis revealed that the identified proteins covered 85 metabolic pathways. A’statistical enrichment test’ showed that 3 BPs and 2 MFs were enriched while only one category was underrepresented for both BP and MF. A ’statistical overrepresentation test’ indicated that 20 BPs,6 MFs, and 15 CCs were overrepresented while only the unclassified category was underrepresented. Further analysis of significantly regulated proteins suggests that selected proteins from two-component system and ABC transport pathways were down-regulated while many proteins from ribosome pathway were up-regulated.In Chapter 4, we systematically compared the protein networks under the regulation of RstA/RstB and PhoP/PhoQ. In all,80 and 85 metabolic pathways were annotated from identified proteins of ArstB and AphoPQ strains, respectively, with 80 pathways shared by the two groups. We found that RstA/RstB and PhoP/PhoQ regulation were partially overlapped, and the PhoP/PhoQ system’s regulatory network was much more extensive compared to that of RstA/RstB system. We identified that PhoP/PhoQ and RstA/RstB co-regulate 16 proteins in the same direction (8 proteins are negative regulated and 8 proteins are positively regulated) and oppositely regulate 15 proteins (up-regulated in ArstB strain and down-regulated in AphoPQ strain). Our quantitative proteomics analysis provides a novel view in the regulation of Salmonella pathogenesis that different TCSs may have cross regulation as well as independent regulatory roles.