Browsing by Author "Kalburge, Sai Siddarth"
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Item Quorum sensing regulators are required for metabolic fitness in Vibrio parahaemolyticus(American Society for Microbiology, 2017-01-09) Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. Fidelma; Sai Siddarth Kalburge, Megan R. Carpenter, Sharon Rozovsky and E. Fidelma Boyd; Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. FidelmaQuorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO, is active, and regulates the two QS master regulators AphA, which is induced and OpaR, which is repressed. At high cell density the opposite occurs, LuxO is inactive, therefore OpaR is induced and AphA is repressed. In V. parahaemolyticus, a significant enteric pathogen of humans, the role of these regulators in pathogenesis is less known. We examined deletion mutants of luxO, opaR and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to wild-type. The opaR mutant did not show any defect in vivo. Colonization was restored to wild-type levels in a luxO/opaR double mutant and was also increased in an opaR/aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. RNA-seq analysis of the wild-type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.Item Quorum sensing regulators are required for metabolic fitness in Vibrio parahaemolyticus(American Society for Microbiology Journals, 2017-01-09) Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. Fidelma; Sai Siddarth Kalburge, Megan R. Carpenter, Sharon Rozovsky, E. Fidelma Boyd; Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. FidelmaQuorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO, is active, and regulates the two QS master regulators AphA, which is induced and OpaR, which is repressed. At high cell density the opposite occurs, LuxO is inactive, therefore OpaR is induced and AphA is repressed. In V. parahaemolyticus, a significant enteric pathogen of humans, the role of these regulators in pathogenesis is less known. We examined deletion mutants of luxO, opaR and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to wild-type. The opaR mutant did not show any defect in vivo. Colonization was restored to wild-type levels in a luxO/opaR double mutant and was also increased in an opaR/aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. RNA-seq analysis of the wild-type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.Item The role of quorum sensing regulators in Vibrio parahaemolyticus(University of Delaware, 2017) Kalburge, Sai SiddarthVibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis worldwide as well as a significant pathogen of shellfish and fish. In addition, the geographic range of this species and others within the genus is spreading globally due to climate change. Our knowledge of how this pathogen colonizes the human intestine, the first step in its pathogenesis, is limited. Some of the most influential factors for survival and pathogenesis of bacteria in changing environments include stress response mechanisms, motility, production of virulence factors and formation of a biofilm. Bacteria possess a communication mechanism, where they synchronize actions with other bacteria in the population by regulating the expression of genes that control these actions. Bacteria do this by secreting molecules called autoinducers that act as extracellular signals that other bacteria in the environment can respond to. This process of bacterial cell to cell communication is called quorum sensing. The work in this dissertation examined the role of the downstream regulators of the quorum sensing pathway in V. parahaemolyticus colonization and fitness. By examining single and double deletion mutants of the three downstream regulators of the quorum sensing pathway, the response regulator luxO and the two quorum sensing master regulators opaR and aphA, we determined that the low cell density master regulator AphA is important for in vivo fitness and that over-expression of the high cell density master regulator opaR is detrimental to in vivo fitness. We then performed RNASeq transcriptome analysis on the wild-type and the luxO mutant, which was found to be defective in vivo and over-expressed the high cell density regulator opaR. The transcriptome analysis revealed that 60% of genes downregulated in the luxO mutant were involved in metabolism and transport, suggesting that the mutant could have a metabolic disadvantage compared to wild-type. This was indeed confirmed by carbon phenotype microarrays, which revealed that the luxO mutant was significantly defective in growth compared to the wild-type when grown in a number of carbon sources. Additionally, the aphA mutant, which was also defective in vivo, exhibited similar growth defects compared to wild-type. Both luxO and aphA mutant strains were defective in 25 carbon sources compared to wild-type. By bioinformatics and biochemical analysis, we further showed that OpaR binds directly to the regulatory regions of genes and operons involved in carbon transport and metabolism thereby indicating a role for this regulator in metabolism in the bacteria. ☐ In Chapter 3, we perform comparative transcriptome analysis of wild-type versus the super-colonizer rpoN mutant. RpoN is a sigma factor that is involved in the transcription of 100s of genes but requires an activator protein to be functional. We examined this regulator since one of its activators is LuxO and together they regulate the expression of the quorum sensing master regulators OpaR and AphA. We found a number of genes were differentially regulated between the wild-type and the rpoN mutant including a number of genes encoding for ribosomal proteins and tRNAs that were upregulated in the rpoN mutant. This was in keeping with the fact that we had previously found the mutant to be metabolically more fit than the wild-type. Expression of the QS master regulators in the rpoN mutant did not match the expression pattern seen in the luxO mutant. We speculate that these differences in expression between the luxO and rpoN mutants might be due to increased expression of one of the quorum regulatory small RNA, qrr2 in the rpoN mutant. ☐ Other work in this dissertation focuses on the acid and osmotic stress response mechanisms in V. parahaemolyticus. As a marine bacteria and a gastrointestinal pathogen, V. parahaemolyticus constantly encounters various salt gradients and is subjected to acid stresses within the gastrointestinal tract. We examined the effect of lethal acid stress on the bacteria under varying degrees of salt concentration. We found that preadaptation of the bacteria to high salt is beneficial for the bacteria as it leads to improved survival under lethal acid stress and other lethal environmental stresses. ☐ Finally, we performed whole genome sequencing on an environmental isolate of V. parahaemolyticus, strain UCM-V493. UCM-V493 was isolated from a sediment sample in Spain in 2002. We were interested in determining differences between pathogenic and non-pathogenic strains. We sequenced this strain to completion and closed both circular chromosomes with a combination of Illumina and Pacbio sequencing. Comparative genomic analysis uncovered many regions of difference between UCM-V493 and RIMD2210633, the canonical clinical isolate.