Browsing by Subject "quorum sensing"
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Item Autoinducer 2-based quorum sensing response of Escherichia coli to sub-therapeutic tetracycline exposure(Texas A&M University, 2006-10-30) Lu, LingengAutoinducer 2 (AI-2) is a quorum sensing signal employed by bacteria to coordinate their response to environmental stresses. The objective of this study was to determine the relationship between presence of AI-2 molecules, exposure to sub-therapeutic tetracycline, the expression of genes associated with the conjugal transfer of antibiotic resistance plasmids, and the conjugal transfer of these plasmids in Escherichia coli. The studies showed that AI-2 activity increased in Tets E. coli in the presence of tetracycline (2 ????g/mL) under both batch and continuous culture conditions. The presence of AI-2 molecules induced tetracycline tolerance development in Tets E. coli. The studies showed that the survival rates of Tets E. coli exposed to AI-2 molecules were significantly higher compared to the cells not exposed to AI-2 molecules or cells that were exposed to only LB (Lauria-Bertani) broth. Molecular analyses using real-time PCR indicate that the expression of at least one conjugation-associated gene (trbC) is increased 9-fold in cells exposed to AI-2 molecules in the presence of sub-therapeutic tetracycline compared to its negative controls. The transconjugation frequency of the plasmid RP4 carrying the tet(A) gene increased between 10-100 fold in the presence of AI-2 molecules. In companion studies, AI-2-like activity was detected in fish, tomatoes, cantaloupes, carrots and milk samples. Interestingly, ground beef and poultry meat contained substances that appear to inhibit AI-2 activity. Collectively, these results highlight the potential importance of bacterial quorum sensing signals such as AI-2 in the response of bacterial cells to environmental stimuli and the possible role of quorum sensing signals in the quality and safety of foods.Item Citrus Bioactive Compounds: Isolation, Characterization and Modulation of Bacterial Intercellular Communication and Pathogenicity(2012-07-16) Vikram, AmitThe secondary metabolites of citrus such as limonoids and flavonoids constitute an important part of human diet. The present work was undertaken to elucidate the effect of citrus limonoids and flavonoids on the bacterial cell-cell signaling in Vibrio harveyi, Escherichia coli O157:H7 and Salmonella Typhimurium LT2. The first experiment was focused on purification of limonoids from grapefruit and sour orange seeds. The limonoids were extracted using organic solvents and purified by chromatographic techniques. A total of ten limonoids (7 aglycones and 3 glucosides) were purified. Currently, simultaneous measurement of aglycones and glucosides of limonoids is not available. To address this limitation, an analytical method using high performance liquid chromatography was developed with the capability of measuring both aglycones and glucosides in a single run. Furthermore, its applicability in the fruit and juice samples was demonstrated. The third study investigated the V. harveyi cell-cell signaling inhibitory potential of purified limonoids. Isolimonic acid, ichangin, obacunone and nomilin were showed potent inhibitory activity. Furthermore, isolimonic acid and ichangin inhibit the signal transduction pathway by up-regulating the response regulator luxO. Isolimonic acid was also found to be a potent inhibitor of Escherichia coli O157:H7 cell-cell signaling in the fourth study. The results demonstrated that isolimonic acid inhibits the autoinducer/epinephrine mediated cell-cell signaling, biofilm and virulence in QseBC and QseA dependent fashion. Further investigations using limonin analogues, in the fifth study, demonstrated that the analogue limonin-7-methoxime inhibited the E. coli biofilm in type 1 pili and antigen 43 dependent-fashion, by preventing the binding of the adhesins to plastic surfaces. Another limonoid, obacunone was demonstrated to attenuate the Salmonella virulence by repressing Salmonella Pathogenicity Island 1 (SPI-1) in EnvZ/OmpR dependent mecahnism. The seventh study showed that naringenin, among the flavonoids, was the most potent inhibitor of V. harveyi and E. coli O157:H7 cell-cell signaling. Furthermore, naringenin was found to repress the (SPI-1) in PstS-HilD dependent fashion in the eighth study. In conclusion, the current project identified several limonoids and flavonoids with cell-cell signaling inhibitory property in three bacterial species.Item Role of new virulence mechanisms/factors (type 3 secretion system and TOX-R regulated lipoprotein [TAGA]) in the pathogenesis of the emerging human pathogen Aeromonas hydrophila(2006-08-10) Lakshmi Pillai; Dr. Ashok K. Chopra, Ph.D. C.Sc.; Dr. Vladimir Motin, Ph.D.; Dr. Golda A.K. Leonard, Ph.D.; Dr. Eric M. Smith, Ph.D.; Dr. Chandrasekha Yallampalli, Ph.D.; Dr. Amy Horneman, Ph.D., SM(ASCP)Aeromonas hydrophila, a gram-negative bacterium that causes gastroenteritis, wound infections, septicemia, and other diseases in humans, produces many different virulence factors. A clinical isolate SSU of A. hydrophila possesses a cytotoxic enterotoxin Act, a potent virulence factor that is secreted into the environment through the bacterium’s type 2 secretion system (T2SS) and possesses several biological activities, including cytotoxicity, enterotoxicity, and lethality in a mouse model. The purpose of this study was to identify new virulence factors that contribute to the pathogenesis of this bacterium. We identified and characterized a type 3 secretion system (T3SS) in A. hydrophila SSU. By marker-exchange mutagenesis of the aopB gene, a crucial gene involved in the formation of the translocon apparatus, the functionality of the T3SS was elucidated, both in in vitro and in vivo models. Further, the characterization of the regulatory gene DNA adenine methyltransferase (Dam) from SSU and its role in modulating the function of both the T3SS and Act was investigated. The role of the T3SS in influencing the phenomenon of quorum sensing (QS) in A. hydrophila SSU was also conducted. This study highlights a unique link between the T3SS and Act of A. hydrophila and the production of QS molecules or lactones. While searching for potential effector proteins secreted through the T3SS of A. hydrophila SSU, the identification of a new virulence factor, ToxR regulated lipoprotein (TagA), was revealed. TagA is a zinc metalloprotease which has only been identified in the gram-negative pathogens, Escherichia coli O157:H7 and Vibrio cholerae. In A. hydrophila, TagA has been shown to play a role in the inhibition of complement by binding to and cleaving the serpin C1-INH. By recruiting C1-INH to the surface of the bacteria and cleaving it, TagA is able to significantly prevent the activation of complement at the cell surface, ultimately increasing the serum resistance of the pathogen. TagA can also target C1-INH to erythrocyte surfaces and decrease the lysis that occurs in the presence of serum. Confocal fluorescence microscopy revealed that the serpin C1-INH binds to TagA on the surface of the bacteria.