Roles of Shigella flexneri H2-oxidizing hydrogenases in acid tolerance
McNorton, Mykeshia Montez'
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NiFe hydrogenases are membrane bound enzymes that catalyze the oxidation or evolution of molecular hydrogen. It has been shown that molecular hydrogen can be used as an energy source. Two uptake (oxidizing) hydrogenases Hya and Hyb are described in Shigella flexneri. Hya and Hyb are expressed in acidic and alkaline conditions, respectively. These uptake enzymes are linked to the electron transport chain through a cytochrome b carrier. It has been shown that deletions in the genes encoding these hydrogenases within several pathogens resulted in strains that were either defective in uptake of nutrients or unable to colonize the host animal. Thus, for this thesis work mutants in the hydrogenases were constructed in S.flexneri to determine their contribution to bacterial physiology, and especially to acid challenge. S. flexneri is an enteric bacterium that is the causative agent of shigellosis. This disease is characterized by diarrhea stools containing blood and mucus. In order to cause disease the bacteria must first survive in the human gastric environment (~pH 2). S. flexneri uses 2 acid resistance pathways to survive in a pH of 2. One which is controlled by RpoS and Crp, and the second which requires glutamate. However, an alternate pathway was described herein whereby S. flexneri requires Hya hydrogenase in absence of exogenous glutamate to survive acidic pH values. Hya was shown to provide a membrane potential. The cationic dye JC-1 and the protonophore CCCP was used to access membrane potential in S.flexneri wild type and Hya mutant strains. Compared to the wild type, the Hya mutant had negligible Δψ activity using JC-1 fluorescence. Acid sensitivity comparable to the Hya mutant was observed when CCCP was added to the wild type strain. A model whereby Hya provides robust periplasmic proton levels to counteract proton (acid) influx is proposed. The deletion of Hya has a global impact on protein expression in S. flexneri. It was observed that proteins involved in glycolysis and fatty acid metabolism increased in expression in the mutant. Decreased expression of proteins associated with detoxification, gluconeogenesis, serine-derived amino acids, and nitrate reductases was observed.