ABSTRACTROLE OF IN VIVO-INDUCED GENES ILVI AND HFQ IN THE SURVIVAL AND VIRULENCE OF ACTINOBACILLUS PLEUROPNEUMONIAE By Sargurunathan SubashchandraboseActinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a contagious and often fatal disease of pigs. Infection with this bacterium leads to the development of a fulminant pleuropneumonia resulting in severe damage to the lungs. A. pleuropneumoniae genes that are up-regulated during the infection of pig lungs were previously identified in our laboratory and designated as in vivo-induced genes. The role of two such in vivo-induced genes, ilvI and hfq, in the pathobiology of A. pleuropneumoniae is the subject of this dissertation.The gene ilvI encodes an enzyme involved in the biosynthesis of branched-chain amino acids (BCAAs). The leucine-responsive regulatory protein (Lrp) is a transcriptional regulator of the ilvIH operon. BCAA biosynthetic genes are associated with virulence in pathogens infecting the respiratory tract and blood stream. Also, twenty five percent of the A. pleuropneumoniae in vivo-induced genes were up-regulated under BCAA limitation, suggesting that these BCAAs may be found at limiting concentrations in certain sites of the mammalian body such as the respiratory tract. The concentration of amino acids in the porcine pulmonary epithelial lining fluid was determined and BCAAs were found at limiting concentration in the respiratory tract. Further, the virulence of two BCAA auxotrophs, an ilvI mutant and an lrp mutant, was tested in a pig infection model and both were found to be attenuated. Finally, inhibitors of BCAA biosynthesis were found to prevent the growth of A. pleuropneumoniae. Hfq, encoding the Host factor Q-beta (Hfq) in A. pleuropneumoniae, was identified as an in vivo-induced gene and is also up-regulated under BCAA limitation. Since Hfq is a global regulator which is induced under one of the signals found in the porcine respiratory tract, the role of Hfq in A. pleuropneumoniae was analyzed. An A. pleuropneumoniae hfq mutant strain failed to form biofilm. Levels of the pgaC transcript, encoding the biofilm matrix biosynthetic enzyme, were ~14-fold lower in the hfq mutant compared to wild-type strain. The hfq mutant displayed enhanced sensitivity to superoxide stress and tellurite. Hfq was found to regulate two virulence-associated phenotypes, biofilm formation and resistance to oxidative stress, in A. pleuropneumoniae.As Hfq was associated with at least two virulence-associated phenotypes, the effect of Hfq on the virulence of A. pleuropneumoniae was tested in a pig infection model. Infection with the hfq mutant did not result in the development of pleuropneumonia while pigs infected with the wild-type strain exhibited classic signs of pleuropneumonia. Competitive index analysis revealed that the hfq mutant is severely attenuated, compared to the wild-type strain. In summary, studies described in this dissertation have uncovered a new host signal found in the porcine respiratory tract and provide evidence for the potential of inhibitors of BCAA biosynthesis as antibacterial agents. Hfq, regulated by BCAA limitation, was found to regulate biofilm formation, resistance to oxidative stress and virulence in A. pleuropneumoniae. The role of Hfq in the virulence of a bacterial pathogen during infection of lungs has been examined in the studies described in this dissertation. The hfq mutant is highly attenuated and is a potential candidate for the development of a live-attenuated vaccine against porcine pleuropneumonia.
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