Branched-Chain Amino Acids Are Required for the Survival and Virulence ofActinobacillus pleuropneumoniaein Swine

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Abstract
InActinobacillus pleuropneumoniae, which causes porcine pleuropneumonia,ilvIwas identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis.ilvIand 7 of 32 additionalivipromoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and thatA. pleuropneumoniaemutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 μmol/liter, which is 10 to 17% of the concentration in plasma. The expression of bothilvIandlrp, a global regulator that is required forilvIexpression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants ofilvIandlrpwere both auxotrophic for BCAA in CDM and attenuated compared to wild-typeA. pleuropneumoniaein competitive index experiments in a pig infection model. Wild-typeA. pleuropneumoniaegrew in CDM+BCAA but not in CDM−BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis thatA. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens.

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