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Infection and Immunity, November 2009, p. 4925-4933, Vol. 77, No. 11
0019-9567/09/$08.00+0     doi:10.1128/IAI.00671-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Branched-Chain Amino Acids Are Required for the Survival and Virulence of Actinobacillus pleuropneumoniae in Swine{triangledown}

Sargurunathan Subashchandrabose,1,5 Rhiannon M. LeVeque,2,5 Trevor K. Wagner,2,5,{dagger} Roy N. Kirkwood,3 Matti Kiupel,4 and Martha H. Mulks1,2,5*

Comparative Medicine and Integrative Biology Program,1 Department of Microbiology and Molecular Genetics,2 Department of Large Animal Clinical Sciences,3 Department of Pathobiology and Diagnostic Investigation,4 Center for Microbial Pathogenesis, Michigan State University, East Lansing, Michigan 488245

Received 11 June 2009/ Returned for modification 1 July 2009/ Accepted 17 August 2009

In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters 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 that A. pleuropneumoniae mutants 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 both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew 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 that A. 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.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, 5193 BPS Building, Michigan State University, East Lansing, MI 48824. Phone: (517) 884-5365. Fax: (517) 353-8957. E-mail: mulks{at}msu.edu

{triangledown} Published ahead of print on 24 August 2009.

Editor: B. A. McCormick

{dagger} Present address: OpGen, Inc., 510 Charmany Road, Madison, WI 53719.

Infection and Immunity, November 2009, p. 4925-4933, Vol. 77, No. 11
0019-9567/09/$08.00+0     doi:10.1128/IAI.00671-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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