Branched-Chain Amino Acids Are Required for the Survival and Virulence of Actinobacillus pleuropneumoniae in Swine |
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| Authors: | Sargurunathan Subashchandrabose Rhiannon M. LeVeque Trevor K. Wagner Roy N. Kirkwood Matti Kiupel Martha H. Mulks |
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| Affiliation: | 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. |
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| Abstract: | ![]()
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.Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a disease of significant economic importance throughout the swine-raising areas of the world (6, 48). This pathogen possesses several well-studied virulence factors, including Apx toxins (20), capsular polysaccharides (57, 58), lipopolysaccharide (1, 17, 41), fimbriae (63), and iron-scavenging proteins (13, 50), which aid in the pathogenesis of acute pleuropneumonia marked by edema, hemorrhage, and necrosis (6, 26). In a search for additional virulence factors of this pathogen, we developed an in vivo expression technology (IVET) system and used this genetic tool to identify A. pleuropneumoniae gene promoters that are upregulated in vivo in the swine lung during infection compared to growth on laboratory media (22, 55).One of the A. pleuropneumoniae in vivo-induced (ivi) promoters that we identified drives the ilvIH operon, which encodes both large and small subunits of acetohydroxy acid synthase isozyme III (AHAS) (55). AHAS enzymes catalyze pivotal steps in the biosynthesis of the branched-chain amino acids (BCAA) isoleucine, leucine, and valine (31). In a survey of IVET, signature-tagged mutagenesis, and microarray studies of other pathogens, we observed that genes involved in BCAA biosynthesis were frequently identified in studies of pathogens that cause pneumonia, meningitis, or septicemia but not in pathogens of the gastrointestinal tract (55). This observation suggests that the ability to synthesize BCAA is critical for pathogens of the respiratory tract but not for gastrointestinal pathogens. BCAA are essential amino acids that must be acquired from ingested food for most mammals, including humans and pigs, and it is possible that fluids in “clean” body sites such as the lungs have only limited supplies of BCAA compared to the digestive tract.To test whether limitation of BCAA affects the expression of A. pleuropneumoniae genes that are induced in vivo, we compared expression from the A. pleuropneumoniae ivi promoters in a chemically defined medium (CDM) containing or lacking BCAA (55). We found that 25% (8 of 32) of the ivi promoters were upregulated during growth in CDM lacking BCAA compared to complete CDM. These included the ilvI promoter, as well as promoters for other genes potentially involved in survival within the host and virulence, such as hfq, a global regulator that binds sRNAs and mRNA and affects expression of virulence-associated genes in many pathogens (9, 49). These results strongly suggest that the environmental conditions encountered by A. pleuropneumoniae during infection of the swine lung include limitation of BCAA.The goals of the present study were to quantify free BCAA in porcine pulmonary secretions, to evaluate the effect of these concentrations of BCAA on expression of genes required for BCAA biosynthesis, and to test whether A. pleuropneumoniae mutants that cannot synthesize BCAA were attenuated. A. pleuropneumoniae deletion-disruption mutants of the ilvI biosynthetic gene and the lrp gene, which encodes a global regulator required for expression of several genes involved in BCAA biosynthesis, were constructed and shown to be attenuated in a porcine infection model. The low levels of available BCAA in pulmonary secretions and the attenuation of these mutants led us to examine the effect of small molecule inhibitors of AHAS on growth of A. pleuropneumoniae in vitro. Several AHAS inhibitors were shown to prevent growth in CDM lacking BCAA but not complete CDM. These results demonstrate that A. pleuropneumoniae, and likely other bacterial pathogens of the respiratory tracts of other mammals, encounter conditions where BCAA are available only in limited supply during infection, that these low levels of BCAA can affect bacterial gene expression, and that these pathogens must be able to synthesize BCAA to survive and cause disease in the lung. |
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