Identification of theActinobacillus pleuropneumoniaeLeucine-Responsive Regulatory Protein and Its Involvement in the Regulation of In Vivo-Induced Genes

Abstract

Actinobacillus pleuropneumoniae is a gram-negative bacterial pathogen that causes a severe hemorrhagic pneumonia in swine. We have previously shown that the limitation of branched-chain amino acids (BCAAs) is a cue that induces the expression of a subset of A. pleuropneumoniae genes identified as specifically induced during infection of the natural host animal by using an in vivo expression technology screen. Leucineresponsive regulatory protein (Lrp) is a global regulator and has been shown in Escherichia coli to regulate many genes, including genes involved in BCAA biosynthesis. We hypothesized that A. pleuropneumoniae contains a regulator similar to Lrp and that this protein is involved in the regulation of a subset of genes important during infection and recently shown to have increased expression in the absence of BCAAs. We report the identification of an A. pleuropneumoniae serotype 1 gene encoding a protein with similarity to amino acid sequence and functional domains of other reported Lrp proteins. We further show that purified A. pleuropneumoniae His 6 -Lrp binds in vitro to the A. pleuropneumoniae promoter regions for ilvI, antisense cps1AB, lrp, and nqr. A genetically defined A. pleuropneumoniae lrp mutant was constructed using an allelic replacement and sucrose counterselection method. Analysis of expression from the ilvI and antisense cps1AB promoters in wild-type, lrp mutant, and complemented lrp mutant strains indicated that Lrp is required for induction of expression of ilvI under BCAA limitation.

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

“…We have previously shown that A. pleuropneumoniae ilvI, which is required for BCAA biosynthesis, is upregulated both in vivo during infection (22) and in CDM lacking BCAA (CDMϪBCAA) compared to complete CDM (CDMϩBCAA) (55) and that leucine-responsive regulatory protein, encoded by the gene lrp, is required for the response of ilvI to BCAA limitation (54). To determine whether expression of ilvI and lrp correlates with the concentrations of BCAA available in the porcine lung, we used luciferase reporter assays and quantitative RT-PCR to measure the expression of these genes in CDM samples containing various concentrations of BCAA.…”

“…Luciferase reporter assays. Expression from the ilvI promoter was quantified by using a luciferase expression plasmid (pIviI) in which the ilvI promoter drives the expression of promoterless luxAB genes (54). Wild-type A. pleuropneumoniae strain AP225 containing pIviI grown overnight on BHI agar supplemented with 10 g of V factor/ml (BHIV) containing 50 g of ampicillin/ml was suspended in CDMϪBCAA and then diluted to an optical density at 520 nm (OD 520 ) of ϳ0.2 in 30 ml of prewarmed CDM containing 50 g of ampicillin/ml and supplemented with 0, 10, 20, 50, or 100% of the concentration of BCAA found in complete CDM.…”

“…To construct an A. pleuropneumoniae ilvI mutant, we used a similar technique to that previously reported by our laboratory for the construction of an lrp mutant (54). The 5Ј and 3Ј ends of the ilvI gene from A. pleuropneumoniae AP100 were amplified from genomic DNA by PCR and cloned into pUC18.…”

“…Group 1 was challenged by percutaneous intratracheal inoculation with 4 ϫ 10 6 CFU of wild-type A. pleuropneumoniae AP225 in 10 ml of PBS, as previously described (23,28). Group 2 received 4 ϫ 10 6 CFU of AP359, an lrp mutant of AP225 (54). Group 3 received 2 ϫ 10 7 CFU of AP359.…”

“…Group 3 received 2 ϫ 10 7 CFU of AP359. Group 4 received 4 ϫ 10 6 CFU of AP359 complemented with plasmid pTW415, which contains the lrp gene in a pGZRS18 vector (54). Group 5 received 10 ml of PBS.…”