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Identification of the Actinobacillus pleuropneumoniae leucine-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. Leucine-responsive 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 His6-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.

SUBMITTER: Wagner TK

PROVIDER: S-EPMC1828405 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Identification of the Actinobacillus pleuropneumoniae leucine-responsive regulatory protein and its involvement in the regulation of in vivo-induced genes.

Wagner Trevor K TK Mulks Martha H MH

Infection and immunity 20061023 1

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. Leucine-responsive regulatory protein (Lrp) is a global regulator and has been shown in ...[more]

PMID: 17060463

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Project description:Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.

Dataset Information

Identification of the Actinobacillus pleuropneumoniae leucine-responsive regulatory protein and its involvement in the regulation of in vivo-induced genes.

Publications

Identification of the Actinobacillus pleuropneumoniae leucine-responsive regulatory protein and its involvement in the regulation of in vivo-induced genes.

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