Identification and regulation of Actinobacillus pleuropneumoniae in vivo induced genes that respond to branched-chain amino acid limitation

Actinobacillus pleuropneumoniae is a Gram-negative bacterial pathogen that is the causative agent of a severe hemorrhagic pleuropneumonia in swine. The severe effect of the disease on the swine industry has led to extensive research on development of improved vaccines. To understand the genetic basis of this disease and to identify potential vaccine targets, an in vivo expression technology (IVET) system was developed for use in A. pleuropneumoniae serotype 1. The IVET system was designed to identify A. pleuropneumoniae in vivo induced ( ivi) genes whose expression is specifically induced during infection of the natural swine host, without the need to identify each individual environmental cue necessary for expression of each gene.; However, to analyze the role of ivi genes, it is important to understand the specific cues that regulate expression of each ivi gene. It was hypothesized that a limitation of branched-chain amino acids (BCAAs) acts as a signal to induce expression of ivi genes in a manner similar to iron limitation. A pool of 32 previously isolated ivi promoter clones were analyzed for increased activity of a reporter under BCAA limiting conditions, and 8 ivi promoters were identified as up-regulated in this study. These data suggest that the limitation of BCAAs is an important cue in the regulation of ivi genes and potentially other virulence genes.; One mechanism known to regulate many Escherichia coli genes in response to BCAA limitation is leucine-responsive regulatory protein (Lrp). An A. pleuropneumoniae gene similar to Lrp was identified and cloned. Purified A. pleuropneumoniae His6-Lrp bound in vitro to 2/8 ivi promoters identified to respond to BCAA limitation. A genetically-defined A. pleuropneumoniae lrp mutant was constructed and used to show the requirement for Lrp in the regulation of several A. pleuropneumoniae genes.; To further understand the role of Lrp in virulence, the A. pleuropneumoniae lrp mutant was analyzed in a swine model of respiratory infection. The lrp mutant was able to cause disease under the conditions tested, with progression of disease and pathology similar to that seen with wild-type A. pleuropneumoniae.; The identification of an environmental stimulus, a regulatory mechanism, and genes regulated by these factors is an important step for understanding the virulence of A. pleuropneumoniae. This research offers insight into new avenues of research to further examine the virulence of A. pleuropneumoniae and other respiratory pathogens.