Transcript profiling of Actinobacillus pleuropneumoniae in bronchoalveolar fluid

Actinobacillus pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses to cause contagious pleuropneumonia. Therefore, the objective of this study was to identify the A. pleuropneumoniae genes that are differentially expressed in a medium mimicking the lung-like environment. Since bronchoalveolar fluid (BALF) contains, in addition to other substances, innate immune components found in the lungs, we examined gene expression of the virulent serovar 1 strain A. pleuropneumoniae CM5 after incubation in BALF. In reverse transcription-PCR differential display (RT-PCR DD) experiments, a 3.3-fold increase was observed in the lamB gene, a component of the maltose regulon, which is involved in maltose and maltodextrin transport and metabolism. Knockout mutations were made in lamB and in malT, which encodes a positive transcriptional regulator of the maltose regulon. In comparison with the wild-type organism and its lamB mutant, the malT mutant grew slowly in complex media and had an increased sensitivity to biological stressors. Transcript profiling of the malT mutant exposed to BALF revealed that knockout of the malT gene invokes a stringent-type gene-expression profile in the bacterium. The stringent response gene, relA, and the genes involved in biofilm formation as well as in amino acid biosynthesis were up-regulated in the malT mutant, whereas the genes involved in energy metabolism, protein biosynthesis, and transport of biomolecules were down-regulated. These results suggest that MalT may be involved in transport of essential nutrient (s) in BALF or may be linked directly or indirectly to the stringent response in A. pleuropneumoniae.;DNA microarray studies showed that 157 genes were differentially expressed by A. pleuropneumoniae in BALF, with an estimated false discovery rate of 1%. FNR-regulated genes encoding reductases for nitrite, periplasmic nitrate and dimethyl sulfoxide were up-regulated in BALF, as were the genes for Na+-translocating NADH-quinone reductase. The sapF gene encoding the peptide transport system ATP-binding protein SapF was also up-regulated. Transcription of a number of known virulence and in vivo expressed genes including apxIVA, was also increased. These studies suggest that, in response to BALF components, A. pleuropneumoniae modulates membrane bioenergetics for energy generation to synthesize proteins involved in survival and expression of virulence factors.