Abstract
BackgroundActinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide. Many virulence factors are involved in the pathogenesis, namely capsular polysaccharides, RTX toxins, LPS and many iron acquisition systems. In order to identify genes that are expressed in vivo during a natural infection, we undertook transcript profiling experiments with an A. pleuropneumoniae DNA microarray, after recovery of bacterial mRNAs from serotype 5b-infected porcine lungs. AppChip2 contains 2033 PCR amplicons based on the genomic sequence of App serotype 5b strain L20, representing more than 95% of ORFs greater than 160 bp in length.ResultsTranscriptional profiling of A. pleuropneumoniae recovered from the lung of a pig suffering from a natural infection or following growth of the bacterial isolate in BHI medium was performed. An RNA extraction protocol combining beadbeating and hot-acid-phenol was developed in order to maximize bacterial mRNA yields and quality following total RNA extraction from lung lesions. Nearly all A. pleuropneumoniae transcripts could be detected on our microarrays, and 150 genes were deemed differentially expressed in vivo during the acute phase of the infection. Our results indicate that, for example, gene apxIVA from an operon coding for RTX toxin ApxIV is highly up-regulated in vivo, and that two genes from the operon coding for type IV fimbriae (APL_0878 and APL_0879) were also up-regulated. These transcriptional profiling data, combined with previous comparative genomic hybridizations performed by our group, revealed that 66 out of the 72 up-regulated genes are conserved amongst all serotypes and that 3 of them code for products that are predicted outer membrane proteins (genes irp and APL_0959, predicted to code for a TonB-dependent receptor and a filamentous hemagglutinin/adhesin respectively) or lipoproteins (gene APL_0920). Only 4 of 72 up-regulated genes had previously been identified in controled experimental infections.ConclusionsThese genes that we have identified as up-regulated in vivo, conserved across serotypes and coding for potential outer membrane proteins represent potential candidates for the development of a cross-protective vaccine against porcine pleuropneumonia.
Highlights
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide
In order to gain new insight into the disease, researchers have relied on techniques that allow for the identification of genes expressed in bacteria during infection of the host [11], namely In Vivo Expression Technology (IVET), Signature Tagged Mutagenesis (STM) and Selective Capture of Transcribed Sequences (SCOTS)
An IVET experiment with A. pleuropneumoniae led to the identification of 10 loci transcribed in vivo [12] and highlighted the importance of genes involved in branched-chain amino acid (BCAA) synthesis [13,14]
Summary
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide. SCOTS experiments were conducted with A. pleuropneumoniae, at two different stages of infection: at the end of the acute stage, in necrotic lung tissues (7 days post-infection) [18], and in the chronic stage (21 days post-infection) [10] These studies demonstrated the importance of anaerobic metabolism and HlyX-regulated mechanisms during A. pleuropneumoniae infection in the lungs, and led to the identification and characterisation of a maturation autotransporter protease, AasP, responsible for cleavage and release of fragments of OmlA from the cell surface [19]. The HlyX regulon was characterized recently, and ironregulated protein B (FrpB) was identified as a potentially important virulence factor up-regulated when A. pleuropneumoniae is grown in oxygen-deprived environments [20] These in vivo studies have been very valuable in gaining a better understanding of the mechanisms involved in A. pleuropneumoniae pathogenesis, limitations associated with the techniques used lead only to a partial overview of the transcriptional events taking place in vivo
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