| Summary: | <p>Abstract</p> <p>Background</p> <p><it>Actinobacillus pleuropneumoniae </it>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 <it>in vivo </it>during a natural infection, we undertook transcript profiling experiments with an <it>A. pleuropneumoniae </it>DNA microarray, after recovery of bacterial mRNAs from serotype 5b-infected porcine lungs. AppChip2 contains 2033 PCR amplicons based on the genomic sequence of <it>App </it>serotype 5b strain L20, representing more than 95% of ORFs greater than 160 bp in length.</p> <p>Results</p> <p>Transcriptional profiling of <it>A. pleuropneumoniae </it>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 <it>A. pleuropneumoniae </it>transcripts could be detected on our microarrays, and 150 genes were deemed differentially expressed <it>in vivo </it>during the acute phase of the infection. Our results indicate that, for example, gene <it>apxIVA </it>from an operon coding for RTX toxin ApxIV is highly up-regulated <it>in vivo</it>, 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 <it>irp </it>and <it>APL_0959</it>, predicted to code for a TonB-dependent receptor and a filamentous hemagglutinin/adhesin respectively) or lipoproteins (gene <it>APL_0920</it>). Only 4 of 72 up-regulated genes had previously been identified in controled experimental infections.</p> <p>Conclusions</p> <p>These genes that we have identified as up-regulated in <it>vivo</it>, conserved across serotypes and coding for potential outer membrane proteins represent potential candidates for the development of a cross-protective vaccine against porcine pleuropneumonia.</p>
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