Transcriptional profiling of Actinobacillus pleuropneumoniae during the acute phase of a natural infection in pigs

Abstract

Background: Actinobacillus 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.

Results: Transcriptional 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.

Conclusions: These 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.

Figure 1

HPS staining of paraffin-embedded infected lung tissue sections, at 100× magnification. Severe fibrinohemorragic pneumonia with infiltration of oat cells.

Figure 2

Hierarchical clustering of reference and field strains of A. pleuropneumoniae, including strain 896-07, based on CGH data obtained with AppChip2. Strains are identified according to "serotype-strain number". Field strains are the same as those used by Gouré et al[25]. R: reference strain. NT: Non-typeable.

Figure 3

Functional classification of A. pleuropneumoniae genes that are differentially expressed in infected pig lungs. AAB, amino acid biosynthesis. BCPGC, biosynthesis of cofactors, prosthetic groups and carriers. CE, cell enveloppe. CP, cellular process. CIM, central intermediary metabolism. DM, DNA metabolism. EM, energy metabolism. FAPM, fatty acid and phospholipid metabolism. HUU, hypothetical/unclassified/unknown. MEE, mobile extrachromosomal element functions. PF, protein fate. PS, protein synthesis. PPNN, purines, pyrimidins, nucleosides and nucleotides. RF, regulatory functions. ST, signal transduction. Tr, transcription. TBP, transport and binding proteins.

Figure 4

Validation of microarray results by qRT-PCR. Results from five up-regulated and six down-regulated genes are presented. Mean log₂ ratios obtained during qRT-PCR experiments are plotted against the mean log₂ ratios obtained with the microarrays. Numbers on the graph refer to the gene numbers in Additional file 2.