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

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.

Adhesion Protein ApfA of Actinobacillus pleuropneumoniae Is Required for Pathogenesis and Is a Potential Target for Vaccine Development

“…This result is in accordance with a previous study, which showed that the type IV fimbrial promoter is induced upon contact with host cells via the luxAB reporter system (22). Besides, two recent studies that looked at A. pleuropneumoniae gene expression during natural and experimental infections in pigs also indicated the upregulation of genes coding for type IV fimbriae (36,37).…”

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

“…We used aerobiosis as it is the typical atmosphere used in experiments involving A. pleuropneumoniae and anaerobiosis as, based on mutant Jacobsen et al 2005;Buettner et al 2009) or transcriptome (Deslandes et al 2010;Klitgaard et al 2012) studies, it is representative of the growth conditions found in necrotic lungs of pigs. As shown in Figure 4 and Table 1, some trans-acting sRNA candidates, like Arrc05 and Arrc08, are located between genes within operons, which could allow them to be mistaken for subproducts of polycistronic mRNA maturation.…”

Porcine Respiratory Pathogens in Swine Farms Environment in Mexico

“…pleuropneumoniae is the etiologic agent of porcine pleuropneumonia in hyperacute, acute or chronic form [36]- [40]. Positive results based on the apxIV gene amplification indicate the presence of this bacterium in almost all samples, with the exception of urine samples.…”