Actinobacillus pleuropneumoniae during a natural infection in pigs
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ABSTRACT: 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.
ORGANISM(S): Actinobacillus pleuropneumoniae serovar 5b str. L20 Actinobacillus pleuropneumoniae
Project description: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. Comparative Genomic Hybridizations between Actinobacillus pleuropneumoniae serotype 5b strain L20 (ref) and serotype 5b fresh field isolate 896-07, recovered from infected pig lung tissues following natural acute infection. Two condition transcript profiling experiments : infectious 5b field strain isolated directly from lungs of naturally deceased pigs after acute infection vs infectious 5b field strain grown in BHI broth to an OD600 of 0.300.
Project description:Actinobacillus pleuropneumoniae is the etiologic agent of contagious pleuropneumonia, an economically important disease of commercially reared swine throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Effects of koromycin, an antimicrobial agent that acts as an noncompetitive inhibitor of the interaction of NQR with its quinone substrate, on the transcriptome of A. pleuropneumoniae was investigated.
Project description:Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae affects pig health status and the swine industry worldwide. Despite of the extensive number of studies focused on A. pleuropneumoniae infection and vaccine development, its exoproteome is still rather unexplored. By combining high-throughput mass spectrometry and immunoproteomic approaches, with our current work we provide an in-depth characterisation of A. pleuropneumoniae serotype 2 exoproteome. Label-free liquid chromatography - tandem mass spectrometry (LC-MS/MS) combined with a comprehensive bioinformatics analysis revealed 484 secreted proteins, of which 84 were predicted to be virulence factors and 142 to be exported via different export mechanisms. The RTX toxins ApxIIA, ApxIIIA and ApxIVA were found to be the most abundant proteins in the A. pleuropneumoniae serotype 2 exoproteome, although ApxIVA is commonly assumed to be expressed exclusively in vivo. Immunoproteomic approaches coupled to LC-MS/MS analysis allowed to portray the immunogenic proteins within the bacterial exoproteome to identify potential vaccine candidates. Using serum pools from uninfected, acutely infected and chronically infected animals, we were able to monitor the seroconversion during disease progression. Overall, our work is expected to contribute to the understanding of the complex pathogenic mechanisms and to facilitate the discovery of potential antimicrobial agents for controlling porcine pleuropneumonia.
Project description:Actinobacillus pleuropneumoniae is the etiologic agent of contagious pleuropneumonia, an economically important disease of commercially reared swine throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune components found in the lung, we examined the gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after exposure to concentrated BALF. This experiment was also carried out with a malT mutant of the same strain.
Project description:Actinobacillus pleuropneumoniae is a gram-negative bacterium that causes porcine pleuropneumonia, which is a widespread, highly contagious, and often fatal respiratory disease in swine. In this experiment pigs were inoculated with A. pleuropneumoniae serotype 5b. Liver samples from three non-inoculated pigs and three experimental inoculated pigs were used to characterize the expression profiles of mRNA and microRNA genes using DNA microarrays and Illumina GA deep sequencing, respectively. The microarray analysis identified a large number of genes, which significantly differed in expression in infected versus non-infected animals. MicroRNAs are short single stranded RNA molecules that regulate gene expression by sequence specific binding to the 3´ untranslated region (3´UTR) of target mRNAs. The deep sequencing analysis determined the identity and abundance of nearly 400 microRNAs, of which a portion was found to significantly differ in expression between the infected and non-infected animals. Target genes for differentially expressed microRNAs were predicted using microCosm Targets, which is based on the miRanda algorithm. Comparison on the two gene lists showed many common genes, which may suggest a causative relationship between changes in microRNA expression and target gene expression.
Project description:Actinobacillus pleuropneumoniae is a gram-negative bacterium that causes porcine pleuropneumonia, which is a widespread, highly contagious, and often fatal respiratory disease in swine. In this experiment pigs were inoculated with A. pleuropneumoniae serotype 5b. Liver samples from three non-inoculated pigs and three experimental inoculated pigs were used to characterize the expression profiles of mRNA and microRNA genes using DNA microarrays and Illumina GA deep sequencing, respectively. The microarray analysis identified a large number of genes, which significantly differed in expression in infected versus non-infected animals. MicroRNAs are short single stranded RNA molecules that regulate gene expression by sequence specific binding to the 3M-BM-4 untranslated region (3M-BM-4UTR) of target mRNAs. The deep sequencing analysis determined the identity and abundance of nearly 400 microRNAs, of which a portion was found to significantly differ in expression between the infected and non-infected animals. Target genes for differentially expressed microRNAs were predicted using microCosm Targets, which is based on the miRanda algorithm. Comparison on the two gene lists showed many common genes, which may suggest a causative relationship between changes in microRNA expression and target gene expression. The expression profiles of mRNA and smallRNA in liver from three experimentally infected pigs were compared with the profiles three non-infected contol pigs.
Project description:The ApxIVA protein belongs to a distinct class of a “clip and link” activity of Repeat-in-ToXin (RTX) exoproteins. Along with three other pore-forming RTX toxins (ApxI, ApxII and ApxIII), ApxIVA serves as a major virulence factor of Actinobacillus pleuropneumoniae, the causative agent of porcine pneumonia. The gene encoding ApxIVA is located on a bicistronic operon downstream of the orf1 gene and is expressed exclusively under in vivo conditions. Both ApxIVA and ORF1 are essential for full virulence, but the molecular mechanisms by which they contribute to the pathogenicity of A. pleuropneumoniae are not yet understood. Here, we provide a comprehensive structural and functional analysis of ApxIVA and ORF1 proteins. Our findings reveal that the N-terminal segment of ApxIVA shares a structural similarity with colicin M (ColM)-like bacteriocins and exhibits antimicrobial activity. The ORF1 protein resembles the colicin M immunity protein (Cmi) and, like Cmi, is exported to the periplasm through its N-terminal signal peptide. Additionally, ORF1 can protect bacterial cells from the antimicrobial activity of ApxIVA, suggesting that ORF1 and ApxIVA function as an antibacterial toxin-immunity pair. Moreover, we demonstrate that fetal calf serum can be utilized as a medium supplement to induce the production of ApxIVA and ORF1 proteins under in vitro conditions. These findings highlight the coordinated action of various RTX determinants, where the fine-tuned spatiotemporal production of ApxIVA may enhance the fitness of A. pleuropneumoniae, facilitating its invasion to a resident microbial community on the surface of airway mucosa.
Project description:Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.