Dataset Information

Profiling microRNAs in lung tissue from pigs infected with Actinobacillus pleuropneumoniae.

ABSTRACT:

Background

MicroRNAs (miRNAs) are a class of non-protein-coding genes that play a crucial regulatory role in mammalian development and disease. Whereas a large number of miRNAs have been annotated at the structural level during the latest years, functional annotation is sparse. Actinobacillus pleuropneumoniae (APP) causes serious lung infections in pigs. Severe damage to the lungs, in many cases deadly, is caused by toxins released by the bacterium and to some degree by host mediated tissue damage. However, understanding of the role of microRNAs in the course of this infectious disease in porcine is still very limited.

Results

In this study, the RNA extracted from visually unaffected and necrotic tissue from pigs infected with Actinobacillus pleuropneumoniae was subjected to small RNA deep sequencing. We identified 169 conserved and 11 candidate novel microRNAs in the pig. Of these, 17 were significantly up-regulated in the necrotic sample and 12 were down-regulated. The expression analysis of a number of candidates revealed microRNAs of potential importance in the innate immune response. MiR-155, a known key player in inflammation, was found expressed in both samples. Moreover, miR-664-5p, miR-451 and miR-15a appear as very promising candidates for microRNAs involved in response to pathogen infection.

Conclusions

This is the first study revealing significant differences in composition and expression profiles of miRNAs in lungs infected with a bacterial pathogen. Our results extend annotation of microRNA in pig and provide insight into the role of a number of microRNAs in regulation of bacteria induced immune and inflammatory response in porcine lung.

SUBMITTER: Podolska A

PROVIDER: S-EPMC3465251 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Publications

Profiling microRNAs in lung tissue from pigs infected with Actinobacillus pleuropneumoniae.

Podolska Agnieszka A Anthon Christian C Bak Mads M Tommerup Niels N Skovgaard Kerstin K Heegaard Peter Mh PM Gorodkin Jan J Cirera Susanna S Fredholm Merete M

BMC genomics 20120906

<h4>Background</h4>MicroRNAs (miRNAs) are a class of non-protein-coding genes that play a crucial regulatory role in mammalian development and disease. Whereas a large number of miRNAs have been annotated at the structural level during the latest years, functional annotation is sparse. Actinobacillus pleuropneumoniae (APP) causes serious lung infections in pigs. Severe damage to the lungs, in many cases deadly, is caused by toxins released by the bacterium and to some degree by host mediated tis ...[more]

PMID: 22953717

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Project description:The gram-negative bacterium Actinobacillus pleuropneumoniae (APP) is an inhabitant of the porcine upper respiratory tract and the causative agent of porcine pleuropneumonia (PP). In recent years, knowledge about the proinflammatory cytokine and chemokine gene expression that occurs in lung and lymph node of the APP-infected swine has been advanced. However, systematic gene expression profiles on hilar nodes from pigs after infection with Actinobacillus pleuropneumoniae have not yet been reported. The transcriptional responses were studied in hilar nodes (HN) from swine experimentally infected with APP and the control groupusing Agilent Porcine Genechip, including 43,603 probe sets. 9,517 transcripts were identified as differentially expressed (DE) at the p ? 0.01 level by comparing the log2 (normalized signal) of the two groups named treatment group (TG) and controls (CG). Eight hundred and fifteen of these DE transcripts were annotated as pig genes in the GenBank database (DB). Two hundred and seventy-two biological process categories (BP), 75 cellular components and 171 molecular functions were substantially altered in the TG compared to CG. Many BP were involved in host immune responses (i.e., signaling, signal transmission, signal transduction, response to stimulus, oxidation reduction, response to stress, immune system process, signaling pathway, immune response, cell surface receptor linked signaling pathway). Seven DE gene pathways (VEGF signaling pathway, Long-term potentiation, Ribosome, Asthma, Allograft rejection, Type I diabetes mellitus and Cardiac muscle contraction) and statistically significant associations with host responses were affected. Many cytokines (including NRAS, PI3K, MAPK14, CaM, HSP27, protein phosphatase 3, catalytic subunit and alpha isoform), mediating the proliferation and migration of endothelial cells and promoting survival and vascular permeability, were activated in TG, whilst many immunomodulatory cytokines were suppressed. The significant changes in the expression patterns of the genes, GO terms, and pathways, led to a decrease of antigenic peptides with antigen presenting cells presented to T lymphocytes via the major histocompatibility complex, and alleviated immune response induced APP of HN. The immune response ability of HN in the APP-infected pigs was weakened; however, cell proliferation and migration ability was enhanced.

Project description: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.

Project description:The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kan(r)) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kan(r) gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Deltacps1N and strain 4074Deltacps1B, respectively. Strain 4074Deltacps1N produced no detectable CP, but strain 4074Deltacps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Deltacps1N to produce 4074Deltacps1N(pABcps101), 4074Deltacps1N(pJMLcps53), and 4074Deltacps1N(pABcps55), respectively. Strain 4074Deltacps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Deltacps1N(pJMLcps53) and 4074Deltacps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Deltacps1N(pABcps101) > or = strain 4074Deltacps1N > strain 4074Deltacps1B. Strain 4074Deltacps1N(pJMLcps53) was less virulent than strain 4074Deltacps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

Dataset Information

Profiling microRNAs in lung tissue from pigs infected with Actinobacillus pleuropneumoniae.

Background

Results

Conclusions

Publications

Profiling microRNAs in lung tissue from pigs infected with Actinobacillus pleuropneumoniae.

Similar Datasets

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