Dataset Information

Analysis of the microRNA Expression Profile of Bovine Monocyte-derived Macrophages Infected with Mycobacterium avium subsp. Paratuberculosis Reveals that miR-150 Suppresses Cell Apoptosis by Targeting PDCD4.

ABSTRACT: M. avium subsp. paratuberculosis (MAP) is the causative pathogen of Johne's disease, a chronic granulomatous enteritis that principally affects ruminants and can survive, proliferate and disseminate in macrophages. MicroRNAs (miRNAs) are important regulators of gene expression and can impact the processes of cells. To investigate the role of miRNAs in monocyte-derived macrophages (MDMs) during MAP infection, we used high-throughput sequencing technology to analyze small RNA libraries of MAP-infected and control MDMs. The results showed that a total of 21 miRNAs were differentially expressed in MDMs after MAP infection, and 8864 target genes were predicted. A functional analysis showed that the target genes were mainly involved in the MAPK signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway and apoptosis. In addition, using a dual-luciferase reporter assay, flow cytometry, and a small interfering (si)RNA knockdown assay, the role of miR-150 in regulating macrophage apoptosis by targeting the programmed cell death protein-4 (PDCD4) was demonstrated. These results provide an experimental basis to reveal the regulatory mechanism of MAP infection and suggest the potential of miRNAs as biomarkers for the diagnosis of Johne's disease in bovines.

SUBMITTER: Wang Z

PROVIDER: S-EPMC6600136 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Analysis of the microRNA Expression Profile of Bovine Monocyte-derived Macrophages Infected with Mycobacterium avium subsp. Paratuberculosis Reveals that miR-150 Suppresses Cell Apoptosis by Targeting PDCD4.

Wang Zi Z Kong Ling Cong LC Jia Bo Yan BY Chen Jing Rui JR Dong Yang Y Jiang Xiu Yun XY Ma Hong Xia HX

International journal of molecular sciences 20190601 11

M. avium subsp. paratuberculosis (MAP) is the causative pathogen of Johne's disease, a chronic granulomatous enteritis that principally affects ruminants and can survive, proliferate and disseminate in macrophages. MicroRNAs (miRNAs) are important regulators of gene expression and can impact the processes of cells. To investigate the role of miRNAs in monocyte-derived macrophages (MDMs) during MAP infection, we used high-throughput sequencing technology to analyze small RNA ...[more]

PMID: 31159463

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Project description:Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen of Johne's disease (paratuberculosis), which mainly causes chronic infectious granulomatous enteritis in ruminants and has brought huge economic losses to animal husbandry. As a specific intracellular pathogen, when MAP invades the body, it is internalized by macrophages where it is able to replicate by inhibition of the phagosome maturation, escaping the host immune system and surviving, which leads to the spread of the disease. More recent studies have shown that circRNA is involved in many pathological and physiological processes of the body as the molecular sponge of miRNA, the scaffold of RNA binding protein and having the characteristic of being able to translate into protein. In this study, the mRNA and circRNA expression profiles of MAP-infected bovine monocyte-macrophages and uninfected bovine cells were analyzed by high-throughput sequencing. A total of 618 differentially expressed mRNA were screened out, including 322 upregulated mRNA and 296 downregulated mRNA. In addition, the analysis of circRNA differential expression profile showed 39 differentially expressed genes including 12 upregulated and 27 downregulated genes. Moreover, differential genes belonging to cytokine activity, chemokine activity, inflammatory reaction, apoptosis, and other functional groups related to macrophage immune response were significantly enriched in Gene Ontology (GO). Multiple signal pathways including NF-κB, MAPK, Toll-like receptor, IL-17, JAK-STAT, and other signaling pathways related to activating macrophage immune response were significantly enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, RT-qPCR technology verified the accuracy of the mRNA sequencing results. In this study, we have obtained the transcriptome information of mRNA and circRNA of bovine monocyte-macrophage infected with MAP. These results will provide data support for the further study of mRNA-miRNA-circRNA network and immune escape mechanism of MAP and will enrich the knowledge of the molecular immune mechanisms of Johne's disease as well.

Project description:Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSP(P)15. We obtained a transposon (Tn) mutant with a disruption in the LSP(P)15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSP(P)15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen.Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely related M. avium subspecies, mycobactin production and iron uptake are different in M. avium subsp. paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSP(P)15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer in M. avium subsp. paratuberculosis evolution.

Project description:BackgroundMycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study.ResultsTo characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical.ConclusionsWe present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.

Project description:The genetic similarity between Mycobacterium avium subsp. paratuberculosis and other mycobacterial species has confounded the development of M. avium subsp. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. avium subsp. paratuberculosis genome in our laboratories has shown >98% sequence identity with Mycobacterium avium subsp. avium in some regions. However, an in silico comparison of the largest annotated M. avium subsp. paratuberculosis contigs, totaling 2,658,271 bp, with the unfinished M. avium subsp. avium genome has revealed 27 predicted M. avium subsp. paratuberculosis coding sequences that do not align with M. avium subsp. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates of M. avium subsp. paratuberculosis. From these analyses, 21 genes were found to be present in all M. avium subsp. paratuberculosis isolates and absent from all other mycobacterial species tested. One region of the M. avium subsp. paratuberculosis genome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only in M. avium subsp. paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novel M. avium subsp. paratuberculosis genes as candidate diagnostic sequences.

Dataset Information

Analysis of the microRNA Expression Profile of Bovine Monocyte-derived Macrophages Infected with Mycobacterium avium subsp. Paratuberculosis Reveals that miR-150 Suppresses Cell Apoptosis by Targeting PDCD4.

Publications

Analysis of the microRNA Expression Profile of Bovine Monocyte-derived Macrophages Infected with <i>Mycobacterium avium</i> subsp. <i>Paratuberculosis</i> Reveals that miR-150 Suppresses Cell Apoptosis by Targeting PDCD4.

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