Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne’s disease, an intestinal disease of ruminants with major economic consequences. Infectious bacilli are phagocytosed by host macrophages upon exposure where they persist, resulting in lengthy subclinical phases of infection that can lead to immunopathology and disease dissemination. Consequently, analysis of the macrophage transcriptome in response to M. avium subsp. paratuberculosis infection can provide valuable insights into the molecular mechanisms that underlie Johne’s disease. Here, we investigate pan-genomic gene expression in bovine monocyte-derived macrophages (MDM) purified from seven age-matched females, in response to in vitro infection with M. avium subsp. paratuberculosis (multiplicity of infection 2:1) at intervals of 2 hours, 6 hours and 24 hours post-infection (hpi). Differentially expressed genes were identified by comparing the transcriptomes of the infected MDM to the non-infected control MDM at each time point (adjusted P-value threshold ≤ 0.10). 1050 differentially expressed unique genes were identified 2 hpi, with 974 and 78 differentially expressed unique genes detected 6 and 24 hpi, respectively. Furthermore, in the infected MDM the number of upregulated genes exceeded the number of downregulated genes at each time point, with the fold-change in expression for the upregulated genes markedly higher than that for the downregulated genes. Inspection and systems biology analysis of the differentially expressed genes revealed an enrichment of genes involved in the inflammatory response, cell signalling pathways and apoptosis. The transcriptional changes associated with cellular signalling and the inflammatory response may reflect different immuno-modulatory mechanisms that underlie host-pathogen interactions during infection.

BackgroundMycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA.ResultsA mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology.ConclusionsThis study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA.

Background Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cell types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2∶1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array.ResultsComparison of M. bovis-challenged MDM gene expression profiles with those from the non-challenged MDM controls at each time point identified 3,064 differentially expressed genes 2 hours post-challenge, with 4,451 and 5,267 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly higher than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors; and (3) apoptosis.ConclusionsThe increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support roles for MyD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis.

Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, (MAP), is a chronic intestinal disease of ruminants with serious economic consequences for cattle production in the United States and elsewhere. During infection, MAP bacilli are phagocytosed and subvert host macrophage processes, resulting in subclinical infections that can lead to immunopathology and dissemination of disease. Analysis of the host macrophage transcriptome during infection can therefore shed light on the molecular mechanisms and host-pathogen interplay associated with Johne’s disease. Here, we describe results of an in vitro study of the bovine monocyte-derived macrophage (MDM) transcriptome response during MAP infection using RNA-seq. MDM were obtained from seven age- and sex-matched Holstein-Friesian cattle and were infected with MAP across a 6-h infection time course with non-infected controls. We observed 245 and 574 differentially expressed (DE) genes in MAP-infected versus non-infected control samples (adjusted P value ≤0.05) at 2 and 6 h post-infection, respectively. Functional analyses of these DE genes, including biological pathway enrichment, highlighted potential functional roles for genes that have not been previously described in the host response to infection with MAP bacilli. In addition, differential expression of pro- and anti-inflammatory cytokine genes, such as those associated with the IL-10 signaling pathway, and other immune-related genes that encode proteins involved in the bovine macrophage response to MAP infection emphasize the balance between protective host immunity and bacilli survival and proliferation. Systematic comparisons of RNA-seq gene expression results with Affymetrix® microarray data generated from the same experimental samples also demonstrated that RNA-seq represents a superior technology for studying host transcriptional responses to intracellular infection.

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SummaryT-cell stimulating protein A (TspA) is an immunogenic, T-cell and B-cell stimulating protein of Neisseria meningitidis. Sequence similarity between TspA and FimV, a Pseudomonas aeruginosa protein involved in twitching motility, suggested a link between TspA and type IV pili (Tfp). To determine the role of TspA an isogenic deletion mutant was created. Loss of TspA did not affect twitching motility or piliation indicating that there are functional differences between TspA and FimV. Mutation of tspA led to a significant reduction in adhesion of meningococci to meningothelial and HEp-2 cells, which was not due to a lack of transcription of adjacent genes or pilC1. Other Tfp-mediated phenotypes (i.e. auto-aggregation and transformation competence) were not altered. Our results indicate that the role of TspA in adhesion is unlikely to be directly linked to the function of Tfp. TspA was expressed by all N. meningitidis and Neisseria polysaccharea strains examined but not by Neisseria gonorrhoeae or Neisseria lactamica, although sequences with homology to tspA were present in their genomes. In summary, TspA is a highly conserved antigen that is required for optimal adhesion of meningococci to human cells.

SummaryMeningococcal secreted proteins (MSPs) have been poorly characterized. We hypothesized that MSPs play essential roles in host-bacterial interactions and in the pathogenesis of disease. In order to test this, we examined differential host gene expression in human meningeal-derived cells, in response to endotoxin-depleted MSPs compared to live bacteria. Using expression arrays, upregulated expression of several pro-inflammatory and apoptosis-related genes was found to be induced by MSPs. The transcription and translation of representative genes was confirmed by using various methods. Increased interleukin 8 (IL-8) and cyclooxygenase 2 (COX-2) gene transcription was confirmed using real-time PCR. Upregulated IL-8, IL-6, ICAM-1 and COX-2 protein expression were confirmed by ELISA, flow cytometry or Western immunoblots. Furthermore, exposure of cells to MSPs or live meningococci induced a small significant resistance effect to staurosporine-induced apoptosis. Secreted meningococcal virulence factors are therefore important in inducing host inflammatory responses and resistance to apoptosis, and they are worthy of extensive investigation.

Bovine tuberculosis (BTB), caused by Mycobacterium bovis, continues to pose a threat to livestock worldwide and, as a zoonotic infection, also has serious implications for human health. The implementation of comprehensive surveillance programmes to detect BTB has been successful in reducing the incidence of infection in many countries, yet BTB has remained recalcitrant to eradication in several EU states, particularly in Ireland and the UK. There are well-recognized limitations in the use of the current diagnostics to detect all infected animals and this has led to renewed efforts to uncover novel diagnostic biomarkers that may serve to enhance the performance of the tests. Studies of single immunological parameters have so far been unable to unlock the complexities of the immune response to mycobacterial infection. However, the development of high-throughput methods including pan-genomic gene expression technologies such as DNA microarrays has facilitated the simultaneous identification and analysis of thousands of genes and their interactions during the immune response. In addition, the application of these new genomic technologies to BTB has identified pathogen-associated immune response signatures of host infection. The objective of these investigations is to understand the changing profile of immune responses throughout the course of infection and to identify biomarkers for sensitive diagnosis, particularly during the early stages of infection. Transcriptional profiling via microarray and more recently via next-generation sequencing technologies may lead to the development of specific and sensitive diagnostics for M. bovis infection and will enhance the prospect of eradication of tuberculosis from cattle populations.