Comparative functional genomics and the bovine macrophage response to strains of the Mycobacterium genus

Mycobacterial infections are major causes of morbidity and mortality in cattle and are also potential zoonotic agents with implications for human health. Despite the implementation of comprehensive animal surveillance programs, many mycobacterial diseases have remained recalcitrant to eradication in several industrialized countries. Two major mycobacterial pathogens of cattle are Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis (MAP), the causative agents of bovine tuberculosis (BTB) and Johne's disease (JD), respectively. BTB is a chronic, granulomatous disease of the respiratory tract that is spread via aerosol transmission, while JD is a chronic granulomatous disease of the intestines that is transmitted via the fecal-oral route. Although these diseases exhibit differential tissue tropism and distinct complex etiologies, both M. bovis and MAP infect, reside, and replicate in host macrophages - the key host innate immune cell that encounters mycobacterial pathogens after initial exposure and mediates the subsequent immune response. The persistence of M. bovis and MAP in macrophages relies on a diverse series of immunomodulatory mechanisms, including the inhibition of phagosome maturation and apoptosis, generation of cytokine-induced necrosis enabling dissemination of infection through the host, local pathology, and ultimately shedding of the pathogen. Here, we review the bovine macrophage response to infection with M. bovis and MAP. In particular, we describe how recent advances in functional genomics are shedding light on the host macrophage-pathogen interactions that underlie different mycobacterial diseases. To illustrate this, we present new analyses of previously published bovine macrophage transcriptomics data following in vitro infection with virulent M. bovis, the attenuated vaccine strain M. bovis BCG, and MAP, and discuss our findings with respect to the differing etiologies of BTB and JD

GOexpress: an R/Bioconductor package for the identification and visualisation of robust gene ontology signatures through supervised learning of gene expression data

Background: Identification of gene expression profiles that differentiate experimental groups is critical for discovery and analysis of key molecular pathways and also for selection of robust diagnostic or prognostic biomarkers. While integration of differential expression statistics has been used to refine gene set enrichment analyses, such approaches are typically limited to single gene lists resulting from simple two-group comparisons or time-series analyses. In contrast, functional class scoring and machine learning approaches provide powerful alternative methods to leverage molecular measurements for pathway analyses, and to compare continuous and multi-level categorical factors. Results: We introduce GOexpress, a software package for scoring and summarising the capacity of gene ontology features to simultaneously classify samples from multiple experimental groups. GOexpress integrates normalised gene expression data (e.g., from microarray and RNA-seq experiments) and phenotypic information of individual samples with gene ontology annotations to derive a ranking of genes and gene ontology terms using a supervised learning approach. The default random forest algorithm allows interactions between all experimental factors, and competitive scoring of expressed genes to evaluate their relative importance in classifying predefined groups of samples. Conclusions: GOexpress enables rapid identification and visualisation of ontology-related gene panels that robustly classify groups of samples and supports both categorical (e.g., infection status, treatment) and continuous (e.g., time-series, drug concentrations) experimental factors. The use of standard Bioconductor extension packages and publicly available gene ontology annotations facilitates straightforward integration of GOexpress within existing computational biology pipelines.Department of Agriculture, Food and the MarineEuropean Commission - Seventh Framework Programme (FP7)Science Foundation IrelandUniversity College Dubli

RNA-seq transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis

Bovine tuberculosis, caused by infection with Mycobacterium bovis, is a major endemic disease affecting cattle populations worldwide, despite the implementation of stringent surveillance and control programs in many countries. The development of high-throughput functional genomics technologies, including gene expression microarrays and RNA-sequencing (RNA-seq), has enabled detailed analysis of the host transcriptome to M. bovis infection, particularly at the macrophage and peripheral blood level. In the present study, we have analyzed the peripheral blood leukocyte (PBL) transcriptome of eight natural M. bovis-infected and eight age- and sex-matched non-infected control Holstein-Friesian animals using RNA-seq. In addition, we compared gene expression profiles generated using RNA-seq with those previously generated using the high-density Affymetrix(®) GeneChip(®) Bovine Genome Array platform from the same PBL-extracted RNA. A total of 3,250 differentially expressed (DE) annotated genes were detected in the M. bovis-infected samples relative to the controls (adjusted P-value ≤0.05), with the number of genes displaying decreased relative expression (1,671) exceeding those with increased relative expression (1,579). Ingenuity(®) Systems Pathway Analysis (IPA) of all DE genes revealed enrichment for genes with immune function. Notably, transcriptional suppression was observed among several of the top-ranking canonical pathways including Leukocyte Extravasation Signaling. Comparative platform analysis demonstrated that RNA-seq detected a larger number of annotated DE genes (3,250) relative to the microarray (1,398), of which 917 genes were common to both technologies and displayed the same direction of expression. Finally, we show that RNA-seq had an increased dynamic range compared to the microarray for estimating differential gene expression

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro

BACKGROUND: Mycobacterium 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. RESULTS: A 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. CONCLUSIONS: This 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

The bovine alveolar macrophage DNA methylome is resilient to infection with Mycobacterium bovis

DNA methylation is pivotal in orchestrating gene expression patterns in various mammalian biological processes. Perturbation of the bovine alveolar macrophage (bAM) transcriptome, due to Mycobacterium bovis (M. bovis) infection, has been well documented; however, the impact of this intracellular pathogen on the bAM epigenome has not been determined. Here, whole genome bisulfite sequencing (WGBS) was used to assess the effect of M. bovis infection on the bAM DNA methylome. The methylomes of bAM infected with M. bovis were compared to those of non-infected bAM 24 hours post-infection (hpi). No differences in DNA methylation (CpG or non-CpG) were observed. Analysis of DNA methylation at proximal promoter regions uncovered >250 genes harbouring intermediately methylated (IM) promoters (average methylation of 33-66%). Gene ontology analysis, focusing on genes with low, intermediate or highly methylated promoters, revealed that genes with IM promoters were enriched for immune-related GO categories; this enrichment was not observed for genes in the high or low methylation groups. Targeted analysis of genes in the IM category confirmed the WGBS observation. This study is the first in cattle examining genome-wide DNA methylation at single nucleotide resolution in an important bovine cellular host-pathogen interaction model, providing evidence for IM promoter methylation in bAM

RNA sequencing (RNA-Seq) reveals extremely low levels of reticulocyte-derived globin gene transcripts in peripheral blood from horses (Equus caballus) and cattle (Bos taurus)

RNA-seq has emerged as an important technology for measuring gene expression in peripheral blood samples collected from humans and other vertebrate species. In particular, transcriptomics analyses of whole blood can be used to study immunobiology and develop novel biomarkers of infectious disease. However, an obstacle to these methods in many mammalian species is the presence of reticulocyte-derived globin mRNAs in large quantities, which can complicate RNA-seq library sequencing and impede detection of other mRNA transcripts. A range of supplementary procedures for targeted depletion of globin transcripts have, therefore, been developed to alleviate this problem. Here, we use comparative analyses of RNA-seq data sets generated from human, porcine, equine, and bovine peripheral blood to systematically assess the impact of globin mRNA on routine transcriptome profiling of whole blood in cattle and horses. The results of these analyses demonstrate that total RNA isolated from equine and bovine peripheral blood contains very low levels of globin mRNA transcripts, thereby negating the need for globin depletion and greatly simplifying blood-based transcriptomic studies in these two domestic species

Comparative 'omics analyses differentiate Mycobacterium tuberculosis and Mycobacterium bovis and reveal distinct macrophage responses to infection with the human and bovine tubercle bacilli

Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis in a range of mammals, including humans. A key feature of MTBC pathogens is their high degree of genetic identity yet distinct host tropism. Notably, while Mycobacterium bovis is highly virulent and pathogenic for cattle, the human pathogen M. tuberculosis is attenuated in cattle. Previous research also suggests that host preference amongst MTBC members has a basis in host innate immune responses. To explore MTBC host tropism, we present in-depth profiling of the MTBC reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv at both the global transcriptional and the translational level via RNA-sequencing and SWATH MS. Furthermore, a bovine alveolar macrophage infection time course model was used to investigate the shared and divergent host transcriptomic response to infection with M. tuberculosis H37Rv or M. bovis AF2122/97. Significant differential expression of virulence-associated pathways between the two bacilli was revealed, including the ESX-1 secretion system. A divergent transcriptional response was observed between M. tuberculosis H37Rv and M. bovis AF2122/97 infection of bovine alveolar macrophages, in particular cytosolic DNA-sensing pathways at 48 h post-infection, and highlights a distinct engagement of M. bovis with the bovine innate immune system. The work presented here therefore provides a basis for the identification of host innate immune mechanisms subverted by virulent host-adapted mycobacteria to promote their survival during the early stages of infection