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ObjectivesImproved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response ofMycobacterium bovis-infected animals after bovine purified protein derivative (PPD-b) stimulation of whole blood—an important component of current bTB diagnostics—will provide new information for development of better diagnostics.MethodsRNA sequencing (RNA-seq) was used to study the peripheral blood transcriptome after stimulation with PPD-b across four time points (-1 wk pre-infection, and +1 wk, +2 wk, and +10 wk post-infection) from a 14-weekM. bovisinfection time course experiment with ten age-matched Holstein-Friesian cattle.ResultsIn vitro PPD-b stimulation of peripheral blood fromM. bovis-infected and non-infected cattle elicited a strong transcriptional response. Comparison of PPD-b stimulated, and unstimulated samples revealed higher expression of genes encoding cytokine receptors, transcription factors, and interferon-inducible proteins. Lower expression was seen for genes encoding proteins involved in antimicrobial activity, C-type lectin receptors, inhibition of signal transduction, and genes encoding metal ion transporters.ConclusionsA transcriptional signature associated with the peripheral blood response to PPD-b stimulation consisting of 170 genes was identified exclusively in the post-infection time points. Therefore, this represents a panel of potential biomarkers ofM. bovisinfection.

Tuberculosis exacts a terrible toll on human and animal health. WhileMycobacterium tuberculosis(Mtb) is restricted to humans,Mycobacterium bovis(Mb) is present in a large range of mammalian hosts. In cattle, bovine TB (bTB) is a notifiable disease responsible for important economic losses in developed countries and underestimated zoonosis in the developing world. Early interactions that take place between mycobacteria and the lung tissue early after aerosol infection govern the outcome of the disease. In cattle, these early steps remain poorly characterized. The precision-cut lung slice (PCLS) model preserves the structure and cell diversity of the lung. We developed this model in cattle in order to study the early lung response to mycobacterial infection.In situimaging of PCLS infected with fluorescent Mb revealed bacilli in the alveolar compartment, adjacent or inside alveolar macrophages (AMPs) and in close contact with pneumocytes. We analyzed the global transcriptional lung inflammation signature following infection of PCLS with Mb and Mtb in two French beef breeds: Blonde d’Aquitaine and Charolaise. Whereas lungs from the Blonde d’Aquitaine produced high levels of mediators of neutrophil and monocyte recruitment in response to infection, such signatures were not observed in the Charolaise in our study. In the Blonde d’Aquitaine lung, whereas the inflammatory response was highly induced by two Mb strains, AF2122 isolated from cattle in the UK and Mb3601 circulating in France, the response against two Mtb strains, H37Rv the reference laboratory strain and BTB1558 isolated from zebu in Ethiopia, was very low. Strikingly, the type I interferon pathway was only induced by Mb but not Mtb strains indicating that this pathway may be involved in mycobacterial virulence and host tropism. Hence, the PCLS model in cattle is a valuable tool to deepen our understanding of early interactions between lung host cells and mycobacteria. It revealed striking differences between cattle breeds and mycobacterial strains. This model could help deciphering biomarkers of resistanceversussusceptibility to bTB in cattle as such information is still critically needed for bovine genetic selection programs and would greatly help the global effort to eradicate bTB.

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. However, the host and pathogen determinants driving host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed both blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated cells (MNCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multi-nucleation. Importantly, we extend our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNCs. Our findings implicate MNC formation in the contrasting pathology between Mtb and Mbv for the bovine host, and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.

Current evolutionary scenarios posit the emergence ofMycobacterium tuberculosis, the deadliest bacterial pathogen for humans globally, from an environmental saprophyte through a cumulative process of genome adaptation.Mycobacterium riyadhenseis a novel non-tuberculous mycobacterium (NTM) that is being increasingly isolated from human clinical cases with tuberculosis (TB)-like symptoms in various parts of the world. We provide evidence here thatM. riyadhenseis likely a ‘missing link’ in our understanding of the evolution ofM. tuberculosis. To elucidate the genomic hallmarks that define the evolutionary relationship betweenM. riyadhenseand other mycobacterial species, including members of theMycobacterium tuberculosiscomplex (MTBC), eight clinical isolates ofM. riyadhensewere sequenced and analyzed. We show, among other features, thatM. riyadhenseshares a large number of conserved orthologues with the MTBC; contains linear and circular plasmids carrying type IV and type VII secretion systems; and shows expansion of toxin/anti-toxin pairs. We conclude thatM. riyadhenseis an emerging mycobacterial pathogen that shares a common ancestor with members of the MTBC and that can serve as an experimental model to study the evolution and pathogenesis of tubercle bacilli.Author summaryMycobacterium tuberculosisis one of the most prolific infectious killers in humans and is a member of theMycobacterium tuberculosiscomplex (MTBC) - a group of genetically related pathogens that cause tuberculosis (TB) in mammalian species. It is postulated that MTBC has evolved from a free-living environmental ancestor into an obligate pathogen. In this evolutionary context, a comprehensive understanding of the genomic hallmarks of the free-living environmental ancestors of the MTBC is of particular scientific interest for better understanding of the evolution of the MTBC.Mycobacterium riyadhenseis a novel environmental mycobacterium, first isolated in 2009 in a hospital in Riyadh, that is increasingly being isolated from clinical cases with typical tuberculosis (TB)-like symptoms in humans. In this study, we report the characterization of eight clinical isolates ofM. riyadhense, compare their genomes to members of the MTBC, and provide a comprehensive insight into the adaptive changes associated with the evolution of the MTBC from environmental mycobacteria. We show thatM. riyadhenseis one of the closest known environmental mycobacteria related to the MTBC, and we provide several lines of molecular evidence thatM. riyadhenseis likely the ‘missing link’ in the evolution ofM. tuberculosis. It shares a common ancestor with members of the MTBC that have evolved through a process of genome reduction, expansion of toxin/antitoxin (T/A) gene systems, and ultimately host adaptation.

BackgroundBovine tuberculosis is caused by infection withMycobacterium bovis, which can also cause disease in a range of other mammals, including humans. Alveolar macrophages are the key immune effector cells that first encounterM. bovisand how the macrophage epigenome responds to mycobacterial pathogens is currently not well understood.ResultsHere, we have used chromatin immunoprecipitation sequencing (ChIP-seq), RNA-seq and miRNA-seq to examine the effect ofM. bovisinfection on the bovine alveolar macrophage (bAM) epigenome. We show that H3K4me3 is more prevalent, at a genome-wide level, in chromatin fromM. bovis-infected bAM compared to control non-infected bAM; this was particularly evident at the transcriptional start sites of genes that determine programmed macrophage responses to mycobacterial infection (e.g. M1/M2 macrophage polarisation). This pattern was also supported by the distribution of RNA Polymerase II (PolII) ChIP-seq results, which highlighted significantly increased transcriptional activity at genes demarcated by permissive chromatin. Identification of these genes enabled integration of high-density GWAS data, which revealed genomic regions associated with resilience to infection withM. bovisin cattle.ConclusionsThrough integration of these data, we show that bAM transcriptional reprogramming occurs through differential distribution of H3K4me3 and PolII at key immune genes. Furthermore, this subset of genes can be used to prioritise genomic variants from a relevant GWAS data set.

We report here an update to the reference genome sequence of the bovine tuberculosis bacillus Mycobacterium bovis AF2122/97, generated using an integrative multiomics approach. The update includes 42 new coding sequences (CDSs), 14 modified annotations, 26 single-nucleotide polymorphism (SNP) corrections, and disclosure that the RD900 locus, previously described as absent from the genome, is in fact present.