Integrative and comparative genomic analyses of mammalian macrophage responses to intracellular mycobacterial pathogens.

Mycobacterium tuberculosis, the causative agent of human tuberculosis (hTB), is a close evolutionary relative of Mycobacterium bovis, which causes bovine tuberculosis (bTB), one of the most damaging infectious diseases to livestock agriculture. Previous studies have shown that the pathogenesis of bTB disease is comparable to hTB disease, and that the bovine and human alveolar macrophage (bAM and hAM, respectively) transcriptomes are extensively reprogrammed in response to infection with these intracellular mycobacterial pathogens. In this study, a multi-omics integrative approach was applied with functional genomics and GWAS data sets across the two primary hosts (Bos taurus and Homo sapiens) and both pathogens (M. bovis and M. tuberculosis). Four different experimental infection groups were used: 1) bAM infected with M. bovis, 2) bAM infected with M. tuberculosis, 3) hAM infected with M. tuberculosis, and 4) human monocyte-derived macrophages (hMDM) infected with M. tuberculosis. RNA-seq data from these experiments 24 h post-infection (24 hpi) was analysed using three computational pipelines: 1) differentially expressed genes, 2) differential gene expression interaction networks, and 3) combined pathway analysis. The results were integrated with high-resolution bovine and human GWAS data sets to detect novel quantitative trait loci (QTLs) for resistance to mycobacterial infection and resilience to disease. This revealed common and unique response macrophage pathways for both pathogens and identified 32 genes (12 bovine and 20 human) significantly enriched for SNPs associated with disease resistance, the majority of which encode key components of the NF-κB signalling pathway and that also drive formation of the granuloma.

Insight into the genetic diversity of Mycobacterium bovis isolated from cattle in Malawi.

We describe the genetic diversity and phylogenetic relationships of Mycobacterium bovis, isolated from cattle in Malawi. Deletion analysis, spoligotyping, and MIRU-VNTR typing were used to genotype the isolates. Combined with a larger dataset from neighboring countries, the overall M. bovis diversity in Southern Africa was contextualized. From the southern and northern regions of Malawi, 24 isolates were confirmed as M. bovis. We pooled data for the central region (60 isolates) from our recent publication to conceptualize the genetic and phylogenetic relationships of M. bovis in Malawi. European 1 was the dominant M. bovis clonal complex, with 10 unique spoligotype patterns, and SB0131 was ubiquitous. High genetic diversity, a low clustering rate, and many singletons, coupled with a low mutation transmission index, infer a low level of recent transmission, and suggest an endemic status of bovine tuberculosis (bTB) in Malawi. M. bovis isolates from Zambia, Mozambique, and South Africa were genetically related to Malawian isolates, whereas Tanzanian isolates were distantly related. The diversity and phylogenetic analysis suggest earlier introductions and maintenance of M. bovis by constant reinfection from reservoir animals. These findings are fundamental to understanding the source and route of infection in order to establish alternative management strategies for bTB.

Exploring virulence in Mycobacterium bovis: clues from comparative genomics and perspectives for the future.

Here we provide a summary of a plenary lecture delivered on Mycobacterium bovis, the bovine TB bacillus, at the M. bovis 2022 meeting held in Galway, Ireland, in June 2022. We focus on the analysis of genetic differences between M. bovis and the human pathogen Mycobacterium tuberculosis as a route to gain knowledge on what makes M. bovis function as an animal pathogen. We provide a brief historical background around M. bovis and comparative virulence experiments with M. tuberculosis, before moving to what we have learned from the studies of the M. bovis genome sequence. We discuss the need to translate knowledge on the molecular basis of virulence in M. bovis into improved control of bovine tuberculosis.

Lack of methoxy-mycolates characterizes the geographically restricted lineage 7 of Mycobacterium tuberculosis complex.

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

Differences in Faecal Microbiome Taxonomy, Diversity and Functional Potential in a Bovine Cohort Experimentally Challenged with Mycobacterium avium subsp. paratuberculosis (MAP).

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease in ruminants, a chronic enteritis which results in emaciation and eventual loss of the animal. Recent advances in metagenomics have allowed a more in-depth study of complex microbiomes, including that of gastrointestinal tracts, and have the potential to provide insights into consequences of the exposure of an animal to MAP or other pathogens. This study aimed to investigate taxonomic diversity and compositional changes of the faecal microbiome of cattle experimentally challenged with MAP compared to an unexposed control group. Faecal swab samples were collected from a total of 55 animals [exposed group (n = 35) and a control group (n = 20)], across three time points (months 3, 6 and 9 post-inoculation). The composition and functional potential of the faecal microbiota differed across time and between the groups (p < 0.05), with the primary differences, from both a taxonomic and functional perspective, occurring at 3 months post inoculation. These included significant differences in the relative abundance of the genera Methanobrevibacter and Bifidobacterium and also of 11 other species (4 at a higher relative abundance in the exposed group and 7 at a higher relative abundance in the control group). Correlations were made between microbiome data and immunopathology measurements and it was noted that changes in the microbial composition correlated with miRNA-155, miR-146b and IFN-É£. In summary, this study illustrates the impact of exposure to MAP on the ruminant faecal microbiome with a number of species that may have relevance in veterinary medicine for tracking exposure to MAP.

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Long term dietary vitamin D3 supplementation impacts both microbicidal and inflammatory responses to ex-vivo Mycobacterium bovis BCG challenge in dairy calves.

Vitamin D deficiency (VDD) is associated with enhanced susceptibility to multiple respiratory diseases in humans, including tuberculosis. However, the consequences of VDD for disease susceptibility in calves are unknown. Previously we developed a model to drive divergent circulating 25OHD concentrations in cattle, where animals were supplemented with vitamin D3 (vit D3) from birth to 7 months of age. Calves in the control group (Ctl) received a diet containing a standard vit D3 concentration, whereas the vit D group (VitD) received a diet with the highest vit D3 concentration allowed under EU guidelines. Here, we assessed the microbicidal activity and immunoregulatory effect of divergent 25OHD circulating levels to Mycobacterium bovis BCG challenge ex-vivo. Blood samples from Ctl and VitD calves were taken at 1-, 3- and 7-months of age. 25OHD concentrations were significantly different at 7 months (but not at 1 or 3 months) with animals from the VitD group having higher serum levels. Differences in microbicidal activity followed the same pattern, with no significant differences observed at 1 and 3 months, but at 7 months a significant increase in the percentage of bacteria killed was detected. Furthermore, analysis of the reactive oxygen species (ROS) and nitric oxide (NO) in serum showed a higher production of ROS and NO in VitD-supplemented calves. In contrast, serum concentrations of IL-1ß and IL-8 were significantly lower. A similar anti-inflammatory profile was observed after gene expression analysis, with a significant downregulation of a cluster of genes including IL1B, IL1R1, CXCL1, CXCL2, CXCL5, MMP9 and COX2 and an upregulation of CXCR1, CX3CR1 and NCF1, in VitD calves after BCG challenge relative to Ctl animals. Collectively, these results suggest that dietary vit D3 boosts antimicrobial and innate immune responses and thereby could improve host anti-mycobacterial immunity.

Vitamin D induced microbicidal activity against Mycobacterium bovis BCG is dependent on the synergistic activity of bovine peripheral blood cell populations.

A growing appreciation is emerging of the beneficial role of vitamin D for health and resistance against infectious diseases, including tuberculosis. However, research has predominantly focused on murine and human species and functional data in bovines is limited. Therefore, the objective of this study was to assess the microbicidal activity and immunoregulatory effect of the vitamin D metabolite 1,25(OH)2D3 on bovine peripheral blood leukocytes (PBL) in response to Mycobacterium bovis BCG (BCG) infection using a combination of functional assays and gene expression profiling. Blood from Holstein-Friesian bull calves with low circulating levels of 25(OH)D was stimulated with 1,25(OH)2D3 for 2 h, and then infected with M. bovis BCG. Results showed that 1,25(OH)2D3 supplementation significantly increased BCG killing by on average 16 %, although responses varied between 1 % and 38 % killing. Serial cell subset depletion was then performed on PBL prior to 1,25(OH)2D3 incubation and BCG infected as before to analyse the contribution of major cell types to mycobacterial growth control. Specific antibodies and either magnetic cell separation or density gradient centrifugation of monocytes, granulocytes, CD3+, CD4+, and CD8+ T lymphocytes were used to capture each cell subset. Results showed that depletion of granulocytes had the greatest impact on BCG growth, leading to a significant enhancement of bacterial colonies. In contrast, depletion of CD4+ or CD8+ T cells individually, or in combination (CD3+), had no impact on mycobacterial growth control. In agreement with our previous data, 1,25(OH)2D3 significantly increased bacterial killing in PBL, in monocyte depleted samples, and a similar trend was observed in the granulocyte depleted subset. In addition, specific analysis of sorted neutrophils treated with 1,25(OH)2D3 showed an enhanced microbicidal activity against both BCG and a virulent strain of M. bovis. Lastly, data showed that 1,25(OH)2D3 stimulation increased reactive oxygen species (ROS) production and the expression of genes encoding host defence peptides (HDP) and pathogen recognition receptors (PRRs), factors that play an important role in the microbicidal activity against mycobacteria. In conclusion, the vitamin D metabolite 1,25(OH)2D3 improves antimycobacterial killing in bovine PBLs via the synergistic activity of monocytes and granulocytes and enhanced activation of innate immunity.

Macrophage innate training induced by IL-4 and IL-13 activation enhances OXPHOS driven anti-mycobacterial responses.

Macrophages are a highly adaptive population of innate immune cells. Polarization with IFNγ and LPS into the 'classically activated' M1 macrophage enhances pro-inflammatory and microbicidal responses, important for eradicating bacteria such as Mycobacterium tuberculosis. By contrast, 'alternatively activated' M2 macrophages, polarized with IL-4, oppose bactericidal mechanisms and allow mycobacterial growth. These activation states are accompanied by distinct metabolic profiles, where M1 macrophages favor near exclusive use of glycolysis, whereas M2 macrophages up-regulate oxidative phosphorylation (OXPHOS). Here, we demonstrate that activation with IL-4 and IL-13 counterintuitively induces protective innate memory against mycobacterial challenge. In human and murine models, prior activation with IL-4/13 enhances pro-inflammatory cytokine secretion in response to a secondary stimulation with mycobacterial ligands. In our murine model, enhanced killing capacity is also demonstrated. Despite this switch in phenotype, IL-4/13 trained murine macrophages do not demonstrate M1-typical metabolism, instead retaining heightened use of OXPHOS. Moreover, inhibition of OXPHOS with oligomycin, 2-deoxy glucose or BPTES all impeded heightened pro-inflammatory cytokine responses from IL-4/13 trained macrophages. Lastly, this work identifies that IL-10 attenuates protective IL-4/13 training, impeding pro-inflammatory and bactericidal mechanisms. In summary, this work provides new and unexpected insight into alternative macrophage activation states in the context of mycobacterial infection.

High-resolution transcriptomics of bovine purified protein derivative-stimulated peripheral blood from cattle infected with Mycobacterium bovis across an experimental time course.

OBJECTIVES: Improved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response of Mycobacterium 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. METHODS: RNA 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-week M. bovis infection time course experiment with ten age-matched Holstein-Friesian cattle. RESULTS: In vitro PPD-b stimulation of peripheral blood from M. 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. CONCLUSIONS: A 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 of M. bovis infection.

Corrigendum: The TbD1 Locus Mediates a Hypoxia-Induced Copper Response in Mycobacterium bovis.

[This corrects the article DOI: 10.3389/fmicb.2022.817952.].

The TbD1 Locus Mediates a Hypoxia-Induced Copper Response in Mycobacterium bovis.

The Mycobacterium tuberculosis complex (MTBC) contains the causative agents of tuberculosis (TB) in mammals. The archetypal members of the MTBC, Mycobacterium tuberculosis and Mycobacterium bovis, cause human tuberculosis and bovine tuberculosis, respectively. Although M. tuberculosis and M. bovis share over 99.9% genome identity, they show distinct host adaptation for humans and animals; hence, while the molecular basis of host adaptation is encoded in their genomes, the mechanistic basis of host tropism is still unclear. Exploration of the in vitro phenotypic consequences of known genetic difference between M. bovis and M. tuberculosis offers one route to explore genotype-phenotype links that may play a role in host adaptation. The TbD1 ("Mycobacterium tuberculosis deletion 1 region") locus encompasses the mmpS6 and mmpL6 genes. TbD1 is absent in M. tuberculosis "modern" lineages (Lineages 2, 3, and 4) but present in "ancestral" M. tuberculosis (Lineages 1 and 7), Mycobacterium africanum lineages (Lineages 5 and 6), newly identified M. tuberculosis lineages (Lineages 8 and 9), and animal adapted strains, such as M. bovis. The function of TbD1 has previously been investigated in M. tuberculosis, where conflicting data has emerged on the role of TbD1 in sensitivity to oxidative stress, while the underlying mechanistic basis of such a phenotype is unclear. In this study, we aimed to shed further light on the role of the TbD1 locus by exploring its function in M. bovis. Toward this, we constructed an M. bovis TbD1 knockout (ΔTbD1) strain and conducted comparative transcriptomics to define global gene expression profiles of M. bovis wild-type (WT) and the ΔTbD1 strains under in vitro culture conditions (rolling and standing cultures). This analysis revealed differential induction of a hypoxia-driven copper response in WT and ΔTbD1 strains. In vitro phenotypic assays demonstrated that the deletion of TbD1 sensitized M. bovis to H2O2 and hypoxia-specific copper toxicity. Our study provides new information on the function of the TbD1 locus in M. bovis and its role in stress responses in the MTBC.

Evidence for local and international spread of Mycobacterium avium subspecies paratuberculosis through whole genome sequencing of isolates from the island of Ireland.

We describe application of whole genome sequencing (WGS) to a collection of 197 Mycobacterium avium subsp paratuberculosis (MAP) isolates gathered from 122 cattle herds across 27 counties of the island of Ireland. We compare WGS to MAP diversity quantified using mycobacterial interspersed random unit - variable number tandem repeats (MIRU-VNTR). While MIRU-VNTR showed only two major types, WGS could split the 197 isolates into eight major groups. We also found six isolates corresponding to INMV 13, a novel MIRU-VNTR type for Ireland. Evidence for dispersal of MAP across Ireland via cattle movement could be discerned from the data, with mixed infections present in several herds. Furthermore, comparisons of MAP WGS data from Ireland to data from Great Britain and continental Europe revealed many instances of close genetic similarity and hence evidence for international transmission of infection. BEAST MASCOT structured coalescent analyses, with relaxed and strict molecular clocks, estimated the substitution rate to be 0.10-0.13 SNPs/site/year and disclosed greater transitions per lineage per year from Europe to Ireland, indicating transmission into Ireland. Our work therefore reveals new insight into the seeding of MAP infection across Ireland, highlighting how WGS can inform policy formulation to ultimately control MAP transmission at local, national and international scales.

Metabolomic changes in Mycobacterium avium subsp. paratuberculosis (MAP) challenged Holstein-Friesian cattle highlight the role of serum amino acids as indicators of immune system activation.

INTRODUCTION: Paratuberculosis, commonly known as Johne's disease, is a chronic granulomatous infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Clinical signs, including reduced milk yields, weight loss and diarrhoea, are typically absent until 2 to 6 years post exposure. OBJECTIVES: To identify metabolomic changes profiles of MAP challenged Holstein-Friesian (HF) cattle and correlate identified metabolites to haematological and immunological parameters. METHODS: At approximately 6 weeks of age, calves (n = 9) were challenged with 3.8 × 109 cells of MAP (clinical isolate CIT003) on 2 consecutive days. Additional unchallenged calves (n = 9) formed the control group. The study used biobanked serum from cattle sampled periodically from 3- to 33-months post challenge. The assessment of sera using flow infusion electrospray high resolution mass spectrometry (FIE-HRMS) for high throughput, sensitive, non-targeted metabolite fingerprinting highlighted differences in metabolite levels between the two groups. RESULTS: In total, 25 metabolites which were differentially accumulated in MAP challenged cattle were identified, including 20 which displayed correlation to haematology parameters, particularly monocyte levels. CONCLUSION: The targeted metabolites suggest shifts in amino acid metabolism that could reflect immune system activation linked to MAP and as well as differences in phosphocholine levels which could reflect activation of the Th1 (tending towards pro-inflammatory) immune response. If verified by future work, selected metabolites could be used as biomarkers to diagnose and manage MAP infected cattle.

Prevalence of Mycobacterium bovis in milk on dairy cattle farms: An international systematic literature review and meta-analysis.

Bovine tuberculosis, caused by Mycobacterium bovis (M. bovis), is a globally distributed chronic disease of animals. The bacteria can be transmitted to humans via the consumption of unpasteurised (raw) milk, thus representing an important public health risk. To investigate the risk of zoonotic transmission of M. bovis via raw milk, this study systematically reviewed published studies to estimate the prevalence of M. bovis in on-farm bulk-tank milk (BTM) and individual cow's milk (IM) by meta-analysis. In total, 1,339 articles were identified through seven electronic databases and initially screened using titles and abstracts. The quality of 108 potentially relevant articles was assessed using full texts, and 67 articles comprising 83 studies (76 IM and 7 BTM), were included in the meta-analysis. The prevalence of M. bovis in IM and BTM was summarised according to the diagnostic test used, and the tuberculin skin test (TST) infection status of the individual cows (for IM) or herds (for BTM). Heterogeneity was quantified using the I-squared statistic. Prediction intervals (95% PIs) were also estimated. For IM, the overall prevalence was summarised at 5% (95%CI: 3%-7%). In TST positive cows, prevalence was summarised at 8% (95%CI: 4%-13%). For BTM, the overall prevalence independent of individual herd TST infection status was summarised at 5% (95%CI: 0%-21%). There was considerable heterogeneity evident among the included studies, while PIs were also wide. Inconsistency in the quality of reporting was also observed resulting in missing information, such as the TST infection status of the individual animal/herd. No study reported the number of M. bovis bacteria in test-positive milk samples. Several studies reported the detection of M. tuberculosis and M. africanum in milk. Despite international efforts to control tuberculosis, this study highlights the risk of zoonotic transmission of M. bovis via unpasteurised milk and dairy products made using raw milk.

Molecular epidemiology of Mycobacterium bovis in central parts of Malawi.

Bovine tuberculosis (bTB) is a neglected disease that affects cattle and humans. The burden of bTB is higher in developing countries as compared to industrialized countries. The reasons behind this discrepancy include the fact that bTB control measures, such as testing and slaughter of infected cattle and pasteurization of milk, are not usually practised in developing countries largely because of their high cost. To improve our understanding of bTB in developing countries, molecular typing studies are essential, in particular in terms of transmission dynamics, infection sources and knowledge of circulating strains of the principal causative agent, Mycobacterium bovis. In this study, we applied a suite of molecular typing techniques encompassing deletion analysis, spoligotyping and MIRU-VNTR to isolates recovered from samples collected during the routine post-mortem of cattle at the cold storage abattoir in Lilongwe, Malawi. Out of 63 isolates, 51 (81%) belonged to the European 1. M. bovis clonal complex. Spoligotyping identified 8 profiles, with SB0131 being the predominant type (56% of isolates). Spoligotypes SB0273 and SB0425 were identified in 14% and 13%, respectively, of the isolates. MIRU-VNTR showed a high discriminatory power of 0.959 and differentiated the 8 spoligotypes to 31 genotypes. The high diversity of M. bovis within the study area suggests the infection has been circulating in the area for a considerable period of time, likely facilitated by the lack of effective control measures. We also observed genetic similarities between isolates from Malawi (this study) to isolates described in previous studies in Zambia and Mozambique, suggesting transmission links in this region. The information provided by this study provides much needed evidence for the formulation of improved bTB control strategies.