Methylation in mycobacterium tuberculosis is lineage specific with associated mutations present globally

DNA methylation is an epigenetic modification of the genome involved in regulating crucial cellular processes, including transcription and chromosome stability. Advances in PacBio sequencing technologies can be used to robustly reveal methylation sites. The methylome of the Mycobacterium tuberculosis complex is poorly understood but may be involved in virulence, hypoxic survival and the emergence of drug resistance. In the most extensive study to date, we characterise the methylome across the 4 major lineages of M. tuberculosis and 2 lineages of M. africanum, the leading causes of tuberculosis disease in humans. We reveal lineage-specific methylated motifs and strain-specific mutations that are abundant globally and likely to explain loss of function in the respective methyltransferases. Our work provides a set of sixteen new complete reference genomes for the Mycobacterium tuberculosis complex, including complete lineage 5 genomes. Insights into lineage-specific methylomes will further elucidate underlying biological mechanisms and other important phenotypes of the epi-genom

Functional genetic variation in pe/ppe genes contributes to diversity in Mycobacterium tuberculosis lineages and potential interactions with the human host.

INTRODUCTION: Around 10% of the coding potential of Mycobacterium tuberculosisis constituted by two poorly understood gene families, the pe and ppe loci, thought to be involved in host-pathogen interactions. Their repetitive nature and high GC content have hindered sequence analysis, leading to exclusion from whole-genome studies. Understanding the genetic diversity of pe/ppe families is essential to facilitate their potential translation into tools for tuberculosis prevention and treatment. METHODS: To investigate the genetic diversity of the 169 pe/ppe genes, we performed a sequence analysis across 73 long-read assemblies representing seven different lineages of M. tuberculosis and M. bovis BCG. Individual pe/ppe gene alignments were extracted and diversity and conservation across the different lineages studied. RESULTS: The pe/ppe genes were classified into three groups based on the level of protein sequence conservation relative to H37Rv, finding that >50% were conserved, with indels in pe_pgrs and ppe_mptr sub-families being major drivers of structural variation. Gene rearrangements, such as duplications and gene fusions, were observed between pe and pe_pgrs genes. Inter-lineage diversity revealed lineage-specific SNPs and indels. DISCUSSION: The high level of pe/ppe genes conservation, together with the lineage-specific findings, suggest their phylogenetic informativeness. However, structural variants and gene rearrangements differing from the reference were also identified, with potential implications for pathogenicity. Overall, improving our knowledge of these complex gene families may have insights into pathogenicity and inform the development of much-needed tools for tuberculosis control

Etiology of Pediatric Bacterial Meningitis Pre- and Post-PCV13 Introduction Among Children Under 5 Years Old in Lomé, Togo.

BACKGROUND: Pediatric bacterial meningitis (PBM) causes severe morbidity and mortality within Togo. Thus, as a member of the World Health Organization coordinated Invasive Bacterial Vaccine Preventable Diseases network, Togo conducts surveillance targeting Streptococcus pneumoniae (pneumococcus), Neisseria meningitidis (meningococcus), and Haemophilus influenzae, at a sentinel hospital within the capital city, Lomé, in the southernmost Maritime region. METHODS: Cerebrospinal fluid was collected from children <5 years with suspected PBM admitted to the Sylvanus Olympio Teaching Hospital. Phenotypic detection of pneumococcus, meningococcus, and H. influenzae was confirmed through microbiological techniques. Samples were shipped to the Regional Reference Laboratory to corroborate results by species-specific polymerase chain reaction. RESULTS: Overall, 3644 suspected PBM cases were reported, and 98 cases (2.7%: 98/3644) were confirmed bacterial meningitis. Pneumococcus was responsible for most infections (67.3%: 66/98), followed by H. influenzae (23.5%: 23/98) and meningococcus (9.2%: 9/98). The number of pneumococcal meningitis cases decreased by 88.1% (52/59) postvaccine introduction with 59 cases from July 2010 to June 2014 and 7 cases from July 2014 to June 2016. However, 5 cases caused by nonvaccine serotypes were observed. Fewer PBM cases caused by vaccine serotypes were observed in infants <1 year compared to children 2-5 years. CONCLUSIONS: Routine surveillance showed that PCV13 vaccination is effective in preventing pneumococcal meningitis among children <5 years of age in the Maritime region. This complements the MenAfriVac vaccination against meningococcal serogroup A to prevent meningitis outbreaks in the northern region of Togo. Continued surveillance is vital for estimating the prevalence of PBM, determining vaccine impact, and anticipating epidemics in Togo

Host-Directed Therapies for tackling Multi-Drug Resistant TB – learning from the Pasteur-Bechamp debates

Tuberculosis (TB) remains a global emergency causing an estimated 1.5 million deaths annually. For several decades the major focus of TB treatment has been on antibiotic development targeting Mycobacterium tuberculosis (M.tb). The lengthy TB treatment duration and poor treatment outcomes associated with multi-drug resistant TB (MDR-TB) are of major concern. The sparse new TB drug pipeline and widespread emergence of MDR-TB signal an urgent need for more innovative interventions to improve treatment outcomes. Building on the historical Pasteur-Bechamp debates on the role of the ‘microbe’ versus the ‘host internal milieu’ in disease causation, we make the case for parallel investments into host-directed therapies (HDTs). A range of potential HDTs are now available which require evaluation in randomized controlled clinical trials as adjunct therapies for shortening the duration of TB therapy and improving treatment outcomes for drug-susceptible TB and MDR-TB. Funder initiatives that may enable further research into HDTs are described

Towards host-directed therapies for tuberculosis

The treatment of tuberculosis is based on combinations of drugs that directly target Mycobacterium tuberculosis. A new global initiative is now focusing on a complementary approach of developing adjunct host-directed therapies. Despite the availability of effective antibiotics for tuberculosis (TB) for the past half century, it remains an important global health problem; there are ~9 million active TB cases and ~1.5 million TB-induced deaths per year (see the World Health Organization (WHO) Global Tuberculosis Report in Further information). Health services around the world face major barriers to achieving optimal outcomes from current TB treatment regimens. These barriers include: the spread of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB); complex and toxic treatment regimens for MDR-TB; HIV co-infection; pharmacokinetic interactions between TB drugs and antiretroviral drugs; relapse; permanent damage to lung and other tissues; long-term functional disability; immune reconstitution inflammatory syndrome (IRIS); and co-morbidity with non-communicable diseases such as diabetes and chronic obstructive airway diseases. Another fundamental problem is the long duration of TB drug treatment (6 months for drug-sensitive TB and at least 18 months for drug-resistant TB) to achieve a cure, owing to the presence of dormant Mycobacterium tuberculosis bacilli that are phenotypically resistant to current classes of anti-TB drugs, which can only target bacterial replication. There is therefore an urgent need for new TB treatments. However, the TB drug pipeline is thin1, 2. For the past 60 years, efforts to develop new treatments have focused on compounds and regimens that target M. tuberculosis directly. Recently, however, attention has focused on investigating a range of adjunct treatment interventions known as host-directed therapies (HDTs) that instead target the host response to infection. Here, we highlight the rationale for HDTs, the current portfolio of HDTs and their mechanisms of action, and a consortium-based approach to drive forward their evaluation in clinical trials

Host Immune Responses Differ between M. africanum- and M. tuberculosis-Infected Patients following Standard Anti-tuberculosis Treatment

Epidemiological differences exist between Mycobacterium africanum (Maf)- and Mycobacterium tuberculosis (Mtb)-infected patients, but to date, contributing host factors have not been characterised. We analysed clinical outcomes, as well as soluble markers and gene expression profiles in unstimulated, and ESAT6/CFP-10-, whole-Maf- and Mtb-stimulated blood samples of 26 Maf- and 49 Mtb-HIV-negative tuberculosis patients before, and after 2 and 6 months of anti-tuberculosis therapy. Before treatment, both groups had similar clinical parameters, but differed in few cytokines concentration and gene expression profiles. Following treatment the body mass index, skinfold thickness and chest X-ray scores showed greater improvement in the Mtb- compared to Maf-infected patients, after adjusting for age, sex and ethnicity (p = 0.02; 0.04 and 0.007, respectively). In addition, in unstimulated blood, IL-12p70, IL12A and TLR9 were significantly higher in Maf-infected patients, while IL-15, IL-8 and MIP-1α were higher in Mtb-infected patients. Overnight stimulation with ESAT-6/CFP-10 induced significantly higher levels of IFN-γ and TNF-α production, as well as gene expression of CCL4, IL1B and TLR4 in Mtb- compared to Maf-infected patients. Our study confirms differences in clinical features and immune genes expression and concentration of proteins associated with inflammatory processes between Mtb- and Maf-infected patients following anti-tuberculosis treatment These findings have public health implications for treatment regimens, and biomarkers for tuberculosis diagnosis and susceptibility

Host and pathogen factors that influence variability of Mycobacterium tuberculosis lipid body content in sputum from patients with tuberculosis: an observational study

Summary Background High proportions of Mycobacterium tuberculosis cells in sputum containing triacylglycerol-rich lipid bodies have been shown to be associated with treatment failure or relapse following antituberculous chemotherapy. Although lipid body determination is a potential biomarker for supporting clinical trial and treatment decisions, factors influencing variability in sputum frequencies of lipid body-positive (%LB+) M tuberculosis in patients are unknown. We aimed to test our hypothesis that exposure to host-generated NO and M tuberculosis strains are factors associated with differences in sputum %LB+. Methods In this observational study, we determined %LB+ frequencies before treatment by microscopy in patients with smear-positive tuberculosis from two separate prospective observational study settings (Gondar, Ethiopia, recruited between May 1, 2010, and April 30, 2011, and Fajara, The Gambia, who provided sputum samples before treatment between May 5, 2010, and Dec 22, 2011). In Ethiopia, fractional exhaled nitric oxide (FeNO) was measured as a biomarker of host NO, and M tuberculosis strain differences were determined by spoligotyping. Treatment response was assessed by percentage weight change after 7 months. In The Gambia, treatment responses were assessed as change in BMI and radiographic burden of disease after 6 months. Sputum M tuberculosis isolates were studied in vitro for their %LB+ and triacylglycerol synthase 1 (tgs1) mRNA responses to NO exposure. Propidium iodide staining was used as a measure of NO strain toxicity. Correlation between in vitro %LB+ frequencies following NO exposure and those of the same strain in sputum was examined with linear regression and Dunnett’s multiple comparison test. Findings In Ethiopia, 73 patients who were smear positive for pulmonary tuberculosis were recruited (43 [59%] were male and 30 [41%] were female). Of these, the %LB+ in the sputum of 59 patients showed linear correlation with log10 FeNO (r2=0·28; p<0·0001) and an association with strain spoligotype was suggested. Seven M tuberculosis strains from The Gambia showed different dose-responses to NO in vitro, demonstrated by changing lipid body content, tgs1 transcription, and bacterial toxicity. In sputum %LB+ frequencies correlated with in vitro %LB+ responses to NO of the corresponding isolate. In a subset of 34 patients across both cohorts, higher sputum %LB+ frequencies before treatment were associated with weaker responses to treatment than lower sputum %LB+ frequencies. Interpretation M tuberculosis strain and exposure to host-generated NO are associated with sputum %LB+. Our results support the use of M tuberculosis strain-dependent sputum %LB+ as a predictive biomarker of treatment response

Methylation in Mycobacterium tuberculosis is lineage specific with associated mutations present globally

Abstract DNA methylation is an epigenetic modification of the genome involved in regulating crucial cellular processes, including transcription and chromosome stability. Advances in PacBio sequencing technologies can be used to robustly reveal methylation sites. The methylome of the Mycobacterium tuberculosis complex is poorly understood but may be involved in virulence, hypoxic survival and the emergence of drug resistance. In the most extensive study to date, we characterise the methylome across the 4 major lineages of M. tuberculosis and 2 lineages of M. africanum, the leading causes of tuberculosis disease in humans. We reveal lineage-specific methylated motifs and strain-specific mutations that are abundant globally and likely to explain loss of function in the respective methyltransferases. Our work provides a set of sixteen new complete reference genomes for the Mycobacterium tuberculosis complex, including complete lineage 5 genomes. Insights into lineage-specific methylomes will further elucidate underlying biological mechanisms and other important phenotypes of the epi-genome

Host and pathogen factors that influence variability of Mycobacterium tuberculosis lipid body content in sputum from patients with tuberculosis: an observational study

Background High proportions of Mycobacterium tuberculosis cells in sputum containing triacylglycerol-rich lipid bodies have been shown to be associated with treatment failure or relapse following antituberculous chemotherapy. Although lipid body determination is a potential biomarker for supporting clinical trial and treatment decisions, factors influencing variability in sputum frequencies of lipid body-positive (%LB+) M tuberculosis in patients are unknown. We aimed to test our hypothesis that exposure to host-generated NO and M tuberculosis strains are factors associated with differences in sputum %LB+. Methods In this observational study, we determined %LB+ frequencies before treatment by microscopy in patients with smear-positive tuberculosis from two separate prospective observational study settings (Gondar, Ethiopia, recruited between May 1, 2010, and April 30, 2011, and Fajara, The Gambia, who provided sputum samples before treatment between May 5, 2010, and Dec 22, 2011). In Ethiopia, fractional exhaled nitric oxide (FeNO) was measured as a biomarker of host NO, and M tuberculosis strain differences were determined by spoligotyping. Treatment response was assessed by percentage weight change after 7 months. In The Gambia, treatment responses were assessed as change in BMI and radiographic burden of disease after 6 months. Sputum M tuberculosis isolates were studied in vitro for their %LB+ and triacylglycerol synthase 1 (tgs1) mRNA responses to NO exposure. Propidium iodide staining was used as a measure of NO strain toxicity. Correlation between in vitro %LB+ frequencies following NO exposure and those of the same strain in sputum was examined with linear regression and Dunnett’s multiple comparison test. Findings In Ethiopia, 73 patients who were smear positive for pulmonary tuberculosis were recruited (43 [59%] were male and 30 [41%] were female). Of these, the %LB+ in the sputum of 59 patients showed linear correlation with log10 FeNO (r2=0·28; p Interpretation M tuberculosis strain and exposure to host-generated NO are associated with sputum %LB+. Our results support the use of M tuberculosis strain-dependent sputum %LB+ as a predictive biomarker of treatment response.</p