Drivers and sites of diversity in the DNA adenine methylomes of 93 Mycobacterium tuberculosis complex clinical isolates.

This study assembles DNA adenine methylomes for 93 Mycobacterium tuberculosis complex (MTBC) isolates from seven lineages paired with fully-annotated, finished, de novo assembled genomes. Integrative analysis yielded four key results. First, methyltransferase allele-methylome mapping corrected methyltransferase variant effects previously obscured by reference-based variant calling. Second, heterogeneity analysis of partially active methyltransferase alleles revealed that intracellular stochastic methylation generates a mosaic of methylomes within isogenic cultures, which we formalize as 'intercellular mosaic methylation' (IMM). Mutation-driven IMM was nearly ubiquitous in the globally prominent Beijing sublineage. Third, promoter methylation is widespread and associated with differential expression in the ΔhsdM transcriptome, suggesting promoter HsdM-methylation directly influences transcription. Finally, comparative and functional analyses identified 351 sites hypervariable across isolates and numerous putative regulatory interactions. This multi-omic integration revealed features of methylomic variability in clinical isolates and provides a rational basis for hypothesizing the functions of DNA adenine methylation in MTBC physiology and adaptive evolution.

Dual-Reporter Mycobacteriophages (Φ2DRMs) Reveal Preexisting Mycobacterium tuberculosis Persistent Cells in Human Sputum.

Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance to a specific bactericidal antibiotic but can survive otherwise lethal concentrations of that antibiotic. In infections with Mycobacterium tuberculosis, such persisters underlie the need for long-term antibiotic therapy and contribute to treatment failure in tuberculosis cases. Here, we demonstrate the value of dual-reporter mycobacteriophages (Φ2DRMs) for characterizing M. tuberculosis persisters. The addition of isoniazid (INH) to exponentially growing M. tuberculosis cells consistently resulted in a 2- to 3-log decrease in CFU within 4 days, and the remaining ≤1% of cells, which survived despite being INH sensitive, were INH-tolerant persisters with a distinct transcriptional profile. We fused the promoters of several genes upregulated in persisters to the red fluorescent protein tdTomato gene in Φ2GFP10, a mycobacteriophage constitutively expressing green fluorescent protein (GFP), thus generating Φ2DRMs. A population enriched in INH persisters exhibited strong red fluorescence, by microscopy and flow cytometry, using a Φ2DRM with tdTomato controlled from the dnaK promoter. Interestingly, we demonstrated that, prior to INH exposure, a population primed for persistence existed in M. tuberculosis cells from both cultures and human sputa and that this population was highly enriched following INH exposure. We conclude that Φ2DRMs provide a new tool to identify and quantitate M. tuberculosis persister cells. IMPORTANCE: Tuberculosis (TB) is again the leading cause of death from a single infectious disease, having surpassed HIV. The recalcitrance of the TB pandemic is largely due to the ability of the pathogen Mycobacterium tuberculosis to enter a persistent state in which it is less susceptible to antibiotics and immune effectors, necessitating lengthy treatment. It has been difficult to study persister cells, as we have lacked tools to isolate these rare cells. In this article, we describe the development of dual-reporter mycobacteriophages that encode a green fluorescent marker of viability and in which the promoters of genes we have identified as induced in the persister state are fused to a gene encoding a red fluorescent protein. We show that these tools can identify heterogeneity in a cell population that correlates with propensity to survive antibiotic treatment and that the proportions of these subpopulations change in M. tuberculosis cells within human sputum during the course of treatment.

Role for Mycobacterium tuberculosis membrane vesicles in iron acquisition.

Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host.

Keto-mycolic acid-dependent pellicle formation confers tolerance to drug-sensitive Mycobacterium tuberculosis.

ABSTRACT The chronic nature of tuberculosis (TB), its requirement of long duration of treatment, its ability to evade immune intervention, and its propensity to relapse after drug treatment is discontinued are reminiscent of other chronic, biofilm-associated bacterial diseases. Historically, Mycobacterium tuberculosis was grown as a pellicle, a biofilm-like structure, at the liquid-air interface in a variety of synthetic media. Notably, the most widely administered human vaccine, BCG, is grown as a pellicle for vaccine production. However, the molecular requirements for this growth remain ill defined. Here, we demonstrate that keto-mycolic acids (keto-MA) are essential for pellicle growth, and mutants lacking in or depleted of this MA species are unable to form a pellicle. We investigated the role of the pellicle biofilm in the reduction of antibiotic sensitivity known as drug tolerance using the pellicle-defective ΔmmaA4 mutant strain. We discovered that the ΔmmaA4 mutant, which is both pellicle defective and highly sensitive to rifampicin (RIF) under planktonic growth, when incorporated within the wild-type pellicle biofilm, was protected from the bactericidal activity of RIF. The observation that growth within the M. tuberculosis pellicle biofilm can confer drug tolerance to a drug-hypersensitive strain suggests that identifying molecular requirements for pellicle growth could lead to development of novel interventions against mycobacterial infections. Our findings also suggest that a class of drugs that can disrupt M. tuberculosis biofilm formation, when used in conjunction with conventional antibiotics, has the potential to overcome drug tolerance. IMPORTANCE Two of the most important questions in tuberculosis (TB) research are (i) how does Mycobacterium tuberculosis persist in the human host for decades in the face of an active immune response and (ii) why does it take six months and four drugs to treat uncomplicated TB. Both these aspects of M. tuberculosis biology are reminiscent of infections caused by organisms capable of forming biofilms. M. tuberculosis is capable of growing as a biofilm-like structure called the pellicle. In this study, we demonstrate that a specific cell wall component, keto-mycolic acid, is essential for pellicle growth. We also demonstrate that a strain of M. tuberculosis that is both drug sensitive and pellicle defective exhibits commensal behavior and becomes drug tolerant by becoming part of a heterogeneous pellicle, a characteristic of multispecies biofilms. These observations could have important implications for identifying novel pathways for M. tuberculosis drug tolerance and the design of new modalities to rapidly treat TB.