Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in Mycobacterium tuberculosis strains with existing resistance to isoniazid and rifampin (i.e., multidrug resistance [MDR]) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotyped pncA and purported alternative PZA-R genes (panD, rpsA, and clpC1) with long-read sequencing of 19 phenotypically PZA-monoresistant isolates collected in Sweden and compared their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDRPZA-R) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter (clpC1p-138) and the N terminus of ClpC1 (ClpC1Val63Ala). Mutations have emerged in both these regions under POA selection in vitro, and the N-terminal region of ClpC1 has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1Val63Ala mutants spanned 4 Indo-Oceanic sublineages. Indo-Oceanic isolates invariably harbored ClpC1Val63Ala and were starkly overrepresented (odds ratio [OR] = 22.2, P < 0.00001) among PZA-monoresistant isolates (11/19) compared to MDRPZA-R isolates (5/80). The genetic basis of Indo-Oceanic isolates' overrepresentation in PZA-monoresistant tuberculosis (TB) remains undetermined, but substantial circumstantial evidence suggests that ClpC1Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development.
ABSTRACT Accurate and timely functional genome annotation is essential for translating basic pathogen research into clinically impactful advances. Here, through literature curation and structure-function inference, we systematically update the functional genome annotation of Mycobacterium tuberculosis virulent type strain H37Rv. First, we systematically curated annotations for 589 genes from 662 publications, including 282 gene products absent from leading databases. Second, we modeled 1,711 under-annotated proteins and developed a semi-automated pipeline that captured shared function between 400 protein models and structural matches of known function on protein data bank, including drug efflux proteins, metabolic enzymes, and virulence factors. In aggregate, these structure- and literature-derived annotations update 940/1,725 under-annotated H37Rv genes and generate hundreds of functional hypotheses. Retrospectively applying the annotation to a recent whole-genome transposon mutant screen provided missing function for 48% (13/27) of under-annotated genes altering antibiotic efficacy and 33% (23/69) required for persistence during mouse TB infection. Prospective application of the protein models enabled us to functionally interpret novel laboratory generated Pyrazinamide-resistant (PZA) mutants of unknown function, which implicated the emerging Coenzyme A depletion model of PZA action in the mutants’ PZA resistance. Our findings demonstrate the functional insight gained by integrating structural modeling and systematic literature curation, even for widely studied microorganisms. Functional annotations and protein structure models are available at https://tuberculosis.sdsu.edu/H37Rv in human- and machine-readable formats. IMPORTANCE Mycobacterium tuberculosis , the primary causative agent of tuberculosis, kills more humans than any other infectious bacteria. Yet 40% of its genome is functionally uncharacterized, leaving much about the genetic basis of its resistance to antibiotics, capacity to withstand host immunity, and basic metabolism yet undiscovered. Irregular literature curation for functional annotation contributes to this gap. We systematically curated functions from literature and structural similarity for over half of poorly characterized genes, expanding the functionally annotated Mycobacterium tuberculosis proteome. Applying this updated annotation to recent in vivo functional screens added functional information to dozens of clinically pertinent proteins described as having unknown function. Integrating the annotations with a prospective functional screen identified new mutants resistant to a first-line TB drug supporting an emerging hypothesis for its mode of action. These improvements in functional interpretation of clinically informative studies underscores the translational value of this functional knowledge. Structure-derived annotations identify hundreds of high-confidence candidates for mechanisms of antibiotic resistance, virulence factors, and basic metabolism; other functions key in clinical and basic tuberculosis research. More broadly, it provides a systematic framework for improving prokaryotic reference annotations.
Abstract Orphan DNA adenine methyltransferases (MTases) of Mycobacterium tuberculosis (Mtb) exhibit diversity across clinical isolates, but the forces driving this variation are not entirely clear. Recently, we observed several isolates exhibiting anomalous hypomethylation by Type I MTase Mycobacterial Adenine Methyltransferase C (MamC) despite a wild-type mamC genotype (‘MamC-anomalous’ isolates). Investigating this hypomethylation through multiple analyses revealed three key findings. First, heterogeneity analysis revealed intercellular mosaic methylation (IMM) in MamC-anomalous isolates. While they often exhibit phase-variable heterogeneity, this is the first report of IMM by a prokaryotic Type I MTase. Second, MamC-anomalous isolates exhibited a large, stable difference in chromosome copy number along the replication axis (a proxy for bacterial growth rate), suggesting a distinct growth phase accompanied by MamC hypomethylation. Third, MamC methylation efficiency decreased progressively with distance from origin of replication on both strands, with a marked exaggeration in MamC-anomalous isolates. In contrast, other Mtb MTases (MamA and MamB) exhibited lower methylation levels away from the origin only on the lagging strand, and without exaggeration in MamC-anomalous isolates. We conclude that, among Mtb MTases, MamC is uniquely linked to growth dynamics.
Point mutations in the rrs gene and the eis promoter are known to confer resistance to the second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer the majority of SLID resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1,184 clinical Mycobacterium tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, and 270 AMK-R, CAP-R, and KAN-R isolates, 274 (84.3%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 103 of the phenotypically resistant isolates. A genome-wide association study identified three genes and promoters with mutations that, on aggregate, were associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 5'-untranslated-region mutations with KAN resistance, supporting clinical relevance for this previously demonstrated mechanism of KAN resistance. We also provide evidence for the novel association of CAP resistance with the promoter of the Rv2680-Rv2681 operon, which encodes an exoribonuclease that may influence the binding of CAP to the ribosome. Aggregating mutations by gene can provide additional insight and therefore is recommended for identifying rare mechanisms of resistance when individual mutations carry insufficient statistical power.
ABSTRACT Pyrazinamide (PZA) plays a crucial role in first-line tuberculosis drug therapy. Unlike other antimicrobial agents, PZA is only active against Mycobacterium tuberculosis at low pH. The basis for this conditional drug susceptibility remains undefined. In this study, we utilized a genome-wide approach to interrogate potentiation of PZA action. We find that mutations in numerous genes involved in central metabolism as well as cell envelope maintenance and stress response are associated with PZA resistance. Further, we demonstrate that constitutive activation of the cell envelope stress response can drive PZA susceptibility independent of environmental pH. Consequently, treatment with peptidoglycan synthesis inhibitors, such as beta-lactams and D-cycloserine, potentiate PZA action through triggering this response. These findings illuminate a regulatory mechanism for conditional PZA susceptibility and reveals new avenues for enhancing potency of this important drug through targeting activation of the cell envelope stress response.
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.
