Pyrazinamide (PZA) plays a crucial role in first-line tuberculosis drug therapy. Unlike other antimicrobial agents, PZA is active against Mycobacterium tuberculosis only 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 found 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, exposure to 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 reveal new avenues for enhancing potency of this important drug through targeting activation of the cell envelope stress response.
Abstract The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of its virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication. Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. We discuss how our results change the picture of virulence attenuation and the power of SMRT sequencing for producing high-quality reference genomes.
Abstract Point mutations in the rrs gene and eis promoter are known to confer resistance to second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer a majority of SLID-resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1184 clinical M. 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, 270 AMK-R, CAP-R, and KAN-R isolates, 264 (81.2%), 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 111 of the phenotypically resistant isolates. A gene-wise method identified three genes and promoters with mutations that, on aggregate, associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 promoter mutations with KAN resistance, supporting clinical relevance for the previously demonstrated role of whiB7 overexpression in KAN resistance. We also provide evidence for the novel association of ppe51 (a gene previously associated with various antimicrobial compounds) with AMK resistance, and for the novel association of thrB with AMK and CAP resistance. The use of gene-wise association can provide additional insight, and therefore is recommended for identification of rare mechanisms of resistance when individual mutations carry insufficient statistical power.
The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication.Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome.Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.
ABSTRACT Whole genome sequencing (WGS) is fundamental to M. tuberculosis basic research and many clinical applications. Coverage across Illumina-sequenced M. tuberculosis genomes is known to vary with sequence context, but this bias is poorly characterized. Here, through a novel application of phylogenomics that distinguishes genuine coverage bias from deletions, we discern Illumina “blind spots” in the M. tuberculosis reference genome for seven sequencing workflows. We find blind spots to be widespread, affecting 529 genes, and provide their exact coordinates, enabling salvage of unaffected regions. Fifty-seven PE/PPE genes (the primary families assumed to exhibit Illumina bias) lack blind spots entirely, while remaining PE/PPE genes account for 55.1% of blind spots. Surprisingly, we find coverage bias persists in homopolymers as short as 6 bp, shorter tracts than previously reported. While GC-rich regions challenge all Illumina sequencing workflows, a modified Nextera library preparation that amplifies DNA with a high-fidelity polymerase markedly attenuates coverage bias in GC-rich and homopolymeric sequences, expanding the “Illumina-sequencable” genome. Through these findings, and by defining workflow-specific exclusion criteria, we spotlight effective strategies for handling bias in M. tuberculosis Illumina WGS. This empirical analysis framework may be used to systematically evaluate coverage bias in other species using existing sequencing data.
Whole-genome sequencing (WGS) is fundamental to Mycobacterium tuberculosis basic research and many clinical applications. Coverage across Illumina-sequenced M. tuberculosis genomes is known to vary with sequence context, but this bias is poorly characterized. Here, through a novel application of phylogenomics that distinguishes genuine coverage bias from deletions, we discern Illumina 'blind spots' in the M. tuberculosis reference genome for seven sequencing workflows. We find blind spots to be widespread, affecting 529 genes, and provide their exact coordinates, enabling salvage of unaffected regions. Fifty-seven pe/ppe genes (the primary families assumed to exhibit Illumina bias) lack blind spots entirely, while the remaining pe/ppe genes account for 55.1 % of blind spots. Surprisingly, we find coverage bias persists in homopolymers as short as 6 bp, shorter tracts than previously reported. While G+C-rich regions challenge all Illumina sequencing workflows, a modified Nextera library preparation that amplifies DNA with a high-fidelity polymerase markedly attenuates coverage bias in G+C-rich and homopolymeric sequences, expanding the 'Illumina-sequenceable' genome. Through these findings, and by defining workflow-specific exclusion criteria, we spotlight effective strategies for handling bias in M. tuberculosis Illumina WGS. This empirical analysis framework may be used to systematically evaluate coverage bias in other species using existing sequencing data.
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 underannotated proteins and developed a semiautomated 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 underannotated 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 underannotated genes altering antibiotic efficacy and 33% (23/69) required for persistence during mouse tuberculosis (TB) infection. Prospective application of the protein models enabled us to functionally interpret novel laboratory generated pyrazinamide (PZA)-resistant 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 bacterium. 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 underscore 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 and other functions key in clinical and basic tuberculosis research. More broadly, they provide a systematic framework for improving prokaryotic reference annotations.
