Identification of bacterial determinants of tuberculosis infection and treatment outcomes: a phenogenomic analysis of clinical strains.

BACKGROUND: Bacterial diversity could contribute to the diversity of tuberculosis infection and treatment outcomes observed clinically, but the biological basis of this association is poorly understood. The aim of this study was to identify associations between phenogenomic variation in Mycobacterium tuberculosis and tuberculosis clinical features. METHODS: We developed a high-throughput platform to define phenotype-genotype relationships in M tuberculosis clinical isolates, which we tested on a set of 158 drug-sensitive M tuberculosis strains sampled from a large tuberculosis clinical study in Ho Chi Minh City, Viet Nam. We tagged the strains with unique genetic barcodes in multiplicate, allowing us to pool the strains for in-vitro competitive fitness assays across 16 host-relevant antibiotic and metabolic conditions. Relative fitness was quantified by deep sequencing, enumerating output barcode read counts relative to input normalised values. We performed a genome-wide association study to identify phylogenetically linked and monogenic mutations associated with the in-vitro fitness phenotypes. These genetic determinants were further associated with relevant clinical outcomes (cavitary disease and treatment failure) by calculating odds ratios (ORs) with binomial logistic regressions. We also assessed the population-level transmission of strains associated with cavitary disease and treatment failure using terminal branch length analysis of the phylogenetic data. FINDINGS: M tuberculosis clinical strains had diverse growth characteristics in host-like metabolic and drug conditions. These fitness phenotypes were highly heritable, and we identified monogenic and phylogenetically linked variants associated with the fitness phenotypes. These data enabled us to define two genetic features that were associated with clinical outcomes. First, mutations in Rv1339, a phosphodiesterase, which were associated with slow growth in glycerol, were further associated with treatment failure (OR 5·34, 95% CI 1·21-23·58, p=0·027). Second, we identified a phenotypically distinct slow-growing subclade of lineage 1 strains (L1.1.1.1) that was associated with cavitary disease (OR 2·49, 1·11-5·59, p=0·027) and treatment failure (OR 4·76, 1·53-14·78, p=0·0069), and which had shorter terminal branch lengths on the phylogenetic tree, suggesting increased transmission. INTERPRETATION: Slow growth under various antibiotic and metabolic conditions served as in-vitro intermediate phenotypes underlying the association between M tuberculosis monogenic and phylogenetically linked mutations and outcomes such as cavitary disease, treatment failure, and transmission potential. These data suggest that M tuberculosis growth regulation is an adaptive advantage for bacterial success in human populations, at least in some circumstances. These data further suggest markers for the underlying bacterial processes that contribute to these clinical outcomes. FUNDING: National Health and Medical Research Council/A∗STAR, National Institutes of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the Wellcome Trust Fellowship in Public Health and Tropical Medicine.

Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs.

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is hypothesized to play a major role in mRNA degradation because of its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the nonpathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in degradation of the transcripts encoded by at least 89% of protein-coding genes, with leadered transcripts often being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in M. smegmatis and Mycobacterium tuberculosis and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negative bacteria. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming that RNase E has a broad impact on the M. tuberculosis transcriptome.

High-throughput phenogenotyping of Mycobacteria tuberculosis clinical strains reveals bacterial determinants of treatment outcomes.

Background: Combatting the tuberculosis (TB) epidemic caused by Mycobacterium tuberculosis ( Mtb ) necessitates a better understanding of the factors contributing to patient clinical outcomes and transmission. While host and environmental factors have been evaluated, the impact of Mtb genetic background and phenotypic diversity is underexplored. Previous work has made associations between Mtb genetic lineages and some clinical and epidemiological features, but the bacterial traits underlying these connections are largely unknown. Methods: We developed a high-throughput functional genomics platform for defining genotype-phenotype relationships across a panel of Mtb clinical isolates. These phenotypic fitness profiles function as intermediate traits which can be linked to Mtb genetic variants and associated with clinical and epidemiological outcomes. We applied this approach to a collection of 158 Mtb strains from a study of Mtb transmission in Ho Chi Minh City, Vietnam. Mtb strains were genetically tagged in multiplicate, which allowed us to pool the strains and assess in vitro competitive fitness using deep sequencing across a set of 14 host-relevant antibiotic and metabolic conditions. Phylogenetic and monogenic associations with these intermediate traits were identified and then associated with clinical outcomes. Findings: Mtb clinical strains have a broad range of growth and drug response dynamics that can be clustered by their phylogenetic relationships. We identified novel monogenic associations with Mtb fitness in various metabolic and antibiotic conditions. Among these, we find that mutations in Rv1339 , a phosphodiesterase, which were identified through their association with slow growth in glycerol, are further associated with treatment failure. We also identify a previously uncharacterized subclade of Lineage 1 strains (L1.1.1.1) that is phenotypically distinguished by slow growth under most antibiotic and metabolic stress conditions in vitro . This clade is associated with cavitary disease, treatment failure, and demonstrates increased transmission potential. Interpretation: High-throughput phenogenotyping of Mtb clinical strains enabled bacterial intermediate trait identification that can provide a mechanistic link between Mtb genetic variation and patient clinical outcomes. Mtb strains associated with cavitary disease, treatment failure, and transmission potential display intermediate phenotypes distinguished by slow growth under various antibiotic and metabolic conditions. These data suggest that Mtb growth regulation is an adaptive advantage for host bacterial success in human populations, in at least some circumstances. These data further suggest markers for the underlying bacterial processes that govern these clinical outcomes. Funding: National Institutes of Allergy and Infectious Diseases: P01 AI132130 (SS, SMF); P01 AI143575 (XW, SMF); U19 AI142793 (QL, SMF); 5T32AI132120-03 (SS); 5T32AI132120-04 (SS); 5T32AI049928-17 (SS) Wellcome Trust Fellowship in Public Health and Tropical Medicine: 097124/Z/11/Z (NTTT) National Health and Medical Research Council (NHMRC)/A*STAR joint call: APP1056689 (SJD) The funding sources had no involvement in study methodology, data collection, analysis, and interpretation nor in the writing or submission of the manuscript. Research in context: Evidence before this study: We used different combinations of the words mycobacterium tuberculosis, tuberculosis, clinical strains, intermediate phenotypes, genetic barcoding, phenogenomics, cavitary disease, treatment failure, and transmission to search the PubMed database for all studies published up until January 20 th , 2022. We only considered English language publications, which biases our search. Previous work linking Mtb determinants to clinical or epidemiological data has made associations between bacterial lineage, or less frequently, genetic polymorphisms to in vitro or in vivo models of pathogenesis, transmission, and clinical outcomes such as cavitary disease, treatment failure, delayed culture conversion, and severity. Many of these studies focus on the global pandemic Lineage 2 and Lineage 4 Mtb strains due in part to a deletion in a polyketide synthase implicated in host-pathogen interactions. There are a number of Mtb GWAS studies that have led to novel genetic determinants of in vitro drug resistance and tolerance. Previous Mtb GWAS analyses with clinical outcomes did not experimentally test any predicted phenotypes of the clinical strains. Published laboratory-based studies of Mtb clinical strains involve relatively small numbers of strains, do not identify the genetic basis of relevant phenotypes, or link findings to the corresponding clinical outcomes. There are two recent studies of other pathogens that describe phenogenomic analyses. One study of 331 M. abscessus clinical strains performed one-by-one phenotyping to identify bacterial features associated with clearance of infection and another details a competition experiment utilizing three barcoded Plasmodium falciparum clinical isolates to assay antimalarial fitness and resistance. Added value of this study: We developed a functional genomics platform to perform high-throughput phenotyping of Mtb clinical strains. We then used these phenotypes as intermediate traits to identify novel bacterial genetic features associated with clinical outcomes. We leveraged this platform with a sample of 158 Mtb clinical strains from a cross sectional study of Mtb transmission in Ho Chi Minh City, Vietnam. To enable high-throughput phenotyping of large numbers of Mtb clinical isolates, we applied a DNA barcoding approach that has not been previously utilized for the high-throughput analysis of Mtb clinical strains. This approach allowed us to perform pooled competitive fitness assays, tracking strain fitness using deep sequencing. We measured the replicative fitness of the clinical strains in multiplicate under 14 metabolic and antibiotic stress condition. To our knowledge, this is the largest phenotypic screen of Mtb clinical isolates to date. We performed bacterial GWAS to delineate the Mtb genetic variants associated with each fitness phenotype, identifying monogenic associations with several conditions. We then defined Mtb phenotypic and genetic features associated with clinical outcomes. We find that a subclade of Mtb strains, defined by variants largely involved in fatty acid metabolic pathways, share a universal slow growth phenotype that is associated with cavitary disease, treatment failure and increased transmission potential in Vietnam. We also find that mutations in Rv1339 , a poorly characterized phosphodiesterase, also associate with slow growth in vitro and with treatment failure in patients. Implications of all the available evidence: Phenogenomic profiling demonstrates that Mtb strains exhibit distinct growth characteristics under metabolic and antibiotic stress conditions. These fitness profiles can serve as intermediate traits for GWAS and association with clinical outcomes. Intermediate phenotyping allows us to examine potential processes by which bacterial strain differences contribute to clinical outcomes. Our study identifies clinical strains with slow growth phenotypes under in vitro models of antibiotic and host-like metabolic conditions that are associated with adverse clinical outcomes. It is possible that the bacterial intermediate phenotypes we identified are directly related to the mechanisms of these outcomes, or they may serve as markers for the causal yet unidentified bacterial determinants. Via the intermediate phenotyping, we also discovered a surprising diversity in Mtb responses to the new anti-mycobacterial drugs that target central metabolic processes, which will be important in considering roll-out of these new agents. Our study and others that have identified Mtb determinants of TB clinical and epidemiological phenotypes should inform efforts to improve diagnostics and drug regimen design.

Arginine methylation sites on SepIVA help balance elongation and septation in Mycobacterium smegmatis.

The growth of mycobacterial cells requires successful coordination between elongation and septation. However, it is not clear which factors mediate this coordination. Here, we studied the function and post-translational modification of an essential division factor, SepIVA, in Mycobacterium smegmatis. We find that SepIVA is arginine methylated, and that alteration of its methylation sites affects both septation and polar elongation of Msmeg. Furthermore, we show that SepIVA regulates the localization of MurG and that this regulation may impact polar elongation. Finally, we map SepIVA's two regulatory functions to different ends of the protein: the N-terminus regulates elongation while the C-terminus regulates division. These results establish SepIVA as a regulator of both elongation and division and characterize a physiological role for protein arginine methylation sites for the first time in mycobacteria.

Spontaneous Control of SIV Replication Does Not Prevent T Cell Dysregulation and Bacterial Dissemination in Animals Co-Infected with M. tuberculosis.

Individuals co-infected with HIV and Mycobacterium tuberculosis (Mtb) are more likely to develop severe tuberculosis (TB) disease than HIV-naive individuals. To understand how a chronic pre-existing Simian immunodeficiency virus (SIV) infection impairs the early immune response to Mtb, we used the Mauritian cynomolgus macaque (MCM) model of SIV/Mtb co-infection. We examined the relationship between peripheral viral control and Mtb burden, Mtb dissemination, and T cell function between SIV+ spontaneous controllers, SIV+ non-controllers, and SIV-naive MCM who were challenged with a barcoded Mtb Erdman strain 6 months post-SIV infection and necropsied 6 weeks post-Mtb infection. Mycobacterial burden was highest in the SIV+ non-controllers in all assessed tissues. In lung granulomas, the frequency of TNF-α-producing CD4+ T cells was reduced in all SIV+ MCM, but IFNγ-producing CD4+ T cells were only lower in the SIV+ non-controllers. Further, while all SIV+ MCM had more PD1+ and TIGIT+ T cells in the lung granulomas relative to SIV-naive MCM, SIV+ controllers exhibited the highest frequency of cells expressing these markers. To measure the effect of SIV infection on within-host bacterial dissemination, we sequenced the molecular barcodes of Mtb present in each tissue and characterized the Mtb population complexity. While Mtb population complexity was not associated with SIV infection group, lymph nodes had increased complexity when compared with lung granulomas across all groups. These results provide evidence that SIV+ animals, independent of viral control, exhibit a dysregulated T cell immune response and enhanced dissemination of Mtb, likely contributing to the poor TB disease course across all SIV/Mtb co-infected animals. IMPORTANCE HIV and TB remain significant global health issues, despite the availability of treatments. Individuals with HIV, including those who are virally suppressed, are at an increased risk to develop and succumb to severe TB disease when compared with HIV-naive individuals. Our study aims to understand the relationship between the extent of SIV replication, mycobacterial growth, and T cell function in the tissues of co-infected Mauritian cynomolgus macaques during the first 6 weeks of Mtb infection. Here we demonstrate that increased viral replication is associated with increased bacterial burden in the tissues and impaired T cell responses, and that the immunological damage attributed to virus infection is not fully eliminated when animals spontaneously control virus replication.

CRISPR Interference Reveals That All-Trans-Retinoic Acid Promotes Macrophage Control of Mycobacterium tuberculosis by Limiting Bacterial Access to Cholesterol and Propionyl Coenzyme A.

Macrophages are a protective replicative niche for Mycobacterium tuberculosis (Mtb) but can kill the infecting bacterium when appropriately activated. To identify mechanisms of clearance, we compared levels of bacterial restriction by human macrophages after treatment with 26 compounds, including some currently in clinical trials for tuberculosis. All-trans-retinoic acid (ATRA), an active metabolite of vitamin A, drove the greatest increase in Mtb control. Bacterial clearance was transcriptionally and functionally associated with changes in macrophage cholesterol trafficking and lipid metabolism. To determine how these macrophage changes affected bacterial control, we performed the first Mtb CRISPR interference screen in an infection model, identifying Mtb genes specifically required to survive in ATRA-activated macrophages. These data showed that ATRA treatment starves Mtb of cholesterol and the downstream metabolite propionyl coenzyme A (propionyl-CoA). Supplementation with sources of propionyl-CoA, including cholesterol, abrogated the restrictive effect of ATRA. This work demonstrates that targeting the coupled metabolism of Mtb and the macrophage improves control of infection and that it is possible to genetically map the mode of bacterial death using CRISPR interference. IMPORTANCE Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, is a leading cause of death due to infectious disease. Improving the immune response to tuberculosis holds promise for fighting the disease but is limited by our lack of knowledge as to how the immune system kills M. tuberculosis. Our research identifies a potent way to make relevant immune cells more effective at fighting M. tuberculosis and then uses paired human and bacterial genomic methods to determine the mechanism of that improved bacterial clearance.

ClpX Is Essential and Activated by Single-Strand DNA Binding Protein in Mycobacteria.

The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we sought to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrated that ClpX is essential for mycobacterial growth, and to understand its essential functions, we identified ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We found an unexpected association between ClpX and proteins involved in DNA replication, and we confirm a physical association between ClpX and the essential DNA maintenance protein single-stranded-DNA binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead, SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB, which had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria.IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX's essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting that the ClpX-SSB complex may play a role in DNA replication in mycobacteria.

Correction: TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities.

[This corrects the article DOI: 10.1371/journal.ppat.1006939.].

Small RNA profiling in Mycobacterium tuberculosis identifies MrsI as necessary for an anticipatory iron sparing response.

One key to the success of Mycobacterium tuberculosis as a pathogen is its ability to reside in the hostile environment of the human macrophage. Bacteria adapt to stress through a variety of mechanisms, including the use of small regulatory RNAs (sRNAs), which posttranscriptionally regulate bacterial gene expression. However, very little is currently known about mycobacterial sRNA-mediated riboregulation. To date, mycobacterial sRNA discovery has been performed primarily in log-phase growth, and no direct interaction between any mycobacterial sRNA and its targets has been validated. Here, we performed large-scale sRNA discovery and expression profiling in M. tuberculosis during exposure to five pathogenically relevant stresses. From these data, we identified a subset of sRNAs that are highly induced in multiple stress conditions. We focused on one of these sRNAs, ncRv11846, here renamed mycobacterial regulatory sRNA in iron (MrsI). We characterized the regulon of MrsI and showed in mycobacteria that it regulates one of its targets, bfrA, through a direct binding interaction. MrsI mediates an iron-sparing response that is required for optimal survival of M. tuberculosis under iron-limiting conditions. However, MrsI is induced by multiple host-like stressors, which appear to trigger MrsI as part of an anticipatory response to impending iron deprivation in the macrophage environment.

Posttranslational modification of a histone-like protein regulates phenotypic resistance to isoniazid in mycobacteria.

There is increasing evidence that phenotypically drug-resistant bacteria may be important determinants of antibiotic treatment failure. Using high-throughput imaging, we defined distinct subpopulations of mycobacterial cells that exhibit heritable but semi-stable drug resistance. These subpopulations have distinct transcriptional signatures and growth characteristics at both bulk and single-cell levels, which are also heritable and semi-stable. We find that the mycobacterial histone-like protein HupB is required for the formation of these subpopulations. Using proteomic approaches, we further demonstrate that HupB is posttranslationally modified by lysine acetylation and lysine methylation. Mutation of a single posttranslational modification site specifically abolishes the formation of one of the drug-resistant subpopulations of cells, providing the first evidence in prokaryotes that posttranslational modification of a bacterial nucleoid-associated protein may epigenetically regulate cell state.

TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities.

Once considered a phenotypically monomorphic bacterium, there is a growing body of work demonstrating heterogeneity among Mycobacterium tuberculosis (Mtb) strains in clinically relevant characteristics, including virulence and response to antibiotics. However, the genetic and molecular basis for most phenotypic differences among Mtb strains remains unknown. To investigate the basis of strain variation in Mtb, we performed genome-wide transposon mutagenesis coupled with next-generation sequencing (TnSeq) for a panel of Mtb clinical isolates and the reference strain H37Rv to compare genetic requirements for in vitro growth across these strains. We developed an analytic approach to identify quantitative differences in genetic requirements between these genetically diverse strains, which vary in genomic structure and gene content. Using this methodology, we found differences between strains in their requirements for genes involved in fundamental cellular processes, including redox homeostasis and central carbon metabolism. Among the genes with differential requirements were katG, which encodes the activator of the first-line antitubercular agent isoniazid, and glcB, which encodes malate synthase, the target of a novel small-molecule inhibitor. Differences among strains in their requirement for katG and glcB predicted differences in their response to these antimicrobial agents. Importantly, these strain-specific differences in antibiotic response could not be predicted by genetic variants identified through whole genome sequencing or by gene expression analysis. Our results provide novel insight into the basis of variation among Mtb strains and demonstrate that TnSeq is a scalable method to predict clinically important phenotypic differences among Mtb strains.

Digitally Barcoding Mycobacterium tuberculosis Reveals In Vivo Infection Dynamics in the Macaque Model of Tuberculosis.

Infection with Mycobacterium tuberculosis causes a spectrum of outcomes; the majority of individuals contain but do not eliminate the infection, while a small subset present with primary active tuberculosis (TB) disease. This variability in infection outcomes is recapitulated at the granuloma level within each host, such that some sites of infection can be fully cleared while others progress. Understanding the spectrum of TB outcomes requires new tools to deconstruct the mechanisms underlying differences in granuloma fate. Here, we use novel genome-encoded barcodes to uniquely tag individual M. tuberculosis bacilli, enabling us to quantitatively track the trajectory of each infecting bacterium in a macaque model of TB. We also introduce a robust bioinformatics pipeline capable of identifying and counting barcode sequences within complex mixtures and at various read depths. By coupling this tagging strategy with serial positron emission tomography coregistered with computed tomography (PET/CT) imaging of lung pathology in macaques, we define a lesional map of M. tuberculosis infection dynamics. We find that there is no significant infection bottleneck, but there are significant constraints on productive bacterial trafficking out of primary granulomas. Our findings validate our barcoding approach and demonstrate its utility in probing lesion-specific biology and dissemination. This novel technology has the potential to greatly enhance our understanding of local dynamics in tuberculosis.IMPORTANCE Classically, M. tuberculosis infection was thought to result in either latent infection or active disease. More recently, the field has recognized that there is a spectrum of M. tuberculosis infection clinical outcomes. Within a single host, this spectrum is recapitulated at the granuloma level, where there can simultaneously be lesional sterilization and poorly contained disease. To better understand the lesional biology of TB infection, we digitally barcoded M. tuberculosis to quantitatively track the fate of each infecting bacterium. By combining this technology with serial PET-CT imaging, we can dynamically track both bacterial populations and granuloma trajectories. We demonstrate that there is little constraint on the bacterial population at the time of infection. However, the granuloma imposes a strong bottleneck on dissemination, and the subset of granulomas at risk of dissemination can be distinguished by physical features.

Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.

The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9Sth1), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9Sth1-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9Sth1 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.

A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis.

Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance.

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape.

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader.

The DNA replication machinery is an important target for antibiotic development in increasingly drug-resistant bacteria, including Mycobacterium tuberculosis. Although blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In Escherichia coli, the proofreading subunit of the replisome, the ɛ exonuclease, is essential for high-fidelity DNA replication; however, we find that the corresponding subunit is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase DnaE1 itself encodes an editing function that proofreads DNA replication, mediated by an intrinsic 3'-5' exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by more than 3,000-fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP domain-mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader.

RNA sequencing for transcript 5'-end mapping in mycobacteria.

Next-generation sequencing technologies facilitate the analysis of multiple important properties of the transcriptome in addition to gene expression levels. Here we describe a method for mapping RNA 5' ends in Mycobacterium tuberculosis, which allows the determination of transcriptional start sites (TSSs), comparative analysis of promoter usage under different conditions, and mapping of endoribonucleolytic processing sites. We describe in detail the procedures for constructing RNA sequencing libraries appropriate for RNA 5' end mapping using an Illumina sequencing platform. We also outline the major steps of data analysis.

The ESX system in Bacillus subtilis mediates protein secretion.

Esat-6 protein secretion systems (ESX or Ess) are required for the virulence of several human pathogens, most notably Mycobacterium tuberculosis and Staphylococcus aureus. These secretion systems are defined by a conserved FtsK/SpoIIIE family ATPase and one or more WXG100 family secreted substrates. Gene clusters coding for ESX systems have been identified amongst many organisms including the highly tractable model system, Bacillus subtilis. In this study, we demonstrate that the B. subtilis yuk/yue locus codes for a nonessential ESX secretion system. We develop a functional secretion assay to demonstrate that each of the locus gene products is specifically required for secretion of the WXG100 virulence factor homolog, YukE. We then employ an unbiased approach to search for additional secreted substrates. By quantitative profiling of culture supernatants, we find that YukE may be the sole substrate that depends on the FtsK/SpoIIIE family ATPase for secretion. We discuss potential functional implications for secretion of a unique substrate.

The mutation rate of mycobacterial repetitive unit loci in strains of M. tuberculosis from cynomolgus macaque infection.

BACKGROUND: Mycobacterial interspersed repetitive units (MIRUs) are minisatellites within the Mycobacterium tuberculosis (Mtb) genome. Copy number variation (CNV) in MIRU loci is used for epidemiological typing, making the rate of variation important for tracking the transmission of Mtb strains. In this study, we developed and assessed a whole-genome sequencing (WGS) approach to detect MIRU CNV in Mtb. We applied this methodology to a panel of Mtb strains isolated from the macaque model of tuberculosis (TB), the animal model that best mimics human disease. From these data, we have estimated the rate of MIRU variation in the host environment, providing a benchmark rate for future epidemiologic work. RESULTS: We assessed variation at the 24 MIRU loci used for typing in a set of Mtb strains isolated from infected cynomolgus macaques. We previously performed WGS of these strains and here have applied both read depth (RD) and paired-end mapping (PEM) metrics to identify putative copy number variants. To assess the relative power of these approaches, all MIRU loci were resequenced using Sanger sequencing. We detected two insertion/deletion events both of which could be identified as candidates by PEM criteria. With these data, we estimate a MIRU mutation rate of 2.70 × 10-03 (95% CI: 3.30 × 10-04- 9.80 × 10-03) per locus, per year. CONCLUSION: Our results represent the first experimental estimate of the MIRU mutation rate in Mtb. This rate is comparable to the highest previous estimates gathered from epidemiologic data and meta-analyses. Our findings allow for a more rigorous interpretation of data gathered from MIRU typing.

Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection.

Tuberculosis poses a global health emergency, which has been compounded by the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains. We used whole-genome sequencing to compare the accumulation of mutations in Mtb isolated from cynomolgus macaques with active, latent or reactivated disease. We sequenced 33 Mtb isolates from nine macaques with an average genome coverage of 93% and an average read depth of 117×. Based on the distribution of SNPs observed, we calculated the mutation rates for these disease states. We found a similar mutation rate during latency as during active disease or in a logarithmically growing culture over the same period of time. The pattern of polymorphisms suggests that the mutational burden in vivo is because of oxidative DNA damage. We show that Mtb continues to acquire mutations during disease latency, which may explain why isoniazid monotherapy for latent tuberculosis is a risk factor for the emergence of isoniazid resistance.