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Successful tuberculosis therapy requires treatment with an unwieldy multidrug combination for several months. Thus, there is a growing need to identify novel genetic vulnerabilities that can be leveraged to develop new, more effective antitubercular drugs. Consequently, recent efforts to optimize tuberculosis (TB) therapy have exploited Mycobacterium tuberculosis (Mtb) chemical genetics to identify pathways influencing antibiotic efficacy, novel mechanisms of antibiotic action, and new targets for TB drug discovery. However, the influence of the complex host environment on these interactions remains largely unknown, leaving the therapeutic potential of the identified targets unclear. In this study, we leveraged a library of conditional mutants targeting 467 essential Mtb genes to characterize the chemical-genetic interactions (CGIs) with TB drugs directly in the mouse infection model. We found that these in vivo CGIs differ significantly from those identified in vitro. Both drug-specific and drug-agnostic effects were identified, and many were preserved during treatment with a multidrug combination, suggesting numerous strategies for enhancing therapy. This work also elucidated the complex effects of pyrazinamide (PZA), a drug that relies on aspects of the infection environment for efficacy. Specifically, our work supports the importance of coenzyme A synthesis- inhibition during infection, as well as the antagonistic effect of iron limitation on PZA activity. In addition, we found that inhibition of thiamine and purine synthesis increases PZA efficacy, suggesting additional therapeutically exploitable metabolic dependencies. Our findings present a map of the unique in vivo CGIs, characterizing the mechanism of PZA activity in vivo and identifying potential targets for TB drug development., Competing Interests: Competing interests statement:The authors declare no competing interest.

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process is not characterized in MTBC ( Mycobacterium tuberculosis complex) adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100× the neutral substitution rate at [Formula: see text] frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC ( P < 0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1-dependent virulence. Our evidence supports the hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.

Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug–drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilized conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment and probe the relationship between Mtb carbon metabolism and chemical–genetic interactions (CGIs). We examined the antitubercular drugs isoniazid, rifampicin, and moxifloxacin and found that CGIs are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo–relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGIs could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of chemical–genetic–environmental interactions that can be used to optimize drug–drug interactions, as well as provide a framework for understanding in vitro correlates of in vivo efficacy.

The outcome of an encounter with Mycobacterium tuberculosis ( Mtb ) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis., Competing Interests: CS, RB, MP, BM, JL, SP, HL, MK, MB, AO, KM, KP, FB, CR, RM, BH, CL, GS, PH, TB, SE, DS, FP, MF, TI, CS No competing interests declared, (© 2022, Smith et al.)

Phage recombination systems have been instrumental in the development of gene modification technologies for bacterial pathogens. In particular, the Che9 phage RecET system has been used successfully for over 10 years for making gene knockouts and fusions in Mycobacterium tuberculosis. This "recombineering" technology typically uses linear dsDNA substrates that contain a drug-resistance marker flanked by (up to) 500 base pairs of DNA homologous to the target site. Less often employed in mycobacterial recombineering is the use of oligonucleotides, which require only the action of the RecT annealase to align oligos to ssDNA regions of the replication fork, for subsequent incorporation into the chromosome. Despite the higher frequency of such events relative to dsDNA-promoted recombineering, oligo-mediated changes generally suffer from the disadvantage of not being selectable, thus making them harder to isolate. This chapter discusses steps and methodologies that increase the frequencies of finding oligo-mediated events, including the transfer of single nucleotide polymorphisms (SNPs) to mycobacterial chromosomes, and the use of oligos in conjunction with the mycobacterial phage Bxb1 site-specific recombination system for the easy generation of knockouts, insertion, and fusions, in a protocol known as ORBIT.

CD8 T cells provide limited protection against Mycobacterium tuberculosis (Mtb) infection in the mouse model. As Mtb causes chronic infection in mice and humans, we hypothesize that Mtb impairs T cell responses as an immune evasion strategy. TB10.4 is an immunodominant antigen in people, nonhuman primates, and mice, which is encoded by the esxH gene. In C57BL/6 mice, 30-50% of pulmonary CD8 T cells recognize the TB10.44-11 epitope. However, TB10.4-specific CD8 T cells fail to recognize Mtb-infected macrophages. We speculate that Mtb elicits immunodominant CD8 T cell responses to antigens that are inefficiently presented by infected cells, thereby focusing CD8 T cells on nonprotective antigens. Here, we leverage naturally occurring polymorphisms in esxH, which frequently occur in lineage 1 strains, to test this "decoy hypothesis". Using the clinical isolate 667, which contains an EsxHA10T polymorphism, we observe a drastic change in the hierarchy of CD8 T cells. Using isogenic Erd.EsxHA10T and Erd.EsxHWT strains, we prove that this polymorphism alters the hierarchy of immunodominant CD8 T cell responses. Our data are best explained by immunodomination, a mechanism by which competition for APC leads to dominant responses suppressing subdominant responses. These results were surprising as the variant epitope can bind to H2-Kb and is recognized by TB10.4-specific CD8 T cells. The dramatic change in TB10.4-specific CD8 responses resulted from increased proteolytic degradation of A10T variant, which destroyed the TB10.44-11epitope. Importantly, this polymorphism affected T cell priming and recognition of infected cells. These data support a model in which nonprotective CD8 T cells become immunodominant and suppress subdominant responses. Thus, polymorphisms between clinical Mtb strains, and BCG or H37Rv sequence-based vaccines could lead to a mismatch between T cells that are primed by vaccines and the epitopes presented by infected cells. Reprograming host immune responses should be considered in the future design of vaccines., Competing Interests: The authors have declared that no competing interests exist.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

Two efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic "targeting oligonucleotide" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a "payload plasmid" that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for " o ligonucleotide-mediated r ecombineering followed by B xb1 i ntegrase t argeting") and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this "drag and drop" strategy by the construction of insertions or deletions in over 100 genes in Mycobacterium tuberculosis and M. smegmatis IMPORTANCE We sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use in M. smegmatis and M. tuberculosis By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1 attP site into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries an attB site and a drug selection marker, we show both formation of a chromosomal attP site and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines., (Copyright © 2018 Murphy et al.)

Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-β-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

M. tuberculosis (Mtb) survives a hostile environment within the host that is shaped in part by oxidative stress. The mechanisms used by Mtb to resist these stresses remain ill-defined because the complex combination of oxidants generated by host immunity is difficult to accurately recapitulate in vitro. We performed a genome-wide genetic interaction screen to comprehensively delineate oxidative stress resistance pathways necessary for Mtb to resist oxidation during infection. Our analysis predicted functional relationships between the superoxide-detoxifying enzyme (SodA), an integral membrane protein (DoxX), and a predicted thiol-oxidoreductase (SseA). Consistent with that, SodA, DoxX, and SseA form a membrane-associated oxidoreductase complex (MRC) that physically links radical detoxification with cytosolic thiol homeostasis. Loss of any MRC component correlated with defective recycling of mycothiol and accumulation of cellular oxidative damage. This previously uncharacterized coordination between oxygen radical detoxification and thiol homeostasis is required to overcome the oxidative environment Mtb encounters in the host., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Identification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis, especially given the increase of resistance worldwide to first- and second-line drugs. Because traditional target-based screening has largely proven unsuccessful for antibiotic discovery, we have developed a scalable platform for target identification in M. tuberculosis that is based on whole-cell screening, coupled with whole-genome sequencing of resistant mutants and recombineering to confirm. The method yields targets paired with whole-cell active compounds, which can serve as novel scaffolds for drug development, molecular tools for validation, and/or as ligands for co-crystallization. It may also reveal other information about mechanisms of action, such as activation or efflux. Using this method, we identified resistance-linked genes for eight compounds with anti-tubercular activity. Four of the genes have previously been shown to be essential: AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. AspS and Pks13 represent novel targets in protein translation and cell-wall biosynthesis. Both MmpL3 and EccB3 are involved in membrane transport. Pks13, AspS, and EccB3 represent novel candidates not targeted by existing TB drugs, and the availability of whole-cell active inhibitors greatly increases their potential for drug discovery.