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Current models of bacterial homologous recombination (HR) posit that extensive resection of a DNA double-strand break (DSB) by a multisubunit helicase-nuclease machine (e.g. RecBCD, AddAB or AdnAB) generates the requisite 3' single-strand DNA substrate for RecA-mediated strand invasion. AdnAB, the helicase-nuclease implicated in mycobacterial HR, consists of two subunits, AdnA and AdnB, each composed of an N-terminal ATPase domain and a C-terminal nuclease domain. DSB unwinding by AdnAB in vitro is stringently dependent on the ATPase activity of the 'lead' AdnB motor translocating on the 3' ssDNA strand, but not on the putative 'lagging' AdnA ATPase. Here, we queried genetically which activities of AdnAB are pertinent to its role in HR and DNA damage repair in vivo by inactivating each of the four catalytic domains. Complete nuclease-dead AdnAB enzyme can sustain recombination in vivo, as long as its AdnB motor is intact and RecO and RecR are available. We conclude that AdnAB's processive DSB unwinding activity suffices for AdnAB function in HR. Albeit not excluding the agency of a backup nuclease, our findings suggest that mycobacterial HR can proceed via DSB unwinding and protein capture of the displaced 3'-OH single strand, without a need for extensive end-resection.

RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy.

Oncolytic viral immunotherapy is an emerging treatment modality for cancer that exploits in vivo replication and other viral properties to enhance immune killing of malignant cells. The potential for horizontal transmission of native or engineered oncolytic viruses creates several unique infection control challenges. In 2015, talimogene laherparepvec (TVEC) became the first agent in this class to gain FDA approval for treatment of melanoma, and several others are being developed. Although some data on the transmissibility of TVEC are available from clinical studies, the aftermarket or real-world experience remains limited. We conducted a PUBMED-based search of the medical literature focusing on the safety and risk of TVEC transmission to close contacts including healthcare workers. The findings are summarized in this review and are intended to provide infection preventionists with practical guidance on handling issues related to administration and care of patients receiving TVEC. Additionally, we describe the current mechanism for evaluating the risk related to similar new agents entering clinical trials at our institution. Development of standarized approaches for the safe administration and precautions for ongoing care, especially in immunocompromised patients, are essential to support the broad adoption of this novel therapy.

Innate immune responses that control early Mtb infection are poorly understood, but understanding these responses may inform vaccination and immunotherapy strategies. Innate T cells that respond to conserved bacterial ligands such as mucosal-associated invariant T (MAIT) and γδ T cells are prime candidates to mediate these early innate responses but have not been examined in subjects who have been recently exposed to Mtb. We recruited a cohort living in the same household with an active tuberculosis (TB) case and examined the abundance and functional phenotypes of 3 innate T cell populations reactive to M. tuberculosis: γδ T, invariant NK T (iNKT), and MAIT cells. Both MAIT and γδ T cells from subjects with Mtb exposure display ex vivo phenotypes consistent with recent activation. However, both MAIT and γδ T cell subsets have distinct response profiles, with CD4+ MAIT and γδ T cells accumulating after infection. Examination of exposed but uninfected contacts demonstrates that resistance to initial infection is accompanied by robust MAIT cell CD25 expression and granzyme B production coupled with a depressed CD69 and IFNγ response. Finally, we demonstrate that MAIT cell abundance and function correlate with the abundance of specific gut microbes, suggesting that responses to initial infection may be modulated by the intestinal microbiome.

Tuberculosis (TB) is an ancient infectious disease of humans that has been extensively studied both clinically and experimentally. Although susceptibility to Mycobacterium tuberculosis infection is clearly influenced by factors such as nutrition, immune status, and both mycobacterial and host genetics, the variable pathogenesis of TB in infected individuals remains poorly understood.Tuberculosis (TB) is an ancient infectious disease of humans that has been extensively studied both clinically and experimentally. Although susceptibility to Mycobacterium tuberculosis infection is clearly influenced by factors such as nutrition, immune status, and both mycobacterial and host genetics, the variable pathogenesis of TB in infected individuals remains poorly understood. During the past two decades, it has become clear that the microbiota—the trillion organisms that reside at mucosal surfaces within and on the body—can exert a major influence on disease outcome through its effects on host innate and adaptive immune function and metabolism. This new recognition of the potentially pleiotropic participation of the microbiome in immune responses has raised the possibility that the microbiota may influence M. tuberculosis infection and/or disease. Similarly, treatment of TB may alter the healthy steady-state composition and function of the microbiome, possibly affecting treatment outcome in addition to other host physiological parameters. Herein, we review emerging evidence for how the microbiota may influence the transition points in the life cycle of TB infection, including (i) resistance to initial infection, (ii) initial infection to latent tuberculosis (LTBI), (iii) LTBI to reactivated disease, and (iv) treatment to cure. A major goal of this review is to frame questions to guide future scientific and clinical studies in this largely unexplored but increasingly important area of TB research.

Infection with Mycobacterium tuberculosis continues to cause substantial human mortality, in part because of the emergence of antimicrobial resistance. Antimicrobial resistance in tuberculosis is solely the result of chromosomal mutations that modify drug activators or targets, yet the mechanisms controlling the mycobacterial DNA-damage response (DDR) remain incompletely defined. Here, we identify RecA serine 207 as a multifunctional signaling hub that controls the DDR in mycobacteria. RecA S207 is phosphorylated after DNA damage, which suppresses the emergence of antibiotic resistance by selectively inhibiting the LexA coprotease function of RecA without affecting its ATPase or strand exchange functions. Additionally, RecA associates with the cytoplasmic membrane during the mycobacterial DDR, where cardiolipin can specifically inhibit the LexA coprotease function of unmodified, but not S207 phosphorylated, RecA. These findings reveal that RecA S207 controls mutagenesis and antibiotic resistance in mycobacteria through phosphorylation and cardiolipin-mediated inhibition of RecA coprotease function.

Physicians who seek to become laboratory-based physician-scientists, especially those without a PhD degree, face substantial challenges during the critical transition period between clinical and laboratory training. These challenges range from the complex process of adopting new logic structures and standards of proof to the more mundane but important challenges that accompany extending training in a new discipline when other career options are available. Discussion of these challenges can inform individual and institutional strategies to enhance entry and retention of physicians in the physician-scientist career path.

Background Peripheral blood transcriptome signatures that distinguish active pulmonary tuberculosis (TB) from control groups have been reported, but correlations of these signatures with sputum mycobacterial load are incompletely defined. Methods We assessed the performance of published TB transcriptomic signatures in Haiti, and identified transcriptomic biomarkers of TB bacterial load in sputum as measured by Xpert® MTB/RIF molecular testing. People in Port au Prince, Haiti, with untreated pulmonary TB (n = 51) formed the study cohort: 19 people with low and 32 with high sputum Mycobacterium tuberculosis load. Peripheral whole blood transcriptomes were generated using RNA sequencing. Results Twenty of the differentially expressed transcripts in TB vs. no TB were differentially expressed in people with low vs. high sputum mycobacterial loads. The difference between low and high bacterial load groups was independent of radiographic severity. In a published data set of transcriptomic response to anti-tuberculosis treatment, this 20-gene subset was more treatment-responsive at 6 months than the full active TB signature. Conclusion We identified genes whose transcript levels in the blood distinguish active TB with high vs. low M. tuberculosis loads in the sputum. These transcripts may reveal mechanisms of mycobacterial control of M. tuberculosis during active infection, as well as identifying potential biomarkers for bacterial response to anti-tuberculosis treatment.

The T cell antigen receptor (TCR) is unique in that its affinity for ligand is unknown before encounter and can vary by orders of magnitude. How the immune system regulates individual T cells that display very different reactivity to antigen remains unclear. Here we found that activated CD4(+) T cells, at the peak of clonal expansion, persistently downregulated their TCR expression in proportion to the strength of the initial antigen recognition. This programmed response increased the threshold for cytokine production and recall proliferation in a clone-specific manner and ultimately excluded clones with the highest antigen reactivity. Thus, programmed downregulation of TCR expression represents a negative feedback mechanism for constraining T cell effector function with a suitable time delay to thereby allow pathogen control while avoiding excess inflammatory damage.

Mycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation of adnAB or recO individually causes partial impairment of HR, but loss of adnAB and recO in combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNA in vitro , also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis of recF and recR in mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair. IMPORTANCE This study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284–2295, 2013, http://dx.doi.org/10.1093/nar/gks1298 ), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.

Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism.

Background Effective treatment of Mycobacterium tuberculosis (Mtb) infection requires at least 6 months of daily therapy with multiple orally administered antibiotics. Although this drug regimen is administered annually to millions worldwide, the impact of such intensive antimicrobial treatment on the host microbiome has never been formally investigated. Here, we characterized the longitudinal outcome of conventional isoniazid-rifampin-pyrazinamide (HRZ) TB drug administration on the diversity and composition of the intestinal microbiota in Mtb-infected mice by means of 16S rRNA sequencing. We also investigated the effects of each of the individual antibiotics alone and in different combinations. Results While inducing only a transient decrease in microbial diversity, HRZ treatment triggered a marked, immediate and reproducible alteration in community structure that persisted for the entire course of therapy and for at least 3 months following its cessation. Members of order Clostridiales were among the taxa that decreased in relative frequencies during treatment and family Porphyromonadaceae significantly increased post treatment. Experiments comparing monotherapy and different combination therapies identified rifampin as the major driver of the observed alterations induced by the HRZ cocktail but also revealed unexpected effects of isoniazid and pyrazinamide in certain drug pairings. Conclusions This report provides the first detailed analysis of the longitudinal changes in the intestinal microbiota due to anti-tuberculosis therapy. Importantly, many of the affected taxa have been previously shown in other systems to be associated with modifications in immunologic function. Together, our findings reveal that the antibiotics used in conventional TB treatment induce a distinct and long lasting dysbiosis. In addition, they establish a murine model for studying the potential impact of this dysbiosis on host resistance and physiology. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0286-2) contains supplementary material, which is available to authorized users.

Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks.

Bacterial antimicrobial resistance is an escalating public health threat, yet the current antimicrobial pipeline remains alarmingly depleted, making the development of new antimicrobials an urgent need. Here, we identify a novel, potent, imidazoline antimicrobial compound, SKI-356313, with bactericidal activity against Mycobacterium tuberculosis and Gram-positive cocci, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). SKI-356313 is active in murine models of Streptococcus pneumoniae and MRSA infection and is potently bactericidal for both replicating and nonreplicating M. tuberculosis. Using a combination of genetics, whole genome sequencing, and a novel target ID approach using real time imaging of core macromolecular biosynthesis, we show that SKI-356313 inhibits DNA replication and displaces the replisome from the bacterial nucleoid. These results identify a new antimicrobial scaffold with a novel mechanism of action and potential therapeutic utility against nonreplicating M. tuberculosis and antibiotic resistant Gram-positive cocci.

Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis , require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δ ku Δ ligD strain), HR (a Δ recA strain), or both (a Δ recA Δ ku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis.

Regulated intramembrane proteolysis of membrane-embedded substrates by site-2 proteases (S2Ps) is a widespread mechanism of transmembrane signal transduction in bacteria and bacterial pathogens. We previously demonstrated that the Mycobacterium tuberculosis S2P Rip1 is required for full virulence in the mouse model of infection. Rip1 controls transcription in part through proteolysis of three transmembrane anti-sigma factors, anti-SigK, -L, and -M, but there are also Rip1-dependent, SigKLM-independent pathways. To determine the contribution of the sigma factors K, L, and M to the Δ rip1 attenuation phenotype, we constructed an M. tuberculosis Δ sigK Δ sigL Δ sigM mutant and found that this strain fails to recapitulate the marked attenuation of Δ rip1 in mice. In a search for additional pathways controlled by Rip1, we demonstrated that the SigD regulon is positively regulated by the Rip1 pathway. Rip1 cleavage of transmembrane anti-SigD is required for expression of SigD target genes. In the absence of Rip1, proteolytic maturation of RsdA is impaired. These findings identify RsdA/SigD as a fourth arm of the branched pathway controlled by Rip1 in M. tuberculosis .

CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis [6]. We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed chromatin immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σA were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σA would be predominantly enriched at promoters based on work in Escherichia coli [3], however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σA. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 [5] as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole.The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48164).

Site-2 proteases (S2Ps) are intramembrane metalloproteases that cleave transmembrane substrates in all domains of life. Many S2Ps, including human S2P and Mycobacterium tuberculosis Rip1, have multiple substrates in vivo, which are often transcriptional regulators. However, S2Ps will also cleave transmembrane sequences of nonsubstrate proteins, suggesting additional specificity determinants. Many S2Ps also contain a PDZ domain, the function of which is poorly understood. Here, we identify an M. tuberculosis protein, PDZ-interacting protease regulator 1 (Ppr1), which bridges between the Rip1 PDZ domain and anti-sigma factor M (Anti-SigM), a Rip1 substrate, but not Anti-SigK or Anti-SigL, also Rip1 substrates. In vivo analyses of Ppr1 function indicate that it prevents nonspecific activation of the Rip1 pathway while coupling Rip1 cleavage of Anti-SigM, but not Anti-SigL, to site-1 proteolysis. Our results support a model of S2P substrate specificity in which a substrate-specific adapter protein tethers the S2P to its substrate while holding the protease inactive through its PDZ domain.

Postsurgical skin and soft tissue infections (SSTIs) caused by nontuberculous mycobacteria (NTM) are uncommon, indolent, difficult to treat, and often mimic pyogenic bacterial infections. Here we present 3 cases of NTM infections following placement of silicone implants for reconstructive breast surgery. These cases emphasize the importance of a high index of suspicion for NTM in patients with SSI after a prosthetic reconstruction refractory to conventional antibiotic therapy and the importance of early investigation with mycobacterial-specific diagnostics.

RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the −10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD. DOI: http://dx.doi.org/10.7554/eLife.22520.001

Bacille Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis that is used widely as a vaccine for tuberculosis and is used as an effective treatment for superficial bladder carcinoma. Despite being the most successful cancer biotherapy, its mechanism of action and response determinants remain obscure. Here, we establish a model system to analyze BCG interaction with bladder cancer cells, using it to show that these cells vary dramatically in their susceptibility to BCG infection. Unexpectedly, the uptake of BCG by bladder cancer cells occurs by macropinocytosis rather than phagocytosis. BCG entry into bladder cancer cells relied upon Rac1, Cdc42, and their effector kinase Pak1. The difference in susceptibility between BCG-permissive and -resistant bladder cancer cells was due to oncogenic activation of signaling pathways that activate macropinocytosis, with phosphoinositide 3-kinase inhibitor activation stimulating BCG uptake independently of Akt. Similarly, activated Ras strongly activated Pak1-dependent uptake of BCG. These results reveal that oncogenic activation of macropinocytosis determines BCG uptake by bladder cancer cells, implying that tumor responsiveness to BCG may be governed by the specific mutations present in the treated cancer cell. Cancer Res; 73(3); 1156–67. ©2013 AACR.

Mycobacteria have two genetically distinct pathways for the homology-directed repair of DNA double-strand breaks: homologous recombination (HR) and single-strand annealing (SSA). HR is abolished by deletion of RecA and reduced in the absence of the AdnAB helicase/nuclease. By contrast, SSA is RecA-independent and requires RecBCD. Here we examine the function of RecO in mycobacterial DNA recombination and repair. Loss of RecO elicits hypersensitivity to DNA damaging agents similar to that caused by deletion of RecA. We show that RecO participates in RecA-dependent HR in a pathway parallel to the AdnAB pathway. We also find that RecO plays a role in the RecA-independent SSA pathway. The mycobacterial RecO protein displays a zinc-dependent DNA binding activity in vitro and accelerates the annealing of SSB-coated single-stranded DNA. These findings establish a role for RecO in two pathways of mycobacterial DNA double-strand break repair and suggest an in vivo function for the DNA annealing activity of RecO proteins, thereby underscoring their similarity to eukaryal Rad52.

During host infection, Mycobacterium tuberculosis (Mtb) encounters several types of stress that impair protein integrity, including reactive oxygen and nitrogen species and chemotherapy. The resulting protein aggregates can be resolved or degraded by molecular machinery conserved from bacteria to eukaryotes. Eukaryotic Hsp104/Hsp70 and their bacterial homologs ClpB/DnaK are ATP-powered chaperones that restore toxic protein aggregates to a native folded state. DnaK is essential in Mycobacterium smegmatis, and ClpB is involved in asymmetrically distributing damaged proteins during cell division as a mechanism of survival in Mtb, commending both proteins as potential drug targets. However, their molecular partners in protein reactivation have not been characterized in mycobacteria. Here, we reconstituted the activities of the Mtb ClpB/DnaK bichaperone system with the cofactors DnaJ1, DnaJ2, and GrpE and the small heat shock protein Hsp20. We found that DnaJ1 and DnaJ2 activate the ATPase activity of DnaK differently. A point mutation in the highly conserved HPD motif of the DnaJ proteins abrogates their ability to activate DnaK, although the DnaJ2 mutant still binds to DnaK. The purified Mtb ClpB/DnaK system reactivated a heat-denatured model substrate, but the DnaJ HPD mutants inhibited the reaction. Finally, either DnaJ1 or DnaJ2 is required for mycobacterial viability, as is the DnaK-activating activity of a DnaJ protein. These studies lay the groundwork for strategies to target essential chaperone-protein interactions in Mtb, the leading cause of death from a bacterial infection.

Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3'-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotides and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologues of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both from a Mycobacterium smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5'-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ.

The tumor suppressor PTEN is a lipid phosphatase that is frequently mutated in various human cancers. PTEN suppresses tumor cell proliferation, survival, and growth mainly by inhibiting the PI3K-Akt signaling pathway through dephosphorylation of phosphatidylinositol 3,4,5-triphosphate. In addition to it role in tumor suppression, the PTEN-PI3K pathway controls many cellular functions, some of which may be important for cellular resistance to infection. Currently, the intersection between tumorigenic signaling pathways and cellular susceptibility to infection is not well defined. In this study we report that PTEN signaling regulates infection of both noncancerous and cancerous cells by multiple intracellular mycobacterial pathogens and that pharmacological modulation of PTEN signaling can affect mycobacterial infection. We found that PTEN deficiency renders multiple types of cells hyper-susceptible to infection by Mycoplasma and Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The lipid phosphatase activity of PTEN is required for attenuating infection. Furthermore, we found mycobacterial infection activates host cell Akt phosphorylation, and pharmacological inhibition of Akt or PI3K activity reduced levels of intracellular infection. Intriguingly, inhibition of mTOR, one of the downstream components of the Akt signaling and a promising cancer therapeutic target, also lowered intracellular Bacillus Calmette-Guérin levels in mammary epithelial cancer MCF-7 cells. These findings demonstrate a critical role of PTEN-regulated pathways in pathogen infection. The relationship of PTEN-PI3K-Akt mTOR status and susceptibility to mycobacterial infection suggests that the interaction of mycobacterial pathogens with cancer cells may be influenced by genetic alterations in the tumor cells.

Opportunistic pulmonary infections are a major cause of post-transplant morbidity and mortality. Among these infections, Aspergillus is a common cause of fatal pneumonia. Owing to the precarious clinical condition of many patients who acquire invasive mold infections, clinicians often treat them on the basis of radiographic findings, such as the halo sign. However, in patients who do not respond to treatment or who have uncommon presentations, bronchoscopy or lung biopsy looking for other pathogens should be considered. This study describes two cases in which the radiographic halo signs characteristic of Aspergillus were in fact due to Legionella jordanis, a pathogen that has been culture proven only in two patients previously (both of whom had underlying lung pathology) and diagnosed by serologic evidence in several other patients. In immunocompromised patients, Legionella can present as a cavitary lesion. Thus, presumptive treatment for this organism should be considered in post-transplant patients who do not have a classic presentation for invasive fungal infection and/or who fail to respond to conventional treatment. These cases illustrate the importance of obtaining tissue cultures to differentiate among the wide variety of pathogens present in this patient population.

Tumor-specific CD8 T-cells in cancers enter a state of dysfunction characterized by the expression of inhibitory receptors and failure to produce effector cytokines and cytotoxic molecules. Here we identify the nuclear factor, Thymocyte selection-associated HMG box protein, TOX, as a master regulator of tumor-specific T-cell dysfunction. TOX is uniquely expressed in dysfunctional CD8 T-cells from mouse and human tumors but absent in functional T-cells. TOX expression is driven by continuous TCR stimulation and NFAT activity. Forced expression of TOX in functional effector T-cells was sufficient to induce a transcriptional program of dysfunction through the concerted expression of genes encoding numerous inhibitory receptors and dysfunction-associated transcription factors. Notably, TOX-deficient tumor-infiltrating T-cells did not upregulate inhibitory receptors such as PD1, LAG3, CD38, or CD39 and maintained high TCF1 expression. Surprisingly, despite their normal, “non-exhausted” phenotype, TOX-deficient T-cells failed to make effector cytokines, suggesting that loss of effector function in tumor-specific T-cells is uncoupled from inhibitory receptor expression. Furthermore, TOX-deficient T-cells failed to persist in tumors, ultimately undergoing activation-induced cell death. We propose that the TOX-induced transcriptional program of hyporesponsiveness is a physiologic negative feedback mechanism that prevents overstimulation; thus TOX is absolutely required for T-cell survival in the setting of chronic antigen stimulation as in cancers. Citation Format: Andrew C. Scott, Steven Camara, Peter Lauer, Alexandra Synder, Dmitriy Zamarin, Tyler Walther, Olivier Levy, Michael Glickman, Jonathan Kaye, Mary Philip, Andrea Schietinger. Thymocyte selection-associated HMG box protein TOX is a master regulator of tumor-specific T-cell dysfunction [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A215.

BCG, a live mycobacterium also used worldwide as a vaccine against tuberculosis, is one of the first successful immunotherapies of cancer. Despite being in clinical use for the treatment of non-muscle-invasive bladder cancer (NMIBC) for 4 decades, the mechanism by which BCG activates the immune system to destroy cancer cells is poorly understood and there are no reliable methods to predict a patient’s response to BCG treatment. Current data suggest that the efficacy of BCG depends on initial attachment of BCG to bladder cancer cells leading to activation of an immune response that is T-cell dependent. It is not known whether bladder cancer cells have a direct role in activating T-cells. Using an orthotopic mouse model of BCG treatment of bladder cancer we show that: 1) BCG induces tumor-specific immunity; 2) CD4+ T-cells are the main requirement for BCG efficacy and for the tumor-specific immunity engendered by BCG therapy;3) bladder cancer cells can directly activate CD4+ T-cells; 4) the efficacy of BCG depends on MHC class II expression on the surface of bladder cancer cells. These data suggest that bladder cancer cells directly activate and drive a CD4+ T-cell dependent immune response that is required for the efficacy of BCG. Current work in the lab is focused on characterizing MHC class II expression on human NMIBC specimens and determining whether expression of MHC class II on bladder cancer cells predicts clinical outcomes after BCG therapy. Citation Format: Gil Redelman-Sidi, Michael Glickman, Anna Binyamin, Anthony Antonelli. MHC class II on cancer cells—Role in response to BCG therapy of bladder cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B087.

Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in Escherichia coli and constitute the major pathway of error-free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via non-homologous end-joining (NHEJ) catalysed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways.

The Mycobacterium tuberculosis cell envelope contains a wide variety of lipids and glycolipids, including mycolic acids, long-chain branched fatty acids that are decorated by cyclopropane rings. Genetic analysis of the mycolate methyltransferase family has been a powerful approach to assign functions to each of these enzymes but has failed to reveal the origin of cis cyclopropanation of the oxygenated mycolates. Here we examine potential redundancy between mycolic acid methyltransferases by generating and analyzing M. tuberculosis strains lacking mmaA2 and cmaA2 , mmaA2 and cmaA1 , or mmaA1 alone. M. tuberculosis lacking both cmaA2 and mmaA2 cannot cis cyclopropanate methoxymycolates or ketomycolates, phenotypes not shared by the mmaA2 and cmaA2 single mutants. In contrast, a combined loss of cmaA1 and mmaA2 had no effect on mycolic acid modification compared to results with a loss of mmaA2 alone. Deletion of mmaA1 from M. tuberculosis abolishes trans cyclopropanation without accumulation of trans -unsaturated oxygenated mycolates, placing MmaA1 in the biosynthetic pathway for trans -cyclopropanated oxygenated mycolates before CmaA2. These results define new functions for the mycolic acid methyltransferases of M. tuberculosis and indicate a substantial redundancy of function for MmaA2 and CmaA2, the latter of which can function as both a cis and trans cyclopropane synthase for the oxygenated mycolates.

Regulated intramembrane proteolysis (RIP) is a mechanism of transmembrane signal transduction that functions through intramembrane proteolysis of substrates. We previously reported that the RIP metalloprotease Rv2869c (Rip1) is a determinant of Mycobacterium tuberculosis (Mtb) cell envelope composition and virulence, but the substrates of Rip1 were undefined. Here we show that Rip1 cleaves three transmembrane anti-sigma factors: anti-SigK, anti-SigL and anti-SigM, negative regulators of Sigma K, L and M. We show that transcriptional activation of katG in response to phenanthroline requires activation of SigK and SigL by Rip1 cleavage of anti-SigK and anti-SigL. We also demonstrate a Rip1-dependent pathway that activates the genes for the mycolic acid biosynthetic enzyme KasA and the resuscitation promoting factor RpfC, but represses the bacterioferritin encoding gene bfrB. Regulation of these three genes by Rip1 is not reproduced by deletion of Sigma K, L or M, either indicating a requirement for multiple Rip1 substrates or additional arms of the Rip1 pathway. These results identify a branched proteolytic signal transduction system in which a single intramembrane protease cleaves three anti-sigma factor substrates to control multiple downstream pathways involved in lipid biosynthesis and defence against oxidative stress.

SummaryMycobacterium tuberculosis infection remains a major global health problem complicated by escalating rates of antibiotic resistance. Despite the established role of mycolic acid cyclopropane modification in pathogenesis, the feasibility of targeting this enzyme family for antibiotic development is unknown. We show through genetics and chemical biology that mycolic acid methyltransferases are essential for M. tuberculosis viability, cell wall structure, and intrinsic resistance to antibiotics. The tool compound dioctylamine, which we show acts as a substrate mimic, directly inhibits the function of multiple mycolic acid methyltransferases, resulting in loss of cyclopropanation, cell death, loss of acid fastness, and synergistic killing with isoniazid and ciprofloxacin. These results demonstrate that mycolic acid methyltransferases are a promising antibiotic target and that a family of virulence factors can be chemically inhibited with effects not anticipated from studies of each individual enzyme.

We report here the use of novel “sloppy” molecular beacon probes in homogeneous PCR screening assays in which thermal denaturation of the resulting probe-amplicon hybrids provides a characteristic set of amplicon melting temperature (Tm) values that identify which species is present in a sample. Sloppy molecular beacons possess relatively long probe sequences, enabling them to form hybrids with amplicons from many different species despite the presence of mismatched base pairs. By using four sloppy molecular beacons, each possessing a different probe sequence and each labeled with a differently colored fluorophore, four different Tm values can be determined simultaneously. We tested this technique with 27 different species of mycobacteria and found that each species generates a unique, highly reproducible signature that is unaffected by the initial bacterial DNA concentration. Utilizing this general paradigm, screening assays can be designed for the identification of a wide range of species. A classic approach for determining the identity of a bacterial species is to employ PCR to exponentially amplify a selected segment of a 16S rRNA gene (26), utilizing a pair of “universal primers” that bind to highly conserved sequences at the ends of the target region, and to then use a sophisticated technique to identify a species-specific sequence in the middle of the resulting amplicons (12). These identification methods include nucleotide sequence analysis (27) and electrophoretic determination of the sizes of fragments produced by incubation of the amplicons with selected restriction endonucleases (17). However, these techniques are time consuming, costly, and labor intensive.

Mycobacterium tuberculosis infection induces complex CD4 T cell responses that include T helper type 1 (Th1) cells and regulatory T cells. Although Th1 cells control infection, they are unable to fully eliminate M. tuberculosis, suggesting that Th1-mediated immunity is restrained from its full sterilizing potential. Investigation into T cell–mediated defense is hindered by difficulties in expanding M. tuberculosis–specific T cells. To circumvent this problem, we cloned CD4+ T cells from M. tuberculosis–infected B6 mice and generated transgenic mice expressing a T cell receptor specific for the immunodominant antigen early secreted antigenic target 6 (ESAT-6). Adoptively transferred naive ESAT-6–specific CD4+ T cells are activated in pulmonary lymph nodes between 7 and 10 d after aerosol infection and undergo robust expansion before trafficking to the lung. Adoptive transfer of activated ESAT-6–specific Th1 cells into naive recipients before aerosol M. tuberculosis infection dramatically enhances resistance, resulting in 100-fold fewer bacteria in infected lungs. However, despite large numbers of Th1 cells in the lungs of mice at the time of M. tuberculosis challenge, protection was not manifested until after 7 d following infection. Our results demonstrate that pathogen-specific Th1 cells can provide protection against inhaled M. tuberculosis, but only after the first week of infection.

Mycobacteria can repair DNA double-strand breaks (DSBs) via a nonhomologous end-joining (NHEJ) system that includes a dedicated DNA ligase (LigD) and the DNA end-binding protein Ku. Here we exploit an improved plasmid-based NHEJ assay and a collection of Mycobacterium smegmatis strains bearing deletions or mutations in Ku or the DNA ligases to interrogate the contributions of LigD’s three catalytic activities (polymerase, ligase, and 3′ phosphoesterase) and structural domains (POL, LIG, and PE) to the efficiency and molecular outcomes of NHEJ in vivo. By analyzing in parallel the repair of blunt, 5′ overhang, and 3′ overhang DSBs, we discovered a novel end-joining pathway specific to breaks with 3′ overhangs that is Ku- and LigD-independent and perfectly faithful. This 3′ overhang NHEJ pathway is independent of ligases B and C; we surmise that it relies on NAD+-dependent LigA, the essential replicative ligase. We find that efficient repair of blunt and 5′ overhang DSBs depends stringently on Ku and the LigD POL domain, but not on the LigD polymerase activity, which mainly serves to promote NHEJ infidelity. The lack of an effect of PE-inactivating LigD mutations on NHEJ outcomes, especially the balance between deletions and insertions at blunt or 5′ overhang breaks, argues against LigD being the catalyst of deletion formation. Ligase-inactivating LigD mutations (or deletion of the LIG domain) have a modest impact on the efficiency of blunt and 5′ overhang DSB repair, because the strand sealing activity can be provided in trans by one of the other resident ATP-dependent ligases (likely LigC). Reliance on the backup ligase is accompanied by a drastic loss of fidelity during blunt end and 5′ overhang DSB repair. We conclude that the mechanisms of mycobacterial NHEJ are many and the outcomes depend on the initial structures of the DSBs and the available ensemble of end-processing and end-sealing components, which are not limited to Ku and LigD.

The capacity to rectify DNA double-strand breaks (DSBs) is crucial for the survival of all species. DSBs can be repaired either by homologous recombination (HR) or non-homologous end joining (NHEJ). The long-standing notion that bacteria rely solely on HR for DSB repair has been overturned by evidence that mycobacteria and other genera have an NHEJ system that depends on a dedicated DNA ligase, LigD, and the DNA-end-binding protein Ku. Recent studies have illuminated the role of NHEJ in protecting the bacterial chromosome against DSBs and other clastogenic stresses. There is also emerging evidence of functional crosstalk between bacterial NHEJ proteins and components of other DNA-repair pathways. Although still a young field, bacterial NHEJ promises to teach us a great deal about the nexus of DNA repair and bacterial pathogenesis.

Mycobacterium tuberculosis and other bacterial pathogens have a Ku-dependent nonhomologous end joining pathway of DNA double-strand break repair. Here we identify mycobacterial UvrD1 as a novel interaction partner for Ku in a genome-wide yeast two-hybrid screen. UvrD1 per se is a vigorous DNA-dependent ATPase but a feeble DNA helicase. Ku stimulates UvrD1 to catalyze ATP-dependent unwinding of 3'-tailed DNAs. UvrD1, Ku, and DNA form a stable ternary complex in the absence of ATP. The Ku binding determinants are located in the distinctive C-terminal segment of UvrD1. A second mycobacterial paralog, UvrD2, is a vigorous Ku-independent DNA helicase. Ablation of UvrD1 sensitizes Mycobacterium smegmatis to killing by ultraviolet and ionizing radiation and to a single chromosomal break generated by I-SceI endonuclease. The physical and functional interactions of bacterial Ku and UvrD1 highlight the potential for cross-talk between components of nonhomologous end joining and nucleotide excision repair pathways.

Regulated intramembrane proteolysis (RIP) is a widely distributed mechanism of signal transduction in which membrane-bound proteases cleave transmembrane domains of substrate proteins. The site-2 protease (S2P) class of RIP metalloproteases is present in most bacterial genomes but is generally of unknown function except for the well-characterized proteases RseP and SpoIVFB. In this review we will discuss the biochemical functions and physiologic roles of S2P proteases in bacteria and highlight recent data implicating S2P family members in host-pathogen interactions.

DNA ligase D (LigD) is a large polyfunctional enzyme involved in nonhomologous end-joining (NHEJ) in mycobacteria. LigD consists of a C-terminal ATP-dependent ligase domain fused to upstream polymerase and phosphoesterase modules. Here we report the 2.4 angstroms crystal structure of the ligase domain of Mycobacterium LigD, captured as the covalent ligase-AMP intermediate with a divalent metal in the active site. A chloride anion on the protein surface coordinated by the ribose 3'-OH and caged by arginine and lysine side chains is a putative mimetic of the 5'-phosphate at a DNA nick. Structure-guided mutational analysis revealed distinct requirements for the adenylylation and end-sealing reactions catalyzed by LigD. We found that a mutation of Mycobacterium LigD that ablates only ligase activity results in decreased fidelity of NHEJ in vivo and a strong bias of mutagenic events toward deletions instead of insertions at the sealed DNA ends. This phenotype contrasts with the increased fidelity of double-strand break repair in deltaligD cells or in a strain in which only the polymerase function of LigD is defective. We surmise that the signature error-prone quality of bacterial NHEJ in vivo arises from a dynamic balance between the end-remodeling and end-sealing steps.

Mycobacterium tuberculosis infection is a continuing global health crisis that kills 2 million people each year. Although the structurally diverse lipids of the M. tuberculosis cell envelope each have non-redundant roles in virulence or persistence, the molecular mechanisms regulating cell envelope composition in M. tuberculosis are undefined. In higher eukaryotes, membrane composition is controlled by site two protease (S2P)-mediated cleavage of sterol regulatory element binding proteins, membrane-bound transcription factors that control lipid biosynthesis. S2P is the founding member of a widely distributed family of membrane metalloproteases that cleave substrate proteins within transmembrane segments. Here we show that a previously uncharacterized M. tuberculosis S2P homologue (Rv2869c) regulates M. tuberculosis cell envelope composition, growth in vivo and persistence in vivo. These results establish that regulated intramembrane proteolysis is a conserved mechanism controlling membrane composition in prokaryotes and show that this proteolysis is a proximal regulator of cell envelope virulence determinants in M. tuberculosis.

Mycobacterium tuberculosis (Mtb) infection remains a global health crisis. Recent genetic evidence implicates specific cell envelope lipids in Mtb pathogenesis, but it is unclear whether these cell envelope compounds affect pathogenesis through a structural role in the cell wall or as pathogenesis effectors that interact directly with host cells. Here we show that cyclopropane modification of the Mtb cell envelope glycolipid trehalose dimycolate (TDM) is critical for Mtb growth during the first week of infection in mice. In addition, TDM modification by the cyclopropane synthase pcaA was both necessary and sufficient for proinflammatory activation of macrophages during early infection. Purified TDM isolated from a cyclopropane-deficient pcaA mutant was hypoinflammatory for macrophages and induced less severe granulomatous inflammation in mice, demonstrating that the fine structure of this glycolipid was critical to its proinflammatory activity. These results established the fine structure of lipids contained in the Mtb cell envelope as direct effectors of pathogenesis and identified temporal control of host immune activation through cyclopropane modification of TDM as a critical pathogenic strategy of Mtb.

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.

Inhaled environmental pollutants, most prominently from cigarettes, confer an increased risk of tuberculosis. A recent study published in Cell by Berg et al., (2016) using the zebrafish model of Mycobacterium marinum infection provides new insights into the role of macrophage lysosomal engorgement in compromising host defense against mycobacteria.

Mycobacterium tuberculosis and Mycobacterium avium are pathogenic slow-growing mycobacteria that cause distinct human diseases. In contrast to recent advances in M. tuberculosis genetics and pathogenesis investigation, M. avium has remained genetically intractable and, consequently, its pathogenic strategies remain poorly understood. Here we report the successful development of efficient allelic exchange and transposon mutagenesis in an opaque clinical strain of M. avium by specialized transduction. Efforts to disrupt the leuD gene of M. avium by specialized transduction were successful but were complicated by inefficient isolation of recombinants secondary to high spontaneous antibiotic resistance. However, by using this leucine auxotroph as a genetic host and the Streptomyces coelicolor leuD gene as a selectable marker, we achieved efficient allelic exchange at the M. avium pcaA locus. A leuD -marked transposon delivered by specialized transduction mutagenized M. avium with efficiencies similar to M. tuberculosis . These results establish a system for random and directed mutagenesis of M. avium . In combination with the forthcoming M. avium genome sequence, these tools will allow the distinct physiologic and pathogenic properties of M. avium to be dissected in molecular detail.

Infection with Mycobacterium tuberculosis (Mtb) remains a severe global health problem that has prompted an aggressive search for new antibiotic targets and vaccine strategies for this persistent pathogen. Recently, a wide variety of genetic determinants of Mtb pathogenicity have been identified, including several genes involved in the biogenesis of the complex Mtb cell envelope. Among these are the mycolic acid cyclopropane synthases, a family of proteins that modify the major cell envelope lipids of Mtb with a diversity of cyclopropane rings. Despite substantial sequence identity, these proteins catalyze highly specific cyclopropane modifications, including proximal modification of the alpha-mycolate (pcaA) and trans-cyclopropane modification (cmaA2). Here we report the mycolic acid modification function of a third cyclopropane synthase, mmaA2, through the creation and analysis of an M. tuberculosis mmaA2 null mutant. Unexpectedly, mmaA2 is essential for the distal cyclopropane modification of the alpha-mycolate, a function previously attributed to cmaA1. alpha-Mycolates of a cmaA1 null mutant were unaffected, demonstrating that cmaA1 is not required for alpha-mycolate modification. Although fully cyclopropanated methoxymycolates are produced in the mmaA2 mutant, cis-cyclopropanation is impaired, leading to accumulation of unsaturated methoxymycolate derivatives. This study establishes mmaA2 as a distal cyclopropane synthase of the alpha-mycolates of M. tuberculosis and the first cyclopropane synthase to modify both alpha- and oxygenated mycolates. These results expand our knowledge of the biosynthesis of the Mtb cell envelope and will allow further elucidation of the relationship between Mtb pathogenesis and the fine structure of mycolic acids.

Mucosal-associated invariant T (MAIT) cells are an abundant class of innate T cells restricted by the MHC I-related molecule MR1. MAIT cells can recognize bacterially-derived metabolic intermediates from the riboflavin pathway presented by MR1 and are postulated to play a role in innate antibacterial immunity through production of cytokines and direct bacterial killing. MR1 tetramers, typically stabilized by the adduct of 5-amino-6-D-ribitylaminouracil (5-A-RU) and methylglyoxal (MeG), are important tools for the study of MAIT cells. A long-standing problem with 5-A-RU is that it is unstable upon storage. Herein we report an efficient synthetic approach to the HCl salt of this ligand, which has improved stability during storage. We also show that synthetic 5-A-RU•HCl produced by this method may be used in protocols for the stimulation of human MAIT cells and production of both human and mouse MR1 tetramers for MAIT cell identification.

Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3′ phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo .