Transcriptional Signatures of Hippocampal Tau Pathology in Primary Age-Related Tauopathy and Alzheimer's Disease.

Background: Tau pathology is common in age-related neurodegenerative diseases. Tau pathology in primary age-related tauopathy (PART) and in Alzheimer's disease (AD) has a similar biochemical structure and anatomic distribution, which is distinct from tau pathology in other diseases. However, the molecular changes associated with intraneuronal tau pathology in PART and AD, and whether these changes are similar in the two diseases, is largely unexplored. Methods: Using GeoMx spatial transcriptomics, mRNA was quantified in CA1 pyramidal neurons with tau pathology and adjacent neurons without tau pathology in 6 cases of PART and 6 cases of AD, and compared to 4 control cases without pathology. Transcriptional changes were analyzed for differential gene expression and for coordinated patterns of gene expression associated with both disease state and intraneuronal tau pathology. Results: Synaptic gene changes and two novel gene expression signatures associated with intraneuronal tau were identified in PART and AD. Overall, gene expression changes associated with intraneuronal tau pathology were similar in PART and AD. Synaptic gene expression was decreased overall in neurons in AD and PART compared to control cases. However, this decrease was largely driven by neurons lacking tau pathology. Synaptic gene expression was increased in tau-positive neurons compared to tau-negative neurons in disease. Two novel gene expression signatures associated with intraneuronal tau were identified by examining coordinated patterns of gene expression. Genes in the up-regulated expression pattern were enriched in calcium regulation and synaptic function pathways, specifically in synaptic exocytosis. These synaptic gene changes and intraneuronal tau expression signatures were confirmed in a published transcriptional dataset of cortical neurons with tau pathology in AD. Conclusions: PART and AD show similar transcriptional changes associated with intraneuronal tau pathology in CA1 pyramidal neurons, raising the possibility of a mechanistic relationship between the tau pathology in the two diseases. Intraneuronal tau pathology was also associated with increased expression of genes associated with synaptic function and calcium regulation compared to tau-negative disease neurons. The findings highlight the power of molecular analysis stratified by pathology in neurodegenerative disease and provide novel insight into common molecular pathways associated with intraneuronal tau in PART and AD.

Hippocampal synaptic alterations associated with tau pathology in primary age-related tauopathy.

Primary age-related tauopathy (PART) is characterized by aggregation of tau in the mesial temporal lobe in older individuals. High pathologic tau stage (Braak stage) or a high burden of hippocampal tau pathology has been associated with cognitive impairment in PART. However, the potential underlying mechanisms are not well understood. Cognitive impairment in many neurodegenerative diseases correlates with synaptic loss, raising the question of whether synaptic loss also occurs in PART. To address this, we investigated synaptic changes associated with tau Braak stage and high tau pathology burden in PART using synaptophysin and phospho-tau immunofluorescence. We compared 12 cases of definite PART with 6 controls and 6 Alzheimer disease cases. In this study, the hippocampal CA2 region showed loss of synaptophysin puncta and intensity in cases of PART with either a high stage (Braak IV) or a high burden of neuritic tau pathology. There was also loss of synaptophysin intensity in CA3 associated with a high stage or high burden of tau pathology. Loss of synaptophysin was present in Alzheimer disease, but the pattern appeared distinct. These novel findings suggest the presence of synaptic loss associated with either a high hippocampal tau burden or a Braak stage IV in PART.

Hippocampal Synaptic Alterations Associated with Tau Pathology in Primary Age-Related Tauopathy.

Primary Age-Related Tauopathy (PART) is characterized by the aggregation of tau in the mesial temporal lobe in older individuals. High pathologic tau stage (Braak stage) or a high burden of hippocampal tau pathology have been associated with cognitive impairment in PART. However, the underlying mechanisms of cognitive impairment in PART are not well understood. Cognitive impairment in many neurodegenerative diseases correlates with synaptic loss, raising the question of whether synaptic loss occurs in PART. To address this, we investigated synaptic changes associated with tau Braak stage and a high tau pathology burden in PART using synaptophysin and phospho-tau immunofluorescence. We compared twelve cases of definite PART with six young controls and six Alzheimer's disease cases. In this study, we identified loss of synaptophysin puncta and intensity in the CA2 region of the hippocampus in cases of PART with either a high stage (Braak IV) or a high burden of neuritic tau pathology. There was also loss of synaptophysin intensity in CA3 associated with a high stage or high burden of tau pathology. Loss of synaptophysin signal was present in AD, but the pattern was distinct from that seen in PART. These novel findings suggest the presence of synaptic loss in PART associated with either a high hippocampal tau burden or a Braak stage IV. These synaptic changes raise the possibility that synaptic loss in PART could contribute to cognitive impairment, though future studies including cognitive assessments are needed to address this question.

Ubiquitin-positive astrogliopathy clinically mimicking Parkinson's disease.

Several neurodegenerative pathologies can clinically mimic Parkinson's disease, including neurodegenerative diseases with glial pathology. However, the glial aggregates are typically composed of known pathogenic proteins and are associated with prominent neuronal loss in the substantia nigra. Here we present an unusual case of a 91-year-old man with a clinical diagnosis of Parkinson's disease, but whose autopsy findings showed a ubiquitin-positive astrogliopathy without significant neuronal loss in the substantia nigra. These glial aggregates affected the basal ganglia, cortex, and cerebellum, and were negative for tau, alpha-synuclein, TDP-43, FUS, and p62. This case is a rare example of an unknown glial neurodegenerative pathology mimicking Parkinson's disease without significant loss of nigral dopaminergic neurons.

Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a 'barcode' to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein-Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

Compartmental Analysis of T-cell Clonal Dynamics as a Function of Pathologic Response to Neoadjuvant PD-1 Blockade in Resectable Non-Small Cell Lung Cancer.

PURPOSE: Neoadjuvant PD-1 blockade is a promising treatment for resectable non-small cell lung cancer (NSCLC), yet immunologic mechanisms contributing to tumor regression and biomarkers of response are unknown. Using paired tumor/blood samples from a phase II clinical trial (NCT02259621), we explored whether the peripheral T-cell clonotypic dynamics can serve as a biomarker for response to neoadjuvant PD-1 blockade. EXPERIMENTAL DESIGN: T-cell receptor (TCR) sequencing was performed on serial peripheral blood, tumor, and normal lung samples from resectable NSCLC patients treated with neoadjuvant PD-1 blockade. We explored the temporal dynamics of the T-cell repertoire in the peripheral and tumoral compartments in response to neoadjuvant PD-1 blockade by using the TCR as a molecular barcode. RESULTS: Higher intratumoral TCR clonality was associated with reduced percent residual tumor at the time of surgery, and the TCR repertoire of tumors with major pathologic response (MPR; <10% residual tumor after neoadjuvant therapy) had a higher clonality and greater sharing of tumor-infiltrating clonotypes with the peripheral blood relative to tumors without MPR. Additionally, the posttreatment tumor bed of patients with MPR was enriched with T-cell clones that had peripherally expanded between weeks 2 and 4 after anti-PD-1 initiation and the intratumoral space occupied by these clonotypes was inversely correlated with percent residual tumor. CONCLUSIONS: Our study suggests that exchange of T-cell clones between tumor and blood represents a key correlate of pathologic response to neoadjuvant immunotherapy and shows that the periphery may be a previously underappreciated originating compartment for effective antitumor immunity.See related commentary by Henick, p. 1205.

Revisiting the ß-Lactams for Tuberculosis Therapy with a Compound-Compound Synthetic Lethality Approach.

The suboptimal effectiveness of ß-lactam antibiotics against Mycobacterium tuberculosis has hindered the utility of this compound class for tuberculosis treatment. However, the results of treatment with a second-line regimen containing meropenem plus a ß-lactamase inhibitor were found to be encouraging in a case study of extensively drug-resistant tuberculosis (M. C. Payen, S. De Wit, C. Martin, R. Sergysels, et al., Int J Tuberc Lung Dis 16:558-560, 2012, https://doi.org/10.5588/ijtld.11.0414). We hypothesized that the innate resistance of M. tuberculosis to ß-lactams is mediated in part by noncanonical accessory proteins that are not considered the classic targets of ß-lactams and that small-molecule inhibitors of those accessory targets might sensitize M. tuberculosis to ß-lactams. In this study, we screened an NIH small-molecule library for the ability to sensitize M. tuberculosis to meropenem. We identified six hit compounds, belonging to either the N-arylindole or benzothiophene chemotype. Verification studies confirmed the synthetic lethality phenotype for three of the N-arylindoles and one benzothiophene derivative. The latter was demonstrated to be partially bioavailable via oral administration in mice. Structure-activity relationship studies of both structural classes identified analogs with potent antitubercular activity, alone or in combination with meropenem. Transcriptional profiling revealed that oxidoreductases, MmpL family proteins, and a 27-kDa benzoquinone methyltransferase could be the targets of the N-arylindole potentiator. In conclusion, our compound-compound synthetic lethality screening revealed novel small molecules that were capable of potentiating the action of meropenem, presumably via inhibition of the innate resistance conferred by ß-lactam accessory proteins. ß-Lactam compound-compound synthetic lethality may be an alternative approach for drug-resistant tuberculosis.

Correction to: persistent mutant oncogene specific T cells in two patients benefitting from anti-PD-1.

.

Persistent mutant oncogene specific T cells in two patients benefitting from anti-PD-1.

BACKGROUND: Several predictive biomarkers are currently approved or are under investigation for the selection of patients for checkpoint blockade. Tumor PD-L1 expression is used for stratification of non-small cell lung (NSCLC) patients, with tumor mutational burden (TMB) also being explored with promising results, and mismatch-repair deficiency is approved for tumor site-agnostic disease. While tumors with high PD-L1 expression, high TMB, or mismatch repair deficiency respond well to checkpoint blockade, tumors with lower PD-L1 expression, lower mutational burdens, or mismatch repair proficiency respond much less frequently. CASE PRESENTATION: We studied two patients with unexpected responses to checkpoint blockade monotherapy: a patient with PD-L1-negative and low mutational burden NSCLC and one with mismatch repair proficient colorectal cancer (CRC), both of whom lack the biomarkers associated with response to checkpoint blockade, yet achieved durable clinical benefit. Both maintained T-cell responses in peripheral blood to oncogenic driver mutations - BRAF-N581I in the NSCLC and AKT1-E17K in the CRC - years after treatment initiation. Mutation-specific T cells were also found in the primary tumor and underwent dynamic perturbations in the periphery upon treatment. CONCLUSIONS: These findings suggest that T cell responses to oncogenic driver mutations may be more prevalent than previously appreciated and could be harnessed in immunotherapeutic treatment, particularly for patients who lack the traditional biomarkers associated with response. Comprehensive studies are warranted to further delineate additional predictive biomarkers and populations of patients who may benefit from checkpoint blockade.

The Mutation-Associated Neoantigen Functional Expansion of Specific T Cells (MANAFEST) Assay: A Sensitive Platform for Monitoring Antitumor Immunity.

Mutation-associated neoantigens (MANA) are a target of antitumor T-cell immunity. Sensitive, simple, and standardized assays are needed to assess the repertoire of functional MANA-specific T cells in oncology. Assays analyzing in vitro cytokine production such as ELISpot and intracellular cytokine staining have been useful but have limited sensitivity in assessing tumor-specific T-cell responses and do not analyze antigen-specific T-cell repertoires. The FEST (Functional Expansion of Specific T cells) assay described herein integrates T-cell receptor sequencing of short-term, peptide-stimulated cultures with a bioinformatic platform to identify antigen-specific clonotypic amplifications. This assay can be adapted for all types of antigens, including MANAs via tumor exome-guided prediction of MANAs. Following in vitro identification by the MANAFEST assay, the MANA-specific CDR3 sequence can be used as a molecular barcode to detect and monitor the dynamics of these clonotypes in blood, tumor, and normal tissue of patients receiving immunotherapy. MANAFEST is compatible with high-throughput routine clinical and lab practices. Cancer Immunol Res; 6(8); 888-99. ©2018 AACR.

Impact of Clofazimine Dosing on Treatment Shortening of the First-Line Regimen in a Mouse Model of Tuberculosis.

The antileprosy drug clofazimine was recently repurposed as part of a newly endorsed short-course regimen for multidrug-resistant tuberculosis. It also enables significant treatment shortening when added to the first-line regimen for drug-susceptible tuberculosis in a mouse model. However, clofazimine causes dose- and duration-dependent skin discoloration in patients, and the optimal clofazimine dosing strategy in the context of the first-line regimen is unknown. We utilized a well-established mouse model to systematically address the impacts of duration, dose, and companion drugs on the treatment-shortening activity of clofazimine in the first-line regimen. In all studies, the primary outcome was relapse-free cure (culture-negative lungs) 6 months after stopping treatment, and the secondary outcome was bactericidal activity, i.e., the decline in the lung bacterial burden during treatment. Our findings indicate that clofazimine activity is most potent when coadministered with first-line drugs continuously throughout treatment and that equivalent treatment-shortening results are obtained with half the dose commonly used in mice. However, our studies also suggest that clofazimine at low exposures may have negative impacts on treatment outcomes, an effect that was evident only after the first 3 months of treatment. These data provide a sound evidence base to inform clofazimine dosing strategies to optimize the antituberculosis effect while minimizing skin discoloration. The results also underscore the importance of conducting long-term studies to allow the full evaluation of drugs administered in combination over long durations.

Neoadjuvant PD-1 Blockade in Resectable Lung Cancer.

BACKGROUND: Antibodies that block programmed death 1 (PD-1) protein improve survival in patients with advanced non-small-cell lung cancer (NSCLC) but have not been tested in resectable NSCLC, a condition in which little progress has been made during the past decade. METHODS: In this pilot study, we administered two preoperative doses of PD-1 inhibitor nivolumab in adults with untreated, surgically resectable early (stage I, II, or IIIA) NSCLC. Nivolumab (at a dose of 3 mg per kilogram of body weight) was administered intravenously every 2 weeks, with surgery planned approximately 4 weeks after the first dose. The primary end points of the study were safety and feasibility. We also evaluated the tumor pathological response, expression of programmed death ligand 1 (PD-L1), mutational burden, and mutation-associated, neoantigen-specific T-cell responses. RESULTS: Neoadjuvant nivolumab had an acceptable side-effect profile and was not associated with delays in surgery. Of the 21 tumors that were removed, 20 were completely resected. A major pathological response occurred in 9 of 20 resected tumors (45%). Responses occurred in both PD-L1-positive and PD-L1-negative tumors. There was a significant correlation between the pathological response and the pretreatment tumor mutational burden. The number of T-cell clones that were found in both the tumor and peripheral blood increased systemically after PD-1 blockade in eight of nine patients who were evaluated. Mutation-associated, neoantigen-specific T-cell clones from a primary tumor with a complete response on pathological assessment rapidly expanded in peripheral blood at 2 to 4 weeks after treatment; some of these clones were not detected before the administration of nivolumab. CONCLUSIONS: Neoadjuvant nivolumab was associated with few side effects, did not delay surgery, and induced a major pathological response in 45% of resected tumors. The tumor mutational burden was predictive of the pathological response to PD-1 blockade. Treatment induced expansion of mutation-associated, neoantigen-specific T-cell clones in peripheral blood. (Funded by Cancer Research Institute-Stand Up 2 Cancer and others; ClinicalTrials.gov number, NCT02259621 .).

Suppressor Cell-Depleting Immunotherapy With Denileukin Diftitox is an Effective Host-Directed Therapy for Tuberculosis.

Host-directed therapies that augment host immune effector mechanisms may serve as important adjunctive therapies for tuberculosis treatment. We evaluated the activity of denileukin diftitox in an acute mouse model of tuberculosis (TB) infection and analyzed the cellular composition and bacterial burden in lungs and spleens. These in vivo studies show that denileukin diftitox potentiates standard TB treatment in the mouse model, an effect which may be due to depletion of T-regulatory and myeloid-derived suppressor cells during TB infection. Our results indicate that denileukin diftitox and other suppressor cell-depleting therapies may be useful adjunctive, host-directed therapies for TB.

Inhibition of innate immune cytosolic surveillance by an M. tuberculosis phosphodiesterase.

Mycobacterium tuberculosis infection leads to cytosolic release of the bacterial cyclic dinucleotide (CDN) c-di-AMP and a host-generated CDN, cGAMP, both of which trigger type I interferon (IFN) expression in a STING-dependent manner. Here we report that M. tuberculosis has developed a mechanism to inhibit STING activation and the type I IFN response via the bacterial phosphodiesterase (PDE) CdnP, which mediates hydrolysis of both bacterial-derived c-di-AMP and host-derived cGAMP. Mutation of cdnP attenuates M. tuberculosis virulence, as does loss of a host CDN PDE known as ENPP1. CdnP is inhibited by both US Food and Drug Administration (FDA)-approved PDE inhibitors and nonhydrolyzable dinucleotide mimetics specifically designed to target the enzyme. These findings reveal a crucial role of CDN homeostasis in governing the outcome of M. tuberculosis infection as well as a unique mechanism of subversion of the host's cytosolic surveillance pathway (CSP) by a bacterial PDE that may serve as an attractive antimicrobial target.

Indole-2-carboxamide-based MmpL3 Inhibitors Show Exceptional Antitubercular Activity in an Animal Model of Tuberculosis Infection.

Our team had previously identified certain indolecarboxamides that represented a new chemical scaffold that showed promising anti-TB activity at both an in vitro and in vivo level. Based on mutational analysis using bacteria found resistant to one of these indolecarboxamides, we identified the trehalose monomycolate transporter MmpL3 as the likely target of these compounds. In the present work, we now further elaborate on the SAR of these compounds, which has led in turn to the identification of a new analog, 4,6-difluoro-N-((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)-1H-indole-2-carboxamide (26), that shows excellent activity against drug-sensitive (MIC = 0.012 µM; SI ≥ 16000), multidrug-resistant (MDR), and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains, has superior ADMET properties, and shows excellent activity in the TB aerosol lung infection model. Compound 26 is also shown to work in synergy with rifampin. Because of these properties, we believe that indolecarboxamide 26 is a possible candidate for advancement to human clinical trials.

A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis.

Detection of cyclic-di-adenosine monophosphate (c-di-AMP), a bacterial second messenger, by the host cytoplasmic surveillance pathway (CSP) is known to elicit type I interferon (IFN) responses, which are crucial to antimicrobial defense. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis requires CSP-mediated detection of c-di-AMP produced by M. tuberculosis and that levels of c-di-AMP modulate the fate of infection. We found that a di-adenylate cyclase (disA or dacA)-overexpressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-ß, elicits increased macrophage autophagy, and exhibits substantial virulence attenuation in mice. We show that c-di-AMP-mediated IFN-ß induction during M. tuberculosis infection requires stimulator of interferon genes (STING)-signaling. We observed that c-di-AMP induction of IFN-ß is independent of the cytosolic nucleic acid receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), but cGAS nevertheless contributes substantially to the overall IFN-ß response to M. tuberculosis infection. In sum, our results reveal c-di-AMP to be a key mycobacterial pathogen-associated molecular pattern (PAMP) driving host type I IFN responses and autophagy. These findings suggest that modulating the levels of this small molecule may lead to novel immunotherapeutic strategies against tuberculosis.

Synthetic lethality reveals mechanisms of Mycobacterium tuberculosis resistance to ß-lactams.

UNLABELLED: Most ß-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe's innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate ß-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem ß-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in ß-lactam resistance. The global transcriptional response of the bacterium to ß-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to ß-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for ß-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. IMPORTANCE: The global emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains has threatened public health worldwide, yet the pipeline of new tuberculosis drugs under development remains limited. One strategy to cope with the urgent need for new antituberculosis agents is to repurpose existing, approved antibiotics. The carbapenem class of ß-lactam antibiotics has been proposed as one such class of drugs. Our study identifies molecular determinants of innate resistance to ß-lactam drugs in M. tuberculosis, and we demonstrate that functional loss of one of these genes enables successful treatment of M. tuberculosis with ß-lactams in the mouse model.

Mycobacterial gene cuvA is required for optimal nutrient utilization and virulence.

To persist and cause disease in the host, Mycobacterium tuberculosis must adapt to its environment during infection. Adaptations include changes in nutrient utilization and alterations in growth rate. M. tuberculosis Rv1422 is a conserved gene of unknown function that was found in a genetic screen to interact with the mce4 cholesterol uptake locus. The Rv1422 protein is phosphorylated by the M. tuberculosis Ser/Thr kinases PknA and PknB, which regulate cell growth and cell wall synthesis. Bacillus subtilis strains lacking the Rv1422 homologue yvcK grow poorly on several carbon sources, and yvcK is required for proper localization of peptidoglycan synthesis. Here we show that Mycobacterium smegmatis and M. tuberculosis strains lacking Rv1422 have growth defects in minimal medium containing limiting amounts of several different carbon sources. These strains also have morphological abnormalities, including shortened and bulging cells, suggesting a cell wall defect. In both mycobacterial species, the Rv1422 protein localizes uniquely to the growing cell pole, the site of peptidoglycan synthesis in mycobacteria. An M. tuberculosis ΔRv1422 strain is markedly attenuated for virulence in a mouse infection model, where it elicits decreased inflammation in the lungs and shows impaired bacterial persistence. These findings led us to name this gene cuvA (carbon utilization and virulence protein A) and to suggest a model in which deletion of cuvA leads to changes in nutrient uptake and/or metabolism that affect cell wall structure, morphology, and virulence. Its role in virulence suggests that CuvA may be a useful target for novel inhibitors of M. tuberculosis during infection.