Computational drug repositioning identifies niclosamide and tribromsalan as inhibitors of Mycobacterium tuberculosis and Mycobacterium abscessus.

Tuberculosis (TB) is still a major global health challenge, killing over 1.5 million people each year, and hence, there is a need to identify and develop novel treatments for Mycobacterium tuberculosis (M. tuberculosis). The prevalence of infections caused by nontuberculous mycobacteria (NTM) is also increasing and has overtaken TB cases in the United States and much of the developed world. Mycobacterium abscessus (M. abscessus) is one of the most frequently encountered NTM and is difficult to treat. We describe the use of drug-disease association using a semantic knowledge graph approach combined with machine learning models that has enabled the identification of several molecules for testing anti-mycobacterial activity. We established that niclosamide (M. tuberculosis IC90 2.95 µM; M. abscessus IC90 59.1 µM) and tribromsalan (M. tuberculosis IC90 76.92 µM; M. abscessus IC90 147.4 µM) inhibit M. tuberculosis and M. abscessus in vitro. To investigate the mode of action, we determined the transcriptional response of M. tuberculosis and M. abscessus to both compounds in axenic log phase, demonstrating a broad effect on gene expression that differed from known M. tuberculosis inhibitors. Both compounds elicited transcriptional responses indicative of respiratory pathway stress and the dysregulation of fatty acid metabolism.

Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

Insight into the on/off switch that regulates expression of the MSMEG-3762/63 efflux pump in Mycobacterium smegmatis.

Drug resistance is one of the most difficult challenges facing tuberculosis (TB) control. Drug efflux is among the mechanisms leading to drug resistance. In our previous studies, we partially characterized the ABC-type MSMEG-3762/63 efflux pump in Mycobacterium smegmatis, which shares high percentage of identity with the Mycobacterium tuberculosis Rv1687/86c pump. MSMEG-3762/63 was shown to have extrusion activity for rifampicin and ciprofloxacin, used in first and second-line anti-TB treatments. Moreover, we described the functional role of the TetR-like MSMEG-3765 protein as a repressor of the MSMEG_3762/63/65 operon and orthologous Rv1687/86/85c in M. tuberculosis. Here we show that the operon is upregulated in the macrophage environment, supporting a previous observation of induction triggered by acid-nitrosative stress. Expression of the efflux pump was also induced by sub-inhibitory concentrations of rifampicin or ciprofloxacin. Both these drugs also prevented the binding of the MSMEG-3765 TetR repressor protein to its operator in the MSMEG_3762/63/65 operon. The hypothesis that these two drugs might be responsible for the induction of the efflux pump operon was assessed by bioinformatics analyses. Docking studies using a structural model of the regulator MSMEG-3765 showed that both antibiotics abolished the ability of this transcriptional repressor to recognize the efflux pump operon by interacting with the homodimer at different binding sites within the same binding pocket. Reduced binding of the repressor leads to induction of the efflux pump in M. smegmatis, and reduced efficacy of these two anti-mycobacterial drugs.

Lack of methoxy-mycolates characterizes the geographically restricted lineage 7 of Mycobacterium tuberculosis complex.

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

Searching for new therapeutic options for the uncommon pathogen Mycobacterium chimaera: an open drug discovery approach.

BACKGROUND: Mycobacterium chimaera is a slowly growing non-tuberculous mycobacterium associated with outbreaks of fatal infections in patients after cardiac surgery, and it is increasingly being detected in patients with chronic lung conditions. M chimaera can cause disseminated disease, osteomyelitis, and chronic skin or soft-tissue infections. We aimed to find new inhibitory compounds and drug repurposing opportunities for M chimaera, as current therapeutic options often result in poor outcomes. METHODS: In an open drug discovery approach, we screened the Medicines for Malaria Venture (MMV) Pathogen Box to assess the in-vitro antimicrobial drug susceptibility of M chimaera compared with the antimicrobial drug susceptibility of the slowly growing, major human pathogen Mycobacterium tuberculosis, and the rapidly growing Mycobacterium abscessus reference strains. Compounds identified from an initial resazurin microtitre cell viability assay screen were further characterised by determining the minimum inhibitory concentration (MIC) of MMV Pathogen Box compounds against M chimaera; and the MICs of a panel of 20 drugs commonly used to treat mycobacterial infections against M tuberculosis, M abscessus, and M chimaera. We also assessed the time-kill kinetics of doxycycline, clarithromycin, ethambutol, and rifabutin against M chimaera. FINDINGS: M chimaera was inhibited by 21 (5%) of 400 compounds in the Pathogen Box. Ten compounds were active against all three mycobacteria. MMV675968, with activity against slowly growing mycobacteria that probably targets folate metabolism, had a mean MIC of 2·22 µM (0·80 µg/mL) against M chimaera. Antimicrobial susceptibility testing showed that oxazolidinones such as linezolid (mean MIC 3·13 µg/mL) were active against M chimaera and that bedaquiline was the most potent compound (mean MIC 0·02 µg/mL). Doxycycline, a broad-spectrum antimicrobial drug with excellent tissue penetration properties, also inhibited M chimaera with a mean MIC of 6·25 µg/mL. INTERPRETATION: Molecular diagnostics present an opportunity for more effective, targeted drug therapies-treating bacterial infections at the species level. Using an open drug discovery platform, we identified compounds that inhibit the newly recognised pathogen M chimaera. The existing evidence base is poor and the option for expensive drug discovery is improbable; therefore, we have also found options for drug repurposing. Future in-vivo efficacy studies will reveal whether these findings result in new, targeted treatment regimens for M chimaera. FUNDING: Wellcome Trust, National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), and the University of Sussex Junior Research Associate scheme.

Characterisation of drug-resistant Mycobacterium tuberculosis mutations and transmission in Pakistan.

Tuberculosis, caused by Mycobacterium tuberculosis, is a high-burden disease in Pakistan, with multi-drug (MDR) and extensive-drug (XDR) resistance, complicating infection control. Whole genome sequencing (WGS) of M. tuberculosis is being used to infer lineages (strain-types), drug resistance mutations, and transmission patterns-all informing infection control and clinical decision making. Here we analyse WGS data on 535 M. tuberculosis isolates sourced across Pakistan between years 2003 and 2020, to understand the circulating strain-types and mutations related to 12 anti-TB drugs, as well as identify transmission clusters. Most isolates belonged to lineage 3 (n = 397; 74.2%) strain-types, and were MDR (n = 328; 61.3%) and (pre-)XDR (n = 113; 21.1%). By inferring close genomic relatedness between isolates (< 10-SNPs difference), there was evidence of M. tuberculosis transmission, with 55 clusters formed consisting of a total of 169 isolates. Three clusters consist of M. tuberculosis that are similar to isolates found outside of Pakistan. A genome-wide association analysis comparing 'transmitted' and 'non-transmitted' isolate groups, revealed the nusG gene as most significantly associated with a potential transmissible phenotype (P = 5.8 × 10-10). Overall, our study provides important insights into M. tuberculosis genetic diversity and transmission in Pakistan, including providing information on circulating drug resistance mutations for monitoring activities and clinical decision making.

Dissecting the Mycobacterium bovis BCG Response to Macrophage Infection to Help Prioritize Targets for Anti-Tuberculosis Drug and Vaccine Discovery.

New strategies are required to reduce the worldwide burden of tuberculosis. Intracellular survival and replication of Mycobacterium tuberculosis after macrophage phagocytosis is a fundamental step in the complex host-pathogen interactions that lead to granuloma formation and disease. Greater understanding of how the bacterium survives and thrives in these environments will inform novel drug and vaccine discovery programs. Here, we use in-depth RNA sequencing of Mycobacterium bovis BCG from human THP-1 macrophages to describe the mycobacterial adaptations to the intracellular environment. We identify 329 significantly differentially regulated genes, highlighting cholesterol catabolism, the methylcitrate cycle and iron homeostasis as important for mycobacteria inside macrophages. Examination of multi-functional gene families revealed that 35 PE/PPE genes and five cytochrome P450 genes were upregulated 24 h after infection, highlighting pathways of potential significance. Comparison of the intracellular transcriptome to gene essentiality and immunogenicity studies identified 15 potential targets that are both required for intracellular survival and induced on infection, and eight upregulated genes that have been demonstrated to be immunogenic in TB patients. Further insight into these new and established targets will support drug and vaccine development efforts.

Biofilms in tuberculosis: What have we learnt in the past decade and what is still unexplored?

Elucidating how Mycobacterium tuberculosis produces biofilms, and its impact for tuberculosis (TB) pathogenesis is gaining momentum. Here, we discuss recent findings reported over the last decade, which help us gain insights into the association between biofilm formation and TB pathogenesis. A new appreciation of extracellular TB phenotypes found in lung lesions will drive drug and vaccine discovery forward to new possibilities.

Lipid droplets and the transcriptome of Mycobacterium tuberculosis from direct sputa: a literature review.

Mycobacterium tuberculosis (Mtb), the main etiology of tuberculosis (TB), is predominantly an intracellular pathogen that has caused infection, disease and death in humans for centuries. Lipid droplets (LDs) are dynamic intracellular organelles that are found across the evolutionary tree of life. This review is an evaluation of the current state of knowledge regarding Mtb-LD formation and associated Mtb transcriptome directly from sputa.Based on the LD content, Mtb in sputum may be classified into three groups: LD positive, LD negative and LD borderline. However, the clinical and evolutionary importance of each state is not well elaborated. Mounting evidence supports the view that the presence of LD positive Mtb bacilli in sputum is a biomarker of slow growth, low energy state, towards lipid degradation, and drug tolerance. In Mtb, LD may serve as a source of chemical energy, scavenger of toxic compounds, prevent destruction of Mtb through autophagy, delay trafficking of lysosomes towards the phagosome, and contribute to Mtb persistence. It is suggest that LD is a key player in the induction of a spectrum of phenotypic and metabolic states of Mtb in the macrophage, granuloma and extracellular sputum microenvironment. Tuberculosis patients with high proportion of LD positive Mtb in pretreatment sputum was associated with higher rate of poor treatment outcome, indicating that LD may have a clinical application in predicting treatment outcome.The propensity for LD formation among Mtb lineages is largely unknown. The role of LD on Mtb transmission and disease phenotype (pulmonary TB vs extra-pulmonary TB) is not well understood. Thus, further studies are needed to understand the relationships between LD positivity and Mtb lineage, Mtb transmission and clinical types.

The Mycobacterium tuberculosis sRNA F6 Modifies Expression of Essential Chaperonins, GroEL2 and GroES.

Almost 140 years after the identification of Mycobacterium tuberculosis as the etiological agent of tuberculosis, important aspects of its biology remain poorly described. Little is known about the role of posttranscriptional control of gene expression and RNA biology, including the role of most of the small RNAs (sRNAs) identified to date. We have carried out a detailed investigation of the M. tuberculosis sRNA F6 and shown it to be dependent on SigF for expression and significantly induced in starvation conditions in vitro and in a mouse model of infection. Further exploration of F6 using an in vitro starvation model of infection indicates that F6 affects the expression of the essential chaperonins GroEL2 and GroES. Our results point toward a role for F6 during periods of low metabolic activity typically associated with long-term survival of M. tuberculosis in human granulomas. IMPORTANCE Control of gene expression via small regulatory RNAs (sRNAs) is poorly understood in one of the most successful pathogens, Mycobacterium tuberculosis. Here, we present an in-depth characterization of the sRNA F6, including its expression in different infection models and the differential gene expression observed upon deletion of the sRNA. Our results demonstrate that deletion of F6 leads to dysregulation of the two essential chaperonins GroEL2 and GroES and, moreover, indicate a role for F6 in the long-term survival and persistence of M. tuberculosis in the human host.

Correction: Lu et al. Weighted Gene Co-Expression Network Analysis Identifies Key Modules and Hub Genes Associated with Mycobacterial Infection of Human Macrophages. Antibiotics 2021, 10, 97.

The authors wish to make the following corrections to this paper [...].

Undetected carriage explains apparent Staphylococcus aureus acquisition in a non-outbreak healthcare setting.

OBJECTIVES: Previous studies have been unable to identify patient or staff reservoirs for the majority of the nosocomial S. aureus acquisitions which occur in the presence of good infection control practice. We set out to establish the extent to which undetected pre-existing carriage explains apparent nosocomial S. aureus acquisition. METHODS: Over two years elective cardiothoracic admissions were screened for S. aureus carriage before and during hospital admission. Routine screening (nose/groin/wound sampling), was supplemented by sampling additional body sites (axilla/throat/rectum) and culture-based methods optimised to detect fastidious phenotypes (small colony variants, cell wall deficient variants) and molecular identification by PCR. RESULTS: 35% of participants (53/151) were S. aureus carriers according to routine pre-healthcare screening; increasing to 42% (63/151) when additional body sites and enhanced cultures were employed. 71% (5/7) of apparent acquisitions were explained by pre-existing carriage using augmented measures. Enhanced culture identified a minority of colonised individuals (3/151 including 1 MRSA carrier) who were undetected by routine and additional screening cultures. 4/14 (29%) participants who became culture-negative during admission had S. aureus genomic material detected at discharge. CONCLUSIONS: Conventional sampling under-estimates carriage of S. aureus and this explains the majority of apparent S. aureus acquisitions among elective cardiothoracic patients.

Identification of a series of hair-cell MET channel blockers that protect against aminoglycoside-induced ototoxicity.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin-Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.

Three-dimensional low shear culture of Mycobacterium bovis BCG induces biofilm formation and antimicrobial drug tolerance.

Mycobacteria naturally grow as corded biofilms in liquid media without detergent. Such detergent-free biofilm phenotypes may reflect the growth pattern of bacilli in tuberculous lung lesions. New strategies are required to treat tuberculosis, which is responsible for more deaths each year than any other bacterial disease. The lengthy 6-month regimen for drug-sensitive tuberculosis is necessary to remove antimicrobial drug tolerant populations of bacilli that persist through drug therapy. The role of biofilm-like growth in the generation of these sub-populations remains poorly understood despite the hypothesised clinical significance and mounting evidence of biofilms in pathogenesis. We adapt a three-dimensional Rotary Cell Culture System to model M. bovis BCG biofilm growth in low-shear detergent-free liquid suspension. Importantly, biofilms form without attachment to artificial surfaces and without severe nutrient starvation or environmental stress. Biofilm-derived planktonic bacilli are tolerant to isoniazid and streptomycin, but not rifampicin. This phenotypic drug tolerance is lost after passage in drug-free media. Transcriptional profiling reveals induction of cell surface regulators, sigE and BCG_0559c alongside the ESX-5 secretion apparatus in these low-shear liquid-suspension biofilms. This study engineers and characterises mycobacteria grown as a suspended biofilm, illuminating new drug discovery pathways for this deadly disease.

Weighted Gene Co-Expression Network Analysis Identifies Key Modules and Hub Genes Associated with Mycobacterial Infection of Human Macrophages.

Tuberculosis (TB) is still a leading cause of death worldwide. Treatments remain unsatisfactory due to an incomplete understanding of the underlying host-pathogen interactions during infection. In the present study, weighted gene co-expression network analysis (WGCNA) was conducted to identify key macrophage modules and hub genes associated with mycobacterial infection. WGCNA was performed combining our own transcriptomic results using Mycobacterium aurum-infected human monocytic macrophages (THP1) with publicly accessible datasets obtained from three types of macrophages infected with seven different mycobacterial strains in various one-to-one combinations. A hierarchical clustering tree of 11,533 genes was built from 198 samples, and 47 distinct modules were revealed. We identified a module, consisting of 226 genes, which represented the common response of host macrophages to different mycobacterial infections that showed significant enrichment in innate immune stimulation, bacterial pattern recognition, and leukocyte chemotaxis. Moreover, by network analysis applied to the 74 genes with the best correlation with mycobacteria infection, we identified the top 10 hub-connecting genes: NAMPT, IRAK2, SOCS3, PTGS2, CCL20, IL1B, ZC3H12A, ABTB2, GFPT2, and ELOVL7. Interestingly, apart from the well-known Toll-like receptor and inflammation-associated genes, other genes may serve as novel TB diagnosis markers and potential therapeutic targets.

Characterization of the Mycobacterial MSMEG-3762/63 Efflux Pump in Mycobacterium smegmatis Drug Efflux.

Multi-drug resistant tuberculosis (MDR-TB) represents a major health problem worldwide. Drug efflux and the activity of efflux transporters likely play important roles in the development of drug-tolerant and drug-resistant mycobacterial phenotypes. This study is focused on the action of a mycobacterial efflux pump as a mechanism of drug resistance. Previous studies demonstrated up-regulation of the TetR-like transcriptional regulator MSMEG_3765 in Mycobacterium smegmatis and its ortholog Rv1685c in Mycobacterium tuberculosis (Mtb) in acid-nitrosative stress conditions. MSMEG-3765 regulates the expression of the MSMEG_3762/63/65 operon, and of the orthologous region in Mtb (Rv1687c/86c/85c). MSMEG-3762 and Rv1687c are annotated as ATP-binding proteins, while MSMEG-3763 and Rv1686c are annotated as trans-membrane polypeptides, defining an ABC efflux pump in both M. smegmatis and Mtb. The two putative efflux systems share a high percentage of identity. To examine the role of the putative efflux system MSMEG-3762/63, we constructed and characterized a MSMEG-3763 deletion mutant in M. smegmatis (∆MSMEG_3763). By comparative analysis of wild type, knockout, and complemented strains, together with structural modeling and molecular docking bioinformatics analyses of the MSMEG-3763 trans-membrane protein, we define the protein complex MSMEG-3762/63 as an efflux pump. Moreover, we demonstrate involvement of this pump in biofilm development and in the extrusion of rifampicin and ciprofloxacin (CIP), antimicrobial drugs used in first- and second-line anti-TB therapies.

Carprofen elicits pleiotropic mechanisms of bactericidal action with the potential to reverse antimicrobial drug resistance in tuberculosis.

BACKGROUND: The rise of antimicrobial drug resistance in Mycobacterium tuberculosis coupled with the shortage of new antibiotics has elevated TB to a major global health priority. Repurposing drugs developed or used for other conditions has gained special attention in the current scenario of accelerated drug development for several global infectious diseases. In a similar effort, previous studies revealed that carprofen, a non-steroidal anti-inflammatory drug, selectively inhibited the growth of replicating, non-replicating and MDR clinical isolates of M. tuberculosis. OBJECTIVES: We aimed to reveal the whole-cell phenotypic and transcriptomic effects of carprofen in mycobacteria. METHODS: Integrative molecular and microbiological approaches such as resazurin microtitre plate assay, high-throughput spot-culture growth inhibition assay, whole-cell efflux inhibition, biofilm inhibition and microarray analyses were performed. Analogues of carprofen were also synthesized and assessed for their antimycobacterial activity. RESULTS: Carprofen was found to be a bactericidal drug that inhibited mycobacterial drug efflux mechanisms. It also restricted mycobacterial biofilm growth. Transcriptome profiling revealed that carprofen likely acts by targeting respiration through the disruption of membrane potential. The pleiotropic nature of carprofen's anti-TB action may explain why spontaneous drug-resistant mutants could not be isolated in practice. CONCLUSIONS: This immunomodulatory drug and its chemical analogues have the potential to reverse TB antimicrobial drug resistance, offering a swift path to clinical trials of novel TB drug combinations.

Spontaneously Occurring Small-Colony Variants of Staphylococcus aureus Show Enhanced Clearance by THP-1 Macrophages.

Staphylococcus aureus is a common cause of chronic and relapsing infection, especially when the ability of the immune system to sterilize a focus of infection is compromised (e.g., because of a foreign body or in the cystic fibrosis lung). Chronic infections are associated with slow-growing colony phenotypes of S. aureus on solid media termed small-colony variants (SCVs). Stable SCVs show characteristic mutations in the electron transport chain that convey resistance to antibiotics, particularly aminoglycosides. This can be used to identify SCVs from within mixed-colony phenotype populations of S. aureus. More recently, populations of SCVs that rapidly revert to a "wild-type" (WT) colony phenotype, in the absence of selection pressure, have also been described. In laboratory studies, SCVs accumulate through prolonged infection of non-professional phagocytes and may represent an adaptation to the intracellular environment. However, data from phagocytic cells are lacking. In this study, we mapped SCV and WT colony populations in axenic growth of multiple well-characterized methicillin-sensitive and methicillin-resistant S. aureus strains. We identified SCVs populations on solid media both in the presence and absence of gentamicin. We generated stable SCVs from Newman strain S. aureus, and infected human macrophages with WT S. aureus (Newman, 8325-4) and their SCV counterparts (SCV3, I10) to examine intracellular formation and survival of SCVs. We show that SCVs arise spontaneously during axenic growth, and that the ratio of SCV:WT morphology differs between strains. Exposure to the intracellular environment of human macrophages did not increase formation of SCVs over 5 days and macrophages were able to clear stable SCV bacteria more effectively than their WT counterparts.

Identification of antigens presented by MHC for vaccines against tuberculosis.

Mycobacterium tuberculosis (M.tb) is responsible for more deaths globally than any other pathogen. The only available vaccine, bacillus Calmette-Guérin (BCG), has variable efficacy throughout the world. A more effective vaccine is urgently needed. The immune response against tuberculosis relies, at least in part, on CD4+ T cells. Protective vaccines require the induction of antigen-specific CD4+ T cells via mycobacterial peptides presented by MHC class-II in infected macrophages. In order to identify mycobacterial antigens bound to MHC, we have immunoprecipitated MHC class-I and class-II complexes from THP-1 macrophages infected with BCG, purified MHC class-I and MHC class-II peptides and analysed them by liquid chromatography tandem mass spectrometry. We have successfully identified 94 mycobacterial peptides presented by MHC-II and 43 presented by MHC-I, from 76 and 41 antigens, respectively. These antigens were found to be highly expressed in infected macrophages. Gene ontology analysis suggests most of these antigens are associated with membranes and involved in lipid biosynthesis and transport. The sequences of selected peptides were confirmed by spectral match validation and immunogenicity evaluated by IFN-gamma ELISpot against peripheral blood mononuclear cell from volunteers vaccinated with BCG, M.tb latently infected subjects or patients with tuberculosis disease. Three antigens were expressed in viral vectors, and evaluated as vaccine candidates alone or in combination in a murine aerosol M.tb challenge model. When delivered in combination, the three candidate vaccines conferred significant protection in the lungs and spleen compared with BCG alone, demonstrating proof-of-concept for this unbiased approach to identifying new candidate antigens.