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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo. We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection. Cumulatively, the results of this work may provide the basis for rational drug design - identifying numerous Mtb proteins, from essential kinases to products involved in metal regulation and cell wall remodeling, all present throughout the course of infection.

Autophagy is a cell biological pathway affecting immune responses. In vitro, autophagy acts as a cell-autonomous defense against Mycobacterium tuberculosis , but its role in vivo is unknown. Here we show that autophagy plays a dual role against tuberculosis: antibacterial and anti-inflammatory. M . tuberculosis infection of Atg5 ᶠˡ/ᶠˡ LysM-Cre ⁺ mice relative to autophagy-proficient littermates resulted in increased bacillary burden and excessive pulmonary inflammation characterized by neutrophil infiltration and IL-17 response with increased IL-1α levels. Macrophages from uninfected Atg5 ᶠˡ/ᶠˡ LysM-Cre ⁺ mice displayed a cell-autonomous IL-1α hypersecretion phenotype, whereas T cells showed propensity toward IL-17 polarization during nonspecific activation or upon restimulation with mycobacterial antigens. Thus, autophagy acts in vivo by suppressing both M . tuberculosis growth and damaging inflammation.

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.

Mucosal-associated invariant T (MAIT) cells exhibit an intrinsic ability to recognize and respond to microbial infections. The semi-invariant antigen recognition receptor of MAIT cells specifically detects the non-polymorphic antigen-presenting molecule, major histocompatibility complex class I-related protein 1 (MR1), which primarily binds riboflavin-derived metabolites of microbial origin. To further interrogate the dependence of these antigens on riboflavin biosynthesis in mycobacteria, we deleted individual genes in the riboflavin biosynthesis pathways in Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) and evaluated the impact thereof on MAIT cell activation. Blocking the early steps of the pathway by deletion of RibA2 or RibG profoundly reduced, but did not completely ablate, MAIT cell activation by Msm or Mtb, whereas deletion of RibC, which catalyzes the last step in the pathway, had no significant effect. Interestingly, deletion of the lumazine synthase (RibH) specifically enhanced MAIT cell recognition of Mtb whereas loss of lumazine synthase activity had no impact on MAIT cell activation by Msm. MAIT cell activation by Msm was likewise unaffected by blocking the production of the MAIT cell antagonist, F o (by inhibiting its conversion from the riboflavin pathway intermediate, 5-amino-6-D-ribitylaminouracil (5-A-RU), through the deletion of fbiC ). Together, these results confirm a central role for 5-A-RU in generating mycobacterial MR1 ligands and reveal similarities and differences between Msm and Mtb in terms of the impact of riboflavin pathway disruption on MAIT cell activation.

Tuberculosis (TB) remains a public health issue causing millions of infections every year. Of these, about 15% ultimately result in death. Efforts to control TB include development of new and more effective vaccines, novel and more effective drug treatments, and new diagnostics that test for both latent TB Infection and TB disease. All of these areas of research benefit from a good understanding of the physiology of Mycobacterium tuberculosis (Mtb), the primary causative agent of TB. Mtb secreted protein antigens have been the focus of vaccine and diagnosis research for the past century. Recently, the discovery of extracellular vesicles (EVs) as an important source of secreted antigens in Mtb has gained attention. Similarly, the discovery that host EVs can carry Mtb products during in vitro and in vivo infection has spiked interest because of its potential use in blood-based diagnostics. Despite advances in understanding the content of Mtb and Mtb-infected host extracellular vesicles, our understanding on the biogenesis and role of Mtb and host extracellular vesicles during Mtb infection is still nascent. Here, we explore the current literature on extracellular vesicles regarding Mtb, discuss the host and Mtb extracellular vesicles as distinct entities, and discuss current gaps in the field.

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1–4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12–80%) and specificities (14–100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis. Plasma from humans with latent tuberculosis (TB) infection (n = 23), active TB disease (n = 40), and uninfected controls (n = 9) were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins). When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10), whole cell lysate (Δ = 0.82 log10), and secreted proteins (Δ = 0.62 log10), though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6) to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ =  -1.53, p = 0.004). Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1), but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1). Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008), foreign-born (Δ = 0.61 log10, p = 0.004), or HIV-seronegative (Δ = 0.60 log10, p = 0.04). Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p < 0.001) and foreign-born (Δ = 0.87, p = 0.01). Humans with active TB disease produce antibodies to the surface of M. tuberculosis with low avidity and with a low IgG/IgM ratio. Highly-avid IgG antibodies to the M. tuberculosis surface may be an appropriate target for future TB vaccines.

In the last decade, there were 10 million new tuberculosis cases per year globally. Around 9.5% of these cases were caused by isoniazid resistant (INHr) Mycobacterium tuberculosis (Mtb) strains. Although isoniazid resistance in Mtb is multigenic, mutations in the catalase-peroxidase (katG) gene predominate among the INHr strains. The effect of these drug-resistance-conferring mutations on Mtb fitness and virulence is variable. Here, we assessed differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of the katG gene. We studied one laboratory and one clinically isolated Mtb clonal pair from different genetic lineages. The INHr strain in each pair had one and two katG mutations with significantly reduced levels of the enzyme and peroxidase activity. Both strains share the V1A mutation, while the double mutant clinical INHr had also the novel E3V katG mutation. Four groups of C57BL/6 mice were infected with one of the Mtb strains previously described. We observed a strong reduction in virulence (reduced bacterial growth), lower induction of proinflammatory cytokines and significantly reduced pathology scores in mice infected with the clinical INHr strain compared to the infection caused by its INHs progenitor strain. On the other hand, there was a subtle reduction of bacteria growth without differences in the pathology scores in mice infected with the laboratory INHr strain. Our results also showed distinct alkyl-hydroperoxidase C (AhpC) levels in the katG mutant strains, which could explain the difference in the virulence profile observed. The difference in the AhpC levels between clonal strains was not related to a genetic defect in the gene or its promoter. Cumulatively, our results indicate that the virulence, pathology and fitness of INHr strains could be negatively affected by multiple mutations in katG, lack of the peroxidase activity and reduced AhpC levels.

MR1-restricted T (MR1T) cells recognize microbial small molecule metabolites presented on the MHC Class I-like molecule MR1 and have been implicated in early effector responses to microbial infection. As a result, there is considerable interest in identifying chemical properties of metabolite ligands that permit recognition by MR1T cells, for consideration in therapeutic or vaccine applications. Here, we made chemical modifications to known MR1 ligands to evaluate the effect on MR1T cell activation. Specifically, we modified 6,7-dimethyl-8- d -ribityllumazine (DMRL) to generate 6,7-dimethyl-8- d -ribityldeazalumazine (DZ), and then further derivatized DZ to determine the requirements for retaining MR1 surface stabilization and agonistic properties. Interestingly, the IFN-γ response toward DZ varied widely across a panel of T cell receptor (TCR)-diverse MR1T cell clones; while one clone was agnostic toward the modification, most displayed either an enhancement or depletion of IFN-γ production when compared with its response to DMRL. To gain insight into a putative mechanism behind this phenomenon, we used in silico molecular docking techniques for DMRL and its derivatives and performed molecular dynamics simulations of the complexes. In assessing the dynamics of each ligand in the MR1 pocket, we found that DMRL and DZ exhibit differential dynamics of both the ribityl moiety and the aromatic backbone, which may contribute to ligand recognition. Together, our results support an emerging hypothesis for flexibility in MR1:ligand-MR1T TCR interactions and enable further exploration of the relationship between MR1:ligand structures and MR1T cell recognition for downstream applications targeting MR1T cells.

Dectin-2 is a C-type lectin involved in the recognition of several pathogens such as Aspergillus fumigatus , Candida albicans , Schistosoma mansonii , and Mycobacterium tuberculosis that triggers Th17 immune responses. Identifying pathogen ligands and understanding the molecular basis of their recognition is one of the current challenges. Purified M . tuberculosis mannose-capped lipoarabinomannan (ManLAM) was shown to induce signaling via Dectin-2, an activity that requires the (α1 → 2)-linked mannosides forming the caps. Here, using isogenic M . tuberculosis mutant strains, we demonstrate that ManLAM is a bona fide and actually the sole ligand mediating bacilli recognition by Dectin-2, although M . tuberculosis produces a variety of cell envelope mannoconjugates, such as phosphatidyl- myo -inositol hexamannosides, lipomannan or manno(lipo)proteins, that bear (α1 → 2)-linked mannosides. In addition, we found that Dectin-2 can recognize lipoglycans from other bacterial species, such as Saccharotrix aerocolonigenes or the human opportunistic pathogen Tsukamurella paurometabola , suggesting that lipoglycans are prototypical Dectin-2 ligands. Finally, from a structure/function relationship perspective, we show, using lipoglycan variants and synthetic mannodendrimers, that dimannoside caps and multivalent interaction are required for ligand binding to and signaling via Dectin-2. Better understanding of the molecular basis of ligand recognition by Dectin-2 will pave the way for the rational design of potent adjuvants targeting this receptor.