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The article discusses the role of autophagy against active tuberculosis. Study shows that autophagy protects against Mycobacterium tuberculosis (M. tuberculosis) infection and excessive inflammation in vivo. It states that macrophages defective for autophagy secrete proinflammatory cytokines and in a cell-autonomous fashion.

Cytokines are key regulators of adequate immune responses to infection with Mycobacterium tuberculosis. We demonstrate that the p110δ catalytic subunit of PI3K acts as a downstream effector of the TLR family member RP105 (CD180) in promoting mycobacteria-induced cytokine production by macrophages. Our data show that the significantly reduced release of TNF and IL-6 by RP105-/- macrophages during mycobacterial infection was not accompanied by diminished mRNA or protein expression. Mycobacteria induced comparable activation of NF-κB and p38 MAPK signaling in wild-type (WT) and RP105-/- macrophages. In contrast, mycobacteria-induced phosphorylation of Akt was abrogated in RP105-/- macrophages. The pi 108-specific inhibitor, Cal-101, and small interfering RNA-mediated knockdown of pllOS diminished mycobacteria-induced TNF secretion by WT but not RP105-/- macrophages. Such interference with p110δ activity led to reduced surface-expressed TNF in WT but not RP105-/- macrophages, while leaving TNF mRNA and protein expression unaffected. Activity of Bruton's tyrosine kinase was required for RP105-mediated activation of Akt phosphorylation and TNF release by mycobacteria-infected macrophages. These data unveil a novel innate immune signaling axis that orchestrates key cytokine responses of macrophages and provide molecular insight into the functions of RP105 as an innate immune receptor for mycobacteria. [ABSTRACT FROM AUTHOR]

Background: 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. Methods: 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). Results: 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). Conclusions/Significance: 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. [ABSTRACT FROM AUTHOR]

Riboflavin (vitamin B 2 ) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo . Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria., Importance: The pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease., Competing Interests: The authors declare no conflict of interest.

Background: Flu-like reactions can occur after exposure to rifampin, rifapentine, or isoniazid. Prior studies have reported the presence of antibodies to rifampin, but associations with underlying pathogenesis are unclear., Methods: We evaluated PREVENT TB study participants who received weekly isoniazid + rifapentine for 3 months (3HP) or daily isoniazid for 9 months (9H) as treatment for M. tuberculosis infection. Flu-like reaction was defined as a grade ≥2 of any of flu-like symptoms. Controls (3HP or 9H) did not report flu-like reactions. We developed a competitive enzyme-linked immunosorbent assays (ELISA) to detect antibodies against rifapentine, isoniazid, rifampin, and rifapentine metabolite., Results: Among 128 participants, 69 received 3HP (22 with flu-like reactions; 47 controls) and 59 received 9H (12 with flu-like reactions; 47 controls). In participants receiving 3HP, anti-rifapentine IgG was identified in 2/22 (9%) participants with flu-like reactions and 6/47 (13%) controls (P = 0.7), anti-isoniazid IgG in 2/22 (9%) participants with flu-like reactions and 4/47 (9%) controls (P = 0.9), and anti-rifapentine metabolite IgG in 2/47 (4%) controls (P = 0.9). Among participants receiving 9H, IgG and IgM anti-isoniazid antibodies were each present in 4/47 (9%) controls, respectively, but none among participants with flu-like reactions; anti-rifapentine IgG antibodies were not present in any participants with flu-like reactions or controls., Conclusions: We detected anti-rifapentine, anti-isoniazid, and anti-rifapentine metabolite antibodies, but the proportions of participants with antibodies were low, and did not differ between participants with flu-like reactions and those without such reactions. This suggests that flu-like reactions associated with 3HP and 9H were not antibody-mediated., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific purposes. Detailed protocols used for production of M. tuberculosis in large scale (under normoxic and hypoxic conditions) in bioreactors and for production of obligate intracellular pathogens such as Mycobacterium leprae and "Mycobacterium lepromatosis" using athymic nude mice and armadillos are provided.

The extraction and separation of native mycobacterial proteins remain necessary for antigen discovery, elucidation of enzymes to improve rational drug design, identification of physiologic mechanisms, use as reagents for diagnostics, and defining host immune responses. In this chapter, methods for the manipulation of whole mycobacterial cells and culture exudates are described in detail as these methods are the requisite first steps towards native protein isolation. Specifically, several methods for the inactivation of viable Mycobacterium tuberculosis along with qualification assays are provided, as this is key to safe manipulation of cell pastes for downstream processes. Next, the concentration of spent culture filtrate media in order to permit separation of soluble, secreted proteins is described followed by the separation of mycobacteria extracellular vesicles (MEV) from the remaining soluble proteins in spent media. We then describe the generation of whole-cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol-enriched proteins. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided. Finally, methods for separation of hydrophobic and hydrophilic proteins from both whole-cell lysate and spent culture media are included. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins.

The use of proteomic technologies to characterize and study the proteome of mycobacteria has provided important information in terms of function, diversity, protein-protein interactions, and host-pathogen interactions in Mycobacterium spp. There are many different mass spectrometry methodologies that can be applied to proteomics studies of mycobacteria and microorganisms in general. Sample processing and appropriate study design are critical to generating high-quality data regardless of the mass spectrometry method applied. Appropriate study design relies on statistical rigor and data curation using bioinformatics approaches that are widely applicable regardless of the organism or system studied. Sample processing, on the other hand, is often a niched process specific to the physiology of the organism or system under investigation. Therefore, in this chapter, we will provide protocols for processing mycobacterial protein samples for the specific application of Top-down and Bottom-up proteomic analyses.

Identification of biomarkers for latent Mycobacterium tuberculosis infection and risk of progression to tuberculosis (TB) disease are needed to better identify individuals to target for preventive therapy, predict disease risk, and potentially predict preventive therapy efficacy. Our group developed multiple reaction monitoring mass spectrometry (MRM-MS) assays that detected M. tuberculosis peptides in serum extracellular vesicles from TB patients. We subsequently optimized this MRM-MS assay to selectively identify 40 M. tuberculosis peptides from 19 proteins that most commonly copurify with serum vesicles of patients with TB. Here, we used this technology to evaluate if M. tuberculosis peptides can also be detected in individuals with latent TB infection (LTBI). Serum extracellular vesicles from 74 individuals presumed to have latent M. tuberculosis infection (LTBI) based on close contact with a household member with TB or a recent tuberculin skin test (TST) conversion were included in this study. Twenty-nine samples from individuals with no evidence of TB infection by TST and no known exposure to TB were used as controls to establish a threshold to account for nonspecific/background signal. We identified at least one of the 40 M. tuberculosis peptides in 70 (95%) individuals with LTBI. A single peptide from the glutamine synthetase (GlnA1) enzyme was identified in 61/74 (82%) individuals with LTBI, suggesting peptides from M. tuberculosis proteins involved in nitrogen metabolism might be candidates for pathogen-specific biomarkers for detection of LTBI. The detection of M. tuberculosis peptides in serum extracellular vesicles from persons with LTBI represents a potential advance in the diagnosis of LTBI., (Copyright © 2020 Mehaffy et al.)

Infection with Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis, remains a global health concern. Both classically and non-classically restricted cytotoxic CD8 + T cells are important to the control of Mtb infection. We and others have demonstrated that the non-classical MHC I molecule HLA-E can present pathogen-derived peptides to CD8 + T cells. In this manuscript, we identified the antigen recognized by an HLA-E-restricted CD8 + T cell clone isolated from an Mtb latently infected individual as a peptide from the Mtb protein, MPT32. Recognition by the CD8 + T cell clone required N-terminal O-linked mannosylation of MPT32 by a mannosyltransferase encoded by the Rv1002c gene. This is the first description of a post-translationally modified Mtb-derived protein antigen presented in the context of an HLA-E specific CD8 + T cell immune response. The identification of an immune response that targets a unique mycobacterial modification is novel and may have practical impact in the development of vaccines and diagnostics.

Background: Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. Mtb resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins., Methods: Multiple reaction monitoring assays were optimized for the enhanced detection of 41 Mtb peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host m / z interference. The final optimized method was tested in 40 exosome samples from TB positive patients., Results: Our enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the Mtb proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the Mtb strains circulating within each given region., Conclusions: An enhanced MRM method to detect ultra-low abundance Mtb peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development.

γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB., (Copyright © 2016 Xia et al.)

The extraction and isolation of native bacterial proteins continue to be valuable technical pursuits in order to understand bacterial physiology, screen for virulence determinants, and describe antigens. In this chapter, methods for the manipulation of whole mycobacterial cells are described in detail. Specifically, the concentration of spent culture filtrate media is described in order to permit separation of soluble, secreted proteins; several discrete separation techniques, including precipitation of protein mixtures with ammonium sulfate and separation of proteins by hydrophobic chromatography are also provided. Similarly, the generation of whole cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol enriched proteins is described. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided, as well as a separation technique (isoelectric focusing) that can be applied to separate hydrophobic proteins. Lastly, two commonly used analytical techniques, in-gel digestion of proteins for LC-MS and analysis of intact proteins by MALDI-ToF MS, are provided for rapid analysis of discrete proteins within subcellular or chromatographic fractions. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins. In addition, a number of these methods may be applied to extract and analyze mycobacterial proteins from cell lines and host derived samples.

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains one of the most prevalent human pathogens in the world. Knowledge regarding the bacilli's physiology as well as its mechanisms of virulence, immunogenicity, and pathogenesis has increased greatly in the last three decades. However, the function of about one-quarter of the Mtb coding genome and the precise activity and protein networks of most of the Mtb proteins are still unknown. Protein mass spectrometry and a new interest in research toward the field of functional proteomics have given a new light to the study of this bacillus and will be the focus of this review. We will also discuss new perspectives in the proteomics field, in particular targeted mass spectrometry methods and their potential applications in TB research and discovery.

Tuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. Specifically, there is no generally accepted early confirmative diagnosis protocol available for TBM. Various Mycobacterium tuberculosis antigens are now recognized as potential markers for diagnosis of TBM. However, their presence remains questionable, and many of these antigens are reported in the blood but not in the cerebrospinal fluid (CSF). This study identifies a specific protein marker in CSF which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% (n = 5) of confirmed and 90% (n = 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa band was excised from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Partially purified proteins from CSF samples of TBM were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. The antigen 85 (Ag 85) complex was detected in CSF of TBM patients by indirect ELISA using antibodies against Ag 85 complex. The results of this study showed the 30-kDa protein band contained MTB proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B), both members of the Ag85 complex. This was also confirmed by using immunotechniques such as indirect ELISA and the dot immunobinding assay. Detection of Ag85 complex was observed in CSF of 89% (71 out of 80) of suspected TBM patients that were 30-kDa protein positive. The observed 30-kDa protein in the CSF is comprised of the MTB Ag85 complex. This protein was earlier reported to be present in the blood of patients with extra-central nervous system tuberculosis. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option for the early and confirmatory diagnosis of TBM.

Trends in increased tuberculosis infection and a fatality rate of approximately 23% have necessitated the search for alternative biomarkers using newly developed postgenomic approaches. Here we provide a systematic analysis of Mycobacterium tuberculosis (Mtb) by directly profiling its gene products. This analysis combines high-throughput proteomics and computational approaches to elucidate the globally expressed complements of the three subcellular compartments (the cell wall, membrane, and cytosol) of Mtb. We report the identifications of 1044 proteins and their corresponding localizations in these compartments. Genome-based computational and metabolic pathways analyses were performed and integrated with proteomics data to reconstruct response networks. From the reconstructed response networks for fatty acid degradation and lipid biosynthesis pathways in Mtb, we identified proteins whose involvements in these pathways were not previously suspected. Furthermore, the subcellular localizations of these expressed proteins provide interesting insights into the compartmentalization of these pathways, which appear to traverse from cell wall to cytoplasm. Results of this large-scale subcellular proteome profile of Mtb have confirmed and validated the computational network hypothesis that functionally related proteins work together in larger organizational structures.

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.

MHC class II (MHC-II)-restricted CD4(+) T cells are essential for control of Mycobacterium tuberculosis infection. This report describes the identification and purification of LprG (Rv1411c) as an inhibitor of primary human macrophage MHC-II Ag processing. LprG is a 24-kDa lipoprotein found in the M. tuberculosis cell wall. Prolonged exposure (>16 h) of human macrophages to LprG resulted in marked inhibition of MHC-II Ag processing. Inhibition of MHC-II Ag processing was dependent on TLR-2. Short-term exposure (<6 h) to LprG stimulated TLR-2-dependent TNF-alpha production. Thus, LprG can exploit TLR-2 signaling to inhibit MHC-II Ag processing in human macrophages. Inhibition of MHC-II Ag processing by mycobacterial lipoproteins may allow M. tuberculosis, within infected macrophages, to avoid recognition by CD4(+) T cells.

Proteomics has been widely applied to develop two-dimensional polyacrylamide gel electrophoresis maps and databases, evaluate gene expression profiles under different environmental conditions, assess global changes associated with specific mutations, and define drug targets of bacterial pathogens. When coupled to immunological assays, proteomics may also be used to identify B-cell and T-cell antigens within complex protein mixtures. This chapter describes the proteomic approaches developed by our laboratories to accelerate the antigen discovery program for Mycobacterium tuberculosis. As presented or with minor modifications, these techniques may be universally applied to other bacterial pathogens or used to identify bacterial proteins possessing other immunological properties.