ABSTRACT
Despite available global efforts and funding, Tuberculosis (TB) continues to affect a considerable number of patients worldwide. Policy makers and stakeholders set clear goals to reduce TB incidence and mortality, but the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) complicate the reach of these goals. Drug-resistance TB needs to be diagnosed rapidly and accurately to effectively treat patients, prevent the transmission of MDR-TB, minimise mortality, reduce treatment costs and avoid unnecessary hospitalisations. In this narrative review, we provide a comprehensive overview of laboratory methods for detecting drug resistance in MTB, focusing on phenotypic, molecular and other drug susceptibility testing (DST) techniques. We found a large variety of methods used, with the BACTEC MGIT 960 being the most common phenotypic DST and the Xpert MTB/RIF being the most common molecular DST. We emphasise the importance of integrating phenotypic and molecular DST to address issues like resistance to new drugs, heteroresistance, mixed infections and low-level resistance mutations. Notably, most of the analysed studies adhered to the outdated definition of XDR-TB and did not consider the pre-XDR definition, thus posing challenges in aligning diagnostic methods with the current landscape of TB resistance.
Subject(s)
Antitubercular Agents , Extensively Drug-Resistant Tuberculosis , Microbial Sensitivity Tests , Mycobacterium tuberculosis , Phenotype , Tuberculosis, Multidrug-Resistant , Humans , Antitubercular Agents/therapeutic use , Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Microbial Sensitivity Tests/methods , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/microbiology , Extensively Drug-Resistant Tuberculosis/diagnosis , Extensively Drug-Resistant Tuberculosis/drug therapy , Extensively Drug-Resistant Tuberculosis/microbiology , Drug Resistance, Multiple, Bacterial/genetics , Molecular Diagnostic Techniques/methods , Predictive Value of TestsABSTRACT
INTRODUCTION: Nontuberculous mycobacteria (NTM) are environmental mycobacteria which may cause pulmonary and extrapulmonary diseases. These organisms are difficult to treat due to their intrinsic drug-resistance. In Italy, no major nationwide study on NTM epidemiology and drug susceptibility was performed. METHODS: Data on the epidemiology of 7,469 NTM clinical isolates identified in Italy in 2016-2020 and on the minimum inhibitory concentrations (MICs) of 1,506 of these strains were analysed. RESULTS: Overall, 63 species were identified in 42 hospital laboratories located in 16 out of 20 regions, with Mycobacterium avium complex (MAC) being the most frequently isolated, followed by M. gordonae, M. xenopi, M. abscessus. The MICs of 12 drugs for MAC, M. xenopi, M. kansasii, M. abscessus, M. fortuitum and M. chelonae were interpreted for clinical significance (susceptible, intermediate, resistant) based on the guidelines published by the Clinical and Laboratory Standards Institute in November 2018. CONCLUSIONS: Our data are in line with other nationwide studies and may be of value for further update of microbiological and clinical guidelines.
Subject(s)
Mycobacterium Infections, Nontuberculous , Nontuberculous Mycobacteria , Humans , Mycobacterium Infections, Nontuberculous/epidemiology , Mycobacterium Infections, Nontuberculous/microbiology , Microbial Sensitivity Tests , Italy/epidemiologyABSTRACT
The lungs of tuberculosis (TB) patients contain a spectrum of granulomatous lesions, ranging from solid and well-vascularized cellular granulomas to avascular caseous granulomas. In solid granulomas, current therapy kills actively replicating (AR) intracellular bacilli, while in low-vascularized caseous granulomas the low-oxygen tension stimulates aerobic and microaerophilic AR bacilli to transit into non-replicating (NR), drug-tolerant and extracellular stages. These stages, which do not have genetic mutations and are often referred to as persisters, are difficult to eradicate due to low drug penetration inside the caseum and mycobacterial cell walls. The sputum of TB patients also contains viable bacilli called differentially detectable (DD) cells that, unlike persisters, grow in liquid, but not in solid media. This review provides a comprehensive update on drug combinations killing in vitro AR and drug-tolerant bacilli (persisters and DD cells), and sterilizing Mycobacterium tuberculosis-infected BALB/c and caseum-forming C3HeB/FeJ mice. These observations have been important for testing new drug combinations in noninferiority clinical trials, in order to shorten the duration of current regimens against TB. In 2022, the World Health Organization, following the results of one of these trials, supported the use of a 4-month regimen for the treatment of drug-susceptible TB as a possible alternative to the current 6-month regimen.
ABSTRACT
Infections caused by Mycobacterium abscessus (Mab), an environmental non-tuberculous mycobacterium, are difficult to eradicate from patients with pulmonary diseases such as cystic fibrosis and bronchiectasis even after years of antibiotic treatments. In these people, the low oxygen pressure in mucus and biofilm may restrict Mab growth from actively replicating aerobic (A) to non-replicating hypoxic (H) stages, which are known to be extremely drug-tolerant. After the exposure of Mab A and H cells to drugs, killing was monitored by measuring colony-forming units (CFU) and regrowth in liquid medium (MGIT 960) of 1-day-old A cells (A1) and 5-day-old H cells (H5). Mab killing was defined as a lack of regrowth of drug-exposed cells in MGIT tubes after >50 days of incubation. Out of 18 drugs tested, 14-day treatments with bedaquiline-amikacin (BDQ-AMK)-containing three-drug combinations were very active against A1 + H5 cells. However, drug-tolerant cells (persisters) were not killed, as shown by CFU curves with typical bimodal trends. Instead, 56-day treatments with the nitrocompounds containing combinations BDQ-AMK-rifabutin-clarithromycin-nimorazole and BDQ-AMK-rifabutin-clarithromycin-metronidazole-colistin killed all A1 + H5 Mab cells in 42 and 56 days, respectively, as shown by lack of regrowth in agar and MGIT medium. Overall, these data indicated that Mab persisters may be killed by appropriate drug combinations.
ABSTRACT
Previous studies on Escherichia coli demonstrated that sub-minimum inhibitory concentration (MIC) of fluoroquinolones induced the SOS response, increasing drug tolerance. We characterized the transcriptional response to moxifloxacin in Mycobacterium tuberculosis. Reference strain H37Rv was treated with moxifloxacin and gene expression studied by qRT-PCR. Five SOS regulon genes, recA, lexA, dnaE2, Rv3074 and Rv3776, were induced in a dose- and time-dependent manner. A range of moxifloxacin concentrations induced recA, with a peak observed at 2 × MIC (0.25 µg/mL) after 16 h. Another seven SOS responses and three DNA repair genes were significantly induced by moxifloxacin. Induction of recA by moxifloxacin was higher in log-phase than in early- and stationary-phase cells, and absent in dormant bacilli. Furthermore, in an H37Rv fluoroquinolone-resistant mutant carrying the D94G mutation in the gyrA gene, the SOS response was induced at drug concentrations higher than the mutant MIC value. The 2 × MIC of moxifloxacin determined no significant changes in gene expression in a panel of 32 genes, except for up-regulation of the relK toxin and of Rv3290c and Rv2517c, two persistence-related genes. Overall, our data show that activation of the SOS response by moxifloxacin, a likely link to increased mutation rate and persister formation, is time, dose, physiological state and, possibly, MIC dependent.
ABSTRACT
The activities of rifampin, nitazoxanide, PA-824, and sutezolid were tested against dormant Mycobacterium tuberculosis under conditions mimicking caseous granulomas (hypoxia at pH 7.3) in comparison with those of the combination rifampin-isoniazid-pyrazinamide-ethambutol (R-I-Z-E), which is used for human therapy. Mycobacterial viability was monitored by CFU and regrowth in MGIT 960. As shown by lack of regrowth in MGIT, rifampin-nitazoxanide-containing combinations, but not R-I-Z-E, killed dormant cells in 28 to 35 days. These observations might be important in designing new tuberculosis therapies.
Subject(s)
Antitubercular Agents/pharmacology , Isoniazid/pharmacology , Mycobacterium tuberculosis/drug effects , Pyrazinamide/pharmacology , Rifampin/pharmacology , Drug Combinations , Drug Therapy, Combination , Ethambutol/pharmacology , Humans , Hydrogen-Ion Concentration , Hypoxia , Microbial Sensitivity Tests , Nitroimidazoles/pharmacology , Oxazolidinones/pharmacology , Tuberculosis/microbiologyABSTRACT
Pyrazinamide (PZA) is a first-line key drug used in combination with other agents for the treatment of tuberculosis (TB). Phenotypic and molecular assays for testing susceptibility of Mycobacterium tuberculosis (Mtb) to PZA have been developed, with the assay in liquid medium at acidic pH in the Bactec MGIT 960 (M960) system being routinely used in the mycobacteriology laboratories. However, false resistance to PZA by this method was reported to occur by several investigators, mostly due to high Mtb inoculum, which may impair drug activity by increasing the pH of the medium. In this study, a revision of the literature on the issue of false resistance in the M960 PZA assay was performed. In the reports examined, all improvements of the M960 test proposed to decrease false resistant results were based on the use of reduced inoculum densities of Mtb cells, to be easily translated into laboratory practice.
Subject(s)
Antitubercular Agents/pharmacology , Microbial Sensitivity Tests/methods , Mycobacterium tuberculosis/drug effects , Pyrazinamide/pharmacology , Culture Media/chemistry , Drug Resistance, Bacterial , False Positive Reactions , Humans , Hydrogen-Ion Concentration , Mycobacterium Infections/microbiology , Reproducibility of Results , Tuberculosis, Multidrug-Resistant/microbiologyABSTRACT
A major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth in vitro. A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies. As part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium, we aimed to optimise the direct MGIA, assess repeatability and reproducibility, and harmonise the assay across different laboratories. We observed an improvement in repeatability with increased cell number and increased mycobacterial input. Furthermore, we determined that co-culturing in static 48-well plates compared with rotating 2â¯ml tubes resulted in a 23% increase in cell viability and a 500-fold increase in interferon-gamma (IFN-γ) production on average, as well as improved reproducibility between replicates, assay runs and sites. Applying the optimised conditions, we report repeatability to be <5% coefficient of variation (CV), intermediate precision to be <20% CV, and inter-site reproducibility to be <30% CV; levels within acceptable limits for a functional cell-based assay. Using relevant clinical samples, we demonstrated comparable results across two shared sample sets at three sites. Based on these findings, we have established a standardised operating procedure (SOP) for the use of the direct PBMC MGIA in TB vaccine development.
Subject(s)
Bacteriological Techniques/standards , Cryopreservation/standards , Drug Development/standards , Interferon-gamma Release Tests/statistics & numerical data , Leukocytes, Mononuclear/microbiology , Mycobacterium bovis/drug effects , Tuberculosis Vaccines/pharmacology , Cell Culture Techniques/standards , Cells, Cultured , Host-Pathogen Interactions , Humans , Interferon-gamma/metabolism , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Mycobacterium bovis/growth & development , Mycobacterium bovis/immunology , Reproducibility of ResultsSubject(s)
Emigrants and Immigrants/statistics & numerical data , Extensively Drug-Resistant Tuberculosis/epidemiology , Rifampin/therapeutic use , Tuberculosis, Multidrug-Resistant/epidemiology , Antitubercular Agents/therapeutic use , Extensively Drug-Resistant Tuberculosis/drug therapy , Humans , Italy/epidemiology , Mycobacterium tuberculosis/isolation & purification , Tuberculosis, Multidrug-Resistant/drug therapyABSTRACT
Mycobacterium tuberculosis (Mtb) is the aetiological agent of tuberculosis, the leading cause of death worldwide from a single infectious agent. Mtb is a highly adaptable human pathogen that might enter a dormant non-replicating (NR), drug-tolerant stage. Reactivation of dormant Mtb can lead to active disease. Antibiotic treatments of active and latent tuberculosis are long, complex and may fail to fully eradicate the infection. Therefore, it is imperative to identify novel compounds with new mechanisms of action active against NR bacilli. Dormant Mtb habitat is mostly thought to be the pH-neutral and hypoxic caseous granuloma. We have used the Wayne culture model to reproduce this environment and tested the activities of two DNA-targeted agents, C8-linked-pyrrolobenzodiazepine(PBD)-polyamide conjugates 1 and 2, against Mtb grown in aerobic and hypoxic conditions in both acidic and pH-neutral media. PBD 2 showed growth inhibitory activity at 5.1 µg/ml against 19-day-old hypoxic NR Mtb cultures with 1.8 log10 CFU reduction on day 21 at pH 7.3. PBD 2 was particularly effective against 5-day-old aerobic cells at pH 7.3, with CFU reduction (>6.8 log10) on day 21 at 5.1 µg/ml being identical to that of rifampin at 8 µg/ml. PBD 2 qualifies as a promising lead against aerobic and NR Mtb.
Subject(s)
Antitubercular Agents/pharmacology , Benzodiazepines/pharmacology , Isoniazid/pharmacology , Mycobacterium tuberculosis/growth & development , Nylons/pharmacology , Pyrroles/pharmacology , Rifampin/pharmacology , Tuberculosis, Pulmonary/drug therapy , Anaerobiosis , Benzodiazepines/chemistry , Cell Line , Humans , Mycobacterium tuberculosis/drug effects , Nylons/chemistry , Pyrroles/chemistrySubject(s)
Antitubercular Agents/pharmacology , Diagnostic Errors/prevention & control , Drug Resistance, Bacterial/drug effects , Microbial Sensitivity Tests/methods , Mycobacterium tuberculosis/drug effects , Pyrazinamide/pharmacology , Amidohydrolases/genetics , Drug Resistance, Bacterial/genetics , Humans , Mutation , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/genetics , Tuberculosis/diagnosis , Tuberculosis/microbiologyABSTRACT
Current tuberculosis (TB) treatment requires 6 months of combination therapy with isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol for active TB and 9 months of INH or 3 months of rifapentine (RFP) + INH for latent TB. The lungs of patients with active and latent TB contain heterogeneous mixtures of cellular and caseous granulomas harboring Mycobacterium tuberculosis bacilli ranging from actively replicating (AR) to nonreplicating (NR), phenotypically drug-resistant stages. Several in vitro models to obtain NR cells were reported, including exposure to hypoxia, nutrient starvation, acid + nitric oxide, and stationary phase. Overall, these models showed that RIF, RFP, PA-824 (PA), metronidazole (MZ), bedaquiline (BQ), and fluoroquinolones were the most active drugs against NR M. tuberculosis. In hypoxia at pH 5.8, some combinations killed AR plus NR cells, as shown by lack of regrowth in liquid media, whereas in hypoxia at pH 7.3 (the pH of the caseum), only RIF and RFP efficiently killed NR bacilli while several other drugs showed little effect. In conventional mouse models, combinations containing RFP, BQ, PA, PZA, moxifloxacin, sutezolid, linezolid, and clofazimine sterilized animals in ≤2 months, as shown by lack of viable bacilli in lung homogenates after 3 months without therapy. Drugs were less effective in C3HeB/FeJ mice forming caseous granulomas. Overall, in vitro observations and in vivo studies suggest that the search for new TB drugs could be addressed to low lipophilic molecules (e.g., new rpoB inhibitors with clogP < 3) killing NR M. tuberculosis in hypoxia at neutral pH and reaching high rates of unbound drug in the caseum.
Subject(s)
Antitubercular Agents/therapeutic use , Latent Tuberculosis/drug therapy , Mycobacterium tuberculosis/drug effects , Tuberculosis/drug therapy , Tuberculosis/microbiology , Animals , Antitubercular Agents/pharmacology , Colony Count, Microbial , Humans , Isoniazid/therapeutic use , Latent Tuberculosis/microbiology , Mice , Mycobacterium tuberculosis/physiology , Pyrazinamide/therapeutic use , Rifampin/analogs & derivatives , Rifampin/therapeutic use , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/microbiologyABSTRACT
The activities of rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide, isoniazid, amikacin, moxifloxacin, niclosamide, thioridazine, and pyrazinamide were tested against nonreplicating (dormant) Mycobacterium tuberculosis H37Rv under conditions of hypoxia at pHs 5.8 and 7.3, mimicking environments of cellular granulomas and caseous granulomas, respectively. At pH 5.8, several drugs killed dormant bacilli, with the best being rifampin and rifapentine. At pH 7.3, only rifampin and rifapentine efficiently killed dormant bacilli, while all other drugs showed little activity.
Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Rifampin/analogs & derivatives , Rifampin/pharmacology , Anaerobiosis , Hydrogen-Ion Concentration , Isonicotinic Acids/pharmacology , Microbial Sensitivity Tests , Mycobacterium tuberculosis/growth & development , Nitroimidazoles/pharmacology , Phenazines/pharmacology , Quinolines/pharmacology , Thiazoles/pharmacologyABSTRACT
The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.
Subject(s)
Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/genetics , Oxygen/metabolism , Anaerobiosis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Developmental , Mycobacterium tuberculosis/metabolism , RegulonSubject(s)
Transients and Migrants , Tuberculosis/epidemiology , Africa , Antitubercular Agents/therapeutic use , Europe , Extensively Drug-Resistant Tuberculosis/epidemiology , Geography , Humans , Italy/epidemiology , Microbial Sensitivity Tests , Mycobacterium tuberculosis , Russia , Tuberculosis, Multidrug-Resistant/epidemiology , World Health OrganizationABSTRACT
The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·(-), with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·(-)-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/metabolism , Hydroxyl Radical/metabolism , Mycobacterium tuberculosis/drug effects , Rifampin/pharmacology , Superoxides/metabolism , Antitubercular Agents/metabolism , Bacterial Proteins/chemistry , Catalase/chemistry , Cyclic N-Oxides , DNA-Directed RNA Polymerases , Electron Spin Resonance Spectroscopy , Hydroxyl Radical/chemistry , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/metabolism , Oxidative Stress , Pentetic Acid/chemistry , Protein Binding , Rifampin/metabolism , Spin Labels , Superoxide Dismutase/chemistry , Superoxides/chemistry , Thiourea/chemistryABSTRACT
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which kills about 2 million people annually. Furthermore, 2 billion people worldwide are latently infected with this organism, with 10% of them reactivating to active TB due to re-growth of nonreplicating (dormant) Mtb residing in their tissues. Because of the huge reservoir of latent TB it is important to find novel drugs/drug combinations killing dormant bacilli (microaerophiles, anaerobes and drug-tolerant persisters) surviving for decades in a wide spectrum of granulomatous lesions in the lungs of TB patients. Antibiotic treatment of drug-susceptible TB requires administration of isoniazid, rifampin, pyrazinamide, ethambutol for 2 months, followed by isoniazid and rifampin for 4 months. To avoid reactivation of dormant Mtb to active pulmonary TB, up to 9 months of treatment with isoniazid is required. Therefore, a strategy to eliminate dormant bacilli needs to be developed to shorten therapy of active and latent TB and reduce the reservoir of people with latent TB. Finding drugs with high rate of penetration into the caseous granulomas and understanding the biology of dormant bacilli and in particular of persister cells, phenotypically resistant to antibiotics, will be essential to eradicate Mtb from humans. In recent years unprecedented efforts have been done in TB drug discovery, aimed at identifying novel drugs and drug combinations killing both actively replicating and nonreplicating Mtb in vitro, in animal models and in clinical trials in humans.
ABSTRACT
Dormancy is defined as a stable but reversible nonreplicating state of Mycobacterium tuberculosis. It is currently thought that dormant M. tuberculosis (D-Mtb) is responsible for latent tuberculosis (TB) infection. Recently, D-Mtb was also shown in sputa of patients with active TB, but the capacity of D-Mtb to stimulate specific immune responses was not investigated. We observed that purified protein derivative-specific human CD4(+) T lymphocytes recognize mycobacterial Ags more efficiently when macrophages are infected with D-Mtb instead of replicating M. tuberculosis (R-Mtb). The different Ag recognition occurs even when the two forms of mycobacteria equally infect and stimulate macrophages, which secrete the same cytokine pattern and express MHC class I and II molecules at the same levels. However, D-Mtb but not R-Mtb colocalizes with mature phagolysosome marker LAMP-1 and with vacuolar proton ATPase in macrophages. D-Mtb, unlike R-Mtb, is unable to interfere with phagosome pH and does not inhibit the proteolytic efficiency of macrophages. We show that D-Mtb downmodulates the gene Rv3875 encoding for ESAT-6, which is required by R-Mtb to block phagosome maturation together with Rv3310 gene product SapM, previously shown to be downregulated in D-Mtb. Thus, our results indicate that D-Mtb cannot escape MHC class II Ag-processing pathway because it lacks the expression of genes required to block the phagosome maturation. Data suggest that switching to dormancy not only represents a mechanism of survival in latent TB infection, but also a M. tuberculosis strategy to modulate the immune response in different stages of TB.
Subject(s)
Latent Tuberculosis/immunology , Lymphocyte Activation/immunology , Mycobacterium tuberculosis/immunology , Phagosomes , T-Lymphocyte Subsets/immunology , Dendritic Cells/immunology , Humans , Immune Evasion , Latent Tuberculosis/microbiology , Latent Tuberculosis/pathology , Macrophages/immunology , Monocytes/immunology , Mycobacterium tuberculosis/growth & development , Phagosomes/immunology , Phagosomes/microbiology , T-Lymphocyte Subsets/microbiology , T-Lymphocyte Subsets/pathologyABSTRACT
The host immune response can limit Mycobacterium tuberculosis (Mtb) spreading in primary tuberculosis (TB) without eradicating all bacilli, which can persist causing latent TB infection and are responsible for reactivation TB. Persistent Mtb is confined to granulomas within phagocytes, but it is also found in other non-immune cells. We focused on fibroblasts since these cells participate to the granuloma formation and were shown to be infected in latent TB infections. We show that in vitro both Mtb and Bacille Calmette-Guérin actively replicate in human fibroblasts. Mycobacterial infection of fibroblasts causes a significant inhibition of interferon (IFN)-γ induced membrane expression of major histocompatibility complex class II molecules in these cells. The functional consequence of in vitro infection is a significant reduction of the fibroblast capacity to present peptides and soluble proteins to autologous specific CD4(+) T cell clones. Moreover, fibroblasts are capable of presenting antigen derived from the processing of heat-killed Mtb, but not from viable Mtb. Data indicate that IFN-γ treated fibroblasts are capable of presenting antigens derived from the processing of whole bacteria in addition to the capacity to present peptides and isolated proteins. Interestingly, Mtb infected fibroblasts lose this capacity, suggesting that Mtb may evade T helper immune surveillance by infecting fibroblasts.