Extracellular Mycobacterium Tuberculosis Research Articles

Uptake‐independent killing of macrophages by extracellular Mycobacterium tuberculosis aggregates

Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular Mtb replication induces the death of the infected macrophages and the release of bacterial aggregates. Here, we show that these aggregates can evade phagocytosis by killing macrophages in a contact‐dependent but uptake‐independent manner. We use time‐lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytosolic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb ESX‐1 secretion system, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface‐exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that blocking ESX‐1‐mediated secretion of the EsxA/EsxB virulence factors does not eliminate the uptake‐independent killing of macrophages and that the 50‐kDa isoform of the ESX‐1‐secreted protein EspB can mediate killing in the absence of EsxA/EsxB secretion. Treatment with an ESX‐1 inhibitor reduces uptake‐independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti‐phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung.

The synthesis and in vitro biological evaluation of novel fluorinated tetrahydrobenzo[j]phenanthridine-7,12-diones against Mycobacterium tuberculosis

Tuberculosis (TB) still has a major impact on public health. In order to efficiently eradicate this life-threatening disease, the exploration of novel anti-TB drugs is of paramount importance. As part of our program to design new 2-azaanthraquinones with anti-mycobacterial activity, various “out-of-plane” tetrahydro- and octahydrobenzo[j]phenanthridinediones were synthesized. In this study, the scaffold of the most promising hits was further optimized in an attempt to improve the bioactivity and to decrease enzymatic degradation. The rudiment bio-evaluation of a small library of fluorinated tetrahydrobenzo[j]phenanthridine-7,12-dione derivatives indicated no significant improvement of the bio-activity against intracellular and extracellular Mycobacterium tuberculosis (Mtb). Though, the derivatives showed an acceptable toxicity against J774A.1 macrophages and early signs of genotoxicity were absent. All derivatives showed to be metabolic stabile in the presence of both phase I and phase II murine or human microsomes. Finally, the onset of reactive oxygen species within Mtb after exposure to the derivatives was measured by electron paramagnetic resonance (EPR). Results showed that the most promising fluorinated derivative is still a possible candidate for the subversive inhibition of mycothione reductase.

Intracellular PD Modelling (PDi) for the Prediction of Clinical Activity of Increased Rifampicin Dosing.

Increasing rifampicin (RIF) dosages could significantly reduce tuberculosis (TB) treatment durations. Understanding the pharmacokinetic-pharmacodynamics (PK–PD) of increasing RIF dosages could inform clinical regimen selection. We used intracellular PD modelling (PDi) to predict clinical outcomes, primarily time to culture conversion, of increasing RIF dosages. PDi modelling utilizes in vitro-derived measurements of intracellular (macrophage) and extracellular Mycobacterium tuberculosis sterilization rates to predict the clinical outcomes of RIF at increasing doses. We evaluated PDi simulations against recent clinical data from a high dose (35 mg/kg per day) RIF phase II clinical trial. PDi-based simulations closely predicted the observed time-to-patient culture conversion status at eight weeks (hazard ratio: 2.04 (predicted) vs. 2.06 (observed)) for high dose RIF-based treatments. However, PDi modelling was less predictive of culture conversion status at 26 weeks for high-dosage RIF (99% predicted vs. 81% observed). PDi-based simulations indicate that increasing RIF beyond 35 mg/kg/day is unlikely to significantly improve culture conversion rates, however, improvements to other clinical outcomes (e.g., relapse rates) cannot be ruled out. This study supports the value of translational PDi-based modelling in predicting culture conversion rates for antitubercular therapies and highlights the potential value of this platform for the improved design of future clinical trials.

MICs and MBCs of levofloxacin, clarithromycin, and KRM-1648 for Mycobacterium tuberculosis and M. avium complex residing in MONO-MAC-6 human macrophage-like cell and A-549 human type II alveolar epithelial cell lines

In this study, we determined the MICs and MBCs of levofloxacin (LVFX), clarithromycin (CAM), and KRM-1648 (KRM) for Mycobacterium tuberculosis (MTB) strain Kurono and M. avium complex (MAC) strain N-444 residing in MONO-MAC-6 human macrophage-like cells (MM6-M phi s) and A-549 human type II alveolar epithelial cells (A-549 cells). First, the MICs of LVFX for MTB replicating in MM6-M phi s (1 microgram/ml) and A-549 cells (2 micrograms/ml) were 4 to 8 times higher than its MICs for extracellular MTB growing in 7HSF medium. In contrast, the MICs of CAM for intracellular MTB residing in both the cells (2-4 micrograms/ml) were 4 to 8 times less than its MICs for extracellular MTB organisms. On the other hand, the MICs of KRM for extracellular MTB were nearly the same as its MICs for intracellular MTB residing in both types of the cells. Secondly, the MICs of LVFX and CAM for extracellular MAC were not significantly different from their MICs for intracellular MAC residing in MM6-M phi s and A-549 cells. The MIC of KRM for MAC residing in A-549 cells was 0.25 microgram/ml, and this value was 32 times higher than its MIC for MAC residing in MM6-M phi s (0.008 microgram/ml). Thirdly, the MBCs of test drugs for intracellular MTB and MAC residing in both types of the cells were somewhat longer than their MBCs for extracellular organisms. These findings indicate that, in the case of pulmonary infections with MTB or MAC, the therapeutic efficacy of a given drug, especially KRM, is more or less influenced by the bacterial location in the host lung tissues where the mycobacterial pathogens survive and multiply, i.e., either alveolar M phi s, type II alveolar epithelial cells, or surrounding environment.

A rapid and sensitive HPLC–MS method for the detection of plasma and cellular rifampicin

Rifampicin is active against both intracellular and extracellular Mycobacterium tuberculosis. The ability to measure rifampicin drug concentrations in both plasma and in cells may be useful in evaluating the suitability of dosage regimens for populations and individuals. Here a novel simple, precise and accurate method for the quantification of rifampicin in both cells and plasma is reported. Sample proteins were precipitated with acetonitrile containing the internal standard and then diluted with water. Aliquots of supernatant were then injected into the HPLC–MS system for chromatographic separation and detection. Rifampicin calibration curves encompassed concentrations from 100 to 12,800 ng/mL. Intra- and inter-assay precision and accuracy were determined using low, medium and high concentration quality control samples and was found to be within 10% in all cases. Rifampicin concentrations were found to be unaffected by freeze–thaw cycles, but were significantly affected by heat-inactivation (58 °C, 40 min). This assay was successfully utilised to determine the pharmacokinetic profile of rifampicin in plasma and peripheral blood mononuclear cells (PBMC) in 8 tuberculosis patients receiving rifampicin over an 8 h period.

Anti- Mycobacterium tuberculosis Activities of New Fluoroquinolones in Combination with Other Antituberculous Drugs

Studies were undertaken in order to assess the anti- Mycobacterium tuberculosis (MTB) activities of newly developed fluoroquinolones in combination with other antituberculous drugs. A new C-8-methoxyl fluoroquinolone, gatifloxacin (GFLX), and a new C-8-chloro fluoroquinolone, sitafloxacin (STFX), in combination with other drugs were examined for their activities against extracellular growing MTB organisms and those replicating in RAW264.7 macrophages (RAW-M phis s). STFX but not GFLX potentiated the activities of rifampin and rifalazil against extracellular MTB. Both GFLX and STFX exhibited combined activities against intramacrophage MTB, when used in combination with rifampin, rifalazil, isoniazid, pyrazinamide, ethambutol, streptomycin, or clofazimine. Although the observed combined effects varied to some extent from case to case depending on drug combinations, the present findings suggest the usefulness of these new fluoroquinolones in multi-drug regimens for tuberculosis patients.

Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vgamma9/Vdelta2 T lymphocytes.

Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

An Antimicrobial Activity of Cytolytic T Cells Mediated by Granulysin

Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.