Is targeting dysregulation in apoptosis splice variants in Mycobacterium tuberculosis (MTB) host interactions and splicing factors resulting in immune evasion by MTB strategies a possibility?

Mycobacterium tuberculosis (Mtb), is one of the foremost organisms causing mortality in humans, and has been for most of human history. When faced with an infection the human immune system is ordinarily very competent in killing both extracellular and intracellular bacilli. However, Mtb is able to evade the host immune system and is even able to establish a persistent infectious reservoir by “hiding” in the immune cells of the host. While the mechanisms by which the bacteria accomplishes this are not fully understood, it is known that the bacterium can subvert cellular processes in cells such as macrophages that prevent the lysis of the bacteria or the cell undergoing apoptosis. They are also able to interfere with immune cell signalling. One of the greatest effects that Mtb has is too alter the transcriptome of the macrophage. An easy way for the bacterium to accomplish this is to alter the alternative splicing patterns of the host. This can lead to a large change in the population of different protein isoforms, some of which have very different functions when compared to the original protein. At the same time the long history of Mtb infecting humans have led to specific immune reactions that occur in the host immune system in order to fight the infection. Many of these specific reactions involve new isoforms of host defence proteins. In this way the human host can use alternate splicing to create new isoforms of immune- related proteins that are more effective in defending against Mtb.The Medical Research Council of South Africahttps://http//www.elsevier.com/locate/tubehj2021Immunolog

Assessment of the pro-inflammatory activity of water sampled from major water treatment facilities in the greater Pretoria region

Notwithstanding direct detection of microbial/viral pathogens or their associated toxins, the quality of drinking water may also be evaluated according to its pro-inflammatory potential. In this latter setting, contamination with pathogens or their products is determined according to the magnitude of activation of blood-derived immune/inflammatory cells following exposure to test water samples in vitro, usually by monitoring the synthesis of pro-inflammatory cytokines. The primary objective of the current study was to apply this procedure to evaluate the pro-inflammatory potential of water sampled at entry, as well as at various stages of treatment, from 3 major water treatment facilities in the greater Pretoria region, viz., the Daspoort, Hartbeespoort, and Rietvlei Water Treatment Facilities. Control water samples included domestic tap water, bottled water from a commercial source, and distilled water. Peripheral blood mononuclear leukocytes (MNL) were isolated from the blood of healthy, adult, human volunteers (n=3), enumerated, suspended in tissue culture medium RPMI 1640 containing antibiotics at a concentration of 1x106/mℓ, and exposed to the various water samples (10%) for 18 h at 37°C. Following incubation, the cell-free supernatants were assayed for the cytokine, interleukin-6 (IL-6), using a quantitative, sandwich, enzyme immunoassay procedure. The mean values for the untreated control system and for a positive control system exposed to bacterial endotoxin (120 ng/mℓ) were 153.5 ±17 and 1 561 ±30 pg/mℓ, respectively (p= 0.03). The production of IL-6 was unaffected following exposure of MNL to the control water samples. However, inlet water sampled from all three facilities, especially Hartbeespoort, resulted in significant activation of production of IL-6 by MNL, which declined with progressive treatment, consistent with removal of pro-inflammatory contaminants. Surprisingly, however, a rebound in pro-inflammatory activity was evident in outlet water sampled from Hartbeespoort. In conclusion, the results of the current study appear to support the efficiency of water treatment procedures at the Daspoort and Rietvlei Treatment Facilities, while confirming the usefulness of IL-6-based assays as adjuncts to conventional water quality testing procedures.http://www.wrc.org.zaam201

Effects of cigarette smoke condensate on growth and biofilm formation by Mycobacterium tuberculosis

BACKGROUND AND OBJECTIVES : Cigarette smoke (CS) is a major risk factor contributing to the burden of tuberculosis. Little is known, however, about the effects of CS exposure on growth and persistence of Mycobacterium tuberculosis (Mtb) organisms. This issue has been addressed in the current study, which is focused on the effects of cigarette smoke condensate (CSC) on the growth and viability of Mtb planktonic and biofilm-forming cultures. MATERIALS AND METHODS : The planktonic and biofilm-forming cultures were prepared in Middlebrook 7H9 and Sauton broth media, respectively, using Mtb strain, H37Rv. The effects of CSC at concentrations of 0.05-3.12 mg/L on growth, biofilm formation and structure were evaluated using microplate Alamar Blue assay, spectrophotometric procedure and scanning electron microscopy (SEM), respectively. Involvement of reactive oxygen species in CSC-mediated biofilm formation was investigated by including catalase in biofilm-forming cultures. RESULTS : CSC did not affect the growth of planktonic bacteria, but rather led to a statistically significant increase in biofilm formation at concentrations of 0.4-3.12 mg/L, as well as in the viability of biofilm-forming bacteria at CSC concentrations of 0.2-1.56 mg/L. SEM confirmed an agglomerated biofilm matrix and irregular bacterial morphology in CSC-treated biofilms. Inclusion of catalase caused significant attenuation of CSC-mediated augmentation of biofilm formation by Mtb, implying involvement of oxidative stress. These findings demonstrate that exposure of Mtb to CSC resulted in increased biofilm formation that appeared to be mediated, at least in part, by oxidative stress, while no effect on planktonic cultures was observed. CONCLUSION : smoking related augmentation of biofilm formation by Mtb may contribute to persistence of the pathogen, predisposing to disease reactivation and counteracting the efficacy of antimicrobial chemotherapy.South African National Research Foundationhttp://www.hindawi.com/journals/bmripm2020ImmunologyMedical Microbiolog

Mechanisms of action and therapeutic efficacies of the lipophilic antimycobacterial agents clofazimine and bedaquiline

Drug-resistant (DR)-tuberculosis (TB) is the major challenge confronting the global tuberculosis (TB) control programme, necessitating treatment with second-line anti- TB drugs, often with limited therapeutic efficacy. This scenario has resulted in the inclusion of Group 5 antibiotics in various therapeutic regimens, two of which promise to impact significantly on the outcome of the therapy of DR-TB. These are the “re-purposed” riminophenazine, clofazimine, and the recently approved diarylquinoline, bedaquiline. Although they differ structurally, both of these lipophilic agents share cationic amphiphilic properties, which enable them to target and inactivate essential ion transporters in the outer membrane of Mycobacterium tuberculosis. In the case of bedaquiline, the primary target is the key respiratory chain enzyme, F1/F0-ATPase, while clofazimine is less selective, apparently inhibiting several targets, which may underpin the extremely low level of resistance to this agent. This review is focused on similarities and differences between clofazimine and bedaquiline, specifically in respect of molecular mechanisms of antimycobacterial action, targeting of quiescent and metabolically-active organisms, therapeutic efficacy in the clinical setting of DR-TB, resistance mechanisms, pharmacodynamics, pharmacokinetics, and adverse events.http://jac.oxfordjournals.org2018-02-28hb2017ImmunologyMedical Microbiolog

Effects of clofazimine on planktonic and biofilm growth of Mycobacterium tuberculosis and Mycobacterium smegmatis

Mycobacteria form lipid-rich biofilms that restrict the efficacy of antimicrobial chemotherapy, possibly necessitating the use of lipophilic antibiotics. In the current study, the activity of one such agent, clofazimine, against Mycobacterium tuberculosis and Mycobacterium smegmatis planktonic cells and biofilms was investigated. Minimum inhibitory concentrations (MICs) of clofazimine were determined for planktonic cultures, whilst minimum bactericidal concentrations (MBCs) were determined for planktonic, biofilm-producing and biofilm-encased organisms using standard bacteriological proce- dures. The effects of clofazimine on biofilm formation and the stability of pre-formed biofilm were measured using a crystal violet-based spectrophotometric procedure. In the case of M. smegmatis, clofazimine was found to be active against planktonic phase (MICs and MBCs of 2.5 mg/L and >20 mg/L, respectively) and biofilm-producing organisms (MBC of 2.5 mg/L); clofazimine demonstrated greater activity against M. tuberculosis, corresponding values of 0.06, 5 and 0.3 mg/L. Although clofazimine inhibited biofilm production both by M. tuberculosis and M. smegmatis (P < 0.05 at 0.07 mg/L and 0.3 mg/L, respectively) and appeared to reduce the pre-formed M. tuberculosis biofilm, addition of antimicrobial agent to pre-existing biofilm matrices failed to kill biofilm-encased organisms. In conclusion, clofazimine is more effective against M. tuberculosis than against M. smegmatis, exhibiting bactericidal activity both for actively growing and slowly replicating bacilli but not for non-replicating organisms of both species.South African Medical Research Council.http://www.elsevier.com/locate/jgar2016-05-31hb201

Expression of the genes encoding the Trk and Kdp potassium transport systems of mycobacterium tuberculosis during growth in vitro

Two potassium (K+)-uptake systems, Trk and Kdp, are operative in Mycobacterium tuberculosis (Mtb), but the environmental factors triggering their expression have not been determined.The current study has evaluated the expression of these genes in the Mtb wildtype and a trk-gene knockout strain at various stages of logarithmic growth in relation to extracellular K+ concentrations and pH. In both strains, mRNA levels of the K+-uptake encoding genes were relatively low compared to those of the housekeeping gene, sigA, at the early- and mid-log phases, increasing during late-log. Increased gene expression coincided with decreased K+ uptake in the context of a drop in extracellular pH and sustained high extracellular K+ concentrations. In an additional series of experiments, the pH of the growth medium was manipulated by the addition of 1N HCl/NaOH. Decreasing the pH resulted in reductions in both membrane potential and K+ uptake in the setting of significant induction of genes encoding both K+ transporters. These observations are consistent with induction of the genes encoding the active K+ transporters of Mtb as a strategy to compensate for loss of membrane potential-driven uptake of K+ at low extracellular pH. Induction of these genes may promote survival in the acidic environments of the intracellular vacuole and granuloma.Part of the work presented herein has been presented at the South African TB Conference, Durban, South Africa, 2014 (http://www.tbconference.co.za/).The South African Medical Research Council and National Research Foundation (Grant no. 87649).http://www.hindawi.com/journals/bmri/am201

Investigation of the anti-mycobacterial mechanism of action of 7-methyljuglone

Although the naphthoquinone, 7-methyljuglone (7-MJ), is active against Mycobacterium tuberculosis (MTB) in vitro, neither the cellular site nor mechanism of anti-mycobacterial action of this agent has been identified. The primary objective of the current study was to investigate the mycobacterial outer membrane as a potential target of 7-MJ by measuring the effects of this agent (0.023 - 1.5 mg/L) on microbial ATP levels and uptake of K+. Methods: Bioluminescence and radiometric (uptake of 86Rb+) procedures were used to assay microbial ATP levels and K+ trans-port respectively. Results: Exposure of MTB (strain H37Rv) to 7-MJ for 60 min resulted in dose-related decreases in both microbial ATP levels and uptake of 86Rb+ which achieved statistical significance (P < 0.05) at concentrations of 0.4 and 0.1 mg/L respectively. Conclusions: These observations are compatible with the mycobacterial membrane as being the putative site of action of 7-MJ, targeting microbial energy metabolism and K+ transport.The National Research Foundation and the Medical Research Council, Pretoria, South Africa.http://www.SciRP.org/journal/ojrd)am2016ImmunologyPlant Scienc

Clofazimine : current status and future prospects

Clofazimine, a lipophilic riminophenazine antibiotic, possesses both antimycobacterial and anti-inflammatory activities. However, its efficacy has been demonstrated only in the treatment of leprosy, not in human tuberculosis, despite the fact that this agent is impressively active in vitro against multidrug-resistant strains of Mycobacterium tuberculosis. Recent insights into novel targets and mechanisms of antimicrobial and antiinflammatory activity coupled with the acquisition of innovative drug delivery technologies have, however, rekindled interest in clofazimine as a potential therapy for multidrug- and extensively multidrug-resistant tuberculosis in particular, as well as several autoimmune diseases. The primary objective of this review is to critically evaluate these recent developments and to assess their potential impact on improving the therapeutic efficacy and versatility of clofazimine.The South African Medical Research Council. Research into the spray-dried formulations of clofazimine is supported by the K-RITH collaborative grant (Howard Hughes Medical Institute and the University of KwaZulu-Natal).http://jac.oxfordjournals.org

Manganese promotes increased formation of hydrogen peroxide by activated human macrophages and neutrophils in vitro

Although pro-inflammatory mechanisms have been implicated in the pathogenesis of manganese (Mn²⁺)-related neurological and respiratory disorders, relatively little is known about the potential of this metal to interact pro-oxidatively with human phagocytes. The primary objective of the current study was to investigate the effects of Mn²⁺ as MnCl₂ (0.5-100 µM) on the generation of the reactive oxygen species (ROS), superoxide, hydrogen peroxide (H₂O₂), and hypohalous acids by isolated human blood neutrophils and monocyte-derived macrophages following activation of these cells with the chemotactic tripeptide, FMLP (1 µM), or the phorbol ester, PMA (25 ng/mL). Generation of ROS was measured using the combination of oxygen consumption, lucigenin/luminol-enhanced chemiluminescence, spectrofluorimetric detection of oxidation of 2,7-dichlorodihydrofluorescein, radiometric assessment of myeloperoxidase (MPO)-mediated protein iodination, release of MPO by ELISA, and spectrophotometric measurement of nitrite formation. Treatment of activated neutrophils with either FMLP or PMA resulted in significantly decreased reactivity of superoxide in the setting of increased formation of H₂O₂ and MPO-mediated iodination, with no detectable effects on either oxygen consumption or MPO release. Similar effects of the metal with respect to superoxide reactivity and H₂O₂ formation were observed with activated macrophages, while generation of NO was unaffected. Taken together with the findings of experiments using cell-free ROS-generating systems, these observations are compatible with a mechanism whereby Mn²⁺, by acting as a superoxide dismutase mimetic, increases the formation of H₂O₂ by activated phagocytes. If operative in vivo, this mechanism may contribute to the toxicity of Mn²⁺http://www.tandfonline.com/loi/iiht20hb2016Immunolog

Submission for special issue : The role of platelet activation in the pathophysiology of HIV, tuberculosis, and pneumococcal disease. Bedaquiline suppresses ADP-mediated activation of human platelets in vitro via interference with phosphatidylinositol 3-kinase

Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concerns remain about the cardiotoxic potential of this agent, albeit by unexplored mechanisms. Accordingly, we have investigated augmentation of the reactivity of human platelets in vitro as a potential mechanism of bedaquiline-mediated cardiotoxicity. Platelet-rich plasma (PRP) or isolated cells prepared from the blood of healthy, adult humans were treated with bedaquiline (0.625–10 μg/ml), followed by activation with adenosine 5’-diphosphate (ADP), thrombin or the thromboxane A2 receptor agonist (U46619). Expression of platelet CD62P (P-selectin), platelet aggregation, Ca2+ fluxes and phosphorylation of Akt1 were measured using flow cytometry, spectrophotometry, fluorescence spectrometry, and by ELISA procedures, respectively. Exposure to bedaquiline caused dose-related inhibition of ADP-activated, but not thrombin- or U46619-activated, expression of CD62P by platelets, achieving statistical significance at a threshold concentration of 5 μg/ml and was paralleled by inhibition of aggregation and Ca2+ mobilization. These ADP-selective inhibitory effects of bedaquiline on platelet activation were mimicked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), implicating PI3-K as being a common target of both agents, a contention that was confirmed by the observed inhibitory effects of bedaquiline on the phosphorylation of Akt1 following activation of platelets with ADP. These apparent inhibitory effects of bedaquiline on the activity of PI3-K may result from the secondary cationic amphiphilic properties of this agent. If operative in vivo, these anti-platelet effects of bedaquiline may contribute to ameliorating the risk of TB-associated cardiovascular disease, but this remains to be explored in the clinical setting.The National Health Laboratory Research Trust of South Africa and the National Research Foundation of South Africa.http://www.frontiersin.org/Immunologyam2022HaematologyInternal Medicin