David Pires

Antimycobacterial evaluation and preliminary phytochemical investigation of selected medicinal plants traditionally used in Mozambique.

ETHNOPHARMACOLOGICAL RELEVANCE Several medicinal plants are traditionally used in Mozambique to treat tuberculosis and related symptoms. AIMS OF THE STUDY It was aimed to assess the in vitro antimycobacterial activity of crude extracts from fifteen medicinal plants and to reveal main classes of compounds which may account for the activity of extracts. METHODS AND MATERIALS The plant materials were sequentially extracted by n-hexane, dichloromethane, ethyl acetate, and 70% ethanol. Decoction of each plant material was also prepared according to traditional use. Broth microdilution method was employed to screen extracts against two mycobacterial species: Mycobacterium smegmatis ATCC 607 and Mycobacterium tuberculosis H37Rv. The extracts with minimum inhibitory concentration(s) (MIC) below 125 μg/mL were considered active and further tested against different mycobacterial species and strains, namely Mycobacterium tuberculosis H37Ra, Mycobacterium bovis BCG ATCC 35734, Mycobacterium smegmatis mc(2) 155, Mycobacterium avium DSM 44156 and DSM 44157. Cytotoxic effect was evaluated against human macrophages from the monocytic THP-1 cells. Main classes of compounds in these active extracts were proposed from their (1)H NMR spectroscopic characterizations. RESULTS n-Hexane extracts of Maerua edulis and Securidaca longepedunculata, ethyl acetate extract of Tabernaemontana elegans and dichloromethane extract of Zanthoxylum capense were found to possess considerable activity against Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with MIC 15.6-62.5 μg/mL. Tabernaemontana elegans ethyl acetate extract displayed strong activity against Mycobacterium tuberculosis H37Rv (MIC 15.6 μg/mL). Except for Tabernaemontana elegans ethyl acetate extract which presented potent cytotoxic effects in THP-1 cells (IC(50)<4 μg/mL), the other three plant extracts showed moderate to none toxicity. Based on (1)H NMR spectroscopic analysis, major components in both Maerua edulis and Securidaca longepedunculata n-hexane extracts were linear chain unsaturated fatty acids. Zanthoxylum capense dichloromethane extract contained more complex constituents (mostly phenolic compounds). In the most potent extract, Tabernaemontana elegans ethyl acetate extract, the prominent compounds were identified as indole alkaloids. CONCLUSIONS The pronounced antimycobacterial activity of the medicinal plants Maerua edulis, Securidaca longepedunculata, Zanthoxylum capense, and Tabernaemontana elegans suggested that they might provide compounds which could be potential anti-TB drug leads.

Zanthoxylum capense constituents with antimycobacterial activity against Mycobacterium tuberculosis in vitro and ex vivo within human macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE Zanthoxylum capense Thunb. (Rutaceae) is a medicinal plant traditionally used in Mozambique to treat tuberculosis. AIMS OF THE STUDY The main aim of the study was to find antimycobacterial lead compounds from Zanthoxylum capense. Another goal was to provide scientific validation for the use of this plant in traditional medicine. METHODS AND MATERIALS By bioassay-guided fractionation, 16 compounds were isolated and screened for their in vitro antimycobacterial activity against two different strains of Mycobacterium tuberculosis. Their in vitro cytotoxicity to human THP-1 macrophages was also assessed. The compounds with favourable selectivity index values (SI>10) were further investigated for their ability to inhibit the growth of Mycobacterium tuberculosis H37Rv in an intracellular macrophage model of infection. RESULTS The best results were obtained for a benzophenanthridine alkaloid, decarine (1), and an N-isobutylamide, N-isobutyl-(2E,4E)-2,4-tetradecadienamide (15), which showed high activity against Mycobacterium tuberculosis H37Rv (MIC of 1.6 μg/ml), and a low macrophage cytotoxicity (IC50>60 μg/ml), indicating considerable selective activity. The benzophenanthridine alkaloid 6-acetonyldihydronitidine (6) revealed cytotoxicity (IC50 1.7 μg/ml), despite the determined MIC of 6.2-12.5 μg/ml. In infected macrophages, decarine (1) was able to reduce bacterial survival by almost two log units at a concentration of 6.2 μg/ml 5 days post-drug exposure. Compound 15 exhibited an intermediate activity at drug concentrations ranging from 6.2 to 25 μg/ml. CONCLUSIONS The high antimycobacterial activity of decarine found, both in vitro and ex vivo against mycobacteria, and the low cytotoxicity towards human macrophages indicate that it may be valuable as a lead scaffold for the development of anti-TB drugs.

Actin-binding protein regulation by microRNAs as a novel microbial strategy to modulate phagocytosis by host cells: the case of N-Wasp and miR-142-3p

Mycobacterium tuberculosis (Mtb) is a successful intracellular pathogen that thrives in macrophages (Mφs). There is a need to better understand how Mtb alters cellular processes like phagolysosome biogenesis, a classical determinant of its pathogenesis. A central feature of this bacterias strategy is the manipulation of Mφ actin. Here, we examined the role of microRNAs (miRNAs) as a potential mechanism in the regulation of actin-mediated events leading to phagocytosis in the context of mycobacteria infection. Given that non-virulent Mycobacterium smegmatis also controls actin filament assembly to prolong its intracellular survival inside host cells, we performed a global transcriptomic analysis to assess the modulation of miRNAs upon M. smegmatis infection of the murine Mφ cell line, J774A.1. This approach identified miR-142-3p as a key candidate to be involved in the regulation of actin dynamics required in phagocytosis. We unequivocally demonstrate that miR-142-3p targets N-Wasp, an actin-binding protein required during microbial challenge. A gain-of-function approach for miR-142-3p revealed a down-regulation of N-Wasp expression accompanied by a decrease of mycobacteria intake, while a loss-of-function approach yielded the reciprocal increase of the phagocytosis process. Equally important, we show Mtb induces the early expression of miR-142-3p and partially down-regulates N-Wasp protein levels in both the murine J774A.1 cell line and primary human Mφs. As proof of principle, the partial siRNA-mediated knock down of N-Wasp resulted in a decrease of Mtb intake by human Mφs, reflected in lower levels of colony-forming units (CFU) counts over time. We therefore propose the modulation of miRNAs as a novel strategy in mycobacterial infection to control factors involved in actin filament assembly and other early events of phagolysosome biogenesis.

Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis

Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their resistance pattern. This work highlights the potential value ion channel blockers as adjuvants of tuberculosis chemotherapy, in particular for the development of new therapeutic strategies, with strong potential for treatment shortening against drug susceptible and resistant forms of tuberculosis. Medicinal chemistry studies are now needed to improve the properties of these compounds, increasing their M. tuberculosis efflux-inhibition and killing-enhancement activity and reduce their toxicity for humans, therefore optimizing their potential for clinical usage.

Thioridazine in PLGA nanoparticles reduces toxicity and improves rifampicin therapy against mycobacterial infection in zebrafish

Abstract Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo.

Immunomodulating microRNAs of mycobacterial infections

MicroRNAs are a class of small non-coding RNAs that have emerged as key regulators of gene expression at the post-transcriptional level by sequence-specific binding to target mRNAs. Some microRNAs block translation, while others promote mRNA degradation, leading to a reduction in protein availability. A single miRNA can potentially regulate the expression of multiple genes and their encoded proteins. Therefore, miRNAs can influence molecular signalling pathways and regulate many biological processes in health and disease. Upon infection, host cells rapidly change their transcriptional programs, including miRNA expression, as a response against the invading microorganism. Not surprisingly, pathogens can also alter the host miRNA profile to their own benefit, which is of major importance to scientists addressing high morbidity and mortality infectious diseases such as tuberculosis. In this review, we present recent findings on the miRNAs regulation of the host response against mycobacterial infections, providing new insights into host-pathogen interactions. Understanding these findings and its implications could reveal new opportunities for designing better diagnostic tools, therapies and more effective vaccines.

Role of Cathepsins in Mycobacterium tuberculosis Survival in Human Macrophages.

Cathepsins are proteolytic enzymes that function in the endocytic pathway, especially in lysosomes, where they contribute directly to pathogen killing or indirectly, by their involvement in the antigen presentation pathways. Mycobacterium tuberculosis (MTB) is a facultative intracellular pathogen that survives inside the macrophage phagosomes by inhibiting their maturation to phagolysosomes and thus avoiding a low pH and protease-rich environment. We previously showed that mycobacterial inhibition of the proinflammatory transcription factor NF-κB results in impaired delivery of lysosomal enzymes to phagosomes and reduced pathogen killing. Here, we elucidate how MTB also controls cathepsins and their inhibitors, cystatins, at the level of gene expression and proteolytic activity. MTB induced a general down-regulation of cathepsin expression in infected cells, and inhibited IFNγ-mediated increase of cathepsin mRNA. We further show that a decrease in cathepsins B, S and L favours bacterial survival within human primary macrophages. A siRNA knockdown screen of a large set of cathepsins revealed that almost half of these enzymes have a role in pathogen killing, while only cathepsin F coincided with MTB resilience. Overall, we show that cathepsins are important for the control of MTB infection, and as a response, it manipulates their expression and activity to favour its intracellular survival.

Esters of Pyrazinoic Acid Are Active against Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Other Naturally Resistant Mycobacteria In Vitro and Ex Vivo within Macrophages

ABSTRACT Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257–263, 2009, http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.

The Mycobacterium tuberculosis outer membrane channel protein CpnT confers susceptibility to toxic molecules

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis, is protected from toxic solutes by an effective outer membrane permeability barrier. Recently, we showed that the outer membrane channel protein CpnT is required for efficient nutrient uptake by M. tuberculosis and Mycobacterium bovis BCG. In this study, we found that the cpnT mutant of M. bovis BCG is more resistant than the wild type to a large number of drugs and antibiotics, including rifampin, ethambutol, clarithromycin, tetracycline, and ampicillin, by 8- to 32-fold. Furthermore, the cpnT mutant of M. bovis BCG was 100-fold more resistant to nitric oxide, a major bactericidal agent required to control M. tuberculosis infections in mice. Thus, CpnT constitutes the first outer membrane susceptibility factor in slow-growing mycobacteria. The dual functions of CpnT in uptake of nutrients and mediating susceptibility to toxic molecules are reflected in macrophage infection experiments: while loss of CpnT was detrimental for M. bovis BCG in macrophages that enable bacterial replication, presumably due to inadequate nutrient uptake, it conferred a survival advantage in macrophages that mount a strong bactericidal response. Importantly, the cpnT gene showed a significantly higher density of nonsynonymous mutations in drug-resistant clinical M. tuberculosis strains, indicating that CpnT is under selective pressure in human tuberculosis and/or during chemotherapy. Our results indicate that the CpnT channel constitutes an outer membrane gateway controlling the influx of nutrients and toxic molecules into slow-growing mycobacteria. This study revealed that reducing protein-mediated outer membrane permeability might constitute a new drug resistance mechanism in slow-growing mycobacteria.

Mycobacterium tuberculosis Modulates miR-106b-5p to Control Cathepsin S Expression Resulting in Higher Pathogen Survival and Poor T-Cell Activation

The success of tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), relies on the ability to survive in host cells and escape to immune surveillance and activation. We recently demonstrated that Mtb manipulation of host lysosomal cathepsins in macrophages leads to decreased enzymatic activity and pathogen survival. In addition, while searching for microRNAs (miRNAs) involved in posttranscriptional gene regulation during mycobacteria infection of human macrophages, we found that selected miRNAs such as miR-106b-5p were specifically upregulated by pathogenic mycobacteria. Here, we show that miR-106b-5p is actively manipulated by Mtb to ensure its survival in macrophages. Using an in silico prediction approach, we identified miR-106b-5p with a potential binding to the 3′-untranslated region of cathepsin S (CtsS) mRNA. We demonstrated by luminescence-based methods that miR-106b-5p indeed targets CTSS mRNA resulting in protein translation silencing. Moreover, miR-106b-5p gain-of-function experiments lead to a decreased CtsS expression favoring Mtb intracellular survival. By contrast, miR-106b-5p loss-of-function in infected cells was concomitant with increased CtsS expression, with significant intracellular killing of Mtb and T-cell activation. Modulation of miR-106b-5p did not impact necrosis, apoptosis or autophagy arguing that miR-106b-5p directly targeted CtsS expression as a way for Mtb to avoid exposure to degradative enzymes in the endocytic pathway. Altogether, our data suggest that manipulation of miR-106b-5p as a potential target for host-directed therapy for Mtb infection.