David Pires

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. Tuberculosis (TB) remains a worldwide health problem with 8 million new cases diagnosed and more than 1 million deaths per year, as reported by the World Health Organization 1. The emergence of multi-(MDR) or extensively drug-resistant (XDR) strains of M. tuberculosis (MTB), the etiologic agent of TB, has brought renewed attention to the dangers of TB spread with the reports estimating 450,000 new cases of MDR-TB annually 1. New strategies that, synergistically fight the disease through both antibiotic treatment and by enhancing the natural ability of the immune system to tackle the pathogen, might facilitate improved MTB clearance and thereby reduce the probability of generating resistant strains. One of the first encounters of the immune system with the pathogen begins in the lungs where macrophages internalize the bacteria 2. These cells are usually able to destroy bacteria upon phagocytosis and exposure to oxida-tive stress at an early stage, and subsequent acidification of the bacteria-containing phagosome upon fusion with late endosomes and lysosomes; there, the bacteria still encounter a toxic environment characterized mainly by the activity of proteolytic and lipolytic enzymes 3. These events will lead to pathogen destruction and processing of its antigens to be presented to lymphocytes through the class II antigen presentation machinery. Pathogenic myco-bacteria, however, impair this process by blocking phagosome maturation and consequent fusion with late endo-somes and lysosomes, avoiding contact with their degradative enzymes 4–7. Despite this capacity, there is evidence that a fraction of phagosomes still become fully mature to process and present mycobacteria antigens to lympho-cytes 8,9. In addition, the arrest of phagosome maturation by MTB may be overcome by macrophage activation through exposure to pro-inflammatory cytokines 10–12 and signalling lipids 13–15 ,or through activation of other cellular processes such as autophagy and apoptosis 10,16–18 , leading to the digestion of the pathogen. Collectively, this infers an important role for lysosomal effectors.

Several medicinal plants are traditionally used in Mozambique to treat tuberculosis and related symptoms.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.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 mc2 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 1H NMR spectroscopic characterizations.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 (IC50 < 4 μg/mL), the other three plant extracts showed moderate to none toxicity. Based on 1H 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.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.Extracts of Maerua edulis, Securidaca longepedunculata, Tabernaemontana elegans and Zanthoxylum capense were found to have considerable antimycobacterial activity.

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.

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, chlorproma-zine, 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 isonia-zid 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 overex-pressed 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 antitu-berculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of

s u m m a r y 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 hostepathogen interactions. Understanding these findings and its implications could reveal new opportunities for designing better diagnostic tools, therapies and more effective vaccines.

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.

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. Tuberculosis (TB) remains a worldwide health problem with 8 million new cases diagnosed and more than 1 million deaths per year, as reported by the World Health Organization 1. The emergence of multi-(MDR) or extensively drug-resistant (XDR) strains of M. tuberculosis (MTB), the etiologic agent of TB, has brought renewed attention to the dangers of TB spread with the reports estimating 450,000 new cases of MDR-TB annually 1. New strategies that, synergistically fight the disease through both antibiotic treatment and by enhancing the natural ability of the immune system to tackle the pathogen, might facilitate improved MTB clearance and thereby reduce the probability of generating resistant strains. One of the first encounters of the immune system with the pathogen begins in the lungs where macrophages internalize the bacteria 2. These cells are usually able to destroy bacteria upon phagocytosis and exposure to oxida-tive stress at an early stage, and subsequent acidification of the bacteria-containing phagosome upon fusion with late endosomes and lysosomes; there, the bacteria still encounter a toxic environment characterized mainly by the activity of proteolytic and lipolytic enzymes 3. These events will lead to pathogen destruction and processing of its antigens to be presented to lymphocytes through the class II antigen presentation machinery. Pathogenic myco-bacteria, however, impair this process by blocking phagosome maturation and consequent fusion with late endo-somes and lysosomes, avoiding contact with their degradative enzymes 4–7. Despite this capacity, there is evidence that a fraction of phagosomes still become fully mature to process and present mycobacteria antigens to lympho-cytes 8,9. In addition, the arrest of phagosome maturation by MTB may be overcome by macrophage activation through exposure to pro-inflammatory cytokines 10–12 and signalling lipids 13–15 ,or through activation of other cellular processes such as autophagy and apoptosis 10,16–18 , leading to the digestion of the pathogen. Collectively, this infers an important role for lysosomal effectors.