Modulating mycobacterial envelope integrity for antibiotic synergy with benzothiazoles.

Developing effective tuberculosis drugs is hindered by mycobacteria's intrinsic antibiotic resistance because of their impermeable cell envelope. Using benzothiazole compounds, we aimed to increase mycobacterial cell envelope permeability and weaken the defenses of Mycobacterium marinum, serving as a model for Mycobacterium tuberculosis Initial hit, BT-08, significantly boosted ethidium bromide uptake, indicating enhanced membrane permeability. It also demonstrated efficacy in the M. marinum-zebrafish embryo infection model and M. tuberculosis-infected macrophages. Notably, BT-08 synergized with established antibiotics, including vancomycin and rifampicin. Subsequent medicinal chemistry optimization led to BT-37, a non-toxic and more potent derivative, also enhancing ethidium bromide uptake and maintaining synergy with rifampicin in infected zebrafish embryos. Mutants of M. marinum resistant to BT-37 revealed that MMAR_0407 (Rv0164) is the molecular target and that this target plays a role in the observed synergy and permeability. This study introduces novel compounds targeting a new mycobacterial vulnerability and highlights their cooperative and synergistic interactions with existing antibiotics.

Uncovering the roles of Mycobacterium tuberculosis melH in redox and bioenergetic homeostasis: implications for antitubercular therapy.

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3, a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD+, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. IMPORTANCE: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

In vitro activity of SPR719 against Mycobacterium ulcerans, Mycobacterium marinum and Mycobacterium chimaera.

Nontuberculosis mycobacterial (NTM) infections are increasing in prevalence across the world. In many cases, treatment options for these infections are limited. However, there has been progress in recent years in the development of new antimycobacterial drugs. Here, we investigate the in vitro activity of SPR719, a novel aminobenzimidazole antibiotic and the active form of the clinical-stage compound, SPR720, against several isolates of Mycobacterium ulcerans, Mycobacterium marinum and Mycobacterium chimaera. We show that SPR719 is active against these NTM species with a MIC range of 0.125-4 µg/ml and that this compares favorably with the commonly utilized antimycobacterial antibiotics, rifampicin and clarithromycin. Our findings suggest that SPR720 should be further evaluated for the treatment of NTM infections.

Glucocorticoid-Induced Exacerbation of Mycobacterial Infection Is Associated With a Reduced Phagocytic Capacity of Macrophages.

Glucocorticoids are effective drugs for treating immune-related diseases, but prolonged therapy is associated with an increased risk of various infectious diseases, including tuberculosis. In this study, we have used a larval zebrafish model for tuberculosis, based on Mycobacterium marinum (Mm) infection, to study the effect of glucocorticoids. Our results show that the synthetic glucocorticoid beclomethasone increases the bacterial burden and the dissemination of a systemic Mm infection. The exacerbated Mm infection was associated with a decreased phagocytic activity of macrophages, higher percentages of extracellular bacteria, and a reduced rate of infected cell death, whereas the bactericidal capacity of the macrophages was not affected. The inhibited phagocytic capacity of macrophages was associated with suppression of the transcription of genes involved in phagocytosis in these cells. The decreased bacterial phagocytosis by macrophages was not specific for Mm, since it was also observed upon infection with Salmonella Typhimurium. In conclusion, our results show that glucocorticoids inhibit the phagocytic activity of macrophages, which may increase the severity of bacterial infections like tuberculosis.

Zn2+ Intoxication of Mycobacterium marinum during Dictyostelium discoideum Infection Is Counteracted by Induction of the Pathogen Zn2+ Exporter CtpC.

Macrophages use diverse strategies to restrict intracellular pathogens, including either depriving the bacteria of (micro)nutrients such as transition metals or intoxicating them via metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of Mycobacterium tuberculosis (Mtb), and its hosts. We use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn2+ during M. marinum infection. We show that M. marinum senses toxic levels of Zn2+ and responds by upregulating one of its isoforms of the Zn2+ efflux transporter CtpC. Deletion of ctpC (MMAR_1271) leads to growth inhibition in broth supplemented with Zn2+ as well as reduced intracellular growth. Both phenotypes were fully rescued by constitutive ectopic expression of the Mtb CtpC orthologue demonstrating that MMAR_1271 is the functional CtpC Zn2+ efflux transporter in M. marinum Infection leads to the accumulation of Zn2+ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn2+ efflux pumps ZntA and ZntB. In cells lacking ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn2+ into the MCV, carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn2+ than in wild-type cells, suggesting restriction by other Zn2+-mediated mechanisms. Absence of CtpC further epistatically attenuates the intracellular proliferation of M. marinum in zntA and zntB KO cells, confirming that mycobacteria face noxious levels of Zn2+IMPORTANCE Microelements are essential for the function of the innate immune system. A deficiency in zinc or copper results in an increased susceptibility to bacterial infections. Zn2+ serves as an important catalytic and structural cofactor for a variety of enzymes including transcription factors and enzymes involved in cell signaling. But Zn2+ is toxic at high concentrations and represents a cell-autonomous immunity strategy that ensures killing of intracellular bacteria in a process called zinc poisoning. The cytosolic and lumenal Zn2+ concentrations result from the balance of import into the cytosol via ZIP influx transporters and efflux via ZnT transporters. Here, we show that Zn2+ poisoning is involved in restricting Mycobacterium marinum infections. Our study extends observations during Mycobacterium tuberculosis infection and explores for the first time how the interplay of ZnT transporters affects mycobacterial infection by impacting Zn2+ homeostasis.

Evaluation of the Anti-Mycobacterial Activity of Newly Synthesized (S)-N-(3-(2-Fluoro-4'-(2-Amino-4-Thiazolyl)Biphenyl-4-Yl)-2-Oxo-1,3-Oxazolidie-5-Ylmethyl) Acetamide Derivative in Vitro and in Mycobacterium marinum-Infected Zebrafish.

Through our previous work, we have identified that novel oxazolidinone structures, the biaryloxazolidinone analogues containing a hydrazone moiety, act as promising antibacterial agents against gram-positive bacterial strains. Based on this active structure, in this study, we synthesized a series of novel oxazolidinones and determined their anti-mycobacterial activities in vitro and in Mycobacterium marinum-infected zebrafish. The in vitro anti-mycobacterial assay demonstrated that all of the synthesized compounds have potent efficacy against both H37Rv and clinical mycobacterial isolates. Among all the generated active agents, (S)-N-(3-(2-fluoro-4'-(2-amino-4-thiazolyl)biphenyl-4-yl)-2-oxo-1,3-oxazolidie-5-ylmethyl)acetamide (compound 7), whose in vitro MIC was 10-fold lower than that of linezolid, showed the strongest bactericidal effects, with ~2.2-log reduction of M. marinum load in zebrafish at 10 mg/kg dosage. Other novel oxazolidinones, compounds 9, 12, 16, and 21, exhibited reduction range of 1.1-1.8 log against M. marinum and displayed better efficacy than linezolid. Our results indicate that these identified compounds have the potential to be further developed as novel anti-mycobacterial agents.

Spreading of a mycobacterial cell-surface lipid into host epithelial membranes promotes infectivity.

Several virulence lipids populate the outer cell wall of pathogenic mycobacteria. Phthiocerol dimycocerosate (PDIM), one of the most abundant outer membrane lipids, plays important roles in both defending against host antimicrobial programs and in evading these programs altogether. Immediately following infection, mycobacteria rely on PDIM to evade Myd88-dependent recruitment of microbicidal monocytes which can clear infection. To circumvent the limitations in using genetics to understand virulence lipids, we developed a chemical approach to track PDIM during Mycobacterium marinum infection of zebrafish. We found that PDIM's methyl-branched lipid tails enabled it to spread into host epithelial membranes to prevent immune activation. Additionally, PDIM's affinity for cholesterol promoted this phenotype; treatment of zebrafish with statins, cholesterol synthesis inhibitors, decreased spreading and provided protection from infection. This work establishes that interactions between host and pathogen lipids influence mycobacterial infectivity and suggests the use of statins as tuberculosis preventive therapy by inhibiting PDIM spread.

Solid lipid nanoparticles containing anti-tubercular drugs attenuate the Mycobacterium marinum infection.

The present study aimed to formulate anti-tubercular drugs (Rifampicin, Isoniazid and Pyrazinamide) loaded solid lipid nanoparticles (ATDs-SLNs) using microemulsion technique for oral administration. Central composite designed (CCD) was applied to study the effect of stearic acid (X1), Compritol® 888 ATO (X2) and equal ratio of poloxamer 188: sodium taurocholate (% w/w) (X3) on particle size, zeta potential and entrapment efficiency. The optimised formulation (SLN8) was found to be spherical in shape with mean particle size 187.9 ± 10.73 nm and zeta potential -47.4 mV. The maximum percentage entrapment of RIF, INH and PYZ in the optimised formulation was found to be 86.40 ± 0.274, 83.84 ± 0.269 and 81.43 ± 0.576, respectively. The in-vitro drug release study demonstrated that the release of drug from SLNs was slow in comparison to marketed formulation and pure ATDs. Cytotoxicity of the ATDs-SLNs was studied on murine macrophage cell line (RAW 264.7) using modified MTT assay demonstrated two folds growth inhibition of M. marinum as compared to standard antitubercular drugs. Overall, the developed SLNs may be considered as a promising anti-mycobacterial nano-drug, providing a new direction to the tuberculosis clinics.

Early cell-autonomous accumulation of neutral lipids during infection promotes mycobacterial growth.

Lipids represent an important source of nutrition for infecting mycobacteria, accumulating within the necrotic core of granulomas and present in foamy macrophages associated with mycobacterial infection. In order to better understand the timing, process and importance of lipid accumulation, we developed methods for direct in vivo visualization and quantification of this process using the zebrafish-M. marinum larval model of infection. We find that neutral lipids accumulate cell-autonomously in mycobacterium-infected macrophages in vivo during early infection, with detectable levels of accumulation by two days post-infection. Treatment with ezetimibe, an FDA-approved drug, resulted in decreased levels of free cholesterol and neutral lipids, and a reduction of bacterial growth in vivo. The effect of ezetimibe in reducing bacterial growth was dependent on the mce4 operon, a key bacterial determinant of lipid utilization. Thus, in vivo, lipid accumulation can occur cell-autonomously at early timepoints of mycobacterial infection, and limitation of this process results in decreased bacterial burden.

Quantification of Natural Growth of Two Strains of Mycobacterium Marinum for Translational Antituberculosis Drug Development.

The zebrafish infected with Mycobacterium marinum (M. marinum) is an attractive tuberculosis disease model, showing similar pathogenesis to Mycobacterium tuberculosis (M. tuberculosis) infections in humans. To translate pharmacological findings from this disease model to higher vertebrates, a quantitative understanding of the natural growth of M. marinum in comparison to the natural growth of M. tuberculosis is essential. Here, the natural growth of two strains of M. marinum, E11 and MUSA , is studied over an extended period using an established model-based approach, the multistate tuberculosis pharmacometric (MTP) model, for comparison to that of M. tuberculosis. Poikilotherm-derived strain E11 and human-derived strain MUSA were grown undisturbed up to 221 days and viability of cultures (colony forming unit (CFU)/mL) was determined by plating at different time points. Nonlinear mixed effects modeling using the MTP model quantified the bacterial growth, the transfer among fast, slow, and non-multiplying states, and the inoculi. Both strains showed initial logistic growth, reaching a maximum after 20-25 days for E11 and MUSA , respectively, followed by a decrease to a new plateau. Natural growth of both E11 and MUSA was best described with Gompertz growth functions. For E11, the inoculum was best described in the slow-multiplying state, for MUSA in the fast-multiplying state. Natural growth of E11 was most similar to that of M. tuberculosis, whereas MUSA showed more aggressive growth behavior. Characterization of natural growth of M. marinum and quantitative comparison with M. tuberculosis brings the zebrafish tuberculosis disease model closer to the quantitative translational pipeline of antituberculosis drug development.

Functional Inhibition of Host Histone Deacetylases (HDACs) Enhances in vitro and in vivo Anti-mycobacterial Activity in Human Macrophages and in Zebrafish.

The rapid and persistent increase of drug-resistant Mycobacterium tuberculosis (Mtb) infections poses increasing global problems in combatting tuberculosis (TB), prompting for the development of alternative strategies including host-directed therapy (HDT). Since Mtb is an intracellular pathogen with a remarkable ability to manipulate host intracellular signaling pathways to escape from host defense, pharmacological reprogramming of the immune system represents a novel, potentially powerful therapeutic strategy that should be effective also against drug-resistant Mtb. Here, we found that host-pathogen interactions in Mtb-infected primary human macrophages affected host epigenetic features by modifying histone deacetylase (HDAC) transcriptomic levels. In addition, broad spectrum inhibition of HDACs enhanced the antimicrobial response of both pro-inflammatory macrophages (Mϕ1) and anti-inflammatory macrophages (Mϕ2), while selective inhibition of class IIa HDACs mainly decreased bacterial outgrowth in Mϕ2. Moreover, chemical inhibition of HDAC activity during differentiation polarized macrophages into a more bactericidal phenotype with a concomitant decrease in the secretion levels of inflammatory cytokines. Importantly, in vivo chemical inhibition of HDAC activity in Mycobacterium marinum-infected zebrafish embryos, a well-characterized animal model for tuberculosis, significantly reduced mycobacterial burden, validating our in vitro findings in primary human macrophages. Collectively, these data identify HDACs as druggable host targets for HDT against intracellular Mtb.

Aryl Hydrocarbon Receptor Modulation by Tuberculosis Drugs Impairs Host Defense and Treatment Outcomes.

Antimicrobial resistance in tuberculosis (TB) is a public health threat of global dimension, worsened by increasing drug resistance. Host-directed therapy (HDT) is an emerging concept currently explored as an adjunct therapeutic strategy for TB. One potential host target is the ligand-activated transcription factor aryl hydrocarbon receptor (AhR), which binds TB virulence factors and controls antibacterial responses. Here, we demonstrate that in the context of therapy, the AhR binds several TB drugs, including front line drugs rifampicin (RIF) and rifabutin (RFB), resulting in altered host defense and drug metabolism. AhR sensing of TB drugs modulates host defense mechanisms, notably impairs phagocytosis, and increases TB drug metabolism. Targeting AhR in vivo with a small-molecule inhibitor increases RFB-treatment efficacy. Thus, the AhR markedly impacts TB outcome by affecting both host defense and drug metabolism. As a corollary, we propose the AhR as a potential target for HDT in TB in adjunct to canonical chemotherapy.

Evaluation of in silico designed inhibitors targeting MelF (Rv1936) against Mycobacterium marinum within macrophages.

We recently identified inhibitors targeting Mycobacterium marinum MelF (Rv1936) by in silico analysis, which exhibited bacteriostatic/bactericidal activity against M. marinum and M. tuberculosis in vitro. Herein, we evaluated the effect of best four inhibitors (# 5175552, # 6513745, # 5255829, # 9125618) obtained from the ChemBridge compound libraries, on intracellular replication and persistence of bacteria within IFN-γ activated murine RAW264.7 and human THP-1 macrophages infected with M. marinum. Inhibitors # 5175552 and # 6513745 significantly reduced (p < 0.05) the intracellular replication of bacilli during day 7 post-infection (p.i.) within RAW264.7 and THP-1 macrophages infected at multiplicity of infection (MOI) of ~1.0. These observations were substantiated by electron microscopy, which revealed the protective effect of # 5175552 in clearing the bacilli inside murine macrophages. Strikingly, # 6513745 displayed synergism with isoniazid against M. marinum in murine macrophages, whereas # 5175552 significantly suppressed (p < 0.05) the persistent bacilli during day 10-14 p.i. in infected RAW264.7 and THP-1 macrophages (MOI of ~ 0.1). Moreover, # 5175552 and # 6513745 were non-cytotoxic to host macrophages at both 1X and 5X MIC. Further validation of these inhibitors against M. tuberculosis-infected macrophages and animal models has potential for development as novel anti-tubercular agents.

Cyclipostins and Cyclophostin Analogues as Multitarget Inhibitors That Impair Growth of Mycobacterium abscessus.

Twelve new Cyclophostin and Cyclipostins analogues (CyC19-30) were synthesized, thus extending our series to 38 CyCs. Their antibacterial activities were evaluated against four pathogenic mycobacteria (Mycobacterium abscessus, Mycobacterium marinum, Mycobacterium bovis BCG, and Mycobacterium tuberculosis) and two Gram negative bacteria. The CyCs displayed very low toxicity toward host cells and were only active against mycobacteria. Importantly, several CyCs were active against extracellular M. abscessus (CyC17/CyC18ß/CyC25/CyC26) or intramacrophage residing mycobacteria (CyC7(α,ß)/CyC8(α,ß)) with minimal inhibitory concentrations (MIC50) values comparable to or better than those of amikacin or imipenem, respectively. An activity-based protein profiling combined with mass spectrometry allowed identification of the potential target enzymes of CyC17/CyC26, mostly being involved in lipid metabolism and/or in cell wall biosynthesis. Overall, these results strengthen the selective activity of the CyCs against mycobacteria, including the most drug-resistant M. abscessus, through the cumulative inhibition of a large number of Ser- and Cys-enzymes participating in key physiological processes.

NaCl triggers the CRP-dependent increase of cAMP in Mycobacterium tuberculosis.

The second messenger 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) has been shown to be involved in the regulation of many biological processes ranging from carbon catabolite repression in bacteria to cell signalling in eukaryotes. In mycobacteria, the role of cAMP and the mechanisms utilized by the bacterium to adapt to and resist immune and pharmacological sterilization remain poorly understood. Among the stresses encountered by bacteria, ionic and non-ionic osmotic stresses are among the best studied. However, in mycobacteria, the link between ionic osmotic stress, particularly sodium chloride, and cAMP has been relatively unexplored. Using a targeted metabolic analysis combined with stable isotope tracing, we show that the pathogenic Mycobacterium tuberculosis but not the opportunistic pathogen Mycobacterium marinum nor the non-pathogenic Mycobacterium smegmatis responds to NaCl stress via an increase in intracellular cAMP levels. We further showed that this increase in cAMP is dependent on the cAMP receptor protein and in part on the threonine/serine kinase PnkD, which has previously been associated with the NaCl stress response in mycobacteria.

Thrombocyte Inhibition Restores Protective Immunity to Mycobacterial Infection in Zebrafish.

BACKGROUND: Infection-induced thrombocytosis is a clinically important complication of tuberculosis infection. Recent studies have highlighted the utility of aspirin as a host-directed therapy modulating the inflammatory response to infection but have not investigated the possibility that the effect of aspirin is related to an antiplatelet mode of action. METHODS: In this study, we utilize the zebrafish-Mycobacterium marinum model to show mycobacteria drive host hemostasis through the formation of granulomas. Treatment of infected zebrafish with aspirin markedly reduced mycobacterial burden. This effect is reproduced by treatment with platelet-specific glycoprotein IIb/IIIa inhibitors demonstrating a detrimental role for infection-induced thrombocyte activation. RESULTS: We find that the reduction in mycobacterial burden is dependent on macrophages and granuloma formation, providing the first in vivo experimental evidence that infection-induced platelet activation compromises protective host immunity to mycobacterial infection. CONCLUSIONS: Our study illuminates platelet activation as an efficacious target of aspirin, a widely available and affordable host-directed therapy candidate for tuberculosis.

The cyclic nitroxide antioxidant 4-methoxy-TEMPO decreases mycobacterial burden in vivo through host and bacterial targets.

Tuberculosis is a chronic inflammatory disease caused by persistent infection with Mycobacterium tuberculosis. The rise of antibiotic resistant strains necessitates the design of novel treatments. Recent evidence shows that not only is M. tuberculosis highly resistant to oxidative killing, it also co-opts host oxidant production to induce phagocyte death facilitating bacterial dissemination. We have targeted this redox environment with the cyclic nitroxide derivative 4-methoxy-TEMPO (MetT) in the zebrafish-M. marinum infection model. MetT inhibited the production of mitochondrial ROS and decreased infection-induced cell death to aid containment of infection. We identify a second mechanism of action whereby stress conditions, including hypoxia, found in the infection microenvironment appear to sensitise M. marinum to killing by MetT both in vitro and in vivo. Together, our study demonstrates MetT inhibited the growth and dissemination of M. marinum through host and bacterial targets.

Antimycobacterial activity in a single-cell infection assay of ellagitannins from Combretum aculeatum and their bioavailable metabolites.

ETHNOPHARMACOLOGICAL RELEVANCE: The water decoction of Combretum aculeatum aerial parts is traditionally used in Senegal to treat tuberculosis (TB). The extract shows significant antimycobacterial activity in a validated single-cell infection assay. AIM OF THE STUDY: The main aim of this study was to identify the antimycobacterial compounds in the water decoction of Combretum aculeatum. Since the traditional preparations are used orally, a bioactivity assessment of the possible bioavailable human metabolites was also performed. MATERIALS AND METHODS: The Combretum aculeatum water decoction extract was first fractionated by flash chromatography. The fractions were submitted to an antibiotic assay against Mycobacterium marinum and to a single-cell infection assay involving Acanthamoeba castellanii as a host. Using these approaches, it was possible to correlate the antimycobacterial activity with two zones of the chromatogram. In parallel with this liquid chromatography (LC)-based activity profiling, high-resolution mass spectrometry (UHPLC-HRMS/MS) revealed the presence of ellagitannin (Et) derivatives in the active zones of the chromatogram. Isolation of the active compounds was performed by preparative chromatography. The structures of the isolated compounds were elucidated by nuclear magnetic resonance (NMR). Additionally, the main human metabolites of commercially available Ets were biologically evaluated in a similar manner. RESULTS: The in vitro bioassay-guided isolation of the Combretum aculeatum water extract led to the identification of three Ets (1-3) and ellagic acid (4). The major compounds 2 and 3 (α- and ß-punicalagin, respectively), exhibited anti-infective activity with an IC50 of 51.48 µM. In view of the documented intestinal metabolism of these compounds, some metabolites, namely, urolithin A (5), urolithin B (6) and urolithin D (7), were investigated for their antimycobacterial activity in the two assays. Urolithin D (7) exhibited the strongest anti-infective activity, with an IC50 of 345.50 µM, but this was moderate compared to the positive control rifampin (IC50 of 6.99 µM). The compounds assayed had no observable cytotoxicity towards the amoeba host cells at concentrations lower than 200 µg/mL. CONCLUSION: The observed antimycobacterial properties of the traditional water decoction of Combretum aculeatum might be related to the activity of Ets derivatives (1-3) and their metabolites, such as ellagic acid (4) and urolithin D (7). Despite the relatively weak activity of these metabolites, the high consumption of tannins achieved by taking the usual traditional decoction doses should lead to an important increase in the plasmatic concentrations of these active and bioavailable metabolites. These results support to some extent the traditional use of Combretum aculeatum to treat tuberculosis.

Intelectin 3 is dispensable for resistance against a mycobacterial infection in zebrafish (Danio rerio).

Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response.

Mycobacterium tuberculosis virulence inhibitors discovered by Mycobacterium marinum high-throughput screening.

High-throughput screening facilities do not generally support biosafety level 3 organisms such as Mycobacterium tuberculosis. To discover not only antibacterials, but also virulence inhibitors with either bacterial or host cell targets, an assay monitoring lung fibroblast survival upon infection was developed and optimized for 384-plate format and robotic liquid handling. By using Mycobacterium marinum as surrogate organism, 28,000 compounds were screened at biosafety level 2 classification, resulting in 49 primary hits. Exclusion of substances with unfavourable properties and known antimicrobials resulted in 11 validated hits of which 7 had virulence inhibiting properties and one had bactericidal effect also in wild type Mycobacterium tuberculosis. This strategy to discover virulence inhibitors using a model organism in high-throughput screening can be a valuable tool for other researchers working on drug discovery against tuberculosis and other biosafety level 3 infectious agents.