Extracellular vesicles in mycobacteria: new findings in biogenesis, host-pathogen interactions, and diagnostics.

Since the discovery of extracellular vesicles (EVs) in mycobacterial species 15 years back, we have learned that this phenomenon is conserved in the Mycobacterium genus and has critical roles in bacterial physiology and host-pathogen interactions. Mycobacterium tuberculosis (Mtb), the tuberculosis (TB) causative agent, produces EVs both in vitro and in vivo including a diverse set of biomolecules with demonstrated immunomodulatory effects. Moreover, Mtb EVs (MEVs) have been shown to possess vaccine properties and carry biomarkers with diagnostic capacity. Although information on MEV biogenesis relative to other bacterial species is scarce, recent studies have shed light on how MEVs originate and are released to the extracellular space. In this minireview, we discuss past and new information about the vesiculogenesis phenomenon in Mtb, including biogenesis, MEV cargo, aspects in the context of host-pathogen interactions, and applications that could help to develop effective tools to tackle the disease.

Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.

Stepwise pathogenic evolution of Mycobacterium abscessus.

Although almost all mycobacterial species are saprophytic environmental organisms, a few, such as Mycobacterium tuberculosis, have evolved to cause transmissible human infection. By analyzing the recent emergence and spread of the environmental organism M. abscessus through the global cystic fibrosis population, we have defined key, generalizable steps involved in the pathogenic evolution of mycobacteria. We show that epigenetic modifiers, acquired through horizontal gene transfer, cause saltational increases in the pathogenic potential of specific environmental clones. Allopatric parallel evolution during chronic lung infection then promotes rapid increases in virulence through mutations in a discrete gene network; these mutations enhance growth within macrophages but impair fomite survival. As a consequence, we observe constrained pathogenic evolution while person-to-person transmission remains indirect, but postulate accelerated pathogenic adaptation once direct transmission is possible, as observed for M. tuberculosis Our findings indicate how key interventions, such as early treatment and cross-infection control, might restrict the spread of existing mycobacterial pathogens and prevent new, emergent ones.

Polymeric nanobiotics as a novel treatment for mycobacterial infections.

Mycobacterium tuberculosis (Mtb) remains a major challenge to global health, made worse by the spread of multi-drug resistance. Currently, the efficacy and safety of treatment is limited by difficulties in achieving and sustaining adequate tissue antibiotic concentrations while limiting systemic drug exposure to tolerable levels. Here we show that nanoparticles generated from a polymer-antibiotic conjugate ('nanobiotics') deliver sustained release of active drug upon hydrolysis in acidic environments, found within Mtb-infected macrophages and granulomas, and can, by encapsulation of a second antibiotic, provide a mechanism of synchronous drug delivery. Nanobiotics are avidly taken up by infected macrophages, enhance killing of intracellular Mtb, and are efficiently delivered to granulomas and extracellular mycobacterial cords in vivo in an infected zebrafish model. We demonstrate that isoniazid (INH)-derived nanobiotics, alone or with additional encapsulation of clofazimine (CFZ), enhance killing of mycobacteria in vitro and in infected zebrafish, supporting the use of nanobiotics for Mtb therapy and indicating that nanoparticles generated from polymer-small molecule conjugates might provide a more general solution to delivering co-ordinated combination chemotherapy.

Evolutionary history of tuberculosis shaped by conserved mutations in the PhoPR virulence regulator.

Although the bovine tuberculosis (TB) agent, Mycobacterium bovis, may infect humans and cause disease, long-term epidemiological data indicate that humans represent a spill-over host in which infection with M. bovis is not self-maintaining. Indeed, human-to-human transmission of M. bovis strains and other members of the animal lineage of the tubercle bacilli is very rare. Here, we report on three mutations affecting the two-component virulence regulation system PhoP/PhoR (PhoPR) in M. bovis and in the closely linked Mycobacterium africanum lineage 6 (L6) that likely account for this discrepancy. Genetic transfer of these mutations into the human TB agent, Mycobacterium tuberculosis, resulted in down-regulation of the PhoP regulon, with loss of biologically active lipids, reduced secretion of the 6-kDa early antigenic target (ESAT-6), and lower virulence. Remarkably, the deleterious effects of the phoPR mutations were partly compensated by a deletion, specific to the animal-adapted and M. africanum L6 lineages, that restores ESAT-6 secretion by a PhoPR-independent mechanism. Similarly, we also observed that insertion of an IS6110 element upstream of the phoPR locus may completely revert the phoPR-bovis-associated fitness loss, which is the case for an exceptional M. bovis human outbreak strain from Spain. Our findings ultimately explain the long-term epidemiological data, suggesting that M. bovis and related phoPR-mutated strains pose a lower risk for progression to overt human TB, with major impact on the evolutionary history of TB.

Multiple deletions in the polyketide synthase gene repertoire of Mycobacterium tuberculosis reveal functional overlap of cell envelope lipids in host-pathogen interactions.

Several specific lipids of the cell envelope are implicated in the pathogenesis of M. tuberculosis (Mtb), including phthiocerol dimycocerosates (DIM) that have clearly been identified as virulence factors. Others, such as trehalose-derived lipids, sulfolipids (SL), diacyltrehaloses (DAT) and polyacyltrehaloses (PAT), are believed to be essential for Mtb virulence, but the details of their role remain unclear. We therefore investigated the respective contribution of DIM, DAT/PAT and SL to tuberculosis by studying a collection of mutants, each with impaired production of one or several lipids. We confirmed that among those with a single lipid deficiency, only strains lacking DIM were affected in their replication in lungs and spleen of mice in comparison to the WT Mtb strain. We found also that the additional loss of DAT/PAT, and to a lesser extent of SL, increased the attenuated phenotype of the DIM-less mutant. Importantly, the loss of DAT/PAT and SL in a DIM-less background also affected Mtb growth in human monocyte-derived macrophages (hMDMs). Fluorescence microscopy revealed that mutants lacking DIM or DAT/PAT were localized in an acid compartment and that bafilomycin A1, an inhibitor of phagosome acidification, rescued the growth defect of these mutants. These findings provide evidence for DIM being dominant virulence factors that mask the functions of lipids of other families, notably DAT/PAT and to a lesser extent of SL, which we showed for the first time to contribute to Mtb virulence.

Indole and aminoimidazole moieties appear as key structural units in antiplasmodial molecules.

From a library of compounds of natural sources, a big series of molecules was chosen by random sampling to evaluate their in vitro antimalarial activity against Plasmodium falciparum and their antifungal activity against Candida sp. From 184 molecules tested, no molecules were active against Candida sp. (MIC>10µg/ml) whereas 13 clearly showed high antiplasmodial activity in vitro, with an IC(50) less than 1µg/ml against the chloroquine-resistant strain of P. falciparum FcM29-Cameroon. The molecules with the best antiplasmodial efficacy were 10-hydroxy-ellipticin (IC(50): 0.08µg/ml), tchibangensin (IC(50): 0.13µg/ml), ellipticin hydrochloride (IC(50): 0.17µg/ml), usambarensin (IC(50): 0.23µg/ml), 7S,3S-ochropposinine oxindole (IC(50): 0.25µg/ml), 3,14-dihydro-ellipticin (IC(50): 0.25µg/ml), tetrahydro-4',5',6'17-usambarensin 17S (IC(50): 0.26µg/ml), ellipticine (IC(50): 0.28µg/ml), aricin (IC(50): 0.3µg/ml), 10-methoxy-ellipticin (IC(50): 0.32µg/ml), aplysinopsin (IC(50): 0.43µg/ml), descarbomethoxydihydrogambirtannin (IC(50): 0.46µg/ml) and ochrolifuanin A (IC(50): 0.47µg/ml). Among these 13 promising molecules, all except descarbomethoxydihydrogambirtannin, ochrolifuanine A and usambarensine presented here novel biological activities since they had never been described in the literature for their antiplasmodial activity. In spite of the large diversity of the molecules which have been tested, it is interesting to note that the ones active against Plasmodium are all indole derivatives (and one is both indolic and aminoimidazolic). To find new antiplasmodial compounds, ethnopharmacological approaches studying traditional medicine treatments for malaria is largely used but random research produced here an interesting yield (7%) of new antiplasmodial hits and appears therefore complementary to the traditional medicine way.