Global spread and genetic monomorphism are hallmarks of Mycobacterium tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii, and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology, are restricted to East-Africa. Here, we sequenced and analyzed the genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5x coverage), 454/Roche (13-18x coverage) and/or Illumina DNA sequencing (45-105x coverage). We show that STB are highly recombinogenic and evolutionary early-branching, with larger genome sizes, 25-fold more SNPs, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse-infection experiments revealed that STB are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral, STB-like pool of mycobacteria by gain of persistence and virulence mechanisms and we provide genome-wide insights into the molecular events involved.

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-tohuman 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 downregulation 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.evolution | phylogeny | adaptation | zoonosis T uberculosis (TB) is caused by bacilli from the genetically compact Mycobacterium tuberculosis complex (MTBC), which gathers eight defined phylogenetic lineages in addition to the more distantly related Mycobacterium canettii group (1-3): M. tuberculosis sensu-stricto from lineages L1-L4 and L7 form a large group of human-adapted strains responsible for the vast majority of global human TB cases, whereas Mycobacterium africanum lineages (L5, L6), which are restricted to humans from West Africa, are phylogenetically linked with the eighth lineage, comprising the various animal-adapted strains, with Mycobacterium bovis as the most downstream member in the phylogeny (Fig. 1A) (4, 5). Animal strains exhibit a wide host range that includes livestock animals in close contact with humans. Episodes of bovine TB in cattle herds have been reported in 128 of 155 countries during the period 2005-2008 (6). Although the bulk of these episodes is mainly found in developing countries (6), bovine TB remains a major problem even in some industrialized countries, best exemplified by the United Kingdom, which has experienced an important resurgence of bovine TB since the 1980s (7). Because M. bovis and other closely related animal-adapted strains are also capable of causing TB in humans, this situation raises concerns regarding the zoonotic risk. Indeed, human TB cases resulting from M. bovis are estimated to be around 2% worldwide (8), with higher incidence (...

The natural resistance of Mycobacterium abscessus to most commonly available antibiotics seriously limits chemotherapeutic treatment options, which is particularly challenging for cystic fibrosis patients infected with this rapid-growing mycobacterium. New drugs with novel molecular targets are urgently needed against this emerging pathogen. However, the discovery of such new chemotypes has not been appropriately performed. Here, we demonstrate the utility of a phenotypic screen for bactericidal compounds against M. abscessus using a library of compounds previously validated for activity against M. tuberculosis. We identified a new piperidinol-based molecule, PIPD1, exhibiting potent activity against clinical M. abscessus strains in vitro and in infected macrophages. Treatment of infected zebrafish with PIPD1 correlated with increased embryo survival and decreased bacterial burden. Whole genome analysis of M. abscessus strains resistant to PIPD1 identified several mutations in MAB_4508, encoding a protein homologous to MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis was unaffected, PIPD1 strongly inhibited the transport of trehalose monomycolate, thereby abrogating mycolylation of arabinogalactan. Mapping the mutations conferring resistance to PIPD1 on a MAB_4508 tridimensional homology model defined a potential PIPD1-binding pocket. Our data emphasize a yet unexploited chemical structure class against M. abscessus infections with promising translational development possibilities.

SummaryMycobacterium abscessus is an emerging pathogen that is increasingly recognized as a relevant cause of human lung infection in cystic fibrosis patients. This highly antibiotic-resistant mycobacterium is an exception within the rapidly growing mycobacteria, which are mainly saprophytic and non-pathogenic organisms. M. abscessus manifests as either a smooth (S) or a rough (R) colony morphotype, which is of clinical importance as R morphotypes are associated with more severe and persistent infections. To better understand the molecular mechanisms behind the S/R alterations, we analysed S and R variants of three isogenic M. abscessus S/R pairs using an unbiased approach involving genome and transcriptome analyses, transcriptional fusions and integrating constructs. This revealed different small insertions, deletions (indels) or single nucleotide polymorphisms within the non-ribosomal peptide synthase gene cluster mps1-mps2-gap or mmpl4b in the three R variants, consistent with the transcriptional differences identified within this genomic locus that is implicated in the synthesis and transport of Glyco-Peptido-Lipids (GPL). In contrast to previous reports, the identification of clearly defined genetic lesions responsible for the loss of GPL-production or transport makes a frequent switching back-and-forth between smooth and rough morphologies in M. abscessus highly unlikely, which is important for our understanding of persistent M. abscessus infections.

Background and Purpose: The efficiency of prehospital care chain response and the adequacy of hospital resources are challenged amid the coronavirus disease 2019 (COVID-19) outbreak, with suspected consequences for patients with ischemic stroke eligible for mechanical thrombectomy (MT). Methods: We conducted a prospective national-level data collection of patients treated with MT, ranging 45 days across epidemic containment measures instatement, and of patients treated during the same calendar period in 2019. The primary end point was the variation of patients receiving MT during the epidemic period. Secondary end points included care delays between onset, imaging, and groin puncture. To analyze the primary end point, we used a Poisson regression model. We then analyzed the correlation between the number of MTs and the number of COVID-19 cases hospitalizations, using the Pearson correlation coefficient (compared with the null value). Results: A total of 1513 patients were included at 32 centers, in all French administrative regions. There was a 21% significant decrease (0.79; [95%CI, 0.76–0.82]; P <0.001) in MT case volumes during the epidemic period, and a significant increase in delays between imaging and groin puncture, overall (mean 144.9±SD 86.8 minutes versus 126.2±70.9; P <0.001 in 2019) and in transferred patients (mean 182.6±SD 82.0 minutes versus 153.25±67; P <0.001). After the instatement of strict epidemic mitigation measures, there was a significant negative correlation between the number of hospitalizations for COVID and the number of MT cases ( R 2 −0.51; P =0.04). Patients treated during the COVID outbreak were less likely to receive intravenous thrombolysis and to have unwitnessed strokes (both P <0.05). Conclusions: Our study showed a significant decrease in patients treated with MTs during the first stages of the COVID epidemic in France and alarming indicators of lengthened care delays. These findings prompt immediate consideration of local and regional stroke networks preparedness in the varying contexts of COVID-19 pandemic evolution.

dThe ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors of Mycobacterium tuberculosis, given that its inactivation severely attenuates virulent mycobacteria. In this work, we show that clinical isolates of M. tuberculosis produce and secrete larger amounts of ESAT-6 than the widely used M. tuberculosis H37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed that whiB6 (rv3862c), a gene upstream of the ESX-1 genetic locus that has not previously been found to be implicated in the regulation of the ESX-1 secretory apparatus, presents a unique single nucleotide insertion in its promoter region in strains H37Rv and H37Ra. This polymorphism is not present in any of the other publicly available M. tuberculosis complex genomes or in any of the 76 clinical M. tuberculosis isolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP downregulates whiB6 expression in H37Rv, while it upregulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy of whiB6 in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that in M. tuberculosis-with the exception of the H37Rv strain-ESX-1 expression is regulated by WhiB6 as part of the PhoP regulon, which adds another level of complexity to the regulation of ESAT-6 secretion with a potential role in virulence adaptation.

Recent insights into the mechanisms by which Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is recognized by cytosolic nucleotide sensors have opened new avenues for rational vaccine design. The only licensed anti-tuberculosis vaccine, Mycobacterium bovis BCG, provides limited protection. A feature of BCG is the partial deletion of the ESX-1 type VII secretion system, which governs phagosomal rupture and cytosolic pattern recognition, key intracellular phenotypes linked to increased immune signaling. Here, by heterologously expressing the esx-1 region of Mycobacterium marinum in BCG, we engineered a low-virulence, ESX-1-proficient, recombinant BCG (BCG::ESX-1) that induces the cGas/STING/TBK1/IRF-3/type I interferon axis and enhances AIM2 and NLRP3 inflammasome activity, resulting in both higher proportions of CD8 T cell effectors against mycobacterial antigens shared with BCG and polyfunctional CD4 Th1 cells specific to ESX-1 antigens. Importantly, independent mouse vaccination models show that BCG::ESX-1 confers superior protection relative to parental BCG against challenges with highly virulent M. tuberculosis.

Horizontal gene transfer (HGT) is a major driving force of bacterial diversification and evolution. For tuberculosis-causing mycobacteria, the impact of HGT in the emergence and distribution of dominant lineages remains a matter of debate. Here, by using fluorescenceassisted mating assays and whole genome sequencing, we present unique experimental evidence of chromosomal DNA transfer between tubercle bacilli of the early-branching Mycobacterium canettii clade. We found that the obtained recombinants had received multiple donor-derived DNA fragments in the size range of 100 bp to 118 kbp, fragments large enough to contain whole operons. Although the transfer frequency between M. canettii strains was low and no transfer could be observed among classical Mycobacterium tuberculosis complex (MTBC) strains, our study provides the proof of concept for genetic exchange in tubercle bacilli. This outstanding, now experimentally validated phenomenon presumably played a key role in the early evolution of the MTBC toward pathogenicity. Moreover, our findings also provide important information for the risk evaluation of potential transfer of drug resistance and fitness mutations among clinically relevant mycobacterial strains.recombination | DNA transfer | tuberculosis | Mycobacterium canettii | evolution