Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients' skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

Mycolactone is a polyketide toxin produced by Mycobacterium ulcerans (Mu), the causative agent of the skin disease Buruli ulcer (BU). Surprisingly, infected tissues lack inflammatory infiltrates. Structural similarities between mycolactone and immunosuppressive agents led us to investigate the immunomodulatory properties of mycolactone on dendritic cells (DCs), the key initiators and regulators of immune responses. At noncytotoxic concentrations, phenotypic and functional maturation of both mouse and human DCs was inhibited by mycolactone. Notably, mycolactone blocked the emigration of mouse-skin DCs to draining lymph nodes, as well as their maturation in vivo. In human peripheral blood-derived DCs, mycolactone inhibited the ability to activate allogeneic T cell priming and to produce inflammatory molecules. Interestingly, production of the cytokines interleukin (IL) 12, tumor necrosis factor alpha, and IL-6 was only marginally affected, whereas production of the chemokines macrophage inflammatory protein (MIP) 1alpha, MIP-1beta, regulated on activation, normal T cell expressed and secreted, interferon gamma-inducible protein 10, and monocyte chemoattractant protein 1 was abolished at nanomolar concentrations. Importantly, mycolactone endogenously expressed by Mu mediated similar inhibitory effects on beta-chemokine production by DCs. In accordance with the histopathological features of BUs, our results suggest that bacterial production of mycolactone may limit both the initiation of primary immune responses and the recruitment of inflammatory cells to the infection site. Moreover, they highlight a potential interest in mycolactone as a novel immunosuppressive agent.

The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes.

Buruli ulcer is a severe human skin disease caused by Mycobacterium ulcerans. This disease is primarily diagnosed in West Africa with increasing incidence. Antimycobacterial drug therapy is relatively effective during the preulcerative stage of the disease, but surgical excision of lesions with skin grafting is often the ultimate treatment. The mode of transmission of this Mycobacterium species remains a matter of debate, and relevant interventions to prevent this disease lack (i) the proper understanding of the M. ulcerans life history traits in its natural aquatic ecosystem and (ii) immune signatures that could be correlates of protection. We previously set up a laboratory ecosystem with predatory aquatic insects of the family Naucoridae and laboratory mice and showed that (i) M. ulcerans-carrying aquatic insects can transmit the mycobacterium through bites and (ii) that their salivary glands are the only tissues hosting replicative M. ulcerans. Further investigation in natural settings revealed that 5%-10% of these aquatic insects captured in endemic areas have M. ulcerans-loaded salivary glands. In search of novel epidemiological features we noticed that individuals working close to aquatic environments inhabited by insect predators were less prone to developing Buruli ulcers than their relatives. Thus we set out to investigate whether those individuals might display any immune signatures of exposure to M. ulcerans-free insect predator bites, and whether those could correlate with protection.

The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models.

Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.

A range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip-OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr , a strong Pip-repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild-type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip-OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole-cell screening assay to identify inhibitors of this enzyme.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom-a skeleton from the Great Chesterford cemetery with a calibrated age of 415-545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide.

Hansen's disease (leprosy), widespread in medieval Europe, is today mainly prevalent in tropical and subtropical regions with around 200,000 new cases reported annually. Despite its long history and appearance in historical records, its origins and past dissemination patterns are still widely unknown. Applying ancient DNA approaches to its major causative agent, Mycobacterium leprae, can significantly improve our understanding of the disease's complex history. Previous studies have identified a high genetic continuity of the pathogen over the last 1500 years and the existence of at least four M. leprae lineages in some parts of Europe since the Early Medieval period.

The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host.

MmpL3 is an inner membrane transporter of Mycobacterium tuberculosis responsible for the export of trehalose momomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. MmpL3 represents an emerging target for tuberculosis therapy. In this paper, we describe the construction and characterization of an mmpL3 knockdown strain of M. tuberculosis. Downregulation of mmpL3 led to a stop in bacterial division and rapid cell death, preceded by the accumulation of TDM precursors. MmpL3 was also shown to be essential for growth in monocyte-derived human macrophages. Using RNA-seq we also found that MmpL3 depletion caused up-regulation of 47 genes and down-regulation of 23 genes (at least 3-fold change and false discovery rate ≤1%). Several genes related to osmoprotection and metal homeostasis were induced, while several genes related to energy production and mycolic acids biosynthesis were repressed suggesting that inability to synthesize a correct outer membrane leads to changes in cellular permeability and a metabolic downshift.

New drugs are needed to control the current tuberculosis (TB) pandemic caused by infection with Mycobacterium tuberculosis We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agents in vitro All five compounds showed good activity against M. tuberculosisin vitro and in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization of M. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in the qcrB gene encoding a subunit of cytochrome bc1 oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochrome bc1-aa3 is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles of M. tuberculosis treated with AX-35 were similar to those generated by other cytochrome bc1 oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochrome bd in respiratory adaptation. In the absence of cytochrome bd oxidase, AX-35 was bactericidal against M. tuberculosis Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.IMPORTANCE New drugs against Mycobacterium tuberculosis are urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potent in vitro and in vivo activities against M. tuberculosis AX compounds target the QcrB subunit of the cytochrome bc1 terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochrome bd oxidase upon QcrB inhibition. In the absence of cytochrome bd oxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the world's leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids, and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.

Tight control of gene expression is crucial for Mycobacterium tuberculosis to adapt to the changing environments encountered when infecting or exiting human cells. While three nucleoid associated proteins (NAPs) EspR, HupB and Lsr2 have been investigated, the role of a fourth, the mycobacterial integration host factor (mIHF), remains elusive. Here, we report a multidisciplinary functional analysis that exploits a conditional mIHF mutant. Gene silencing was bactericidal and resulted in elongated cells devoid of septa, with only one nucleoid. ChIP-sequencing identified 153 broad peaks distributed around the chromosome, which were often situated upstream of transcriptional start sites where EspR also bound. RNA-sequencing showed expression of 209 genes to be heavily affected upon mIHF depletion, including those for many tRNAs, DNA synthesis and virulence pathways. Consistent with NAP function, mIHF acts as a global regulator by directly and indirectly controlling genes required for pathogenesis and for housekeeping functions.

Mycobacterium leprae, the causative agent of leprosy, is an unculturable bacterium with a considerably reduced genome (3.27 Mb) compared to homologues mycobacteria from the same ancestry. In 2001, the genome of M. leprae was first described and subsequently four genotypes (1-4) and 16 subtypes (A-P) were identified providing means to study global transmission patterns for leprosy. In order to understand the role of asymptomatic carriers we investigated M. leprae carriage as well as infection in leprosy patients (n = 60) and healthy household contacts (HHC; n = 250) from Bangladesh using molecular detection of the bacterial element RLEP in nasal swabs (NS) and slit skin smears (SSS). In parallel, to study M. leprae genotype distribution in Bangladesh we explored strain diversity by whole genome sequencing (WGS) and Sanger sequencing. In the studied cohort in Bangladesh, M. leprae DNA was detected in 33.3% of NS and 22.2% of SSS of patients with bacillary index of 0 whilst in HHC 18.0% of NS and 12.3% of SSS were positive. The majority of the M. leprae strains detected in this study belonged to genotype 1D (55%), followed by 1A (31%). Importantly, WGS allowed the identification of a new M. leprae genotype, designated 1B-Bangladesh (14%), which clustered separately between the 1A and 1B strains. Moreover, we established that the genotype previously designated 1C, is not an independent subtype but clusters within the 1D genotype. Intraindividual differences were present between the M. leprae strains obtained including mutations in hypermutated genes, suggesting mixed colonization/infection or in-host evolution. In summary, we observed that M. leprae is present in asymptomatic contacts of leprosy patients fueling the concept that these individuals contribute to the current intensity of transmission. Our data therefore emphasize the importance of sensitive and specific tools allowing post-exposure prophylaxis targeted at M. leprae-infected or -colonized individuals.

The role of biofilms in the pathogenesis of mycobacterial diseases remains largely unknown. Mycobacterium ulcerans, the etiological agent of Buruli ulcer, a disfiguring disease in humans, adopts a biofilm-like structure in vitro and in vivo, displaying an abundant extracellular matrix (ECM) that harbors vesicles. The composition and structure of the ECM differs from that of the classical matrix found in other bacterial biofilms. More than 80 proteins are present within this extracellular compartment and appear to be involved in stress responses, respiration, and intermediary metabolism. In addition to a large amount of carbohydrates and lipids, ECM is the reservoir of the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, and purified vesicles extracted from ECM are highly cytotoxic. ECM confers to the mycobacterium increased resistance to antimicrobial agents, and enhances colonization of insect vectors and mammalian hosts. The results of this study support a model whereby biofilm changes confer selective advantages to M. ulcerans in colonizing various ecological niches successfully, with repercussions for Buruli ulcer pathogenesis.

To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.

The EspB protein of Mycobacterium tuberculosis is a 60 kDa virulence factor, implicated in conjugation and exported by the ESX-1 system of which it may also be a component. Previous attempts to obtain high-resolution maps of EspB by cryo-electron microscopic examination of single particles have been thwarted by severe orientation bias of the particles. This was overcome by using detergent as a surfactant thereby allowing reconstruction of the EspB structure at 3.37 Å resolution. The final structure revealed the N-terminal domain of EspB to be organized as a cylindrical heptamer with dimensions of 90 Å x 90 Å and a central channel of 45 Å diameter whereas the C-terminal domain was unstructured. New atomic insight was obtained into the helical packing required for protomer interactions and the overall electrostatic potential. The external surface is electronegatively charged while the channel is lined with electropositive patches. EspB thus has many features of a pore-like transport protein that might allow the passage of an ESX-1 substrate such as the 35 Å diameter EsxA-EsxB heterodimer or B-form DNA consistent with its proposed role in DNA uptake.

Human settlement of Madagascar traces back to the beginning of the first millennium with the arrival of Austronesians from Southeast Asia, followed by migrations from Africa and the Middle East. Remains of these different cultural, genetic, and linguistic legacies are still present in Madagascar and other islands of the Indian Ocean. The close relationship between human migration and the introduction and spread of infectious diseases, a well-documented phenomenon, is particularly evident for the causative agent of leprosy, Mycobacterium leprae. In this study, we used whole-genome sequencing (WGS) and molecular dating to characterize the genetic background and retrace the origin of the M. leprae strains circulating in Madagascar (n = 30) and the Comoros (n = 3), two islands where leprosy is still considered a public health problem and monitored as part of a drug resistance surveillance program. Most M. leprae strains (97%) from Madagascar and Comoros belonged to a new genotype as part of branch 1, closely related to single nucleotide polymorphism (SNP) type 1D, named 1D-Malagasy. Other strains belonged to the genotype 1A (3%). We sequenced 39 strains from nine other countries, which, together with previously published genomes, amounted to 242 genomes that were used for molecular dating. Specific SNP markers for the new 1D-Malagasy genotype were used to screen samples from 11 countries and revealed this genotype to be restricted to Madagascar, with the sole exception being a strain from Malawi. The overall analysis thus ruled out a possible introduction of leprosy by the Austronesian settlers and suggests a later origin from East Africa, the Middle East, or South Asia.

The ESX-1, type VII, secretion system represents the major virulence determinant of Mycobacterium tuberculosis, one of the most successful intracellular pathogens. Here, by combining genetic and high-throughput approaches, we show that EspL, a protein of 115 amino acids, is essential for mediating ESX-1-dependent virulence and for stabilization of EspE, EspF and EspH protein levels. Indeed, an espL knock-out mutant was unable to replicate intracellularly, secrete ESX-1 substrates or stimulate innate cytokine production. Moreover, proteomic studies detected greatly reduced amounts of EspE, EspF and EspH in the espL mutant as compared to the wild type strain, suggesting a role for EspL as a chaperone. The latter conclusion was further supported by discovering that EspL interacts with EspD, which was previously demonstrated to stabilize the ESX-1 substrates and effector proteins, EspA and EspC. Loss of EspL also leads to downregulation in M. tuberculosis of WhiB6, a redox-sensitive transcriptional activator of ESX-1 genes. Overall, our data highlight the importance of a so-far overlooked, though conserved, component of the ESX-1 secretion system and begin to delineate the role played by EspE, EspF and EspH in virulence and host-pathogen interaction.