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Successful control of Mycobacterium tuberculosis (Mtb) infection by macrophages relies on immunometabolic reprogramming, where the role of fatty acids (FAs) remains poorly understood. Recent studies unraveled Mtb’s capacity to acquire saturated and monounsaturated FAs via the Mce1 importer. However, upon activation macrophages produce polyunsaturated FAs (PUFAs), mammal-specific FAs mediating the generation of key immunomodulatory eicosanoids. Here, we asked whether de novo synthesis of PUFAs is modulated in Mtb-infected macrophages and benefits host or pathogen. Quantitative lipidomics revealed that Mtb infection activates the early PUFA biosynthetic pathway for production of eicosanoids. While PUFA synthesis blockade significantly impaired the inflammatory and antimicrobial responses of infected macrophages, it had no effect on Mtb growth in vivo. Using a click-chemistry approach, we found that Mtb efficiently imports PUFAs of the ω6 subset via Mce1 in axenic culture, including the eicosanoid precursor arachidonic acid (AA). Notably, Mtb preferentially internalized AA over all other FAs within infected macrophages, but here Mtb’s import of AA was largely Mce1-independent and correlated with elevated AA uptake by host cells. Together, these findings reveal AA as a major FA substrate for intracellular Mtb. They suggest that Mtb’s hijacking of host-derived AA may counteract its stimulatory effect on anti-mycobacterial immune responses.

Previously, we have identified a putative novel rapidly growing Mycobacterium species, referred to as TNTM28, recovered from the sputum of an apparently immunocompetent young man with an underlying pulmonary disease. Here we provide a thorough characterization of TNTM28 genome sequence, which consists of one chromosome of 5,526,191 bp with a 67.3% G + C content, and a total of 5193 predicted coding sequences. Phylogenomic analyses revealed a deep-rooting relationship to the Mycobacterium fortuitum complex, thus suggesting a new taxonomic entity. TNTM28 was predicted to be a human pathogen with a probability of 0.804, reflecting the identification of several virulence factors, including export systems (Sec, Tat, and ESX), a nearly complete set of Mce proteins, toxin-antitoxins systems, and an extended range of other genes involved in intramacrophage replication and persistence (hspX, ahpC, sodA, sodC, katG, mgtC, ClpR, virS, etc.), some of which had likely been acquired through horizontal gene transfer. Such an arsenal of potential virulence factors, along with an almost intact ESX-1 locus, might have significantly contributed to TNTM28 pathogenicity, as witnessed by its ability to replicate efficiently in macrophages. Overall, the identification of this new species as a potential human pathogen will help to broaden our understanding of mycobacterial pathogenesis.

Mycobacterium microti is an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC), which was originally isolated from voles, but has more recently also been isolated from other selected mammalian hosts, including occasionally from humans. Here, we have generated and analysed the complete genome sequences of five representative vole and clinical M. microti isolates using PacBio- and Illumina-based technologies, and have tested their virulence and vaccine potential in SCID (severe combined immune deficient) mouse and/or guinea pig infection models. We show that the clinical isolates studied here cluster separately in the phylogenetic tree from vole isolates and other clades from publicly available M. microti genome sequences. These data also confirm that the vole and clinical M. microti isolates were all lacking the specific RD1mic region, which in other tubercle bacilli encodes the ESX-1 type VII secretion system. Biochemical analysis further revealed marked phenotypic differences between isolates in type VII-mediated secretion of selected PE and PPE proteins, which in part were attributed to specific genetic polymorphisms. Infection experiments in the highly susceptible SCID mouse model showed that the clinical isolates were significantly more virulent than the tested vole isolates, but still much less virulent than the M. tuberculosis H37Rv control strain. The strong attenuation of the ATCC 35872 vole isolate in immunocompromised mice, even compared to the attenuated BCG (bacillus Calmette–Guérin) vaccine, and its historic use in human vaccine trials encouraged us to test this strain’s vaccine potential in a guinea pig model, where it demonstrated similar protective efficacy as a BCG control, making it a strong candidate for vaccination of immunocompromised individuals in whom BCG vaccination is contra-indicated. Overall, we provide new insights into the genomic and phenotypic variabilities and particularities of members of an understudied clade of the MTBC, which all share a recent common ancestor that is characterized by the deletion of the RD1mic region.

Mycobacterium tuberculosis (Mtb) strains are classified into different phylogenetic lineages (L), three of which (L2/L3/L4) emerged from a common progenitor after the loss of the MmpS6/MmpL6-encoding Mtb-specific deletion 1 region (TbD1). These TbD1-deleted “modern” lineages are responsible for globally-spread tuberculosis epidemics, whereas TbD1-intact “ancestral” lineages tend to be restricted to specific geographical areas, such as South India and South East Asia (L1) or East Africa (L7). By constructing and characterizing a panel of recombinant TbD1-knock-in and knock-out strains and comparison with clinical isolates, here we show that deletion of TbD1 confers to Mtb a significant increase in resistance to oxidative stress and hypoxia, which correlates with enhanced virulence in selected cellular, guinea pig and C3HeB/FeJ mouse infection models, the latter two mirroring in part the development of hypoxic granulomas in human disease progression. Our results suggest that loss of TbD1 at the origin of the L2/L3/L4 Mtb lineages was a key driver for their global epidemic spread and outstanding evolutionary success., Mycobacterium tuberculosis (Mtb) modern strains emerged from a common progenitor after the loss of Mtb-specific deletion 1 region (TbD1). Here, the authors show that deletion of TbD1 correlates with enhanced Mtb virulence in animal models, mirroring the development of hypoxic granulomas in human disease progression.

Background Human tuberculosis (TB) is caused by a plethora of Mycobacterium tuberculosis complex (MTBC) strains belonging to seven phylogenetic branches. Lineages 2, 3 and 4 are considered “modern” branches of the MTBC responsible for the majority of worldwide TB. Since the current BCG vaccine confers variable protection against pulmonary TB, new candidates are investigated. MTBVAC is the unique live attenuated vaccine based on M. tuberculosis in human clinical trials. Methods MTBVAC was originally constructed by unmarked phoP and fadD26 deletions in a clinical isolate belonging to L4. Here we construct new vaccines based on isogenic gene deletions in clinical isolates of the L2 and L3 modern lineages. These three vaccine candidates were characterized at molecular level and also in animal experiments of protection and safety. Findings Safety studies in immunocompromised mice showed that MTBVAC-L2 was less attenuated than BCG Pasteur, while the original MTBVAC was found even more attenuated than BCG and MTBVAC-L3 showed an intermediate phenotype. The three MTBVAC candidates showed similar or superior protection compared to BCG in immunocompetent mice vaccinated with each MTBVAC candidate and challenged with three representative strains of the modern lineages. Interpretation MTBVAC vaccines, based on double phoP and fadD26 deletions, protect against TB independently of the phylogenetic linage used as template strain for their construction. Nevertheless, lineage L4 confers the best safety profile. Funding European Commission (TBVAC2020, H2020-PHC-643381), Spanish Ministry of Science (RTI2018-097625-B-I00), Instituto de Salud Carlos III (PI18/0336), Gobierno de Aragón/Fondo Social Europeo and the French National Research Council (ANR-10-LABX-62-IBEID, ANR-16-CE35-0009, ANR-16-CE15-0003)., Graphical abstract Image, graphical abstract

The fatty acid synthase type II (FAS-II) multienzyme system builds the main chain of mycolic acids (MAs), important lipid pathogenicity factors of Mycobacterium tuberculosis (Mtb). Due to their original structure, the identification of the (3 R)-hydroxyacyl-ACP dehydratases, HadAB and HadBC, of Mtb FAS-II complex required in-depth work. Here, we report the discovery of a third dehydratase protein, HadDMtb (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto- and methoxy-MAs. HadDMtb deletion triggered a marked change in Mtb keto-MA content and size distribution, deeply impacting the production of full-size molecules. Furthermore, abnormal MAs, likely generated from 3-hydroxylated intermediates, accumulated. These data strongly suggest that HadDMtb catalyzes the 3-hydroxyacyl dehydratation step of late FAS-II elongation cycles during keto-MA biosynthesis. Phenotyping of Mtb hadD deletion mutant revealed the influence of HadDMtb on the planktonic growth, colony morphology and biofilm structuration, as well as on low temperature tolerance. Importantly, HadDMtb has a strong impact on Mtb virulence in the mouse model of infection. The effects of the lack of HadDMtb observed both in vitro and in vivo designate this protein as a bona fide target for the development of novel anti-TB intervention strategies.

Understanding the molecular strategies used by Mycobacterium tuberculosis to invade and persist within the host is of paramount importance to tackle the tuberculosis pandemic. Comparative genomic surveys have revealed that hadC, encoding a subunit of the HadBC dehydratase, is mutated in the avirulent M. tuberculosis H37Ra strain. We show here that mutation or deletion of hadC affects the biosynthesis of oxygenated mycolic acids, substantially reducing their production level. Additionally, it causes the loss of atypical extra-long mycolic acids, demonstrating the involvement of HadBC in the late elongation steps of mycolic acid biosynthesis. These events have an impact on the morphotype, cording capacity and biofilm growth of the bacilli as well as on their sensitivity to agents such as rifampicin. Furthermore, deletion of hadC leads to a dramatic loss of virulence: an almost 4-log drop of the bacterial load in the lungs and spleens of infected immunodeficient mice. Both its unique function and importance for M. tuberculosis virulence make HadBC an attractive therapeutic target for tuberculosis drug development.

Summary The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs) of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II., Graphical Abstract, Highlights • T cell hybridomas detect individual mycobacterial proteins without cross-reactivity • Detection of mycobacterial proteins by T cells allows visualization of their cellular topography • Measurement of intraphagocyte mycobacterial proteins can be performed with T cells • A multiplexed assay of mycobacterial protein quantitation has numerous applications, Sayes et al. develop an approach to express distinct fluorescent reporters that is based on the recognition of specific Mycobacterium tuberculosis MHC class II epitopes by highly discriminative T cell hybridomas. This multiplexed technology allows the study of secretion, subcellular location, and regulation patterns of these instrumental protein members.

Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium tuberculosis uses different ESX/Type VII secretion (T7S) systems to transport proteins important for virulence and host immune responses. We recently reported that secretion of T7S substrates belonging to the mycobacteria-specific Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins of the PGRS (polymorphic GC-rich sequences) and MPTR (major polymorphic tandem repeat) subfamilies required both a functional ESX-5 system and a functional PPE38/71 protein for secretion. Inactivation of ppe38/71 and the resulting loss of PE_PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE_PGRS/PPE-MPTR surface proteins are not exported by certain animal-adapted strains of the M. tuberculosis complex including M. bovis. This Δppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be restored by introduction of the M. tuberculosis ppe38-locus. Epitope mapping of the PPE-MPTR protein PPE10, further allowed us to monitor T-cell responses in splenocytes from BCG/M. tuberculosis immunized mice, confirming the dependence of PPE10-specific immune-induction on ESX-5/PPE38-mediated secretion. Restoration of PE_PGRS/PPE-MPTR secretion in recombinant BCG neither altered global antigenic presentation or activation of innate immune cells, nor protective efficacy in two different mouse vaccination-infection models. This unexpected finding stimulates a reassessment of the immunomodulatory properties of PE_PGRS/PPE-MPTR proteins, some of which are contained in vaccine formulations currently in clinical evaluation.

Sensing and response to changes in nutrient availability are essential for the lifestyle of environmental and pathogenic bacteria. Serine/threonine protein kinase G (PknG) is required for virulence of the human pathogen Mycobacterium tuberculosis, and its putative substrate GarA regulates the tricarboxylic acid cycle in M. tuberculosis and other Actinobacteria by protein-protein binding. We sought to understand the stimuli that lead to phosphorylation of GarA, and the roles of this regulatory system in pathogenic and non-pathogenic bacteria. We discovered that M. tuberculosis lacking garA was severely attenuated in mice and macrophages and furthermore that GarA lacking phosphorylation sites failed to restore the growth of garA deficient M. tuberculosis in macrophages. Additionally we examined the impact of genetic disruption of pknG or garA upon protein phosphorylation, nutrient utilization and the intracellular metabolome. We found that phosphorylation of GarA requires PknG and depends on nutrient availability, with glutamate and aspartate being the main stimuli. Disruption of pknG or garA caused opposing effects on metabolism: a defect in glutamate catabolism or depletion of intracellular glutamate, respectively. Strikingly, disruption of the phosphorylation sites of GarA was sufficient to recapitulate defects caused by pknG deletion. The results suggest that GarA is a cellular target of PknG and the metabolomics data demonstrate that the function of this signaling system is in metabolic regulation. This function in amino acid homeostasis is conserved amongst the Actinobacteria and provides an example of the close relationship between metabolism and virulence. ISSN:1553-7374 ISSN:1553-7366

The 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.

International audience; Global spread and limited genetic variation are hallmarks of M. 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 whole genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5× coverage), 454/Roche (13-18× coverage) and/or Illumina DNA sequencing (45-105× coverage). We show that STB isolates are highly recombinogenic and evolutionarily early branching, with larger genome sizes, higher rates of genetic variation, 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 showed that STB strains 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 insights into the molecular events involved.

Mycobacterium tuberculosis (Mtb), possesses at least three type VII secretion systems, ESX-1, -3 and -5 that are actively involved in pathogenesis and host-pathogen interaction. We recently showed that an attenuated Mtb vaccine candidate (Mtb Δppe25-pe19), which lacks the characteristic ESX-5-associated pe/ppe genes, but harbors all other components of the ESX-5 system, induces CD4+ T-cell immune responses against non-esx-5-associated PE/PPE protein homologs. These T cells strongly cross-recognize the missing esx-5-associated PE/PPE proteins. Here, we characterized the fine composition of the functional cross-reactive Th1 effector subsets specific to the shared PE/PPE epitopes in mice immunized with the Mtb Δppe25-pe19 vaccine candidate. We provide evidence that the Mtb Δppe25-pe19 strain, despite its significant attenuation, is comparable to the WT Mtb strain with regard to: (i) its antigenic repertoire related to the different ESX systems, (ii) the induced Th1 effector subset composition, (iii) the differentiation status of the Th1 cells induced, and (iv) its particular features at stimulating the innate immune response. Indeed, we found significant contribution of PE/PPE-specific Th1 effector cells in the protective immunity against pulmonary Mtb infection. These results offer detailed insights into the immune mechanisms underlying the remarkable protective efficacy of the live attenuated Mtb Δppe25-pe19 vaccine candidate, as well as the specific potential of PE/PPE proteins as protective immunogens., Author Summary Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is one of the most widely spread human pathogens, responsible for more than 9.6 million of new tuberculosis cases and 1.5 million deaths, annually. The resurgence of pulmonary tuberculosis in immuno-compromised patients, including HIV-co-infected populations, and increasing spread of drug-resistant Mtb strains are worrying. Given the estimated 2 billion cases of latent Mtb infections and the only partial efficacy of the unique, currently available tuberculosis-vaccine Mycobacterium bovis BCG (Bacille Calmette-Guerin) it is necessary to develop improved vaccines. Here, we demonstrate that the host cellular immunity, mediated by CD4+ T lymphocytes, specific to the “PE/PPE” families of mycobacterial antigens, contribute to the protection against Mtb-induced disease. We revealed the fine composition of the PE/PPE-specific T cells by characterizing their effector functions and differentiation status. We previously described a live attenuated mycobacterial strain as a vaccine candidate that is able to induce such CD4+ T cells and which displays particular properties at stimulating the cells of the innate immune system. These responses play a central role in the initiation of the host defense and in the protection against tuberculosis. Our results pave the way for further development of candidates in preclinical models of anti-tuberculosis vaccination.

International audience; Mycobacterium tuberculosis is a major, globally spread, aerosol-transmitted human pathogen, thought to have evolved by clonal expansion from a Mycobacterium canettii-like progenitor. In contrast, extant M. canettii strains are rare, genetically diverse, and geographically restricted mycobacteria of only marginal epidemiological importance. Here, we show that the contrasting evolutionary success of these two groups is linked to loss of lipooligosaccharide biosynthesis and subsequent morphotype changes. Spontaneous smooth-to-rough M. canettii variants were found to be mutated in the polyketide-synthase-encoding pks5 locus and deficient in lipooligosaccharide synthesis, a phenotype restored by complementation. Importantly, these rough variants showed an altered host–pathogen interaction and increased virulence in cellular- and animal-infection models. In one variant, lipooligosaccharide deficiency occurred via homologous recombination between two pks5 genes and removal of the intervening acyltransferase-encoding gene. The resulting single pks5 configuration is similar to that fixed in M. tuberculosis, which is known to lack lipooligosaccharides. Our results suggest that pks5-recombination-mediated bacterial surface remodelling increased virulence, driving evolution from putative generalist mycobacteria towards professional pathogens of mammalian hosts.Tuberculosis is a major human infectious disease. Although many aspects of the disease-causing potential of its aetiological agent M. tuberculosis are known1, our understanding of the molecular events that favoured its evolutionary success as one of the most widely distributed human pathogens remains scant. New insights into this question are important for uncovering the mechanisms of pathogenesis and new drug targets2. Strains of the closely related and phylogenetically early branching M. canettii, also named ‘smooth tubercle bacilli’ (STB)3, are powerful resources to investigate the evolution of M. tuberculosis and the M. tuberculosis complex (MTBC)3. The first strain of M. canettii was isolated by Georges Canetti in 1969, and since then, fewer than 100 isolates have been described, most of which have been isolated from tuberculosis patients with a connection to the Horn of Africa4,​5,​6,​7. Despite their geographic restriction, M. canettii strains show much greater genetic variability and are less virulent/persistent than M. tuberculosis. Genome comparisons suggest that M. tuberculosis evolved by clonal expansion from a pool of M. canettii-like tubercle bacilli through the gain of virulence and persistence mechanisms3,8,9. Although some genomic differences have been found to be specific for a single M. canettii strain, apparently due to isolated horizontal gene transfer (for example, the mce5 operon3 or the eptABCD operon10, present exclusively in strain STB-J)3, others are conserved throughout all M. canettii strains as a result of phylogenetic ancestry (for example, cobF, which is present in M. canettii and deleted from the MTBC)3.Here, we investigate phenotypic differences between M. canettii and M. tuberculosis and focus on the unique, conserved, smooth (S) colony morphotype of M. canettii that contrasts with the rough (R) morphotype of MTBC members. Previously, S morphotypes of non-tuberculous mycobacterial species such as Mycobacterium avium11, Mycobacterium abscessus12, Mycobacterium kansasii13 or Mycobacterium marinum14 have been found to be less virulent than R morphotypes, raising the question of whether the highly conserved M. tuberculosis R morphotype might have been the result of evolutionary selection, based on host–pathogen interactions favouring a more virulent or persistent phenotype. In other mycobacterial species, S/R variation is often attributed to different kinds of cell surface glycolipid, such as glycopeptidolipid (GPL) for M. avium15 and M. abscessus16,17 or lipooligosaccharide (LOS) for M. kansasii13 and M. marinum14,18,19. Insights from earlier studies of M. canettii have remained abstruse, as no specific lipid exclusively present in the S morphotype has been identified20, nor has the genetic basis for morphotype variation been determined4.Building on recent data from several M. canettii genomes3, we studied smooth and spontaneously converted R variants of two different M. canettii strains, STB-K (CIPT 140070010) and STB-I (CIPT 140070007)3, hereafter termed KS/R and IS/R, respectively. We used whole genome sequencing (WGS) and identified differences in the genes of the pks5 locus, which in Mycobacterium smegmatis, M. marinum or M. kansasii are implicated in LOS biosynthesis14,21,​22,​23. In this Article, we uncover the mechanisms underlying the S-to-R morphology change of tubercle bacilli and explore the biological consequences with emphasis on the patho-evolution of M. tuberculosis.

The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1-ESX-5). While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA(5), eccD(5), rv1794 and esxM genes or the ppe25-pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD(5) ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the MtbeccD(5) ko and MtbΔppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model. Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.

Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis.

The severe skin-destructive disease caused by Mycobacterium ulcerans, named Buruli ulcer, is the third most important mycobacterial disease in humans after tuberculosis and leprosy. Recently we demonstrated that M. ulcerans could colonize the salivary glands of the water bug, Naucoris cimicoides. In this study, we report that M. ulcerans may be delivered from the digested prey aspirate to the coelomic cavity via a unique headspace, the head capsule (HC). During the infected meal, we observed that M. ulcerans clusters adhered to the stylets that were retracted in the HC at the end of the meal. M. ulcerans was able to translocate from the HC to the coelomic cavity where it is phagocytosed by the plasmatocytes. These cells are subverted as shuttle cells and deliver M. ulcerans to the salivary glands. At this early stage of its parasitic life style, two other important features of M. ulcerans can be documented: first, mycolactone is not required for translocation of M. ulcerans into the HC, in contrast to the next step, colonization of the salivary glands; second, M. ulcerans clusters bind a member of the serpin protein family present in the salivary gland homogenate.

AgMnII3 (MnIII0,26Al0,74) (MoO4)5 is prepared by solid state reaction of a 1:1:3:5 molar mixture of Al2O3, AgNO3, Mn(OAc)3·2H2O, and (NH4)2Mo4O13 (porcelain crucible, 1143 K, 4 weeks).

KNa5Mn3Mo6O24 is prepared by solid state reaction of a mixture of Na2CO3, K2CO3, Mn(OAc)3, and (NH4)2Mo4O13 in the molar ratio of Na:K:Mn:Mo = 1:1:2:3 (porcelain crucible, 950 K, 3 weeks; cooling to 900 K at a rate of 5 K/d).

Background Mycobacterium bovis BCG is presently the only available anti-tuberculosis vaccine used, worldwide. While BCG protects against miliary tuberculosis (TB) and tuberculoid meningitis in children, it often fails to protect against adult pulmonary TB. It is thus imperative that new improved anti-TB vaccines are developed. The integration of the ESX-1 secretion system, absent from BCG due to the deletion of region of difference 1 (RD1), into the genome of BCG has been shown to confer to BCG::ESX-1 enhanced protection against TB as compared to BCG. Methods In the present study, to counterbalance the increase in virulence resulting from the integration of the RD1 region into BCG, we have constructed and evaluated several BCG::ESX-1 variants that carry selected amino-acid changes in the ESX-1-secreted antigen ESAT-6. In order to find the candidate that combines low virulence with high protective efficacy, these novel recombinant BCG::ESX-1 strains were tested for their virulence properties and their protective efficacy against Mycobacterium tuberculosis in two different animal models (mouse and guinea-pig). Results Among several candidates tested, the BCG::ESAT-L28A/L29S strain, carrying modifications at residues Leu 28 -Leu 29 of the ESAT molecule, showed strong attenuation in mice and high protective efficiency both in mouse and guinea-pig vaccination-infection models. Conclusion This strain thus represents a promising candidate that merits further investigations and development. Our research also provides the proof of concept that selected ESX-1-complemented BCG strains may show low virulence and increased protective potential over parental strains.

International audience; Pathogenesis of Mycobacterium tuberculosis depends on the secretion of key virulence factors, such as the 6 kDa early secreted antigenic target ESAT-6 (EsxA) and its protein partner, the 10 kDa culture filtrate protein CFP-10 (EsxB), via the ESX-1 secretion system. ESX-1 represents the prototype system of the recently named type VII secretion systems that exist in a range of actinobacteria. The M. tuberculosis genome harbours a total of five gene clusters potentially coding for type VII secretion systems, designated ESX-1 - ESX-5, with ESX-4 being the most ancient system from which other ESX systems seem to have evolved by gene duplication and gene insertion events. The five ESX systems show similarity in gene content and gene order but differ in function. ESX-1 and ESX-5 are both crucial virulence determinants of M. tuberculosis, but with different mechanisms. While ESX-1 is implicated in the lysis of the host cell phagosomes, ESX-5 is involved in secretion of the mycobacteria specific PE and PPE proteins and cell wall stability. Research on type VII secretion systems has thus become a large and competitive research topic that is tightly linked to studies of host-pathogen interaction of pathogenic mycobacteria. Insights into this matter are of relevance for redrawing the patho-evolution of M. tuberculosis, which might help improving current strategies for prevention, diagnostics and therapy of tuberculosis as well as elucidating the virulence mechanisms employed by this important human pathogen.

In the new molybdenum oxide potassium penta­sodium trimanganese hexa­kis­(molybdate), KNa5Mn3Mo6O24, the structure is composed of M 2O10 (M = Mn, Na) dimers and MoO4 tetra­hedra sharing corners and forming layers which are linked via common corners of another type of MO4 tetra­hedra, forming a three-dimensional structure with two types of large channels in which the Na+ and K+ cations are located., The new phase potassium penta­sodium trimanganese hexa­kis(molybdate), KNa5Mn3Mo6O24, has been synthesized using solid-state methods. The structure is composed of M 2O10 (M = Mn, Na) dimers and MoO4 tetra­hedra (point group symmetry 2) sharing corners and forming layers parallel to (100), which are linked via common corners of another type of MO4 tetra­hedra, forming a three-dimensional structure with two types of large channels along [001] in which two types of Na+ cations (one with site symmetry 2, one with -1) and K+ cations (site symmetry 2, half-occupation) are located. Mn2+ and the third type of Na+ cations are located at the same site M with occupancies of 0.75 and 0.25, respectively. A comparative structural description is provided between the structure of the title compound and those of the related phases Cu1.35Fe3(PO4)3 and NaAgFeMn2(PO4)3.

The title compound is prepared by solid state reaction of Na2CO3, Mn(OAc)3·2H2O, and (NH4)2Mo4O13 in the molar ratio Na:Mn:Mo = 2:2:3 (porcelain crucible, 998 K, 3 weeks).

The title compound, disodium dimanganese(II) tris[orthomolybdate(VI)], was prepared by solid-state reactions. The structure can be described as being composed of Mn2Mo2O14double-chains that are interconnected by corner-sharing with MoO4tetrahedra, leading to a three-dimensional framework with channels propagating in [100] and [001] in which the Na+counter-cations are located. One of these Na sites is located on an inversion centre, one is partially occupied [occupancy 0.341 (9)], and one is statistically occupied by Na and Mn in a ratio of 0.829 (5):0.171 (5). Na1.67Mn2.17(MoO4)3is isotypic with structures of the Ag2M2(MoO4)3(M= Zn, Mg, Co, Mn) family. A comparative structural description is provided between the structure of the title compound and those of related phases containing (MXO8)nchains (M= Mo, Mn andX= As) orM2O10(M= Mo, Mn, Nb, V) dimers.

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra “knock-ins” that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis., Author Summary Mycobacterium tuberculosis, the causative agent of human tuberculosis is an extremely successful human pathogen in spite of its lack of classical virulence factors, such as toxins. The pathogenesis of this bacterium is closely linked with its ability to circumvent destruction by the host cell, which depends on a large variety of mycobacterial lipids and secreted proteins. Genome comparison of fully virulent strains with closely related, but attenuated strains that have lost their ability to cause disease is a powerful approach to identify factors contributing to mycobacterial virulence. In this study we have compared the virulent paradigm strain of tuberculosis research M. tuberculosis H37Rv, with the widely used, attenuated laboratory variant, H37Ra. This analysis, combined with gene complementation studies, has identified a mutation in the two-component system regulator PhoP that impacts on the secretion and T cell recognition of the 6-kD early-secreted antigenic target ESAT-6. In previous studies, both PhoP and the ESAT-6 secretion system ESX-1 have been identified independently as major virulence determinants of M. tuberculosis, so the finding that their action is interconnected opens exciting new insights and perspectives into the virulence regulation of tubercle bacilli.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette–Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2 , is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding σ-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

The 174-kb virulence plasmid pMUM001 inMycobacterium ulceransepidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation ofM. ulceransAgy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverseM. ulceransstrains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in allM. ulceransstrains. Physical mapping of these plasmids revealed that likeM. ulceransAgy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis thatM. ulceransdiverged from the closely related organismMycobacterium marinumby acquiring pMUM. Our results suggest that pMUM is a defining characteristic ofM. ulceransbut that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.

The title compound, tetrasodium lithium cobalt aluminium hexa-(orthoarsenate), was synthesized by a solid state reaction route. In the crystal structure, Co(2+) ions are partially substituted by Al(3+) in an octa-hedral environment [M1 with site symmetry 2/m; occupancy ratio Co:Al = 0.286 (10):0.714 (10)]. The charge compensation is ensured by Li(+) cations sharing a tetra-hedral site with Co(2+) ions [M2 with site symmetry 2; occupancy ratio Co:Li = 0.690 (5):0.310 (5)]. The anionic unit is formed by two octa-hedra and three tetra-hedra linked only by corners. The CoM1M2As2O19 units associate to an open three-dimensional framework containing tunnels propagating along the a-axis direction. One Na(+) cation is located in the periphery of the tunnels while the other two are situated in the centres: all Na(+) cations exhibit half-occupancy. The structure of the studied material is compared with those of various related minerals reported in the literature.

Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a Mycobacterium , and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.