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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 RD1 mic 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 RD1 mic region.

Tuberculosis (TB) claims 1.5 million lives per year. This situation is largely due to the low efficacy of the only licensed TB vaccine, Bacillus Calmette-Guérin (BCG) against pulmonary TB. The metabolic disease type 2 diabetes (T2D) is a risk factor for TB and the mechanisms underlying increased TB susceptibility in T2D are not well understood. Furthermore, it is unknown if new TB vaccines will provide protection in the context of T2D. Here we used a diet-induced murine model of T2D to investigate the underlying mechanisms of TB/T2D comorbidity and to evaluate the protective capacity of two experimental TB vaccines in comparison to conventional BCG. Our data reveal a distinct immune dysfunction that is associated with diminished recognition of mycobacterial antigens in T2D. More importantly, we provide compelling evidence that mucosal delivery of recombinant BCG strains expressing the Mycobacterium tuberculosis ( Mtb ) ESX-1 secretion system (BCG::RD1 and BCG::RD1 ESAT-6 ∆92-95) are safe and confer superior immunity against aerosol Mtb infection in the context of T2D. Our findings suggest that the remarkable anti-TB immunity by these recombinant BCG strains is achieved via augmenting the numbers and functional capacity of antigen presenting cells in the lungs of diabetic mice., Competing Interests: The authors declare no competing interest.

Mycobacterium tuberculosis ( Mtb ) synthesizes a variety of atypical lipids that are exposed at the cell surface and help the bacterium infect macrophages and escape elimination by the cell's immune responses. In the present study, we investigate the mechanism of action of one family of hydrophobic lipids, the phthiocerol dimycocerosates (DIM/PDIM), major lipid virulence factors. DIM are transferred from the envelope of Mtb to host membranes during infection. Using the polarity-sensitive fluorophore C-Laurdan, we visualized that DIM decrease the membrane polarity of a supported lipid bilayer put in contact with mycobacteria, even beyond the site of contact. We observed that DIM activate the complement receptor 3, a predominant receptor for phagocytosis of Mtb by macrophages. DIM also increased the activity of membrane-permeabilizing effectors of Mtb , among which the virulence factor EsxA. This is consistent with previous observations that DIM help Mtb disrupt host cell membranes. Taken together, our data show that transferred DIM spread within the target membrane, modify its physical properties and increase the activity of host cell receptors and bacterial effectors, diverting in a non-specific manner host cell functions. We therefore bring new insight into the molecular mechanisms by which DIM increase Mtb's capability to escape the cell's immune responses., (Copyright © 2020 Augenstreich, Haanappel, Sayes, Simeone, Guillet, Mazeres, Chalut, Mourey, Brosch, Guilhot and Astarie-Dequeker.)

Antibiotics are widely used in the treatment of bacterial infections. Although known for their microbicidal activity, antibiotics may also interfere with the host's immune system. Here, we analyzed the effects of bedaquiline (BDQ), an inhibitor of the mycobacterial ATP synthase, on human macrophages. Genome-wide gene expression analysis revealed that BDQ reprogramed cells into potent bactericidal phagocytes. We found that 579 and 1,495 genes were respectively differentially expressed in naive- and M. tuberculosis -infected macrophages incubated with the drug, with an over-representation of lysosome-associated genes. BDQ treatment triggered a variety of antimicrobial defense mechanisms, including phagosome-lysosome fusion, and autophagy. These effects were associated with activation of transcription factor EB, involved in the transcription of lysosomal genes, resulting in enhanced intracellular killing of different bacterial species that were naturally insensitive to BDQ. Thus, BDQ could be used as a host-directed therapy against a wide range of bacterial infections., Competing Interests: AG, JC, AM, AB, MT, EB, JM, MG, GL, RB, BG, LT No competing interests declared, (© 2020, Giraud-Gatineau et al.)

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)., Competing Interests: Declaration Competing of Interest C.M., A.A. and J.G.-A. are co-inventors on the patent “Tuberculosis vaccine”. N.A., C.M. and J. G-A. are co-inventors on the patent “Compositions for use as a prophylactic agent to those at risk of infection of tuberculosis, or as secondary agents for treating infected tuberculosis patients”. Both patents were filled by the University of Zaragoza, (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Tuberculosis (TB) is the deadliest infectious disease worldwide. Bacille-Calmette-Guérin (BCG), the only licensed TB vaccine, affords variable protection against TB but remains the gold standard. BCG improvement is focused around three strategies: recombinant BCG strains, heterologous routes of administration, and booster vaccination. It is currently unknown whether combining these strategies is beneficial. The preclinical evaluation for new TB vaccines is heavily skewed toward immunogenicity and efficacy; however, safety and efficacy are the dominant considerations in human use. To facilitate stage gating of TB vaccines, we developed a simple empirical model to systematically rank vaccination strategies by integrating multiple measurements of safety, immunogenicity, and efficacy. We assessed 24 vaccination regimens, composed of three BCG strains and eight combinations of delivery. The model presented here highlights that mucosal booster vaccination may cause adverse outcomes and provides a much needed strategy to evaluate and rank data obtained from TB vaccine studies using different routes, strains, or animal models., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

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, HadD Mtb (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto- and methoxy-MAs. HadD Mtb 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 HadD Mtb 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 HadD Mtb on the planktonic growth, colony morphology and biofilm structuration, as well as on low temperature tolerance. Importantly, HadD Mtb has a strong impact on Mtb virulence in the mouse model of infection. The effects of the lack of HadD Mtb observed both in vitro and in vivo designate this protein as a bona fide target for the development of novel anti-TB intervention strategies.

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.

The borderline between virulence and efficacy in live attenuated vaccine strains is often blurred and this is also the case for the Bacillus Calmette-Guérin (BCG), the only currently licensed anti-tuberculosis vaccine used on a large, global scale, which was obtained almost 100 years ago. While BCG is more than 99% identical at the genome level to Mycobacterium tuberculosis , the causative pathogen of human tuberculosis, some important differences in virulence factors cause naturally irreversible attenuation and safety of this vaccine in the immunocompetent host. Some of these virulence factors are involved in persistence capacities of the vaccine strains and also represent strong immunogens, responsible for inducing different host signaling pathways, which have to be taken into consideration for the development of revised and new vaccine strains. Here we discuss a number of selected mycobacterial features in relation to their biological functions and potential impact on virulence and vaccine efficacy., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed., (Copyright: © 2019 Kroesen VM et al.)

Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival., Competing Interests: The authors have declared that no competing interests exist.

Tuberculosis remains one of the deadliest infectious diseases of humanity. To better understand the evolutionary history of host-adaptation of tubercle bacilli (MTB), we sought for mycobacterial species that were more closely related to MTB than the previously used comparator species Mycobacterium marinum and Mycobacterium kansasii. Our phylogenomic approach revealed some recently sequenced opportunistic mycobacterial pathogens, Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense, to constitute a common clade with MTB, hereafter called MTB-associated phylotype (MTBAP), from which MTB have emerged. Multivariate and clustering analyses of genomic functional content revealed that the MTBAP lineage forms a clearly distinct cluster of species that share common genomic characteristics, such as loss of core genes, shift in dN/dS ratios, and massive expansion of toxin-antitoxin systems. Consistently, analysis of predicted horizontal gene transfer regions suggests that putative functions acquired by MTBAP members were markedly associated with changes in microbial ecology, for example adaption to intracellular stress resistance. Our study thus considerably deepens our view on MTB evolutionary history, unveiling a decisive shift that promoted conversion to host-adaptation among ancestral founders of the MTBAP lineage long before Mycobacterium tuberculosis has adapted to the human host., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Over the long course of evolution from a probable environmental reservoir, the pathogen that we know today as Mycobacterium tuberculosis has become fully capable of adapting to the life inside host cells by evading and modifying their responses to infection. Factors contributing to the success of this pathogen are numerous and thanks to a large body of work accumulated over the past decades, we are closer to understanding the remarkable complexity of tuberculosis pathogenesis. The unique type VII secretion systems and various complex lipids of the cell envelope have emerged as some of the most important and most studied factors in this regard. This review attempts to summarize recent findings on these and other virulence factors, while discussing their evolution in different closely related tuberculosis-causing bacteria as well, with the aim of exploring the processes which led M. tuberculosis to becoming one of the deadliest infections agents., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The causative agent of human tuberculosis, Mycobacterium tuberculosis , has a complex lipid-rich diderm envelope, which acts as a major barrier protecting the bacterium against the hostile environment inside the host cells. For the transfer of diverse molecules across this complex cell envelope, M. tuberculosis has a series of general and specialized protein secretion systems, characterized by the SecA general secretion pathway, the twin-arginine translocation pathway, and five specific ESX type VII secretion systems. In this review, we focus on the latter systems, known as ESX-1 to ESX-5, which were first discovered almost 20 years ago during the in silico analysis of the genome sequence of M. tuberculosis H37Rv. Since then, these systems have been the subject of highly dynamic research due to their involvement in several key biological processes and host-pathogen interactions of the tubercle bacilli.

Multi-drug-resistant tuberculosis (TB) is a major public health problem, concerning about half a million cases each year. Patients hardly adhere to the current strict treatment consisting of more than 10 000 tablets over a 2-year period. There is a clear need for efficient and better formulated medications. We have previously shown that nanoparticles made of cross-linked poly-β-cyclodextrins (pβCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pβCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pβCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pβCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pβCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pβCD nanoparticles loaded or not with antibiotics have an antibacterial action on their own and could be used as a carrier in drug regimen formulations effective against TB.

PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mycobacterium africanum consists of Lineages L5 and L6 of the Mycobacterium tuberculosis complex (MTBC) and causes human tuberculosis in specific regions of Western Africa, but is generally not transmitted in other parts of the world. Since M. africanum is evolutionarily closely placed between the globally dispersed Mycobacterium tuberculosis and animal-adapted MTBC-members, these lineages provide valuable insight into M. tuberculosis evolution. Here, we have collected 15 M. africanum L5 strains isolated in France over 4 decades. Illumina sequencing and phylogenomic analysis revealed a previously underappreciated diversity within L5, which consists of distinct sublineages. L5 strains caused relatively high levels of extrapulmonary tuberculosis and included multi- and extensively drug-resistant isolates, especially in the newly defined sublineage L5.2. The specific L5 sublineages also exhibit distinct phenotypic characteristics related to in vitro growth, protein secretion and in vivo immunogenicity. In particular, we identified a PE_PGRS and PPE-MPTR secretion defect specific for sublineage L5.2, which was independent of PPE38. Furthermore, L5 isolates were able to efficiently secrete and induce immune responses against ESX-1 substrates contrary to previous predictions. These phenotypes of Type VII protein secretion and immunogenicity provide valuable information to better link genome sequences to phenotypic traits and thereby understand the evolution of the MTBC.

Visualization is defined as the production of mental images in the process of reading (Esrock 2005: 633). This article is concerned with varieties of visualization during an absorbing reading of a fictional narrative, the mental images that range from an indistinct and largely automatic default visualization to the much more vivid images that occur at significant stages in the narrative. Neuroscientific studies of vision have collected a large and impressively varied body of experimental evidence for two major processing streams - the dorsal and the ventral-specialized for vision-for-action and vision for-perception respectively. Further experiments distinguish different dispositional specializations: visualizers with a high spatial visualizing ability demonstrating a more efficient use of resources in the dorsal pathway, and those with a high object visualization and more efficient use of the ventral pathway (Kozhevnikov et al., 2010: 29). We can assume that both types of mental processing will be prompted in fictional narratives with differences in prominence depending on their authors' inclinations and the design and purpose of the narrative text. According to Amedeo D'Angiulli (2013: 7), who conducted elaborate tests of vividness in mental imagery using written descriptive passages as stimulus, dynamic imagery was significantly less vivid than static imagery. These results confirm traditional literary criticism based on introspection which argues that detailed description of static objects elicits an especially lively imagination. However, narratives can provoke even stronger visualizations by rendering subjective moments of seeing in which a fictional character is emotionally involved. In encouraging readers to shift now and then from the default mode of motion-oriented visualizing to a more affective and more conscious object visualization, literary fictions exercise their power to evoke imaginings that one would not generate by oneself. This may indicate that literary narratives can prove a training ground for expanding one's visualizing capacities., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 partially 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., Competing Interests: LM and RB are named inventors on a patent related to RD1, RD5 and RD8 regions of BCG. MAB is a named inventor on a separate patent related to genomic differences of the Mycobacterium tuberculosis complex. The other authors declare that no financial or competing interests exist.

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., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis, the causative agent of human tuberculosis is one of the most widely spread human pathogens. It has succeeded to infect a quarter of the global human population by developing most sophisticated ways to circumvent innate and adaptive immune defences. This highly specialized, major human pathogen has evolved from a pool of ancestral environmental mycobacteria, whose extant representatives are known under the name of Mycobacterium canettii. Recent whole genome analyses in combination with different phenotypic screens have provided key insights into the evolution of M. tuberculosis and closely related members regrouped in the M. tuberculosis complex (MTBC). They have also elucidated novel virulence determinants that are essential for these obligate pathogens. In this review, we present the most recent evolutionary models of the MTBC and various factors that have contributed to the outstanding evolutionary success of the tuberculosis agent., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems 1,2 . The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus 3,4 . However, the role of these proteins remains largely elusive 1 . Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion 5 . Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the 'modern' Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution 6,7 .

Mycobacterium abscessus , a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. M. abscessus can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the M. abscessus genes required for replication within amoebae. To this end, we constructed and screened a transposon ( Tn ) insertion library of an M. abscessus subsp e cies massiliense clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in M. abscessus encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to M. abscessus growth and survival in amoebae and macrophages, we generated a deletion mutant of eccB 4 that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates., Competing Interests: The authors declare no conflict of interest.

The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

Mycobacterium tuberculosis ( Mtb ), the etiological agent of human tuberculosis (TB), has plagued humans for thousands of years. TB still remains a major public health problem in our era, causing more than 4,400 deaths worldwide every day and killing more people than HIV. After inhaling Mtb -contaminated aerosols, TB primo-infection starts in the terminal lung airways, where Mtb is taken up by alveolar macrophages. Although macrophages are known as professional killers for pathogens, Mtb has adopted remarkable strategies to circumvent host defenses, building suitable conditions to survive and proliferate. Within macrophages, Mtb initially resides inside phagosomes, where its survival mostly depends on its ability to take control of phagosomal processing, through inhibition of phagolysosome biogenesis and acidification processes, and by progressively getting access to the cytosol. Bacterial access to the cytosolic space is determinant for specific immune responses and cell death programs, both required for the replication and the dissemination of Mtb . Comprehension of the molecular events governing Mtb survival within macrophages is fundamental for the improvement of vaccine-based and therapeutic strategies in order to help the host to better defend itself in the battle against the fierce invader Mtb . In this mini-review, we discuss recent research exploring how Mtb conquers and transforms the macrophage into a strategic base for its survival and dissemination as well as the associated defense strategies mounted by host.

Pathogens have evolved a range of mechanisms to counteract host defenses, notably to survive harsh acidic conditions in phagosomes. In the case of Mycobacterium tuberculosis, it has been shown that regulation of phagosome acidification could be achieved by interfering with the retention of the V-ATPase complexes at the vacuole. Here, we present evidence that M. tuberculosis resorts to yet another strategy to control phagosomal acidification, interfering with host suppressor of cytokine signaling (SOCS) protein functions. More precisely, we show that infection of macrophages with M. tuberculosis leads to granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion, inducing STAT5-mediated expression of cytokine-inducible SH2-containing protein (CISH), which selectively targets the V-ATPase catalytic subunit A for ubiquitination and degradation by the proteasome. Consistently, we show that inhibition of CISH expression leads to reduced replication of M. tuberculosis in macrophages. Our findings further broaden the molecular understanding of mechanisms deployed by bacteria to survive., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Tuberculosis (TB) is a deadly infectious disease, which kills millions of people every year. The causative pathogen, Mycobacterium tuberculosis (MTB), is estimated to have infected up to a third of the world's population; however, only approximately 10% of infected healthy individuals progress to active TB. Despite evidence for heritability, it is not currently possible to predict who may develop TB. To explore approaches to classify susceptibility to TB, we infected with MTB dendritic cells (DCs) from putatively resistant individuals diagnosed with latent TB, and from susceptible individuals that had recovered from active TB. We measured gene expression levels in infected and non-infected cells and found hundreds of differentially expressed genes between susceptible and resistant individuals in the non-infected cells. We further found that genetic polymorphisms nearby the differentially expressed genes between susceptible and resistant individuals are more likely to be associated with TB susceptibility in published GWAS data. Lastly, we trained a classifier based on the gene expression levels in the non-infected cells, and demonstrated reasonable performance on our data and an independent data set. Overall, our promising results from this small study suggest that training a classifier on a larger cohort may enable us to accurately predict TB susceptibility.

Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT-6). This small protein, which is secreted by the type VII secretion system ESX-1 (T7SS/ESX-1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock-out or knock-in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX-1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb., (© 2017 John Wiley & Sons Ltd.)

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.

Available chemotherapeutic options are very limited against Mycobacterium abscessus , which imparts a particular challenge in the treatment of cystic fibrosis (CF) patients infected with this rapidly growing mycobacterium. New drugs are urgently needed against this emerging pathogen, but the discovery of active chemotypes has not been performed intensively. Interestingly, however, the repurposing of thiacetazone (TAC), a drug once used to treat tuberculosis, has increased following the deciphering of its mechanism of action and the detection of significantly more potent analogues. We therefore report studies performed on a library of 38 TAC-related derivatives previously evaluated for their antitubercular activity. Several compounds, including D6, D15, and D17, were found to exhibit potent activity in vitro against M. abscessus , Mycobacterium massiliense , and Mycobacterium bolletii clinical isolates from CF and non-CF patients. Similar to TAC in Mycobacterium tuberculosis , the three analogues act as prodrugs in M. abscessus , requiring bioactivation by the EthA enzyme, MAB_0985. Importantly, mutations in the transcriptional TetR repressor MAB_4384, with concomitant upregulation of the divergently oriented adjacent genes encoding an MmpS5/MmpL5 efflux pump system, accounted for high cross-resistance levels among all three compounds. Overall, this study uncovered a new mechanism of drug resistance in M. abscessus and demonstrated that simple structural optimization of the TAC scaffold can lead to the development of new drug candidates against M. abscessus infections., (Copyright © 2017 American Society for Microbiology.)

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 Mmar ) 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 Mmar confers superior protection relative to parental BCG against challenges with highly virulent M. tuberculosis., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis and Mycobacterium marinum are thought to exert virulence, in part, through their ability to lyse host cell membranes. The type VII secretion system ESX-1 [6-kDa early secretory antigenic target (ESAT-6) secretion system 1] is required for both virulence and host cell membrane lysis. Both activities are attributed to the pore-forming activity of the ESX-1-secreted substrate ESAT-6 because multiple studies have reported that recombinant ESAT-6 lyses eukaryotic membranes. We too find ESX-1 of M. tuberculosis and M. marinum lyses host cell membranes. However, we find that recombinant ESAT-6 does not lyse cell membranes. The lytic activity previously attributed to ESAT-6 is due to residual detergent in the preparations. We report here that ESX-1-dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores. ESX-1-mediated lysis is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems. Our findings suggest redirection of research to understand the mechanism of ESX-1-mediated lysis., Competing Interests: The authors declare no conflict of interest.

Mycobacterium tuberculosis, the etiological agent of human tuberculosis, harbours five ESAT-6/type VII secretion (ESX/T7S) systems. The first esx gene clusters were identified during the genome-sequencing project of M. tuberculosis H37Rv. Follow-up studies revealed additional genes playing important roles in ESX/T7S systems. Among the latter genes, one can find those that encode Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins as well as a gene cluster that is encoded >260 kb upstream of the esx-1 locus and encodes ESX-1 secretion-associated proteins EspA (Rv3616c), EspC (Rv3615c) and EspD (Rv3614c). The espACD cluster has been suggested to have an important function in ESX-1 secretion since EspA-EspC and EsxA-EsxB are mutually co-dependent on each other for secretion. However, the molecular mechanism of this co-dependence and interaction between the substrates remained unknown. In this issue of Molecular Microbiology, Lou and colleagues show that EspC forms high-molecular weight polymerization complexes that resemble selected components of type II, III and/or IV secretion systems of Gram-negative bacteria. Indeed, EspC-multimeric complexes form filamentous structures that could well represent a secretion needle of ESX-1 type VII secretion systems. This exciting observation opens new avenues for research to discover and characterize ESX/T7S components and elucidates the co-dependence of EsxA/B secretion with EspA/C., (© 2016 John Wiley & Sons Ltd.)

Bacterial secretion systems are sophisticated molecular machines that fulfil a wide range of important functions, which reach from export/secretion of essential proteins or virulence factors to the implication in conjugation processes. In contrast to the widely distributed Sec and Twin Arginine Translocation (TAT) systems, the recently identified ESX/type VII systems show a more restricted distribution and are typical for mycobacteria and other high-GC Actinobacteria. Similarly, type VII-like secretion systems have been described in low-GC Gram-positive bacteria belonging to the phylum Firmicutes. While the most complex organization of type VII secretion systems currently known is found in slow-growing mycobacteria, which harbour up to 5 chromosomal-encoded systems (ESX-1 to ESX-5), much simpler organization is reported for type VII-like systems in Firmicutes. In this chapter, we describe common and divergent features of type VII- and type VII-like secretion pathways and also comment on their biological key roles, many of which are related to species-/genus-specific host-pathogen interactions and/or virulence mechanisms.

Genome-based insights into the evolution of Mycobacterium tuberculosis and other tuberculosis-causing mycobacteria are constantly increasing. In particular, the recent genomic and functional characterization of several Myocbacterium canettii strains, which are thought to resemble in many aspects the putative common ancestor of the members of the M. tuberculosis complex (MTBC), has consolidated a plausible scenario of the early evolution of tuberculosis-causing mycobacteria, in which the clonal MTBC, comprising numerous key pathogens of mammalian hosts, has evolved from a generalist mycobacterium living in the environment. These studies also have considerably enriched our knowledge on selected molecular events that likely have contributed to the incursion, maintenance and spread of the MTBC members in diverse mammalian hosts. Here, we summarize and discuss recently revealed molecular and evolutionary aspects and emphasize the vast utility of M. canettii strains for identifying the mechanisms that contributed to the global emergence of M. tuberculosis as one of the most important human pathogens.

Despite intense research, PE_PGRS proteins still represent an intriguing aspect of mycobacterial pathogenesis. These cell surface proteins influence virulence in several pathogenic species, but their diverse and exact functions remain unclear. Herein, we focussed on a PE_PGRS member from Mycobacterium marinum, MMAR_0242, characterized by an extended and unique C-terminal domain. We demonstrate that an M. marinum mutant carrying a transposon insertion in MMAR_0242 is highly impaired in its ability to replicate in macrophages and amoebae, because of its inability to inhibit lysosomal fusion. As a consequence, this mutant failed to survive intracellularly as evidenced by a reduced number of cytosolic actin tail-forming bacteria and by quantitative electron microscopy, which mainly localized MMAR_0242::Tn within membrane-defined vacuoles. Functional complementation studies indicated that the C-terminus, but not the N-terminal PE_PGRS domain, is required for intracellular growth/survival. In line with these findings, disruption of MMAR_0242 resulted in a highly attenuated virulence phenotype in zebrafish embryos, characterized by restricted bacterial loads and a failure to produce granulomas. Furthermore, expression of MMAR_0242 in Mycobacterium smegmatis, a non-pathogenic species naturally deficient in PE_PGRS production, resulted in increased survival in amoebae with enhanced cytotoxic cell death and increased survival in infected mice with splenomegaly. Overall, these results indicate that MMAR_0242 is required for full virulence of M. marinum and sufficient to confer pathogenic properties to M. smegmatis., (© 2016 John Wiley & Sons Ltd.)

Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF). Mycobacterium abscessus is known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show that M. abscessus S and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome-cytosol communication. In conclusion, M. abscessus S exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species., (© 2016 The Authors.)

Mycobacterium tuberculosis uses sophisticated secretion systems, named 6 kDa early secretory antigenic target (ESAT6) protein family secretion (ESX) systems (also known as type VII secretion systems), to export a set of effector proteins that helps the pathogen to resist or evade the host immune response. Since the discovery of the esx loci during the M. tuberculosis H37Rv genome project, structural biology, cell biology and evolutionary analyses have advanced our knowledge of the function of these systems. In this Review, we highlight the intriguing roles that these studies have revealed for ESX systems in bacterial survival and pathogenicity during infection with M. tuberculosis. Furthermore, we discuss the diversity of ESX systems that has been described among mycobacteria and selected non-mycobacterial species. Finally, we consider how our knowledge of ESX systems might be applied to the development of novel strategies for the treatment and prevention of disease.

The tuberculosis agent Mycobacterium tuberculosis has undergone a long and selective evolution toward human infection and represents one of the most widely spread pathogens due to its efficient aerosol-mediated human-to-human transmission. With the availability of more and more genome sequences, the evolutionary trajectory of this obligate pathogen becomes visible, which provides us with new insights into the molecular events governing evolution of the bacterium and its ability to accumulate drug-resistance mutations. In this review, we summarize recent developments in mycobacterial research related to this matter that are important for a better understanding of the current situation and future trends and developments in the global epidemiology of tuberculosis, as well as for possible public health intervention possibilities.

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 fluorescence-assisted 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., Competing Interests: The authors declare no conflict of interest.

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., (© 2016 John Wiley & Sons Ltd.)

Mycobacterium tuberculosis, the infectious agent of human tuberculosis is a master player in circumventing the defense mechanisms of the host immune system. The host-pathogen interaction in the case of an infection with M. tuberculosis is highly complex, involving dedicated mycobacterial virulence factors as well as the action of the innate and adapted immune systems, which determine the outcome of infection. Macrophages play a key role in this process through internalizing the bacterium in a phagosomal vacuole. While this action has normally the function of eliminating invading bacteria, M. tuberculosis employs efficient strategies to prevent its extermination. The question on how-and-where the bacterium succeeds in doing so has interested generations of scientists and still remains a fascinating and important research subject focused on mycobacterial lipids, secretion systems and other contributing factors. This topic is also central to the longstanding and partially controversial discussion on mycobacterial phagosomal rupture and vacuole-to-cytosol translocation, to be reviewed here in more detail., (© 2016 John Wiley & Sons Ltd.)

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.

Lineage 7 of the Mycobacterium tuberculosis complex has recently been identified among strains originating from Ethiopia. Using different DNA typing techniques, this study provides additional information on the genetic heterogeneity of five lineage 7 strains collected in the Amhara Region of Ethiopia. It also confirms the phylogenetic positioning of these strains between the ancient lineage 1 and TbD1-deleted, modern lineages 2, 3 and 4 of Mycobacterium tuberculosis . Four newly identified large sequence polymorphisms characteristic of the Amhara Region lineage 7 strains are described. While lineage 7 strains have been previously identified in the Woldiya area, we show that lineage 7 strains circulate in other parts of the Amhara Region and also among foreign-born individuals from Eritrea and Somalia in The Netherlands. For ease of documenting future identification of these strains in other geographical locations and recognizing the place of origin, we propose to assign lineage 7 strains the lineage name ' Aethiops vetus ' .

IFN-γ is a critical mediator of host defense against Mycobacterium tuberculosis (Mtb) infection. Antigen-specific CD4+ T cells have long been regarded as the main producer of IFN-γ in tuberculosis (TB), and CD4+ T cell immunity is the main target of current TB vaccine candidates. However, given the recent failures of such a TB vaccine candidate in clinical trials, strategies to harness CD4-independent mechanisms of protection should be included in future vaccine design. Here, we have reported that noncognate IFN-γ production by Mtb antigen-independent memory CD8+ T cells and NK cells is protective during Mtb infection and evaluated the mechanistic regulation of IFN-γ production by these cells in vivo. Transfer of arenavirus- or protein-specific CD8+ T cells or NK cells reduced the mortality and morbidity rates of mice highly susceptible to TB in an IFN-γ-dependent manner. Secretion of IFN-γ by these cell populations required IL-18, sensing of mycobacterial viability, Mtb protein 6-kDa early secretory antigenic target-mediated (ESAT-6-mediated) cytosolic contact, and activation of NLR family pyrin domain-containing protein 3 (NLRP3) inflammasomes in CD11c+ cell subsets. Neutralization of IL-18 abrogated protection in susceptible recipient mice that had received noncognate cells. Moreover, improved Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine-induced protection was lost in the absence of ESAT-6-dependent cytosolic contact. Our findings provide a comprehensive mechanistic framework for antigen-independent IFN-γ secretion in response to Mtb with critical implications for future intervention strategies against TB.

In mycobacteria, MmpL proteins represent key components that participate in the biosynthesis of the complex cell envelope. Whole genome analysis of a spontaneous rough morphotype variant of Mycobacterium abscessus subsp. bolletii identified a conserved tyrosine that is crucial for the function of MmpL family proteins. Isogenic smooth (S) and rough (R) variants differed by a single mutation linked to a Y842H substitution in MmpL4a. This mutation caused a deficiency in glycopeptidolipid production/transport in the R variant and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was found, involving massive production of serpentine cords, abscess formation and rapid larval death. Importantly, this finding allowed us to demonstrate an essential role of Tyr842 in several different MmpL proteins, including Mycobacterium tuberculosis MmpL3. Structural homology models of MmpL4a and MmpL3 identified two additional critical residues located in the transmembrane regions TM10 and TM4 that are facing each other. We propose that these central residues are part of the proton-motive force that supplies the energy for substrate transport. Hence, we provide important insights into mechanistic/structural aspects of MmpL proteins as lipid transporters and virulence determinants in mycobacteria., (© 2015 John Wiley & Sons Ltd.)

Background: In mycobacteria, conjugation differs from the canonical Hfr model, but is still poorly understood. Here, we quantified this evolutionary processe in a natural mycobacterial population, taking advantage of a large clinical strain collection of the emerging pathogen Mycobacterium abscessus (MAB)., Results: Multilocus sequence typing confirmed the existence of three M. abscessus subspecies, and unravelled extensive allelic exchange between them. Furthermore, an asymmetrical gene flow occurring between these main lineages was detected, resulting in highly admixed strains. Intriguingly, these mosaic strains were significantly associated with cystic fibrosis patients with lung infections or chronic colonization. Genome sequencing of those hybrid strains confirmed that half of their genomic content was remodelled in large genomic blocks, leading to original tri-modal 'patchwork' architecture. One of these hybrid strains acquired a locus conferring inducible macrolide resistance, and a large genomic insertion from a slowly growing pathogenic mycobacteria, suggesting an adaptive gene transfer. This atypical genomic architecture of the highly recombinogenic strains is consistent with the distributive conjugal transfer (DCT) observed in M. smegmatis. Intriguingly, no known DCT function was found in M. abscessus chromosome, however, a p-RAW-like genetic element was detected in one of the highly admixed strains., Conclusion: Taken together, our results strongly suggest that MAB evolution is sporadically punctuated by dramatic genome wide remodelling events. These findings might have far reaching epidemiological consequences for emerging mycobacterial pathogens survey in the context of increasing numbers of rapidly growing mycobacteria and M. tuberculosis co-infections.

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., (© 2015 John Wiley & Sons Ltd.)