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Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pidot et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis ( Mtb ) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens , Mycobacterium lacus , Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb . We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb , was present only in M. decipiens , which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis ( Mtb ) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum . We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb ., Competing Interests: The authors declare no conflict of interest.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Maure et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tuberculosis (TB) remains one of the deadliest infectious diseases in human history, prevailing even in the 21st century. The causative agents of TB are represented by a group of closely related bacteria belonging to the Mycobacterium tuberculosis complex (MTBC), which can be subdivided into several lineages of human- and animal-adapted strains, thought to have shared a last common ancestor emerged by clonal expansion from a pool of recombinogenic Mycobacterium canettii-like tubercle bacilli. A better understanding of how MTBC populations evolved from less virulent mycobacteria may allow for discovering improved TB control strategies and future epidemiologic trends. In this review, we highlight new insights into the evolution of mycobacteria at the genus level, describing different milestones in the evolution of mycobacteria, with a focus on the genomic events that have likely enabled the emergence and the dominance of the MTBC. We also review the recent literature describing the various MTBC lineages and highlight their particularities and differences with a focus on host preferences and geographic distribution. Finally, we discuss on putative mechanisms driving the evolution of tubercle bacilli and mycobacteria in general, by taking the mycobacteria-specific distributive conjugal transfer as an example., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The molecular factors and genetic adaptations that contributed to the emergence of Mycobacterium tuberculosis (MTB) from an environmental Mycobacterium canettii-like ancestor, remain poorly investigated. In MTB, the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Here, we describe that several mutations, present in phoR of M. canettii relative to MTB, impact the expression of the PhoP regulon and the pathogenicity of the strains. First, we establish a molecular model of PhoR and show that some substitutions found in PhoR of M. canettii are likely to impact the structure and activity of this protein. Second, we show that STB-K, the most attenuated available M. canettii strain, displays lower expression of PhoP-induced genes than MTB. Third, we demonstrate that genetic swapping of the phoPR allele from STB-K with the ortholog from MTB H37Rv enhances expression of PhoP-controlled functions and the capacities of the recombinant strain to colonize human macrophages, the MTB target cells, as well as to cause disease in several mouse infection models. Fourth, we extended these observations to other M. canettii strains and confirm that PhoP-controlled functions are expressed at lower levels in most M. canettii strains than in M. tuberculosis. Our findings suggest that distinct PhoR variants have been selected during the evolution of tuberculosis bacilli, contributing to higher pathogenicity and persistence of MTB in the mammalian host., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Malaga et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In 2020, a record number of tick-borne encephalitis (TBE) cases was reported in major endemic areas in Germany, i.e., the southern federal states of Baden-Wuerttemberg and Bavaria. Most cases were unvaccinated. Other tick-borne diseases (TBDs), including Lyme borreliosis and tularemia, are rising, too. Thus, strategies are needed to increase TBE vaccination uptake in risk areas and promote education on TBD prevention. Primary care physicians are key providers of both vaccinations and TBD education. The TBD-Prevention (TBD-Prev) study aimed to investigate the knowledge, attitudes and behaviors of primary care physicians in Baden-Wuerttemberg and Bavaria with regard to TBE vaccination and prevention of TBDs and to derive strategies for increasing vaccination rates and improving knowledge about TBE and other TBDs in the population and among primary care physicians. We invited all primary care physicians (N = 14,046) in both states to participate by mail. Using standardized, self-administered questionnaires, available both on paper and online, we asked physicians anonymously about their knowledge, attitudes and behaviors with respect to TBE vaccination and TBD prevention and their need for further information/educational materials. A total of 2321 physicians participated between May and September 2022 (response rate 17%), of whom 1222 (53%) worked in Baden-Wuerttemberg and 1067 (46%) in Bavaria. Among the participating physicians, 56% were male, 71% were >50 years and 51% worked in an individual practice. Furthermore, 91% were aware of the German national vaccination guidelines, and 98% perceived their knowledge of the risks and benefits of vaccination as adequate. A total of 97% offer TBE vaccinations, 67% provide vaccination counselling during initial consultations with new patients and 64% actively remind patients about due vaccinations. In addition, 24% expressed a need for further information materials, mainly traditional, analogue media such as flyers (82%) and posters (50%), and named timeliness, quality assurance, easy comprehensibility and independence from the pharmaceutical industry as the most important characteristics of such materials. Almost all participating physicians reported offering TBE vaccinations and feeling well-informed about TBE vaccination and TBDs. However, active offering of vaccinations and education could be further improved, and additional, low-threshold information materials are needed. Based on these results, we will develop and provide various materials on TBE vaccination and TBDs, in particular flyers and posters, for use by physicians during consultations.

H37Rv is the most widely used Mycobacterium tuberculosis strain, and its genome is globally used as the M. tuberculosis reference sequence. Here, we present Bact-Builder, a pipeline that uses consensus building to generate complete and accurate bacterial genome sequences and apply it to three independently cultured and sequenced H37Rv aliquots of a single laboratory stock. Two of the 4,417,942 base-pair long H37Rv assemblies are 100% identical, with the third differing by a single nucleotide. Compared to the existing H37Rv reference, the new sequence contains ~6.4 kb additional base pairs, encoding ten new regions that include insertions in PE/PPE genes and new paralogs of esxN and esxJ, which are differentially expressed compared to the reference genes. New sequencing and de novo assemblies with Bact-Builder confirm that all 10 regions, plus small additional polymorphisms, are also present in the commonly used H37Rv strains NR123, TMC102, and H37Rv1998. Thus, Bact-Builder shows promise as an improved method to perform accurate and reproducible de novo assemblies of bacterial genomes, and our work provides important updates to the primary M. tuberculosis reference genome., (© 2022. The Author(s).)

Lentiviral vectors (LVs) are highly efficient at inducing CD8 + T cell responses. However, LV-encoded antigens are processed inside the cytosol of antigen-presenting cells, which does not directly communicate with the endosomal major histocompatibility complex class II (MHC-II) presentation pathway. LVs are thus poor at inducing CD4 + T cell response. To overcome this limitation, we devised a strategy whereby LV-encoded antigens are extended at their N-terminal end with the MHC-II-associated light invariant chain (li), which contains an endosome-targeting signal sequence. When evaluated with an LV-encoded polyantigen composed of CD4 + T cell targets from Mycobacterium tuberculosis, intranasal vaccination in mice triggers pulmonary polyfunctional CD4 + and CD8 + T cell responses. Adjuvantation of these LVs extends the mucosal immunity to Th17 and Tc17 responses. A systemic prime and an intranasal boost with one of these LV induces protection against M. tuberculosis. This strategy improves the protective power of LVs against infections and cancers, where CD4 + T cell immunity plays an important role., Competing Interests: Declaration of interests P.C. is the founder and CSO of TheraVectys. J.L., F.A., P.A., M.-W.K., F.M., and A.N. are employees of TheraVectys. J.L., F.A., C.B., F.M., L.M., and P.C. are inventors of a pending patent directed to LV immunization able to induce CD4(+) T cells., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Most viral vectors, including the potently immunogenic lentiviral vectors (LVs), only poorly direct antigens to the MHC-II endosomal pathway and elicit CD4 + T cells. We developed a new generation of LVs encoding antigen-bearing monomers of collectins substituted at their C-terminal domain with the CD40 ligand ectodomain to target and activate antigen-presenting cells. Host cells transduced with such optimized LVs secreted soluble collectin-antigen polymers with the potential to be endocytosed in vivo and reach the MHC-II pathway. In the murine tuberculosis model, such LVs induced efficient MHC-II antigenic presentation and triggered both CD8 + and CD4 + T cells at the systemic and mucosal levels. They also conferred a significant booster effect, consistent with the importance of CD4 + T cells for protection against Mycobacterium tuberculosis. Given the pivotal role of CD4 + T cells in orchestrating innate and adaptive immunity, this strategy could have a broad range of applications in the vaccinology field., (© 2022. The Author(s).)

Since 2004, a tuberculosis surveillance protocol has been carried out in Aragon, thereby managing to detect all tuberculosis outbreaks that take place in the community. The largest outbreak was caused by a strain named Mycobacterium tuberculosis Zaragoza (MtZ), causing 242 cases as of 2020. The main objective of this work was to analyze this outbreak and the molecular characteristics of this successful strain that could be related to its greater transmission. To do this, we first applied whole-genome sequencing to 57 of the isolates. This revealed two principal transmission clusters and six subclusters arising from them. The MtZ strain belongs to L4.8 and had eight specific single nucleotide polymorphisms (SNPs) in genes considered to be virulence factors [ ptpA , mc3D , mc3F , VapB41 , pks15 (two SNPs), virS , and VapC50 ]. Second, a transcriptomic study was carried out to better understand the multiple IS 6110 copies present in its genome. This allowed us to observe three effects of IS 6110 : the disruption of the gene in which the IS 6110 is inserted ( desA3 ), the overexpression of a gene ( ppe38 ), and the absence of transcription of genes ( cut1:Rv1765c ) due to the recombination of two IS 6110 copies. Finally, because of the disruption of ppe38 and ppe71 genes by an IS 6110 , a study of PE_PGRS secretion was carried out, showing that MtZ secretes these factors in higher amounts than the reference strain, thereby differing from the hypervirulent phenotype described for the Beijing strains. In conclusion, MtZ consists of several SNPs in genes related to virulence, pathogenesis, and survival, as well as other genomic polymorphisms, which may be implicated in its success among our population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Comín, Madacki, Rabanaque, Zúñiga-Antón, Ibarz, Cebollada, Viñuelas, Torres, Sahagún, Klopp, Gonzalo-Asensio, Brosch, Iglesias and Samper.)

SignificanceTuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis , and its close pathogenic relative Mycobacterium marinum , initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes. Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed to the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.

Cyclic (di)nucleotides act as universal second messengers endogenously produced by several pathogens. Specifically, the roles of c-di-AMP in Mycobacterium tuberculosis immunity and virulence have been largely explored, although its contribution to the safety and efficacy of live tuberculosis vaccines is less understood. In this study, we demonstrate that the synthesis of c-di-AMP is negatively regulated by the M. tuberculosis PhoPR virulence system. Accordingly, the live attenuated tuberculosis vaccine candidate M. tuberculosis vaccine (MTBVAC), based on double phoP and fadD26 deletions, produces more than 25- and 45-fold c-di-AMP levels relative to wild-type M. tuberculosis or the current vaccine bacille Calmette-Guérin (BCG), respectively. Secretion of this second messenger was exclusively detected in MTBVAC but not in M. tuberculosis or in BCG. We also demonstrate that c-di-AMP synthesis during in vitro cultivation of M. tuberculosis is a growth-phase- and medium-dependent phenotype. To uncover the role of this metabolite in the vaccine properties of MTBVAC, we constructed and validated knockout and overproducing/oversecreting derivatives by inactivating the disA or cnpB gene, respectively. All MTBVAC derivatives elicited superior interleukin-1β (IL-1β) responses compared with BCG during an in vitro infection of human macrophages. However, both vaccines failed to elicit interferon β (IFNβ) activation in this cellular model. We found that increasing c-di-AMP levels remarkably correlated with a safer profile of tuberculosis vaccines in the immunodeficient mouse model. Finally, we demonstrate that overproduction of c-di-AMP due to cnpB inactivation resulted in lower protection of MTBVAC, while the absence of c-di-AMP in the MTBVAC disA derivative maintains the protective efficacy of this vaccine in mice., Competing Interests: C.M. and J.G.-A. are coinventors on the patent “Tuberculosis vaccine”. N.A., C.M., and J.G.-A. are coinventors 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 filed by the University of Zaragoza., (© 2022 The Authors.)

Pathogenomic evidence suggests that Mycobacterium tuberculosis (MTB) evolved from an environmental ancestor similar to Mycobacterium canettii, a rare human pathogen. Although the adaptations responsible for this transition are poorly characterized, the ability to persist in humans seems to be important. We set out to identify the adaptations contributing to the evolution of persistence in MTB. We performed an experimental evolution of eight M. canettii populations in mice; four populations were derived from the isolate STB-K (phylogenomically furthest from MTB) and four from STB-D (closest to MTB), which were monitored for 15 and 6 cycles, respectively. We selected M. canettii mutants with enhanced persistence in vivo compared with the parental strains, which were phenotypically closer to MTB. Genome sequencing of 140 mutants and complementation analysis revealed that mutations in two loci were responsible for enhanced persistence. Most of the tested mutants were more resistant than their parental strains to nitric oxide, an important effector of immunity. Modern MTB were similarly more resistant to nitric oxide than M. canettii. Our findings demonstrate phenotypic convergence during experimental evolution of M. canettii, which mirrors natural evolution of MTB. Furthermore, they indicate that the ability to withstand host-induced stresses was key for the emergence of persistent MTB., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis's pathogenic hallmarks is its ability to persist in a dormant state in the host. Thus, this pathogen has developed mechanisms to withstand stressful conditions found in the human host. Particularly, the Ser/Thr-protein kinase PknG has gained relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomic analyses of protein extracts from M. tuberculosis H37Rv and a mutant lacking pknG. We found that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance compared with the wild-type. The main biological processes affected by pknG deletion were cell envelope components biosynthesis and response to hypoxia. Thirteen DosR-regulated proteins were underrepresented in the pknG deletion mutant, including Hrp-1, which was 12.5-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, our results allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival. SIGNIFICANCE: PknG is a Ser/Thr kinase from Mycobacterium tuberculosis with key roles in bacterial metabolism and bacterial survival within the host. However, at present the molecular mechanisms underlying these functions remain largely unknown. In this work, we evaluate the effect of pknG deletion on M. tuberculosis proteome using different approaches. Our results clearly show that the global proteome was remodeled in the absence of PknG and shed light on new molecular mechanism underlying PknG role. Altogether, this work contributes to a better understanding of the molecular bases of the adaptation of M. tuberculosis, one of the most deadly human pathogens, to its host., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The interactions between microbes and their hosts are among the most complex biological phenomena known today. The interaction may reach from overall beneficial interaction, as observed for most microbiome/microbiota related interactions to interaction with virulent pathogens, against which host cells have evolved sophisticated defence strategies. Among the latter, the confinement of invading pathogens in a phagosome plays a key role, which often results in the destruction of the invader, whereas some pathogens may counteract phagosomal arrest and survive by gaining access to the cytosol of the host cell. In the current review, we will discuss recent insights into this dynamic process of host-pathogen interaction, using Mycobacterium tuberculosis and related pathogenic mycobacteria as main examples., (© 2021 John Wiley & Sons Ltd.)

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.

Rationale & Objective: Hyperphosphatemia is a risk factor for poor clinical outcomes in patients with kidney failure receiving maintenance dialysis. Opinion-based clinical practice guidelines recommend the use of phosphate binders and dietary phosphate restriction to lower serum phosphate levels toward the normal range in patients receiving maintenance dialysis, but the benefits of these approaches and the optimal serum phosphate target have not been tested in randomized trials. It is also unknown if aggressive treatment that achieves unnecessarily low serum phosphate levels worsens outcomes., Study Design: Multicenter, pragmatic, cluster-randomized clinical trial., Setting & Participants: HiLo will randomize 80-120 dialysis facilities operated by DaVita Inc and the University of Utah to enroll 4,400 patients undergoing 3-times-weekly, in-center hemodialysis., Intervention: Phosphate binder prescriptions and dietary recommendations to achieve the "Hi" serum phosphate target (≥6.5 mg/dL) or the "Lo" serum phosphate target (<5.5 mg/dL)., Outcomes: Primary outcome: Hierarchical composite outcome of all-cause mortality and all-cause hospitalization. Main secondary outcomes: Individual components of the primary outcome., Results: The trial is currently enrolling., Limitations: HiLo will not adjudicate causes of hospitalizations or mortality and does not protocolize use of specific phosphate binder classes., Conclusions: HiLo aims to address an important clinical question while more generally advancing methods for pragmatic clinical trials in nephrology by introducing multiple innovative features including stakeholder engagement in the study design, liberal eligibility criteria, use of electronic informed consent, engagement of dietitians to implement the interventions in real-world practice, leveraging electronic health records to eliminate dedicated study visits, remote monitoring of serum phosphate separation between trial arms, and use of a novel hierarchical composite outcome., Trial Registration: Registered at ClinicalTrials.gov with study number NCT04095039., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Current models of horizontal gene transfer (HGT) in mycobacteria are based on "distributive conjugal transfer" (DCT), an HGT type described in the fast-growing, saprophytic model organism Mycobacterium smegmatis , which creates genome mosaicism in resulting strains and depends on an ESX-1 type VII secretion system. In contrast, only few data on interstrain DNA transfer are available for tuberculosis-causing mycobacteria, for which chromosomal DNA transfer between two Mycobacterium canettii strains was reported, a process which, however, was not observed for Mycobacterium tuberculosis strains. Here, we have studied a wide range of human- and animal-adapted members of the Mycobacterium tuberculosis complex (MTBC) using an optimized filter-based mating assay together with three selected strains of M. canettii that acted as DNA recipients. Unlike in previous approaches, we obtained a high yield of thousands of recombinants containing transferred chromosomal DNA fragments from various MTBC donor strains, as confirmed by whole-genome sequence analysis of 38 randomly selected clones. While the genome organizations of the obtained recombinants showed mosaicisms of donor DNA fragments randomly integrated into a recipient genome backbone, reminiscent of those described as being the result of ESX-1-mediated DCT in M. smegmatis , we observed similar transfer efficiencies when ESX-1-deficient donor and/or recipient mutants were used, arguing that in tubercle bacilli, HGT is an ESX-1-independent process. These findings provide new insights into the genetic events driving the pathoevolution of M. tuberculosis and radically change our perception of HGT in mycobacteria, particularly for those species that show recombinogenic population structures despite the natural absence of ESX-1 secretion systems. IMPORTANCE Data on the bacterial sex-mediated impact on mycobacterial evolution are limited. Hence, our results presented here are of importance as they clearly demonstrate the capacity of a wide range of human- and animal-adapted Mycobacterium tuberculosis complex (MTBC) strains to transfer chromosomal DNA to selected strains of Mycobacterium canettii Most interestingly, we found that interstrain DNA transfer among tubercle bacilli was not dependent on a functional ESX-1 type VII secretion system, as ESX-1 deletion mutants of potential donor and/or recipient strains yielded numbers of recombinants similar to those of their respective parental strains. These results argue that HGT in tubercle bacilli is organized in a way different from that of the most widely studied Mycobacterium smegmatis model, a finding that is also relevant beyond tubercle bacilli, given that many mycobacteria, like, for example, Mycobacterium avium or Mycobacterium abscessus , are naturally devoid of an ESX-1 secretion system but show recombinogenic, mosaic-like genomic population structures., (Copyright © 2021 Madacki et al.)

Mycobacterium tuberculosis infections claim more than a million lives each year, and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from phagolysosomes to the cytosol upon phagocytosis by macrophages. Translocation from the phagolysosome to the cytosol is an ESX-1-dependent process, as previously shown in vitro Here, we show that in vivo , mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillos, zebrafish, and patient material infected with M. tuberculosis , M. marinum , or M. leprae In contrast, when innate or adaptive immunity was compromised, as in severe combined immunodeficiency (SCID) or interleukin-1 receptor 1 (IL-1R1)-deficient mice, significant numbers of cytosolic M. tuberculosis bacilli were detected in the lungs of infected mice. Taken together, in vivo , translocation to the cytosol of M. tuberculosis is controlled by adaptive immune responses as well as IL-1R1-mediated signals. IMPORTANCE For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is subcellular localization, as M. tuberculosis can translocate from phagolysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study, we establish that high-level cytosolic escape of mycobacteria can indeed occur in vivo but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in the clearance of cells containing cytosolic mycobacteria connects two important fields, cell biology and immunology, which is vital for the understanding of the pathology of M. tuberculosis ., (Copyright © 2021 van der Niet et al.)

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&#95;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&#95;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&#95;PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE&#95;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&#95;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&#95;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&#95;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.)

Epidemiological and experimental observations suggest that chronic microbial colonization can impact the immune control of other unrelated pathogens contracted in a concomitant or sequential manner. Possible interactions between Mycobacterium tuberculosis infection and persistence of other bacteria have scarcely been investigated. Here we demonstrated that natural colonization of the digestive tract with Helicobacter hepaticus in mice is concomitant with modification of the gut microbiota, subclinical inflammation, and drastic impairment of immune control of the growth of subsequently administered M. tuberculosis, which results in severe lung tissue injury. Our results provided insights upon the fact that this prior H. hepaticus colonization leads to failures in the mechanisms that could prevent the otherwise balanced cross-talk between M. tuberculosis and the immune system. Such disequilibrium ultimately leads to the inhibition of control of mycobacterial growth, outbreak of inflammation, and lung pathology. Among the dysregulated immune signatures, we noticed a correlation between the detrimental lung injury and the accumulation of activated T-lymphocytes. Our findings suggest that the impact of prior Helicobacter spp. colonization and subsequent M. tuberculosis parasitism might be greater than previously thought, which is a key point given that both species are among the most frequent invasive bacteria in human populations.

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.