The Polyketide Synthase Pks13 Catalyzes a Novel Mechanism of Lipid Transfer in Mycobacteria

SummaryMycolate-containing compounds constitute major strategic elements of the protective coat surrounding the tubercle bacillus. We have previously shown that FAAL32-Pks13 polyketide synthase catalyzes the condensation reaction, which produces α-alkyl β-ketoacids, direct precursors of mycolic acids. In contrast to the current biosynthesis model, we show here that Pks13 catalyzes itself the release of the neosynthesized products and demonstrate that this function is carried by its thioesterase-like domain. Most importantly, in agreement with the prediction of a trehalose-binding pocket in its catalytic site, this domain exhibits an acyltransferase activity and transfers Pks13’s products onto an acceptor molecule, mainly trehalose, leading to the formation of the trehalose monomycolate precursor. Thus, this work allows elucidation of the hinge step of the mycolate-containing compound biosynthesis pathway. Above all, it highlights a unique mechanism of transfer of polyketide synthase products in mycobacteria, which is distinct from the conventional intervention of the discrete polyketide-associated protein (Pap)-type acyltransferases

Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria

Rôle des acides mycoliques oxygénés dans la physiopathologie de la tuberculose et inhibition de la méthyltransférase Hma impliquée dans leur biosynthèse

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Rapports entre timing pubertaire perçu et estime de soi chez des adolescents belges et français

Les questions de l’identité personnelle et de l’identité sexuée ne peuvent être élucidées par l’adolescent sans en référer au corps, même si elles ne s’y limitent pas. Le processus de maturation pubertaire a des répercussions sur la représentation de soi (Blyth et al. 1982) et sur l’établissement des relations sociales à l’adolescence (Alsaker,1995). La poussée staturo-pondérale, comme l’atteinte de la maturité sexuelle, ne se produisent pas en même temps chez tous, certains ont déjà atteint une pleine maturité physiologique, alors que d’autres n’ont pas encore abordé le développement pubertaire. Cette variabilité interindividuelle renforcée par des différences entre les sexes (tant par le moment où débute le développement pubertaire que la façon dont sont vécues les transformations corporelles) a des effets au niveau de la construction identitaire, de la perception de soi et du rapport aux autres (Glowacz et al. 2008, Williams & Currie, 2000, Seigel et al. 1999). Le timing pubertaire perçu reposant sur la perception de l’adolescent de sa maturation pubertaire comparée à celle de ses pairs intègre à la fois les dimensions physiologiques et psychologiques, intra-individuelles et inter-individuelles. Cette communication vise à étudier en quoi la perception d’une synchronisation ou désynchronisation de sa maturation pubertaire par rapport aux pairs influence l’estime de soi durant la phase de transition entre l’enfance et l’adolescence et en cours d’adolescence. 249 collégiens et lycéens belges (12 à 18 ans) et 200 collégiens français (11 à 16 ans) ont été interrogés. Le timing perçu est évalué par la méthode de Berg-Kelly & Erdes (1997) faisant appel au vécu de l’adolescent par rapport à son développement pubertaire, à l’intégration de ces changements ou non-changements physiques au niveau de son image, aux impacts dans son rapport à l’autre ainsi qu’à l’intégration du regard de l’autre. L’estime de soi est mesurée par l’échelle de Harter (1988) et par l’échelle d’estime de soi toulousaine (Bardou et al. 2012). Nos résultats confirment une plus faible estime de soi globale, une perception négative et une insatisfaction par rapport à l’apparence physique chez les garçons au timing pubertaire retardé. Chez les filles, contrairement à nos hypothèses, il n’est pas relevé de différences au niveau de l’estime de soi globale selon le timing pubertaire pour l’échantillon belge tandis que pour l’échantillon français sont relevées des différences au niveau de l’estime de soi physique et de l’estime de soi scolaire

Post-traumatic stress disorders in women victims-survivors of intimate partner violence: a mixed-methods pilot study in a French coordinated structure

Objectives This study aimed to examine the prevalence of post-traumatic stress disorder (PTSD) in victims-survivors of intimate partner violence (IPV) consulting at the specialised and original facility ‘Maison des Femmes’ (MdF) or in two close municipal health centres (MHCs).Design A mixed-methods study using a convergent parallel design from July 2020 to June 2021.Setting/participants A questionnaire was proposed to women aged 18 years and over having suffered from IPV, in the MdF and in two MHCs. We also conducted qualitative interviews with a subsample of the women, asking for victim-survivors’ perceptions of the effect of the MdF’s care.Primary and secondary outcome measures The presence of a PTSD using the PTSD self-report checklist of symptoms, possibility of reaching women by phone 6 months after the inclusion visit, level of self-rated global health, number of emergency visits in the past 6 months, substances use, readiness to change and safety behaviours.Results A total of 67 women (mean age: 34 years (SD=9.7)) responded to our questionnaire. PTSD diagnosis was retained for 40 women (59.7%). Around 30% of participants self-rated their global health as bad. Less than 30% (n=18) of women were regular smokers, and only 7.5% of participants had a problematic alcohol use (Alcohol Use Disorders Identification Test-Consumption score ≥4), 19.4% women used psychotropic drugs. Six months after inclusion, half of participants had been reached by phone. Analysis of the qualitative interviews clarified victim-survivors’ perceptions of the MdF’s specific care: social networking, multidisciplinary approach, specialised listening, healthcare facilities, evasion and ‘feeling at home’.Conclusions The high prevalence of PTSD at inclusion was nearly the same between the three centres. This mixed-methods comparison will serve as a pilot study for a larger comparative trial to assess the long-term impact of the MdF’s specialised care on victims-survivors’ mental health, compared with the care of uncoordinated structures.Trial registration number NCT04304469

Pleiotropic effect of AccD5 and AccE5 depletion in acyl-coenzyme A carboxylase activity and in lipid biosynthesis in mycobacteria.

Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs

Correction: Pleiotropic Effect of AccD5 and AccE5 Depletion in Acyl-Coenzyme A Carboxylase Activity and in Lipid Biosynthesis in Mycobacteria.

[This corrects the article DOI: 10.1371/journal.pone.0099853.]

Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits

Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two β subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24–26-CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen.Fil: Bazet Lyonnet, Bernardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Diacovich, Lautaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gago, Gabriela Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Spina, Lucie. Universite de Toulouse; FranciaFil: Bardou, Fabienne. Universite de Toulouse; FranciaFil: Lemassu, Anne. Universite de Toulouse; FranciaFil: Quemard, Annaïk. Universite de Toulouse; FranciaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

<i>accD5-accE5</i> conditional mutant D5 MUT.

<p>A) Schematic representation of the genetic organization of the <i>M. smegmatis</i> conditional mutant D5 MUT. In pBB25, transcription of the <i>accD5-accE5</i> operon is controlled by P<i>_tr</i>, which can be repressed with the addition of ATc to the media. B) <i>M. smegmatis</i> mc²155 (WT), Isogenic (ISO-D5) and D5 MUT strains were grown on plates with 200 ng ml⁻¹ (+ATc) or without ATc (−ATc). C) Growth curves of D5 MUT in the presence or absence of ATc 200 ng ml⁻¹. A saturated culture of D5 MUT grown at 37°C was diluted in fresh 7H9 medium to an OD_{600 nm} of 0.01 and further incubated at 37°C. After 5, 8, 11 and 21 h; an aliquot of the main culture was separated and supplemented with ATc 200 ng ml⁻¹. Growth was followed by measuring OD_{600 nm}.</p