Etude du rôle des polykétides mycobactériens dans la biogénèse de l'enveloppe et la virulence des mycobactéries : caractérisation de l'étape de condensation des acides mycoliques

Certaines mycobactéries sont responsables à l'heure actuelle et depuis l'antiquité de pathologies infectieuses graves comme la tuberculose, résultante d'une infection par Mycobacterium tuberculosis. En dépit d'une médecine efficace liée à l'avènement des antibiotiques, cette infection bactérienne est à l'échelle mondiale, la première cause de mortalité due à un agent infectieux. Deux millions de décès seraient annuellement liés à cette pathologie selon les données de l'organisation mondiale de la santé (OMS, 2002). La tuberculose est un véritable fléau dans la majorité des pays en voie de développement où son endémie corrèle celle liée à l'épidémie du VIH, et le nombre de tuberculeux recensé en Europe comme dans d'autres pays développés reste non négligeable. La pathogénie du bacille tuberculeux laisse apparaître un équilibre quasi-parasitique avec un tiers de la population mondiale infectée. L'apparition de souches multi-résistantes aux chimiothérapies actuelles inquiète le milieu médical en réduisant le nombre de médicaments disponibles. Une stratégie de prise en charge mondiale par l'OMS a récemment été adoptée pour tenter d'endiguer l'épidémie de tuberculose. En parallèle, des efforts en recherche fondamentale et clinique sont entrepris afin de décortiquer la pathogénie du bacille tuberculeux pour mieux le combattre. Dans cet objectif, le séquençage récent de son génome a représenté une avancée majeure. Il dévoile notamment 250 gènes relatifs à la biosynthèse lipidique, soit cinq fois plus que chez E.coli, dont 24 coderaient des polykétides synthases (Pks). Ces enzymes sont sources de molécules complexes souvent biologiquement actives, dont la toxine de Mycobacterium ulcerans, la mycolactone, est particulièrement représentative chez les mycobactéries. Récemment, nombre de produits de polykétides synthases se sont révélés être des composantes de l'enveloppe de Mycobacterium tuberculosis. Cette enveloppe représente une arme remarquable pour le bacille tuberculeux tant par la barrière d'imperméabilité qu'elle constitue que par ses propriétés immunologiques vraisemblablement impliquées dans sa résistance à l'immunité de l'hôte. L'approche génétique abordée dans ce travail a permis d'évaluer la contribution des différents polykétides mycobactériens dans la virulence de M.bovis BCG et M.tuberculosis en modèle murin. De plus, nous avons pu caractériser la fonction d'un locus comprenant la polykétide synthase 13 ainsi qu'une acyl-AMP synthase et une acyl-coA carboxylase dans la dernière étape de la biosynthèse des acides mycoliques, lipides majeurs de l'enveloppe mycobactérienne et essentiels pour la survie des mycobactéries. Ainsi, les enzymes caractérisées dans ce travail constituent des cibles de choix pour le développement de nouveaux antituberculeux.Since the antiquity, mycobacteria have been involved in lethal pathology such as leprosy or tuberculosis, the latter caused by Mycobacterium tuberculosis and the former by Mycobacterium leprae. Despite modern medicine including antibiotics, tuberculosis keep on being the first cause of mortality due to a single infectious agent with an average of two millions deaths per year, according to the world health organisation (WHO, 2002). Tuberculosis is a real plague in the majority of developping countries where its endemy correlates those of the HIV epidemy. Furthermore the number of tuberculosis patient number in Europe as well as in other developping countries is still not negligible. Moreover, the emergence of strains resistant to the currently used antibiotics is worrying the medical profession by reducing avalaible active molecules. Without the huge mortality associated with tuberculosis, this disease would be considered as a parasitic infection regarding the estimated number of infected but asymptomatic people (around 33% of the world population). Recently, the WHO has initiated a global approach to control the epidemy. In parallel, efforts in fondamental and clinical investigation are ongoing to understand tuberculosis pathogeny and to developp new clinical tools. Recently, the entire genome of M.tuberculosis was sequenced. It revealed 250 genes related to lipid biosynthesis, which represents a considerable involvment regarding the approximativly 50 genes found in E.coli genome. Remarkably 24 of this 250 genes encode polyketide synthase (Pks). In other bacteria, these enzymes are known to produce complex molecules with pharmaceutical interest. In mycobacteria, the mycolactone, is a potent toxin produced by Mycobacterium ulcerans. Recently, numerous polyketides has been shown to be components of the M.tuberculosis envelop. This envelop represents a powerfull weapon for the tuberculous bacillus constituting a permeability barrier and delivering immunological advantages. The genetic approach used in this work allowed us to evaluate the contribution of the various polyketides in the virulence of M.bovis BCG and M.tuberculosis in a mouse model. Furthermore, we caracterised the fonction of the locus including the polyketide synthase 13, an acyl-AMP synthase and an acyl-coA carboxylase in the last step of mycolic acid biosynthesis. Mycolic acids are major constituent of the mycobacterial cell envelop and are essential for mycobacterial viability, therefore the enzymes caracterised in the second part of this work constitutes promising targets for developping new antituberculous drugs

TNF-α antagonists differentially induce TGF-β1-dependent resuscitation of dormant-like Mycobacterium tuberculosis

TNF-α- as well as non-TNF-α-targeting biologics are prescribed to treat a variety of immune-mediated inflammatory disorders. The well-documented risk of tuberculosis progression associated with anti-TNF-α treatment highlighted the central role of TNF-α for the maintenance of protective immunity, although the rate of tuberculosis detected among patients varies with the nature of the drug. Using a human, in-vitro granuloma model, we reproduce the increased reactivation rate of tuberculosis following exposure to Adalimumab compared to Etanercept, two TNF-α-neutralizing biologics. We show that Adalimumab, because of its bivalence, specifically induces TGF-β1-dependent Mycobacterium tuberculosis (Mtb) resuscitation which can be prevented by concomitant TGF-β1 neutralization. Moreover, our data suggest an additional role of lymphotoxin-α-neutralized by Etanercept but not Adalimumab-in the control of latent tuberculosis infection. Furthermore, we show that, while Secukinumab, an anti-IL-17A antibody, does not revert Mtb dormancy, the anti-IL-12-p40 antibody Ustekinumab and the recombinant IL-1RA Anakinra promote Mtb resuscitation, in line with the importance of these pathways in tuberculosis immunity

Functional characterization and phenotypic monitoring of human hematopoietic stem cell expansion and differentiation of monocytes and macrophages by whole-cell mass spectrometry

The different facets of macrophages allow them to play distinct roles in tissue homeostasis, tissue repair and in response to infections. Individuals displaying dysregulated macrophage functions are proposed to be prone to inflammatory disorders or infections. However, this being a cause or a consequence of the pathology remains often unclear. In this context, we isolated and expanded CD34+ HSCs from healthy blood donors and derived them into CD14+ myeloid progenitors which were further enriched and differentiated into macrophages. Aiming for a comprehensive phenotypic profiling, we generated whole-cell mass spectrometry (WCMS) fingerprints of cell samples collected along the different stages of the differentiation process to build a predictive model using a linear discriminant analysis based on principal components. Through the capacity of the model to accurately predict sample's identity of a validation set, we demonstrate that WCMS profiles obtained from bona fide blood monocytes and respectively derived macrophages mirror profiles obtained from equivalent HSC derivatives. Finally, HSC-derived macrophage functionalities were assessed by quantifying cytokine and chemokine responses to a TLR agonist in a 34-plex luminex assay and by measuring their capacity to phagocytise mycobacteria. These functional read-outs could not discriminate blood monocytes-derived from HSC-derived macrophages. To conclude, we propose that this method opens new avenues to distinguish the impact of human genetics on the dysregulated biological properties of macrophages in pathological conditions

Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity

BackgroundThe epidemiology of Mycobacterium tuberculosis complex (MTBC) lineage 5 (L5) infections in Ghana revealed a significantly increased prevalence in Ewes compared to other self-reported ethnic groups. In that context, we sought to investigate the early phase of tuberculosis (TB) infection using ex vivo infection of macrophages derived from the blood of Ewe and Akan ethnic group volunteers with MTBC L4 and L5 strains.MethodsThe study participants consisted of 16 controls, among which self-reported Akan and Ewe ethnicity was equally represented, as well as 20 cured TB cases consisting of 11 Akans and 9 Ewes. Peripheral blood mononuclear cells were isolated from both healthy controls and cured TB cases. CD14+ monocytes were isolated and differentiated into monocyte-derived macrophages (MDMs) before infection with L4 or L5 endemic strains. The bacterial load was assessed after 2 hours (uptake) as well as 3 and 7 days post-infection.ResultsWe observed a higher capacity of MDMs from Ewes to phagocytose L4 strains (p < 0.001), translating into a higher bacillary load on day 7 (p < 0.001) compared to L5, despite the higher replication rate of L5 in Ewe MDMs (fold change: 1.4 vs. 1.2, p = 0.03) among the controls. On the contrary, within macrophages from Akans, we observed a significantly higher phagocytic uptake of L5 (p < 0.001) compared to L4, also translating into a higher load on day 7 (p = 0.04). However, the replication rate of L4 in Akan MDMs was higher than that of L5 (fold change: L4 = 1.2, L4 = 1.1, p = 0.04). Although there was no significant difference in the uptake of L4 and L5 among cured TB cases, there was a higher bacterial load of both L4 (p = 0.02) and L5 (p = 0.02) on day 7 in Ewe MDMs.ConclusionOur results suggest that host ethnicity (driven by host genetic diversity), MTBC genetic diversity, and individual TB infection history are all acting together to modulate the outcome of macrophage infections by MTBC

Mixed Th1 and Th2 Mycobacterium tuberculosis-specific CD4 T cell responses in patients with active pulmonary tuberculosis from Tanzania.

Mycobacterium tuberculosis (Mtb) and helminth infections elicit antagonistic immune effector functions and are co-endemic in several regions of the world. We therefore hypothesized that helminth infection may influence Mtb-specific T-cell immune responses. We evaluated the cytokine profile of Mtb-specific T cells in 72 individuals with pulmonary TB disease recruited from two Sub-Saharan regions with high and moderate helminth burden i.e. 55 from Tanzania (TZ) and 17 from South Africa (SA), respectively. We showed that Mtb-specific CD4 T-cell functional profile of TB patients from Tanzania are primarily composed of polyfunctional Th1 and Th2 cells, associated with increased expression of Gata-3 and reduced expression of T-bet in memory CD4 T cells. In contrast, the cytokine profile of Mtb-specific CD4 T cells of TB patients from SA was dominated by single IFN-γ and dual IFN-γ/TNF-α and associated with TB-induced systemic inflammation and elevated serum levels of type I IFNs. Of note, the proportion of patients with Mtb-specific CD8 T cells was significantly reduced in Mtb/helminth co-infected patients from TZ. It is likely that the underlying helminth infection and possibly genetic and other unknown environmental factors may have caused the induction of mixed Th1/Th2 Mtb-specific CD4 T cell responses in patients from TZ. Taken together, these results indicate that the generation of Mtb-specific CD4 and CD8 T cell responses may be substantially influenced by environmental factors in vivo. These observations may have major impact in the identification of immune biomarkers of disease status and correlates of protection

Quantitative whole-cell MALDI-TOF MS fingerprints distinguishes human monocyte sub-populations activated by distinct microbial ligands

Conventionally, human monocyte sub-populations are classified according to surface marker expression into classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)) and non-classical (CD14(+)CD16(++)) lineages. The involvement of non-classical monocytes, also referred to as proinflammatory monocytes, in the pathophysiology of diseases including diabetes mellitus, atherosclerosis or Alzheimer's disease is well recognized. The development of novel high-throughput methods to capture functional states within the different monocyte lineages at the whole cell proteomic level will enable real time monitoring of disease states.; We isolated and characterized (pan-) monocytes, mostly composed of classical CD16(-) monocytes, versus autologous CD16(+) subpopulations from the blood of healthy human donors (n = 8) and compared their inflammatory properties in response to lipopolysaccharides and M.tuberculosis antigens by multiplex cytokine profiling. Following resting and in vitro antigenic stimulation, cells were recovered and subjected to whole-cell mass spectrometry analysis. This approach identified the specific presence/absence of m/z peaks and therefore potential biomarkers that can discriminate pan-monocytes from their CD16 counterparts. Furthermore, we found that semi-quantitative data analysis could capture the subtle proteome changes occurring upon microbial stimulation that differentiate resting, from lipopolysaccharides or M. tuberculosis stimulated monocytic samples.; Whole-cell mass spectrometry fingerprinting could efficiently distinguish monocytic sub-populations that arose from a same hematopoietic lineage. We also demonstrate for the first time that mass spectrometry signatures can monitor semi-quantitatively specific activation status in response to exogenous stimulation. As such, this approach stands as a fast and efficient method for the applied immunology field to assess the reactivity of potentially any immune cell types that may sustain health or promote related inflammatory diseases

Etude du rôle des polykétides mycobactériens dans la biogénèse de l'enveloppe et la virulence des mycobactéries (caractérisation de l'étape de condensation des acides mycoliques)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Generating three-dimensional human granulomas in vitro to study Mycobacterium tuberculosis-host interaction

Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma’s environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the pathogen. In addition to the incorporation of a physiological extracellular matrix, this model has the advantage of recapitulating dormant-like Mtb features, as well as reproducing Mtb resuscitation observed under immunomodulatory treatments

IL-2 induction of NKp44 expression on CD56^bright NK cells is inhibited by mycobacteria.

<p>A) FACS plot analysis of NKp44 expression from NK cells cultured in the presence or the absence of IL-2 (100U/ml) and or <i>M. bovis</i> BCG (MOI 1:5) for 5 days. B) Bar graphs showing B) significant increase of CD56 MFI of the NKp44⁺ NK cell population and C) significant decreased frequencies of IL-2 induced NKp44⁺ NK cell in the presence of mycobacteria from three independent experiments and donors (Paired t test, *p<0.05, **p<0.01).</p

<i>M. bovis</i> BCG inhibits IL-2 induced proliferation of CD56^bright NK cells.

<p>A) Purified NK cells from a healthy human donor were labelled with CFSE and cultured for 7 days in the presence of IL-2 (100U/ml) +/- <i>M. bovis</i> BCG at various MOI before flow cytometry analysis. Dose dependent inhibition of NK cell proliferation by mycobacteria was observed. B) Joined dot plot illustrating the reproducibility of NK cell proliferation inhibition by mycobacteria across different NK cell preparation across independent donors (n=4, paired t test, **p<0.01).</p