Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives.

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor α, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection

Reprogramming of endothelial gene expression by tamoxifen inhibits angiogenesis and ERα-negative tumor growth.

peer reviewedRationale: 17β-estradiol (E2) can directly promote the growth of ERα-negative cancer cells through activation of endothelial ERα in the tumor microenvironment, thereby increasing a normalized tumor angiogenesis. ERα acts as a transcription factor through its nuclear transcriptional AF-1 and AF-2 transactivation functions, but membrane ERα plays also an important role in endothelium. The present study aims to decipher the respective roles of these two pathways in ERα-negative tumor growth. Moreover, we delineate the actions of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) in ERα-negative tumors growth and angiogenesis, since we recently demonstrated that tamoxifen impacts vasculature functions through complex modulation of ERα activity. Methods: ERα-negative B16K1 cancer cells were grafted into immunocompetent mice mutated for ERα-subfunctions and tumor growths were analyzed in these different models in response to E2 and/or tamoxifen treatment. Furthermore, RNA sequencings were analyzed in endothelial cells in response to these different treatments and validated by RT-qPCR and western blot. Results: We demonstrate that both nuclear and membrane ERα actions are required for the pro-tumoral effects of E2, while tamoxifen totally abrogates the E2-induced in vivo tumor growth, through inhibition of angiogenesis but promotion of vessel normalization. RNA sequencing indicates that tamoxifen inhibits the E2-induced genes, but also initiates a specific transcriptional program that especially regulates angiogenic genes and differentially regulates glycolysis, oxidative phosphorylation and inflammatory responses in endothelial cells. Conclusion: These findings provide evidence that tamoxifen specifically inhibits angiogenesis through a reprogramming of endothelial gene expression via regulation of some transcription factors, that could open new promising strategies to manage cancer therapies affecting the tumor microenvironment of ERα-negative tumors

Introduction to IWFOS'2008

International audienc

Effets membranaires du récepteur alpha des œstrogènes

Les récepteurs des œstrogènes (ER) sont utilisés comme cibles thérapeutiques soit en les activant (par exemple dans le cas de la contraception ou de la ménopause), soit en les inhibant (comme pour le cancer du sein). Cependant, l’idéal serait de cibler chaque tissu spécifiquement. Certains modulateurs (les SERM, selective estrogen receptor modulator), spécifiques du récepteur alpha des œstrogènes (ERα), sont capables partiellement de réaliser ce ciblage spécifique. Il est donc important de comprendre la spécificité tissulaire de l’action de ERα afin d’optimiser le rapport bénéfice/risque de ces modulateurs. À côté de sa fonction nucléaire classique comme facteur de transcription, une fraction extranucléaire d’ERα relaye des actions rapides membranaires des œstrogènes. De nouveaux modèles de souris transgéniques ont récemment permis de dévoiler, dans chaque tissu, les rôles physiologiques et spécifiques des effets nucléaires et des effets membranaires d’ERα

Vers une optimisation de la modulation du récepteur des œstrogènes dans le traitement hormonal de la ménopause

Les femmes vivent désormais plus d’un tiers de leur vie après la survenue de la ménopause. Le déclin de la production d’œstrogènes endogènes au cours de cette période s’accompagne fréquemment de troubles fonctionnels qui affectent la qualité de vie. Ces symptômes peuvent être soulagés par un traitement hormonal (THM) initialement fondé sur l’administration d’œstrogènes conjugués équins (principalement aux États-Unis, par voie orale) ou d’un œstrogène naturel, le 17β-estradiol (en Europe, notamment par voie transdermique). Le récepteur des œstrogènes α (REα) relaye la majorité des effets physiologiques des œstrogènes. REα appartient à la superfamille des récepteurs nucléaires. Il régule la transcription de gènes via ses fonctions activatrices (AF1 et AF2). Outre ces actions génomiques classiques, les œstrogènes peuvent aussi activer une sous-population de récepteurs REα présents à la membrane des cellules et ainsi induire des signaux rapides. Dans cette revue, nous résumerons l’évolution des THM depuis les débuts de la substitution hormonale jusqu’aux nouvelles molécules émergentes fondées sur une modulation sélective du REα. Nous décrirons également les progrès récents sur la compréhension des mécanismes d’action des œstrogènes, en détaillant les rôles respectifs des REα nucléaire et membranaire et les développements thérapeutiques possibles qui pourraient en découler

Signature-Tagged Transposon Mutagenesis Identifies Novel Mycobacterium tuberculosis Genes Involved in the Parasitism of Human Macrophages

Using signature-tagged transposon mutagenesis, we isolated 23 Mycobacterium tuberculosis mutants, corresponding to 21 genes or genetic regions, attenuated in their ability to parasitize human macrophages. Mutants disrupted in the ABC transporter-encoding genes Rv0986 and Rv0987 were further characterized as being impaired in their ability to bind to host cells

A Point Mutation in the Two-Component Regulator PhoP-PhoR Accounts for the Absence of Polyketide-Derived Acyltrehaloses but Not That of Phthiocerol Dimycocerosates in Mycobacterium tuberculosis H37Ra▿ §

Similarities between Mycobacterium tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis revealed a base substitution resulting in a one-amino-acid change in the likely DNA-binding region of PhoP in H37Ra relative to H37Rv. Using gel-shift assays, we show that this mutation abrogates the ability of the H37Ra PhoP protein to bind to a 40-bp segment of its own promoter. Consistent with this result, the phoP gene from H37Rv but not that from H37Ra was able to restore the synthesis of sulfolipids, diacyltrehaloses and polyacyltrehaloses in an isogenic phoP-phoR knock-out mutant of M. tuberculosis Moreover, complementation of H37Ra with phoP from H37Rv fully restored sulfolipid, diacyltrehalose and polyacyltrehalose synthesis, clearly indicating that the lack of production of these lipids in H37Ra is solely due to the point mutation in phoP. Using a pks2-3/4 knock-out mutant of M. tuberculosis H37Rv, evidence is further provided that the above-mentioned polyketide-derived acyltrehaloses do not significantly contribute to the virulence of the tubercle bacillus in a mouse model of infection. Reasons for the attenuation of H37Ra thus most likely stand in other virulence factors, many of which are expected to belong to the PhoP regulon and another of which, unrelated to PhoP, appears to be the lack of production of phthiocerol dimycocerosates in this strain

Genetic Basis for the Biosynthesis of Methylglucose Lipopolysaccharides in Mycobacterium tuberculosis

International audienceMycobacteria produce two unusual polymethylated polysaccharides, the 6-O-methylglucosyl-containing lipopolysaccharides (MGLP) and the 3-O-methylmannose polysaccharides, which have been shown to regulate fatty acid biosynthesis in vitro. A cluster of genes dedicated to the synthesis of MGLP was identified in Mycobacterium tuberculosis and Mycobacterium smegmatis. Overexpression of the putative glycosyltransferase gene Rv3032 in M. smegmatis greatly stimulated MGLP production, whereas the targeted disruption of Rv3032 in M. tuberculosis and that of the putative methyltransferase gene MSMEG2349 in M. smegmatis resulted in a dramatic reduction in the amounts of MGLP synthesized and in the accumulation of precursors of these molecules. Disruption of Rv3032 also led to a significant decrease in the glycogen content of the tubercle bacillus, indicating that the product of this gene is likely to be involved in the elongation of more than one alpha-(1-->4)-glucan in this bacterium. Results thus suggest that Rv3032 encodes the alpha-(1-->4)-glucosyltransferase responsible for the elongation of MGLP, whereas MSMEG2349 encodes the O-methyltransferase required for the 6-O-methylation of these compounds