Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the bhydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors

Bioinformatic and functional approach for the characterization of secreted proteins in Trypanosomatids (implications for the biology and virulence of the parasite)

Les Trypanosomatidés Leishmania spp., Trypanosoma cruzi et Trypanosoma brucei sont responsables de pathologies humaines, dont le cycle de vie alterne entre un insecte vecteur et un hôte vertébré. Ces pathogènes ont développé différentes stratégies pour assurer leur survie chez leurs hôtes. Des facteurs de sécrétion jouent un rôle important dans l'infection et dans la modulation de la réponse immune de l'hôte. Les protéines sécrétées représentent donc des cibles potentielles pour la conception de nouvelles thérapies et/ou de nouveaux vaccins. Nous avons développé une approche basée sur des analyses bioinformatiques afin d'identifier des protéines conservées chez les Trypanosomatidés, impliquées dans la voie de sécrétion classique. Cette méthode nous a permis d'identifier trois nouvelles protéines conservées, sécrétées dans le milieu extracellulaire. L'étude des propriétés biologiques de ces protéines suggère que l'une d'entre elles est impliquée dans un processus favorisant la survie du parasite Leishmania et sa replication à l'intérieur de la cellule hôte. Les études portant sur l'immunogénicité des protéines sécrétées par T. cruzi ont montré que l'une des protéines est très antigénique chez l'homme. L'ensemble de ces résultats démontre l'utilité des analyses bioinformatiques couplées à des tests fonctionnels pour l'identification de nouvelles protéines sécrétées, représentant des facteurs de virulence ou des facteurs antigéniques potentiels des Trypanosomatidés.The trypanosomatid parasites, Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei are protozoa that complete their life cycle in an insect vector and a vertebrate host. These pathogens have developed various strategies to modify their environment, influence host immune responses, or invade target cells. Materials secreted by these parasites are involved in such processes and may represent targets for vaccines and/or rational drug design. In this thesis, we designed an experimental approach based on bioinformatic analyses to identify conserved trypanosomatid proteins involved in the endoplasmic reticulum/Golgi-dependent secretory pathway. This method allowed us to identify three new trypanosomatid conserved proteins, demonstrating the utility of this approach for the identification of secreted proteins. Studies of the biological properties of these proteins suggest that one of them is involved in a process increasing survival and replication of Leishmania parasites inside its target cell. Immunogenicity studies of T. cruzi secreted proteins showed that one of these proteins is antigenic, suggesting its suitability for the development of a new diagnostic tool for Chagas disease. Altogether, these results indicate the utility of bioinformatic analyses combined with functional tests for the identification of novel secreted proteins, representing potential virulence factors or antigens in trypanosomatidsMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Deciphering the Leishmania exoproteome : what we know and what we can learn

Parasitic protozoa of the genus Leishmania are the causative agents of leishmaniasis. Survival and transmission of these parasites in their different hosts require membrane-bound or extracellular factors to interact with and modify their host environments. Over the last decade, several approaches have been applied to study all the extracellular proteins exported by an organism at a particular time or stage in its life cycle and under defined conditions, collectively termed the secretome or the exoproteome. In this review, we focus on emerging data shedding light on the secretion mechanisms involved in the production of the Leishmania exoproteome. We also describe other methodologies currently available that could be used to analyse the Leishmania exoproteome. Understanding the complexity of the Leishmania exoproteome is a key component to elucidating the mechanisms used by these parasites for exporting proteins to the extracellular space during its life cycle. Given the importance of extracellular factors, a detailed knowledge of the Leishmania exoproteome may provide novel targets for rational drug design and/or a source of antigens for vaccine development

Mycobacterium tuberculosis S-adenosyl-l-homocysteine hydrolase is negatively regulated by Ser/Thr phosphorylation

International audienc

An experimental approach for the identification of conserved secreted proteins in trypanosomatids

Extracellular factors produced by Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei are important in the host-parasite relationship. Here, we describe a genome-based approach to identify putative extracellular proteins conserved among trypanosomatids that are likely involved in the classical secretory pathway. Potentially secreted proteins were identified by bioinformatic analysis of the T. cruzi genome. A subset of thirteen genes encoding unknown proteins with orthologs containing a signal peptide sequence in L. infantum, L. major, and T. brucei were transfected into L. infantum. Tagged proteins detected in the extracellular medium confirmed computer predictions in about 25% of the hits. Secretion was confirmed for two L. infantum orthologs proteins using the same experimental system. Infectivity studies of transgenic Leishmania parasites suggest that one of the secreted proteins increases parasite replication inside macrophages. This methodology can identify conserved secreted proteins involved in the classical secretory pathway, and they may represent potential virulence factors in trypanosomatids

Phosphorylation of Mycobacterial PcaA Inhibits Mycolic Acid Cyclopropanation

International audienc

Congenital Chagas disease involves Trypanosoma cruzi sub-lineage IId in the northwestern province of Salta, Argentina

Trypanosoma cruzi is genetically classified into six discrete phylogenetic lineages on the basis of different genetic markers. Identifying lineages circulating among humans in different areas is essential to understand the molecular epidemiology of Chagas disease. In the present study, 18 T. cruzi isolates from congenitally infected newborns in the northwestern province of Salta-Argentina were studied by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD). All isolates were typed by MLEE and RAPID as belonging to T. cruzi lid. Analysis of minor variants of TcIId using probes hybridizing with hypervariable domains of kDNA minicircles, detected three variants with a similar distribution among the isolates. Our findings confirm the presence of T. cruzi IId among congenitally infected newborns in northwestern Argentina and support the assumption that human infection by T. cruzi in the Southern Cone countries of Latin America is due principally to T. cruzi II

Mycolic acid profile of the parental <i>M. tuberculosis</i> strain and independent TAC-resistant derived mutants.

<p>FAMEs and MAMEs were extracted from exponentially growing cultures and resolved by single dimension TLC in hexane/ethyl acetate (19/1, v/v) prior to visualization using molybdophosphoric acid and charring. α, α-mycolic acids; methoxy, methoxy-mycolic acids; keto, keto-mycolic acids.</p