Clonación y expresión en Escherichia coli del gen ctpE Rv0908) de Mycobacterium tuberculosis: determinación preliminar de su posible actividad Na+ATPasa

La tuberculosis es uno de los desafíos más importantes para la salud pública en el mundo. Actualmente hay una prevalencia de 2 billones de personas infectadas con Mycobacterium tuberculosis, lo que representa el 30% de la población mundial. En la actualidad, entre los blancos terapéuticos de mayor interés para el control de patógenos intracelulares se encuentran las proteínas de membrana plasmática encargadas del transporte iónico: ATPasas, transportadores ABC y sistemas antiporter ion/H+ (Novoa et. al. 2012). De hecho, la regulación de la concentración de iones metálicos es central en la fisiología de la interacción hospedero- patógeno y ambos han desarrollado mecanismos sofisticados para el transporte de iones hacia el interior o el exterior celular dependiendo de sus necesidades (Soldati y Neyrolles, 2012). Estudios genómicos de M. tuberculosis evidenciaron que existen 28 secuencias que codifican para transportadores putativos de iones metálicos, entre ellos 12 ATPasas tipo P (Novoa et. al., 2012; Agranoff y Krishna, 2004, 1998). Este gran número de ATPasas (el mayor número reportado para una bacteria), puede dar la versatilidad requerida al bacilo para adaptarse a las condiciones intra- y extracelulares durante las diferentes etapas de la infección, las cuales difieren marcadamente en osmolaridad, concentraciones de iones metálicos, pH y disponibilidad de nutrientes. Estudios bioinformáticos, permitieron clasificar a las ATPasas tipo P de M. tuberculosis CtpA, CtpB, CtpC, CtpD, CtpG, CtpJ y CtpV como transportadores de metales pesados (Cu2+, Cu+, Cu+/ Cu2+, Zn2+ y Co2+/Ni2+); a CtpE, CtpF, CtpH y CtpI como transportadores de iones de metales alcalinos y alcalinotérreos (Na+, K+, Ca2+, H+ y Mg2+); y a KdpB, como una proteína que tiene alta identidad con la subunidad ß de una ATPasa multimérica transportadora de K+ de E. coli (Novoa et. al. 2012). Sin embargo, la especificidad de sustrato para estas ATPasas solo ha sido establecida experimentalmente para tres de ellas. A partir de la creación de mutantes se ha logrado establecer que CtpD es una Co2+/Ni2+ - ATPasa (Raimunda et. al. 2012) y que CtpV y CtpC son ATPasas transportadoras de Cu2+ y Zn2+, respectivamente. (Ward S. et.al. 2010 y Botella H. et.al. 2011). En nuestro grupo de investigación con anterioridad estableció la actividad ATPasa dependiente de Na+ en la membrana plasmática de M. tuberculosis. Dicha actividad se asoció a la presencia de una Na+ ATPasa tipo P, pues es inhibida por vanadato, un reconocido inhibidor de este tipo de bombas (Cuesta, 2010). Los estudios bioinformáticos también identificaron en el genoma del bacilo, al gen ctpE (Rv0908) de M. tuberculosis como posible codificante de una Na+ ATPasa tipo P. El presente trabajo busca establecer la especificidad iónica de la proteína codificada por el gen ctpE de M. tuberculosis. Específicamente, se logró la clonación del gen en el vector de expresión pBADA2 y la expresión heteróloga en células de E. coli del producto génico CtpE. También, los resultados de algunos ensayos bioquímicos y medidas electrofisiológicas, realizados en vesículas de membrana de E. coli, sugieren que la proteína CtpE corresponde a un transportador de Na+ y/o K+.Abstract. Tuberculosis is one of the most important challenges for global public health. Currently, there is a prevalence of 2 billion people infected with Mycobacterium tuberculosis, which represents 30% of world´s population. Among the new therapeutic targets of highest interest to control intracellular pathogens, there are plasma membrane proteins involved in ionic transport: ATPases, ABC transporters and antiporter systems ion/H+ (Novoa et. al. 2012). In fact, the regulation of metallic ions concentration plays an important role in the physiology of host- pathogen interaction and both have developed sophisticated mechanisms for ion transport to the intra- or extracellular space according to their needs. (Soldati and Neyrolles, 2012). Different studies have shown that M. tuberculosis genome possess 28 sequences that codify putative trasnsporters of metallic ions, including 12 P type ATPases (Novoa et. al., 2012; Agranoff and Krishna, 2004, 1998). This is the highest number reported for any bacteria, and might give the bacillum versatility to survive to different confitions during the infection, which differ in osmolarity, metallic ion concentration, pH and nutrient availability. Bioinformatic studies allowed to classify P type ATPases of M. tuberculosis CtpA, CtpB, CtpC, CtpD, CtpG, CtpJ and CtpV as heavy metal transporters (Cu2+, Cu+, Cu+/ Cu2+, Zn2+ and Co2+/Ni2+); CtpE, CtpF, CtpH and CtpI as alkaline and alkaline earth metal transporters (Na+, K+, Ca2+, H+ and Mg2+); and KdpB, as a protein with high identity with ß subunit of a multimeric ATPase transporter of K+ in E. coli (Novoa et. al. 2012). However, ion specificity has only been achieved for three of them. From mutagenesis studies it has been possible to establish that CtpD is a Co2+/Ni2+ - ATPase (Raimunda et. al. 2012) while CtpV and CtpC are transporters of Cu2+ and Zn2+, respectively. (Ward S. et.al. 2010 and Botella H. et.al. 2011). Within our research group it has been stablished the ATPase activity stimulated by Na+ in the plasma membrane of M. tuberculosis. This activity was associated to the presence of a Na+ type P ATPase, due to it is inhibited by vanadate, a recognized inhibitor of this kind of pumps (Cuesta, 2010). Bioinformatic studies also identified the gene ctpE (Rv0908) in M. tuberculosis genome as the codifying gene of a Na+ type P ATPase. The objective of this work is to stablish the ion specificity of the protein codified by the gene ctpE of M. tuberculosis. Specifically, cloning the gene in the expression vector pBADA2 and heterologous expression of CtpE in E. coli was achieved. The obtained results from biochemical assays and electrophysiological measurements made on membrane vesicles of E. coli, suggest that the protein CtpE corresponds to a Na+ and/or K+ transporter.Maestrí

Pharmaceutical Polymorph Control in a Drug-Mimetic Supramolecular Gel

We report the synthesis of a bis(urea) gelator designed to specifically mimic the chemical structure of the highly polymorphic drug substance ROY. Crystallization of ROY from toluene gels of this gelator results in the formation of the metastable red form instead of the thermodynamic yellow polymorph. In contrast, all other gels and solution control experimetns give the yellow form. Conformational and crystal structure prediction methods have been used to propose the structure of the gel and shows that the templation of the red form by the targetted gel results from conformational matching of the gelator to the ROY substrate coupled with overgorwth of ROY onto the the local periodic structure of the gel fibres

Characterization and Molecular Determinants for β-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium

Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium

Characterization and molecular determinants for β-lactam specificity of the multidrug efflux pump AcrD from Salmonella typhimurium

Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium

Approved Drugs Containing Thiols as Inhibitors of Metallo-β-lactamases: Strategy To Combat Multidrug-Resistant Bacteria

Resistance to β-lactam antibiotics can be mediated by metallo-β-lactamase enzymes (MBLs). An MBL inhibitor could restore the effectiveness of β-lactams. We report on the evaluation of approved thiol-containing drugs as inhibitors of NDM-1, VIM-1, and IMP-7. Drugs were assessed by a novel assay using a purchasable fluorescent substrate and thermal shift. Best compounds were tested in antimicrobial susceptibility assay. Using these orthogonal screening methods, we identified drugs that restored the activity of imipenem