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Resumen del póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014., Antibiotic resistance has become a major health issue. Unfortunately, the development of new antibiotics is being very slow and expensive and we are running out of weapons to fi ght against the appearance of multi-resistant bugs. Bacterial conjugation is the main mechanism for the wide spread dissemination of antibiotic resistance genes and, therefore, the search of specific inhibitors of conjugation (COINs) could become a brand new strategy in this warfare. In the search of potential COINs, previous studies have reported that unsaturated fatty acids (uFAs) were able to inhibit conjugation. Based on these studies we have looked for the molecular targets of these compounds and we have found that the Type IV secretion ATPase TrwD is inhibited by linoleic acid and 2-alkynoic fatty acids, such as the 2-hexadecynoic acid. These two uFAs compounds were the most effective inhibitors in R388 plasmid conjugation. The inhibitory effect is specific for the traffic ATPase TrwD, as the remaining ATPases of the Type IV secretion system are unaffected by both uFAs and 2-AFAs. We have characterized the inhibition mechanism and we have found that in both cases it is a non-competitive inhibition, as the affinities for the substrate (ATP) and product (ADP) are not altered in the presence of the fatty acids. Altogether, these results could open new avenues in the development of new strategies to prevent the dissemination of antibiotic resistance genes.

Los sistemas de secreción tipo IV (T4SS) son usados por las bacterias Gram-negativas bien para el intercambio genético entre ellas (proceso denominado conjugación) o bien, en el caso de bacterias patogénicas, para secretar efectores a sus células hospedadoras. Los T4SS son sistemas multiproteicos, formados por al menos doce proteínas, que se expanden a través de las membranas interna y externa de la célula donadora. Tanto para la biogénesis del sistema como para la transferencia del sustrato, se necesita la energía proporcionada por los tres motores situados en la base del canal (VirD4, VirB4 y VirB11). Este trabajo se ha centrado en el estudio de TrwD, un homólogo de VirB11 en nuestro sistema modelo, el plásmido conjugativo R388. Esta tesis aporta evidencias bioquímicas que apoyan que la familia de las proteínas VirB11 pertenecen a la superfamilia de ATPasas implicadas en el tráfico de proteínas durante la secreción mediada por Sistemas de Tipo IV. En este contexto se ha caracterizado la interacción con las otras dos ATPasas del sistema corroborando el papel esencial de esta proteína regulando tanto la biogénesis del sistema como la transferencia de sustrato. Por último se han evaluado inhibidores de la conjugación bacteriana, como alternativa al uso de antibióticos, que actúan específicamente sobre la actividad de esta proteína.

TrwD, the VirB11 homologue in conjugative plasmid R388, is a member of the large secretion ATPase superfamily, which includes ATPases from bacterial type II and type IV secretion systems, type IV pilus, and archaeal flagellae assembly. Based on structural studies of the VirB11 homologues in Helicobacter pylori and Brucella suis and the archaeal type II secretion ATPase GspE, a unified mechanism for the secretion ATPase superfamily has been proposed. Here, we have found that the ATP turnover of TrwD is down-regulated by physiological concentrations of magnesium. This regulation is exerted by increasing the affinity for ADP, hence delaying product release. Circular dichroism and limited proteolysis analysis indicate that magnesium induces conformational changes in the protein that promote a more rigid, but less active, form of the enzyme. The results shown here provide new insights into the catalytic mechanism of the secretion ATPase superfamily. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported by the Ministerio de Ciencia e Innovación (MCINN, Spain) Grant BFU2008-00806 (to E. C. and I. A.), Grant BFU2008-00995/BMC from the MCINN, Grant RD06/0008/1012 from the Instituto de Salud Carlos III, and Grant LSHM-CT-2005_019023 from the European VI Framework Program (to F. d. l. C.), and Grant BFU2010-15443 (to F. M.) from the MCINN.

Peptide ligation chemistry has revolutionized protein science by facilitating access to synthetic proteins. Here, we describe the development of additive-free ligation-deselenization chemistry at ?-selenoaspartate and ?-selenoglutamate that enables the generation of native polypeptide products on unprecedented timescales. The deselenization step is chemoselective in the presence of unprotected selenocysteine, which is highlighted in the synthesis of selenoprotein K. The power of the methodology is also showcased through the synthesis of three tick-derived thrombin-inhibiting proteins, each of which were assembled, purified, and isolated for biological assays within a few hours. The methodology described here should serve as a powerful means of accessing synthetic proteins, including therapeutic leads, in the future.