Máquinas moleculares que sintetizan anhidridos fosfóricos.

RESUMEN Esta tesis versa sobre el estudio de cuatro enzimas que tienen como función específica la síntesis y liberación del anhidridos fosfóricos, y que son cruciales para la biosíntesis de los aminoácidos ornitina/arginina, prolina, lisina e isoleucina, y para la síntesis microbiana de nucleótidos de pirimidina. Previamente a mi incorporación en el laboratorio, éste habia identificado un plegamiento característico y novedoso común a los enzimas carbamato quinasa (Marina, A. et al., 1999; Ramón-Maiques, S. et al., 2000) y acetilglutamato quinasa (Ramón-Maiques, S. et al., 2002). Ambos enzimas, pertenecen al grupo de la Enzyme Commission EC 2.7.2, transferidores de un fosforilo a un grupo carboxilato, y por tanto, con la misión específica de sintetizar anhidridos mixtos carboxílico-fosfórico, en rutas de biosíntesis de aminoácidos. Carbamato quinasa y acetilglutamato quinasa, pertenecen a la familia estructural aminoácido quinasa (Base de datos PFAM, familia PF00696; http://www.sanger.ac.uk/Software/Pfam), familia que por similitud de secuencia de aminoácidos, incluye además, a los enzimas aspartatoquinasa, glutamato 5-quinasa, UMP quinasa bacteriana y N-acetil L-glutamato sintasa. Dada la similitud de secuencia entre los enzimas de la familia aminoácido quinasa, el laboratorio propuso que el plegamiento de carbamato quinasa y acetilglutamato quinasa iba a ser común al resto de enzimas de la familia y la puesta a prueba experimental de esta hipótesis ha sido uno de los objetivos principales del laboratorio y de mi trabajo. Parte de mi trabajo inicial, se centró en la acetilglutamato quinasa (NAGK), enzima cuya estructura había sido ya determinada a alta resolución (Ramon-Maiques, et al., 2002), por otros miembros del grupo en el que he desarrollado mi trabajo. Mi trabajo en relación con este enzima fue corroborar mediante mutagénesis dirigida, las inferencias funcionales derivadas del estudio estructural previo. Dada la similitud entre acetilglutamato quinasa y aspartoquinasa (AK), otro enzima de la familia amino ácido quinasa, clave en la síntesis de treonina, metionina, lisina e isoleucina, y con gran potencial desde el punto de vista biotecnológico, utilizamos la información estructural de NAGK, para realizar un trabajo de mutagénesis dirigida de aspartoquinasa, cuyos resultados nos permitieron construir un modelo estructural de AK. El tercer enzima que he estudiado, la UMP quinasa, es clave en la síntesis de nucleótidos de pirimidina y presenta la peculiaridad dentro de los enzimas de la familia aminoácido quinasa de sintetizar la formación de un anhidrido fosfórico-fosfórico. Mi trabajo con este enzima se ha centrado en determinar su estructura mediante difracción de rayos X. Por último he estudiado el enzima glutamato 5-quinasa, clave en la síntesis de prolina en microorganismos y plantas, que es tambien clave para la síntesis de ornitina en animales. He determinado la estructura tridimensional de la glutamato 5-quinasa de E. coli mediante difracción de rayos X. El ámbito de estudio con los cuatro enzimas objeto de esta tesis, ha sido siempre el estructural, buscando conectar y asociar rasgos estructurales con comportamiento funcional. El trabajo que he realizado, ha dado lugar a las siguientes publicaciones: - Marco-Marín, C. et al (2003). Journal of Molecular Biology (2003) 334, 459-476. - Marco-Marín, C. et al (2005). Biochim. Biophys. Acta. (2005) 1747, 271-275. - Marco-Marín, C. et al (2005). Journal of Molecular Biology (2005) 352, 438-454. - Marco-Marín, C. et al (2007). Journal of Molecular Biology (2007) 367, 1431-1446. __________________________________________________________________________________________________This thesis is about four enzymes that synthesize phosphoric anhydrides, and which have key roles on the biosynthetic pathways of ornithine/arginine, proline, lysine and isoleucine, and on the microbial synthesis of pyrimidine nucleotides. A novel and characteristic fold common to the enzymes carbamate kinase (CK) and acetylglutamate kinase (NAGK) had been identified by other members of the laboratory, previously to my incorporation. CK and NAGK belong to the amino acid kinase structural family (PFAM database, PF00696; http://www.sanger.ac.uk/Software/Pfam), that also includes aspartokinase (AK), glutamate 5-kinase, bacterial UMP kinase and N-acetyl L-glutamate synthase. Based on the sequence similarity of these enzymes, the laboratory proposed a common fold for all of them, equivalent to that previously described for CK and NAGK. One of the main objectives of the laboratory and of my work has been to probe this hypothesis experimentally. Initially, I studied NAGK from E. coli and I corroborated by site-directed mutagenesis, functional inferences derived from the previous structural work. The high similitude between NAGK and AK allowed to perform a site-directed mutagenesis study of AK, and to use the results of this study to build a molecular model of the AKIII from E. coli. The third enzyme that I have studied, UMP kinase, is a key enzyme of the pathway of biosynthesis of pyrimidine nucleotides in bacteria, that different to the other enzymes of the amino acid kinase family, synthetizes a phosphoric-phosphoric anhydride. I have determined the 3D-structure of the UMP kinase from Pyrococcus furiosus using X-ray crystallography, and also of the glutamate 5-kinase fom E. coli, which is a key enzyme of the biosynthesic pathway of proline in microorganisms and plants, and also of ornithine in animals. The work of this thesis is included in the following publications: - Marco-Marín, C. et al (2003). Journal of Molecular Biology (2003) 334, 459-476. - Marco-Marín, C. et al (2005). Biochim. Biophys. Acta. (2005) 1747, 271-275. - Marco-Marín, C. et al (2005). Journal of Molecular Biology (2005) 352, 438-454. - Marco-Marín, C. et al (2007). Journal of Molecular Biology (2007) 367, 1431-1446

Draft Genome Sequences of 12 Monophasic Salmonella enterica subsp. enterica Serotype Typhimurium 1,4,[5], 12:i:- Strains Isolated from Wild Griffon Vultures in Eastern Spain

[EN] Monophasic Salmonella enterica subsp. enterica serovar Typhimurium is one of the most common zoonotic pathogens. Salmonella species reside in a wide variety of hosts, including wild animals. Thus, we report here the genome sequences of 12 monophasic S. Typhimurium strains isolated from healthy wild vultures to gain better insight into their epidemiology and host-pathogen interactions.This work was funded by Generalitat Valenciana (Government of Valencia) and by CEU-UCH (Consolidacion de Indicadores INDI15/16, INDI16/20, and INDI17/25).Marín, C.; D'auria, G.; Martínez-Priego, L.; Marco-Jiménez, F. (2019). Draft Genome Sequences of 12 Monophasic Salmonella enterica subsp. enterica Serotype Typhimurium 1,4,[5], 12:i:- Strains Isolated from Wild Griffon Vultures in Eastern Spain. Microbiology Resource Announcements. 8(42):1-3. https://doi.org/10.1128/MRA.00570-19S13842Blanco, G. (2018). Supplementary feeding as a source of multiresistantSalmonellain endangered Egyptian vultures. Transboundary and Emerging Diseases, 65(3), 806-816. doi:10.1111/tbed.12806Krawiec, M., Kuczkowski, M., Kruszewicz, A., & Wieliczko, A. (2015). Prevalence and genetic characteristics of Salmonella in free-living birds in Poland. BMC Veterinary Research, 11(1), 15. doi:10.1186/s12917-015-0332-xMolina-López, R. A., Vidal, A., Obón, E., Martín, M., & Darwich, L. (2015). Multidrug-resistantSalmonella entericaSerovar Typhimurium Monophasic Variant 4,12:i:- Isolated from Asymptomatic Wildlife in a Catalonian Wildlife Rehabilitation Center, Spain. Journal of Wildlife Diseases, 51(3), 759-763. doi:10.7589/2015-01-019Marin, C., Torres, C., Marco-Jiménez, F., Cerdà-Cuéllar, M., Sevilla, S., Ayats, T., & Vega, S. (2018). Supplementary feeding stations for conservation of vultures could be an important source of monophasic Salmonella typhimurium 1,4,[5],12:i:-. Science of The Total Environment, 636, 449-455. doi:10.1016/j.scitotenv.2018.04.310Marin, C., Palomeque, M.-D., Marco-Jiménez, F., & Vega, S. (2014). Wild Griffon Vultures (Gyps fulvus) as a Source of Salmonella and Campylobacter in Eastern Spain. PLoS ONE, 9(4), e94191. doi:10.1371/journal.pone.0094191Schmieder, R., & Edwards, R. (2011). Quality control and preprocessing of metagenomic datasets. Bioinformatics, 27(6), 863-864. doi:10.1093/bioinformatics/btr026Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114-2120. doi:10.1093/bioinformatics/btu170Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., … Homer, N. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics, 25(16), 2078-2079. doi:10.1093/bioinformatics/btp352Quinlan, A. R., & Hall, I. M. (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics, 26(6), 841-842. doi:10.1093/bioinformatics/btq033Seemann, T. (2014). Prokka: rapid prokaryotic genome annotation. Bioinformatics, 30(14), 2068-2069. doi:10.1093/bioinformatics/btu153Lima, T., Auchincloss, A. H., Coudert, E., Keller, G., Michoud, K., Rivoire, C., … Bairoch, A. (2009). HAMAP: a database of completely sequenced microbial proteome sets and manually curated microbial protein families in UniProtKB/Swiss-Prot. Nucleic Acids Research, 37(Database), D471-D478. doi:10.1093/nar/gkn661Finn, R. D., Coggill, P., Eberhardt, R. Y., Eddy, S. R., Mistry, J., Mitchell, A. L., … Bateman, A. (2015). The Pfam protein families database: towards a more sustainable future. Nucleic Acids Research, 44(D1), D279-D285. doi:10.1093/nar/gkv1344Seribelli, A. A., Frazão, M. R., Gonzales, J. C., Cao, G., Leon, M. S., Kich, J. D., … Falcão, J. P. (2018). Draft Genome Sequences of 20 Salmonella enterica subsp. enterica Serovar Typhimurium Strains Isolated from Swine in Santa Catarina, Brazil. Genome Announcements, 6(16), e00232-18. doi:10.1128/genomea.00232-1

A Novel Two-domain Architecture Within the Amino Acid Kinase Enzyme Family Revealed by the Crystal Structure of Escherichia coli Glutamate 5-kinase

Glutamate 5-kinase (G5K) makes the highly unstable product glutamyl 5-phosphate (G5P) in the initial, controlling step of proline/ornithine synthesis, being feedback-inhibited by proline or ornithine, and causing, when defective, clinical hyperammonaemia. We determined two crystal structures of G5K from Escherichia coli, at 2.9 Å and 2.5 Å resolution, complexed with glutamate and sulphate, or with G5P, sulphate and the proline analogue 5-oxoproline. E. coli G5K presents a novel tetrameric (dimer of dimers) architecture. Each subunit contains a 257 residue AAK domain, typical of acylphosphate-forming enzymes, with characteristic α3β8α4 sandwich topology. This domain is responsible for catalysis and proline inhibition, and has a crater on the β sheet C-edge that hosts the active centre and bound 5-oxoproline. Each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic β5β4 sandwich fold and three α helices. The AAK and PUA domains of one subunit associate non-canonically in the dimer with the same domains of the other subunit, leaving a negatively charged hole between them that hosts two Mg ions in one crystal, in line with the G5K requirement for free Mg. The tetramer, formed by two dimers interacting exclusively through their AAK domains, is flat and elongated, and has in each face, pericentrically, two exposed active centres in alternate subunits. This would permit the close apposition of two active centres of bacterial glutamate-5-phosphate reductase (the next enzyme in the proline/ornithine-synthesising route), supporting the postulated channelling of G5P. The structures clarify substrate binding and catalysis, justify the high glutamate specificity, explain the effects of known point mutations, and support the binding of proline near glutamate. Proline binding may trigger the movement of a loop that encircles glutamate, and which participates in a hydrogen bond network connecting active centres, which is possibly involved in the cooperativity for glutamate. © 2007 Elsevier Ltd. All rights reserved.This work was supported by grants BFU2004-05159, BFU2004-04472 from the Spanish Ministry of Education and Science, and PI052838 from the Spanish Ministry of Health.Peer Reviewe

The PII-NAGK-PipX-NtcA Regulatory Axis of Cyanobacteria: A Tale of Changing Partners, Allosteric Effectors and Non-covalent Interactions

PII, a homotrimeric very ancient and highly widespread (bacteria, archaea, plants) key sensor-transducer protein, conveys signals of abundance or poorness of carbon, energy and usable nitrogen, converting these signals into changes in the activities of channels, enzymes, or of gene expression. PII sensing is mediated by the PII allosteric effectors ATP, ADP (and, in some organisms, AMP), 2-oxoglutarate (2OG; it reflects carbon abundance and nitrogen scarcity) and, in many plants, L-glutamine. Cyanobacteria have been crucial for clarification of the structural bases of PII function and regulation. They are the subject of this review because the information gathered on them provides an overall structure-based view of a PII regulatory network. Studies on these organisms yielded a first structure of a PII complex with an enzyme, (N-acetyl-Lglutamate kinase, NAGK), deciphering how PII can cause enzyme activation, and how it promotes nitrogen stockpiling as arginine in cyanobacteria and plants. They have also revealed the first clear-cut mechanism by which PII can control gene expression. A small adaptor protein, PipX, is sequestered by PII when nitrogen is abundant and is released when is scarce, swapping partner by binding to the 2OG-activated transcriptional regulator NtcA, co-activating it. The structures of PII-NAGK, PII-PipX, PipX alone, of NtcA in inactive and 2OG-activated forms and as NtcA-2OG-PipX complex, explain structurally PII regulatory functions and reveal the changing shapes and interactions of the T-loops of PII depending on the partner and on the allosteric effectors bound to PII. Cyanobacterial studies have also revealed that in the PII-PipX complex PipX binds an additional transcriptional factor, PlmA, thus possibly expanding PipX roles beyond NtcA-dependency. Further exploration of these roles has revealed a functional interaction of PipX with PipY, a pyridoxal-phosphate (PLP) protein involved in PLP homeostasis whose mutations in the human ortholog cause epilepsy. Knowledge of cellular levels of the different components of this PII-PipX regulatory network and of KD values for some of the complexes provides the basic background for gross modeling of the system at high and low nitrogen abundance. The cyanobacterial network can guide searches for analogous components in other organisms, particularly of PipX functional analogs

Experimental evidence reveals both cross-infection and cross-contamination risk of embryos storage in liquid nitrogen biobanks

[EN] This study was conducted to demonstrate the potential hazards of cross-infection and cross-contamination of embryos during storage in liquid nitrogen biobanks. For the harmless and successful cryopreservation of embryos, the vitrification method must be chosen meticulously to guarantee not only a high post-thaw survival of embryos, but also to reduce the risk of disease transmission when those embryos are in storage for long periods. In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the effects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae-early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures.This research was supported by the Ministry of Economy, Industry and Competitiveness (MICINN), through a Spanish research project (AGL2017-85162-C2-1-R).Marín, C.; Garcia-Dominguez, X.; Montoro-Dasí, L.; Lorenzo-Rebenaque, L.; Vicente Antón, JS.; Marco-Jiménez, F. (2020). Experimental evidence reveals both cross-infection and cross-contamination risk of embryos storage in liquid nitrogen biobanks. Animals. 10(4):1-13. https://doi.org/10.3390/ani10040598S113104Kushnir, V. A., Barad, D. H., Albertini, D. F., Darmon, S. K., & Gleicher, N. (2017). Systematic review of worldwide trends in assisted reproductive technology 2004–2013. 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Reproductive Medicine and Biology, 13(3), 107-117. doi:10.1007/s12522-014-0176-2Marco-Jiménez, F., Jiménez-Trigos, E., Almela-Miralles, V., & Vicente, J. S. (2016). Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method. PLOS ONE, 11(2), e0148661. doi:10.1371/journal.pone.0148661Cobo, A., Romero, J. L., Pérez, S., de los Santos, M. J., Meseguer, M., & Remohí, J. (2010). Storage of human oocytes in the vapor phase of nitrogen. Fertility and Sterility, 94(5), 1903-1907. doi:10.1016/j.fertnstert.2009.10.042Nakashima, A., Ino, N., Kusumi, M., Ohgi, S., Ito, M., Horikawa, T., … Saito, H. (2010). Optimization of a novel nylon mesh container for human embryo ultrarapid vitrification. Fertility and Sterility, 93(7), 2405-2410. doi:10.1016/j.fertnstert.2009.01.063Chen, Y., Zheng, X., Yan, J., Qiao, J., & Liu, P. (2013). Neonatal outcomes after the transfer of vitrified blastocysts: closed versus open vitrification system. Reproductive Biology and Endocrinology, 11(1). doi:10.1186/1477-7827-11-107Panagiotidis, Y., Vanderzwalmen, P., Prapas, Y., Kasapi, E., Goudakou, M., Papatheodorou, A., … Maroulis, G. (2013). Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study. Reproductive BioMedicine Online, 26(5), 470-476. doi:10.1016/j.rbmo.2013.01.016Cai, H., Niringiyumukiza, J. D., Li, Y., Lai, Q., Jia, Y., Su, P., & Xiang, W. (2018). Open versus closed vitrification system of human oocytes and embryos: a systematic review and meta-analysis of embryologic and clinical outcomes. Reproductive Biology and Endocrinology, 16(1). doi:10.1186/s12958-018-0440-0Paffoni, A., Guarneri, C., Ferrari, S., Restelli, L., Nicolosi, A. E., Scarduelli, C., & Ragni, G. (2011). Effects of two vitrification protocols on the developmental potential of human mature oocytes. 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The PII-NAGK-PipX-NtcA Regulatory Axis of Cyanobacteria: A Tale of Changing Partners, Allosteric Effectors and Non-covalent Interactions

PII, a homotrimeric very ancient and highly widespread (bacteria, archaea, plants) key sensor-transducer protein, conveys signals of abundance or poorness of carbon, energy and usable nitrogen, converting these signals into changes in the activities of channels, enzymes, or of gene expression. PII sensing is mediated by the PII allosteric effectors ATP, ADP (and, in some organisms, AMP), 2-oxoglutarate (2OG; it reflects carbon abundance and nitrogen scarcity) and, in many plants, L-glutamine. Cyanobacteria have been crucial for clarification of the structural bases of PII function and regulation. They are the subject of this review because the information gathered on them provides an overall structure-based view of a PII regulatory network. Studies on these organisms yielded a first structure of a PII complex with an enzyme, (N-acetyl-Lglutamate kinase, NAGK), deciphering how PII can cause enzyme activation, and how it promotes nitrogen stockpiling as arginine in cyanobacteria and plants. They have also revealed the first clear-cut mechanism by which PII can control gene expression. A small adaptor protein, PipX, is sequestered by PII when nitrogen is abundant and is released when is scarce, swapping partner by binding to the 2OG-activated transcriptional regulator NtcA, co-activating it. The structures of PII-NAGK, PII-PipX, PipX alone, of NtcA in inactive and 2OG-activated forms and as NtcA-2OG-PipX complex, explain structurally PII regulatory functions and reveal the changing shapes and interactions of the T-loops of PII depending on the partner and on the allosteric effectors bound to PII. Cyanobacterial studies have also revealed that in the PII-PipX complex PipX binds an additional transcriptional factor, PlmA, thus possibly expanding PipX roles beyond NtcA-dependency. Further exploration of these roles has revealed a functional interaction of PipX with PipY, a pyridoxal-phosphate (PLP) protein involved in PLP homeostasis whose mutations in the human ortholog cause epilepsy. Knowledge of cellular levels of the different components of this PII-PipX regulatory network and of KD values for some of the complexes provides the basic background for gross modeling of the system at high and low nitrogen abundance. The cyanobacterial network can guide searches for analogous components in other organisms, particularly of PipX functional analogs.Supported by grants BFU2014-58229-P and BFU2017-84264-P from the Spanish Government

Genetic Heterogeneity Underlying Phenotypes with Early-Onset Cerebellar Atrophy

Cerebellar atrophy (CA) is a frequent neuroimaging finding in paediatric neurology, usually associated with cerebellar ataxia. The list of genes involved in hereditary forms of CA is continuously growing and reveals its genetic complexity. We investigated ten cases with early-onset cerebellar involvement with and without ataxia by exome sequencing or by a targeted panel with 363 genes involved in ataxia or spastic paraplegia. Novel variants were investigated by in silico or experimental approaches. Seven probands carry causative variants in well-known genes associated with CA or cerebellar hypoplasia: SETX, CACNA1G, CACNA1A, CLN6, CPLANE1, and TBCD. The remaining three cases deserve special attention; they harbour variants in MAST1, PI4KA and CLK2 genes. MAST1 is responsible for an ultrarare condition characterised by global developmental delay and cognitive decline; our index case added ataxia to the list of concomitant associated symptoms. PIK4A is mainly related to hypomyelinating leukodystrophy; our proband presented with pure spastic paraplegia and normal intellectual capacity. Finally, in a patient who suffers from mild ataxia with oculomotor apraxia, the de novo novel CLK2 c.1120T>C variant was found. The protein expression of the mutated protein was reduced, which may indicate instability that would affect its kinase activity

One Health Approach : Invasive California Kingsnake (Lampropeltis californiae) as an Important Source of Antimicrobial Drug-Resistant Salmonella Clones on Gran Canaria Island

The aim of this study was to investigate the invasive species Lampropeltis californiae (California kingsnake) as a reservoir of Salmonella and its ability to spread different clones of the bacterium with zoonotic potential into the environment, as well as study its antimicrobial resistance patterns in Gran Canaria (Spain). The main results showed that a high diversity of Salmonella subsp. salamae strains circulate in Gran Canaria with a high prevalence of resistance shown for antimicrobials of public health importance, as summarised in the European Decision 2013/652/EU. The increase in the reptile population has led to a rise in the number of zoonotic infections due to close contact with reptiles, with reptile-associated salmonellosis being particularly relevant. California kingsnake invasion not only threatens the endemic reptile population of the island of Gran Canaria (Spain) but also poses serious public health problems by spreading zoonotic pathogens and their antimicrobial resistance (AMR) to the environment. Thus, the aim of this study was to assess the occurrence, genetic diversity, and AMR among Salmonella spp. strains isolated from California kingsnakes in Gran Canaria Island (Spain). Of 73 invasive individuals captured, 20.5% carried Salmonella spp., belonging to different subspecies and serovars, with subsp. salamae as the most abundant. Pulsed-field electrophoresis showed high genetic diversity among subsp. salamae isolates, and among these, 73.3% showed resistance to at least one of the antimicrobials tested. In conclusion, the present study revealed the importance of wild invasive California kingsnakes as reservoirs of drug-resistant Salmonella spp. that could pose a direct threat to livestock and humans. Identification of drug-resistant Salmonella strains in wildlife provides valuable information on potential routes of transmission that involve risks to public and animal health

CryoEM structures of the SARS-CoV-2 spike bound to antivirals

(Póster 63) Background: Single-particle cryoelectron microscopy (cryoEM) has played a key role in the fight against COVID-19. The molecular mechanisms for the action of some of the currently approved drugs targeting the SARS-CoV-2 RNA-dependent RNA polymerase, the fast developments of the current available vaccines and antibody therapies are examples of the impact of the knowledge gained from the cryoEM structures of SARS-CoV-2 proteins in complex with proteins (ACE2 or antibodies/nanobodies) or small compounds. Our aim is to use this technology to understand structurally how certain antiviral compounds and proteins targeting the spike may inhibit viral entry. Methods: 1) Production of wild-type and mutated spike and ACE2 proteins using baculovirus/insect cells. 2) Spike binding kinetics: protein-protein and protein-small compound interactions measured by BLI Biolayer interferometry (BLI) and/or microscale Thermophoresis (MST). 3) Buffer optimization for cryoEM grid preparation of spike variants by thermal shift assays and negative-staining electron microscopy (NSEM). These techniques are also used to adjust the molar ratio of spike:ACE2 and spike:small-compound complexes. 4) Structural characterization by cryoEM. Results: At IBV-CSIC we have created a pipeline for the production and characterization of several spike variants and ACE2 decoys. While this pipeline is described in detail in other oral/poster communications, this communication is centered around one of the pillars within this pipeline; the structural characterization of possible drug candidates bound to the SARS-CoV-2 spike by cryoEM. In this way, we have successfully solved structures of the spike bound to: A) protein inhibitors as ACE2 decoys; B) a small inhibitory compound; C) mixtures of proteins and small-compound (nanobody-heparan derivative) working cooperatively as inhibitors. These protein/drug candidates were previously selected based on the results obtained in our interactomics platform, whereas their concentration and the buffer conditions for cryoEM grids preparation were established based on thermal shift assays and NSEM. Conclusion: CryoEM is a powerful tool to directly visualize the effect caused by a potential drug on a protein target. In a short period of time we have developed this technique in our institute to be applied to the SARS-CoV-2 spike protein, not only to obtain high-resolution structures of SARS- CoV-2 spike variants of concern (see WP4) but also to obtain the structures of complexes of the spike with various inhibitory compounds of very different nature

Un ataque combinado químico, virológico, biofísico y estructural hace posible la obtención de nuevos inhibidores de entrada celular de SARS-CoV-2 y la caracterización de su mecanismo de inhibición

Resumen del trabajo presentado al 45º Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Zaragoza del 5 al 8 de septiembre de 2023.IBV-COVID19 Pipeline: C.Espinosa, N.Gougeard, M.P.Hernández-Sierra, A.Rubio-del-Campo, R.Ruiz-Partida, L.Villamayor.El virus SARS-CoV-2 causa el COVID-19 al infectar las células a través de la interacción de la proteína de su espícula (S) con el receptor celular enzima convertidora de angiotensina 2 (ACE2). Para buscar inhibidores de este paso clave en la infección viral, examinamos una biblioteca interna (IQM-CSIC, Madrid) de compuestos multivalentes derivados de triptófano, primero usando pseudopartículas de Virus de Estomatits Vesicular que expresaban S (I2SysBio, UV y CSIC, Valencia), identificando un compuesto como potente inhibidor de entrada no citotóxico. La optimización química (IQM-CSIC) generó otros dos potentes inhibidores de entrada no citotóxicos que, como 2, también inhibieron la entrada celular de SARS-CoV-2 genuino (I2SysBio). Los estudios con proteínas recombinantes puras (IBV-CSIC, Valencia) usando termofluor y termoforesis de microescala revelaron la unión de estos compuestos a S, y a su dominio de unión al receptor producido separadamente, probando interferencia con la interacción con ACE2. La criomicroscopía electrónica de S (IBV-CSIC), libre o unido al compuesto activo, arrojó luz sobre los mecanismos de inhibición por estos compuestos de la entrada viral a la célula. Esta actividad triinstitucional combinada ha identificado y caracterizado una nueva clase de inhibidores de entrada de SARS-CoV-2 de claro potencial preventivo o terapéutico de COVID-19.ECNextGeneration EUfund 2020/2094 de CSIC/PTI Salud Global; Crue/CSIC/Santander Fondo Supera Covid-19;CSIC-COV19-082; CIBERER-ISCIIICOV20/00437; Covid19-SCI/GValenciana (RG);PID2020- 120322RB-C21 (VR) y PID2020-116880GB-I00 (JLLl) Agenc. Estat Investig.Peer reviewe