BUSCADOR RECOLECTA

In recent years, the Smart City concept has grown in popularity, and a significant number of cities around the world have adopted smart city strategies. Smart and sustainable cities are an emerging urban development approach due to their immense potential to improve environmental sustainability. The Smart City concept is based on collecting, analyzing, and displaying a large amount of data and information concerning urban systems and subsystems. As time goes by, the capacity of smart cities for generating digital information has grown exponentially. However, this digital information is heterogeneous, massive, collected from different sources, generated in different formats, and in most cases not structured, which exacerbates the situation of extracting valuable knowledge from data. Therefore, it is fundamental to handle the significant volumes of heterogeneous sensed data and to integrate such data along with information and analysis tools into a comprehensive platform. This can ensure the security, efficiency, and performance of the different Smart City tasks. A comprehensive software platform could provide services such as facilities for application development, integration of heterogeneous data sources, deployment, and management to ease the construction of sophisticated Smart Cities’ applications. In this context, the work begins with a concise description of the concept of smart city and the technologies involved in it. It addresses the development of an urban data platform along with how to obtain and integrate information from sensors and other data sources, in order to provide aggregated and intelligent views of raw data to support various domains within the city; in our case, smart mobility. The platform architecture is implemented following a five-layer model that considers elements from perception, sensing to data management, processing, and visualization. With the aim of evaluating the efficiency of the developed platform, three different use cases are described and analyzed, which have been implemented in the city of Pamplona, Spain, as vertical services linked to the platform: intelligent urban mobility-bike handling, bike-2-bike communication, and restricted vehicle access zone control system. Ultimately, this work provides an experiment to assess different long-range wireless communication technologies to enable their implementation within an urban environment., This work was partially supported by the European Union’s Horizon 2020 Research and Innovation Programme (STARDUST-Holistic and Integrated Urban Model for Smart Cities) under Grant 774094, by the Ministerio de Ciencia, Innovación y Universidades, Gobierno de España (Agencia Estatal de Investigación, Fondo Europeo de Desarrollo Regional-FEDER, European Union) under Grant RTI2018-095499-B-C31 IoTrain, and by Gobierno de Navarra under Grant PC183-184 (Securing information and applications in V2G environments, SECV2G)., Programa de Doctorado en Tecnologías de las Comunicaciones, Bioingeniería y de las Energías Renovables (RD 99/2011), Bioingeniaritzako eta Komunikazioen eta Energia Berriztagarrien Teknologietako Doktoretza Programa (ED 99/2011)

La tutora de esta tesis es Inmaculada Farrán Blanch, La brucelosis es una zoonosis mundialmente extendida con importantes repercusiones económicas y sanitarias tanto para la ganadería como para el ser humano. Existen doce especies distintas de Brucella con distinta preferencia de hospedador; entre ellas, B. melitensis (bovino), B. abortus (pequeños rumiantes) y B. suis (porcino) son las más frecuentes en el ganado doméstico, y a excepción de B. suis bv2, suponen un grave riesgo para el ser humano. La vacunación de los animales es una herramienta esencial para reducir la infección en los mismos y, así, evitar la transmisión al ser humano. La infección en pequeños rumiantes es ocasionada por las especies Brucella melitensis y B. ovis; la primera de ellas produce abortos a final de gestación en hembras gestantes y la segunda, orquitis y epididimitis en machos con la consecuente reducción de la fertilidad. La cepa atenuada B. melitensisRev1 es la única vacuna recomendada desde hace más de 60 años contra la brucelosis en pequeños rumiantes. Pese a su extendido empleo y demostrada eficacia, Rev1 posee ciertos inconvenientes que recomiendan la búsqueda de una vacuna alternativa más segura que, idealmente, pueda aplicarse en animales gestantes. En este contexto, en el grupo de Brucelosis del IdAB-CSIC, se han desarrollado dos candidatos de Brucella melitensis mediante deleción en fase del gen wzm (16MΔwzmy Rev1Δwzm) involucrado en el sistema de dos componentes Wzm/Wztr es responsable de la exportación del polisacárido O al periplasma bacteriano para completar la biosíntesis del LPS liso. En los Capítulos 1 y 2 de esta Tesis Doctoral, se ha estudiado la seguridad, respuesta inmune y patogénesis placentaria de ambos candidatos vacunales, tanto en modelos preclínicos (i.e. cultivos de células trofoblásticas y ratona gestante) como en ganado ovino de distintos sexos y en distintos tercios de la gestación. Además, se han realizado estudios novedosos de patogénesis placentaria y respuesta inmune a lo largo de la gestación en ratonas inoculadas con distintas cepas de referencia de Brucella como son B. melitensis16M (virulenta) y Rev1 (atenuada) y B. ovis PA. Los principales resultados del Capítulo 1 indicaron que, en comparación con sus parentales, ambos mutantes rugosos poseían menor capacidad de invasión, replicación y efecto citotóxico en trofoblastos BeWo, menor capacidad de colonización placentaria en ratones con resolución de la infección a este nivel en los últimos estadíos de la gestación, ausencia de lesiones placentarias y elevada viabilidad fetal, así como menor respuesta proinflamatoria a lo largo de la gestación murina. En el Capítulo 2, ambos mutantes mostraron atenuación y buena respuesta serológica en carneros, ovejas no gestantes y ovejas gestantes inoculadas por las vías subcutánea o conjuntival, en distintos tercios de gestación. Aunque ninguno de los candidatos indujo abortos, Rev1∆wzmse consideró más seguro que 16M∆wzmpara ovejas gestantes, puesto que este último mostró una reactivación de la infección en el momento del parto, que conllevó la excreción transitoria por placenta/fluido vaginal y/o leche en algunos animales. En conjunto, los Capítulos 1 y 2 nos han permitido establecer una relación entre la multiplicación de Brucella en trofoblastos BeWo y placentas murinas, y su capacidad para inducir abortos y transmitirse a la descendencia en ovejas inoculadas a mitad de gestación. Asimismo, pese a la gran utilidad del modelo de ratona gestante para seleccionar candidatos más seguros, la evaluación final de ciertos parámetros de seguridad debe realizarse en el hospedador natural. Futuros ensayos de eficacia y respuesta inmune en ovino permitirán determinar la utilidad real de Rev1∆wzm, como alternativa a la actual vacuna Rev1.Trascomprobar las buenas propiedades vacunales de Rev1∆wzm, en el Capítulo 3 de esta Tesis se propuso construir un mutante en B. suis bv2 para abordar la lucha contra la brucelosis porcina, mediante la construcción y caracterización de Bs2Δwzm. Este mutante mostró propiedades bioquímicas distintas a 16M∆wzmo Rev1∆wzm, así como menor atenuación de la esperada. Durante estos estudios, detectamos que la cepa parental Bs2 presentaba una marcada inestabilidad de fase L→R no reportada anteriormente. Además, aplicando el modelo de ratona gestante puesto a punto en el Capítulo 1, estudiamos la patogénesis placentaria y respuesta inmune de Bs2 a lo largo de la gestación en ratonas. Por lo tanto, este Capítulo 3 se completó con un detallado análisis genético y fenotípico de la cepa Bs2, con el objetivo de determinar el alcance de dicha inestabilidad de fase tanto in vitro como in vivo, y las bases moleculares responsables de la misma. Los principales resultados indicaron que, en ausencia de escisiones o mutaciones en las regiones genéticas wboy wbk portadoras de genes implicados en la biosíntesis del LPS, este fenómeno podría deberse a variaciones en la expresión bacteriana, en función de las condiciones ambientales. De hecho, los cultivos con un elevado grado de disociación bacteriana mostraron una reducción significativa de la expresión de los genes codificantes de distintas manosil-y glicosil-transferasas, junto con un aumento de la expresión de la proteína de membrana externa Omp2b. Debido a su complejidad, en esta Tesis no ha sido posible determinar con exactitud los mecanismos moleculares implicados en la disociación espontánea de B. suisbv2, por lo que son necesarios más estudios que permitan comprender estos mecanismos, su implicación biológica, y con ello, construir una cepa B. suisbv2 estable sobre la que poder obtener candidatos vacunales adecuados frente a la brucelosis porcina. Uno de los resultados más interesantes de esta Tesis Doctoral ha sido evidenciar, por primera vez, que la disociación de Bs2 observada in vitro tiene una implicación in vivo, encontrando en un mismo animal bacterias con fenotipos lisos en tejidos placentarios y rugosos en tejidos esplénicos de ratones. Futuros estudios en cerdas gestantes permitirán dilucidar si este fenómeno de adaptación bacteriana ocurre también en el hospedador natural., Brucellosis is a worldwide zoonosis with important economic and health implications for both livestock and humans. There are twelve different Brucella species with different host preference; among them, B. melitensis(ovine), B. abortus(bovine) and B. suis(porcine) are the most frequent in domestic livestock,supposinga serious risk to humans. Vaccination of animals is an essential tool to reduce infection and thus, toprevent transmission to humans. Infection in small ruminants is caused by Brucella melitensisand B. ovisspecies; the former causes abortions at the end of gestation in pregnant females and the latter, orchitis and epididymitis in males with the consequent reduction of fertility. The attenuated strain B. melitensisRev1 is the only vaccine recommended for more than 60 years tocontrol of brucellosis in small ruminants. Despite its widespread use and proven efficacy, Rev1 has certain drawbacks that recommend the search for a safer alternative vaccine that, ideally, can be applied in pregnant animals. In this context, in the Brucellosis group of the IdAB-CSIC, two Brucella melitensiscandidates have been developed by phase-deletion of the wzmgene (16MΔwzmand Rev1Δwzm) involved in the two-component Wzm/Wzt system responsible for the export of the O polysaccharide to the bacterial periplasm forbiosynthesis of the smooth LPS. In Chapters 1 and 2 of this PhD Thesis, the safety, immune response and placental pathogenesis of both vaccine candidates have been studied in both preclinical models (i.e. trophoblast cell cultures and pregnant mouse) and sheep of different sex and periodof pregnancy. In addition, novel studies of placental pathogenesis and immune response throughout gestation have been performed in mice inoculated with different Brucellareference strains such as B. melitensis16M (virulent) and Rev1 (attenuated) and B. ovisPA. The main results of the first Chapter indicated that, compared to their parentals, both rough mutants had lower invasiveness, replication and cytotoxic effect on BeWo trophoblasts, lower placental colonization ability in mice with resolution of infection at late pregnancy, absence of placental injuriesand high fetal viability, as well as lower pro-inflammatory response throughout murine gestation. ThesecondChapter discusses how both mutants showed attenuation withgood serological response in rams, non-pregnant ewes and pregnant ewes inoculated by the subcutaneous or conjunctival routes, at different stagesof gestation. Although none of the candidates induced abortions, Rev1∆wzmwas considered safer than 16M∆wzmin pregnant ewes, since the latter showed reactivation of infection at parturition, leading to transient excretion byplacenta/vaginal fluid and/or milk in some animals. Taken together, Chapters 1 and 2 have allowed us to establish a relationship between Brucella multiplication in BeWo trophoblasts and murine placentas, and their ability to induce abortions and transmissionto offspring in ewes inoculated at mid-pregnancy. Also, notwithstandingthe great utility of the pregnant mouse model for selecting safer candidates, the final evaluation of certain safety parameters must be performed in the natural host. Future efficacy and immune response trials in sheep will allow us to determine the actual usefulness of Rev1∆wzm, as an alternative to the current Rev1 vaccine.After verifying the positivevaccinal properties of Rev1∆wzm, in the third Chapter of this Thesis we proposed to construct a mutant in B. suisbv2 to address the control of porcine brucellosis, by constructing and characterizing Bs2Δwzm. This mutant showed distinct biochemical properties from 16M∆wzmor Rev1∆wzm, as well as less attenuation than expected. During these studies, we detected that the parental Bs2 strain exhibited a marked S→R phase instability not previously reported. Furthermore, applying the pregnant mouse model describedin the first Chapter, we studied the placental pathogenesis and immune responseof Bs2 throughout murine pregnancy. Accordingly,this third Chapter was completed with a detailed genetic and phenotypic analysis of the Bs2 strain, with the aim of determining the extent of such phase instability both in vitroand in vivo, and the molecular basis responsible for it. The main results indicated that, in the absence of deletionsor mutations in the wboand wbk genetic regions carrying genes involved in LPS biosynthesis, the variations of the S-LPS to R-LPS in B. suisbv2could be due to variations in bacterial expression, depending on environmental conditions. In fact, cultures with a high degree of bacterial dissociation showed a significant reduction in the expression of genes encoding different mannosyl-and glycosyl-transferases, together with an increase in the expression of the outer membrane protein Omp2b. Due to the complexityof this topic, it has not been possible to determine in this Thesis the exactly molecular mechanisms involved in the spontaneous dissociation of B. suisbv2, so further studies should be performedto understand these mechanisms, their biological implication, and with this, to construct a stable B. suisbv2 strain suitable for vaccine candidates construction against swine brucellosis. One of the most interesting results of this Thesis has been to demonstrate, for the first time, that the dissociation of Bs2 observed in vitrohas an implication in vivo, finding in the same animal bacteria with smoothphenotypes in placental tissues and rough phenotype in splenic tissues of mice. Future studies in pregnant sows will clarifywhether this phenomenon of bacterial adaptation occurs also in the natural host., Contrato de formación de Doctorados Industriales de Gobierno de Navarra (convocatoria 2016), cofinanciado por CSIC y desarrollado en el IdAB. Los trabajos realizados han sido financiados por los proyectos de investigación RTI2018-098658-B-C21 del Ministerio de Ciencia e Innovación (Agencia Estatal de Investigación) y PT068-2018 y PT007 2019 del Departamento de Universidades, Innovación y Transformación Digital de Gobierno de Navarra., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

The lipopolysaccharide (LPS) O-polysaccharide (O-PS) is the main virulence factor in Brucella. After synthesis in the cytoplasmic membrane, O-PS is exported to the periplasm by the Wzm/Wzt system, where it is assembled into a LPS. This translocation also engages a bactoprenol carrier required for further biosynthesis pathways, such as cell wall biogenesis. Targeting O-PS export by blockage holds great potential for vaccine development, but little is known about the biological implications of each Wzm/Wzt moiety. To improve this knowledge and to elucidate its potential application as a vaccine, we constructed and studied wzm/wzt single- and double-deletion mutants, using the attenuated strain Brucella melitensis Rev1 as the parental strain. This allowed us to describe the composition of Brucella peptidoglycan for the first time. We observed that these mutants lack external O-PS yet trigger changes in genetic transcription and in phenotypic properties associated with the outer membrane and cell wall. The three mutants are highly attenuated; unexpectedly, Rev1Δwzm also excels as an immunogenic and effective vaccine against B. melitensis and Brucella ovis in mice, revealing that low persistence is not at odds with efficacy. Rev1Δwzm is attenuated in BeWo trophoblasts, does not infect mouse placentas, and is safe in pregnant ewes. Overall, these attributes and the minimal serological interference induced in sheep make Rev1Δwzm a highly promising vaccine candidate., This work was funded by Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21), Gobierno de Navarra (PT040-2018 and PT007-2019) projects. SM-B contracts were granted by the FEDER 2016–2018 program of Garantía Juvenil and by an UPNA pre-doctoral fellowship 2018–2022. IP-U Doctorados Industriales contract was cofounded by Gobierno de Navarra and CSIC. Research in the FC lab was supported by The Swedish Research Council (VR), The Knut and Alice Wallenberg Foundation (KAW), The Laboratory of Molecular Infection Medicine Sweden (MIMS), and The Kempe Foundation.

La tutora de la tesis es Inmaculada Farrán Blanch, La brucelosis es una enfermedad zoonótica causada por bacterias del género Brucella que, además, conlleva grandes pérdidas económicas. La principal fuente de infección para el ser humano son los rumiantes, en los que la enfermedad cursa con aborto y otras alteraciones reproductivas. En la mayoría de las regiones afectadas, la vacunación de los animales es la única estrategia realista para controlar la infección. La cepa atenuada B. melitensis Rev1 es la única vacuna recomendada frente a la brucelosis ovina y caprina, pero posee ciertos inconvenientes, entre los que destacan la virulencia en ovejas gestantes, la interferencia serológica que generan, la posibilidad de infectar al ser humano y la resistencia a los tratamientos con estreptomicina, que instan la búsqueda de nuevas vacunas. El lipopolisacárido liso (S-LPS) de Brucella es un conocido factor de virulencia y también el antígeno inmunodominante en las pruebas de diagnóstico, por lo que sus distintas modificaciones representan una interesante aproximación en la búsqueda de una vacuna segura. Entre las rutas de biosíntesis del S-LPS, el sistema de dos componentes Wzm/Wzt es el responsable de transportar el O-PS sintetizado en la cara citoplasmática de la membrana interna hasta el espacio periplasmático, para su posterior ensamblaje al core-lípido A para formar el S-LPS. El bloqueo de esta ruta conlleva la creación de mutantes con LPS rugoso (R-LPS) capaces de acumular un O-PS dentro de las bacterias que puede ser decisivo para generar una respuesta inmune efectiva frente a la infección. Sin embargo, aún se desconocen las implicaciones biológicas de cada proteína del transportador. En particular, el bloqueo del O-PS podría acarrear modificaciones en la envuelta bacteriana, ya que su transporte requiere la participación del bactoprenol como molécula portadora, el cual también está involucrado en la biosíntesis de otras estructuras celulares. Con el objetivo de profundizar en el conocimiento de este sistema transportador de Brucella y su potencial aplicación en el desarrollo de vacunas, en el capítulo 1 de esta tesis se presenta la construcción y caracterización de mutantes Δwzm/Δwzt simples y doble derivados de Rev1. Además, con fines comparativos, se construyeron mutantes ∆wzm derivados de B. abortus 2308 y S19, y se utilizó un mutante 16M∆wzm derivado de la cepa virulenta B. melitensis 16M previamente desarrollado en el grupo de Sanidad Animal del Instituto de Agrobiotecnología (IdAB). Como resultado, los mutantes carentes de O-PS externo exhibieron cambios de expresión génica, propiedades antigénicas del O-PS interno y cambios fenotípicos asociados a modificaciones de la membrana externa y/o de la pared celular. Además, los mutantes Rev1 ∆wzm/∆wzt fueron muy susceptibles
tanto a polimixinas como a los antibióticos utilizados para el tratamiento de la brucelosis humana, en particular, a la estreptomicina; y mostraron una gran atenuación en ratón. Entre ellos, Rev1∆wzm destacó por inducir un pico de esplenomegalia transitoria, ser altamente inmunogénico y generar una protección eficaz frente a la infección virulenta por B. melitensis y por B. ovis en el modelo murino. En el capítulo 2, antes de estudiar la seguridad de Rev1∆wzm en su hospedador natural, se determinó la capacidad de este candidato vacunal para crecer en los medios de cultivo selectivos Farrell (FM) y CITA (CM), actualmente recomendados para el aislamiento primario de Brucella. La fuerte susceptibilidad del mutante a las polimixinas y la sinergia observada entre colistina y vancomicina conllevaron una severa inhibición de este candidato vacunal en ambos medios selectivos. Para resolverlo, se reformuló y evaluó el denominado Brucella Selective Medium (BSM) que, tras su evaluación con más de 1.700 muestras ovinas de campo, demostró ser capaz de mejorar el rendimiento de los medios anteriores. Además, profundizando en la productividad de BSM para otras brucellae, se observó que tanto BSM como CM inhibían el crecimiento de una colección de cepas B. abortus bv1. El análisis de las posibles causas llevó al desarrollo un segundo medio selectivo denominado Brucella Selective Improved Culture (BruSIC) que resultó permisivo para todas las cepas de Brucella analizadas y tan inhibidor como CM para las bacterias contaminantes habituales en muestras veterinarias. BruSIC ofrece la ventaja adicional de no precisar suero bovino para el aislamiento de cepas de Brucella suero-dependientes, lo que simplifica la preparación del medio, abarata costes y evita el uso de compuestos de origen animal. En el capítulo 3, se analizó la seguridad y respuesta inmune inducidas en corderos inoculados con Rev1∆wzm, demostrando que se activa la formación de anticuerpos sin interferir en las pruebas serológicas estándar para el diagnóstico de la brucelosis. Estos resultados, más otros logrados por el grupo de investigación en ganado adulto y en ovejas gestantes, hacen de este mutante un prometedor candidato vacunal para el control de la infección por B. melitensis y B. ovis en pequeños rumiantes. El contenido de estos tres capítulos de tesis ha sido publicado en las revistas científicas Frontiers in Microbiology (grupo Frontiers) y Microbiology Spectrum (ASM)., Brucellosis is a zoonotic disease caused by bacteria of the genus Brucella, that also entails large economic losses. The main infection source for humans are ruminants, in which the disease induces abortion and other reproductive disorders. In most affected regions, vaccinating animals is the only realistic strategy to control the infection. The attenuated strain B. melitensis Rev1 is the only recommended vaccine against ovine and caprine brucellosis, but it has several drawbacks, such as virulence when administered in pregnant animals, serological interference, possibility of infecting humans, and resistance to treatments including streptomycin, that urge the search for new vaccines. The smooth lipopolysaccharide (S-LPS) is a well-known virulence factor and the immunodominant antigen in diagnostic tests, thus, its modifications represent an interesting approach in the research for a safe vaccine. Among S-LPS biosynthesis pathways, the Wzm/Wzt two-component system is the responsible of transporting the O-PS synthesised onto the cytoplasmic side of the inner membrane to the periplasm, for its subsequent assembly to the core-lipid A to form the S-LPS. Blocking this pathway leads to the generation of rough LPS (R-LPS) mutants capable of accumulating O-PS within the bacteria, that may be decisive for an effective immune response against the infection. However, the biological implications of each transporter protein are still unknown. In particular, the O-PS blockage could lead to modifications in bacterial envelope, since the O-PS transport requires the participation of bactoprenol as a carrier molecule, that is also involved in the biosynthesis of other cellular structures. Aiming to deepen the knowledge about this system and its potential application in vaccines development, in Chapter 1 of this thesis, it is presented the construction and characterisation of single and double Δwzm/Δwzt mutants derived from Rev1. In addition, for comparative purposes, ∆wzm mutants were constructed from B. abortus 2308 and S19 and studied together with a 16M∆wzm mutant, derived from the B. melitensis 16M virulent strain previously developed in the Animal Health Group of the Institute of Agrobiotechnology (IdAB). As a result, the mutants lacking outer O-PS exhibited changes in gene expression, antigenic properties of inner O-PS, and phenotypic changes associated with outer membrane and/or cell wall modifications. In addition, Rev1 ∆wzm/∆wzt mutants were highly susceptible to polymyxins and other antibiotics used in human brucellosis treatment, particularly, to streptomycin; and they showed a large attenuation in mouse. Among them, Rev1∆wzm stood out for inducing a transient peak of splenomegaly, being highly immunogenic, and generating effective protection against B. melitensis and B. ovis virulent infections in murine model. In Chapter 2, prior to studying the safety of Rev1∆wzm in the natural host, it was determined the ability of this vaccine candidate to grow in Farrell (FM) and CITA (CM) selective culture media, currently recommended for Brucella primary isolation. The mutant strong susceptibility to polymyxins and the synergy between colistin and vancomycin observed led to the severe inhibition of the vaccine candidate in both selective media. To solve this, the named Brucella Selective Medium (BSM) was reformulated and evaluated with more than 1,700 ovine field samples, being able to improve the previous media performance. Furthermore, by exploring the productivity of BSM for other brucellae, it was observed that both BSM and CM inhibited the growth of a B. abortus bv1 strains collection. The analysis of the possible causes led to the development of a second selective medium called Brucella Selective Improved Culture (BruSIC), which was permissive for all the Brucella strains analysed, and as inhibitory as CM for the usual contaminant bacteria in veterinary samples. In addition, BruSIC offers the advantage of not requiring bovine serum to isolate serum-dependent Brucella strains, simplifying medium preparation, lowering costs and avoiding the use of compounds from animal sources. In Chapter 3, it was analysed the safety and immune response induced in lambs inoculated with Rev1∆wzm, demonstrating the activation of antibodies development without interfering in the standard serological tests for brucellosis diagnosis. These results, as well as others achieved by the research group in adult cattle and pregnant sheep, make this mutant a promising vaccine candidate to control B. melitensis and B. ovis infections in small ruminants. The content of these three thesis chapters has been published in the scientific journals Frontiers in Microbiology (Frontiers group) and Microbiology Spectrum (ASM)., La realización de la presente tesis doctoral ha sido posible gracias a la obtención de un contrato del Programa de Ayudas para la Formación de Personal Investigador Predoctoral de la UPNA 2018-2022, disfrutada en el Instituto de Agrobiotecnología (IdAB; CSIC-Gobierno de Navarra).Los trabajos realizados han sido financiados por los proyectos de investigación del Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R y RTI2018-098658-B-C21) y del Gobierno de Navarra (PT040-2018 y PT007-2019)., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

The lipopolysaccharide (LPS) O-polysaccharide (O-PS) is the main virulence factor in Brucella. After synthesis in the cytoplasmic membrane, O-PS is exported to the periplasm by the Wzm/Wzt system, where it is assembled into a LPS. This translocation also engages a bactoprenol carrier required for further biosynthesis pathways, such as cell wall biogenesis. Targeting O-PS export by blockage holds great potential for vaccine development, but little is known about the biological implications of each Wzm/Wzt moiety. To improve this knowledge and to elucidate its potential application as a vaccine, we constructed and studied wzm/wzt single- and double-deletion mutants, using the attenuated strain Brucella melitensis Rev1 as the parental strain. This allowed us to describe the composition of Brucella peptidoglycan for the first time. We observed that these mutants lack external O-PS yet trigger changes in genetic transcription and in phenotypic properties associated with the outer membrane and cell wall. The three mutants are highly attenuated; unexpectedly, Rev1Δwzm also excels as an immunogenic and effective vaccine against B. melitensis and Brucella ovis in mice, revealing that low persistence is not at odds with efficacy. Rev1Δwzm is attenuated in BeWo trophoblasts, does not infect mouse placentas, and is safe in pregnant ewes. Overall, these attributes and the minimal serological interference induced in sheep make Rev1Δwzm a highly promising vaccine candidate., This work was funded by Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21), Gobierno de Navarra (PT040-2018 and PT007-2019) projects. SM-B contracts were granted by the FEDER 2016–2018 program of Garantía Juvenil and by an UPNA pre-doctoral fellowship 2018–2022. IP-U Doctorados Industriales contract was cofounded by Gobierno de Navarra and CSIC. Research in the FC lab was supported by The Swedish Research Council (VR), The Knut and Alice Wallenberg Foundation (KAW), The Laboratory of Molecular Infection Medicine Sweden (MIMS), and The Kempe Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Abortion and reproductive failures induced by Brucella are the main symptoms of animal brucellosis. Laboratory animal models are essential tools of research to study the Brucella pathogenesis before experimentation in natural hosts. To extend the existing knowledge, we studied B. melitensis 16M (virulent) and Rev1 (attenuated) as well as B. suis bv2 infections in pregnant mice. Here, we report new information about kinetics of infection (in spleens, blood, placentas, vaginal shedding, and foetuses), serum cytokine profiles, and histopathological features in placentas and the litter throughout mice pregnancy. Both B. melitensis strains showed a marked placental tropism and reduced viability of pups (mainly in 16M infections), which was preceded by an intense Th1-immune response during placental development. In contrast, B. suis bv2 displayed lower placental tropism, mild proinflammatory immune response, and scarce bacterial transmission to the litter, thus allowing foetal viability. Overall, our studies revealed three different smooth Brucella patterns of placental and foetal pathogenesis in mice, providing a useful animal model for experimental brucellosis., This research was funded by Agencia Española de Investigación of theMinisterio de Ciencia,
Innovación y Universidades de España, through the R&D project reference RTI2018-098658-B-C21.

Brucellosis, a re-emerging zoonotic infection, threatens animal welfare and public health with serious economic consequences. A definitive diagnosis requires Brucella isolation by culturing field specimens in specific media. This study aimed to (i) assess the effectivity of recommended Farrell’s médium (FM) and CITA medium (CM) for the isolation of four Brucella melitensis strains (16M, Rev1, and the 16MDwzm and Rev1Dwzm in-frame deletion mutants) with variable susceptibility to polymyxins; (ii) develop a Brucella selective medium (BSM) suitable for these strains; (iii) test BSM, FM, and CM with other Brucella species; and (iv) develop an improved selective culture medium (BruSIC) for all brucellae, including B. abortus bv1. The four B. melitensis strains were strongly inhibited in FM and (except Rev1) CM. Since Rev1Dwzm’s CM inhibition was due to a synergistic effect of colistin and vancomycin, we formulated BSM with half the concentrations of both antibiotics, achieving a similar growth of B. melitensis to blood agar base (BAB) and an inhibition of contaminant microorganisms comparable to CM; CM performance was surpassed by BSM for the primary isolation of B. melitensis when tested in 1,789 real sheep samples. For other brucellae, BSM and CM were more inhibitory than FM for B. abortus bv1 when using plates immediately after preparation but not after $4 weeks of storage. To address this, we developed the improved solid medium BruSIC by replacing the calf serum in BSM with activated charcoal. BruSIC yielded faster colony growth than BSM and CM and similar CFU numbers than BAB (including for B. ovis in BAB-Serum) and inhibited accompanying microorganisms in sheep and cow samples as effectively as BSM., This study was funded by the Department of Industry of the Gobierno de Navarra (PT068-2018 and PT007-2019) and Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21). The contract of S.M.-B. was granted by a UPNA 2018-2022 predoctoral fellowship, and that of I.P.-U. was granted by the Doctorados Industriales program of the Gobierno de Navarra cofunded by CSIC. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We thank the staff of Laboratorio Central de Sanidad Animal (LCSA; Granada, Spain) for supplying some of the strains, and the staff of VISAVET (UCM, Madrid, Spain) and INTIA (Navarra Government, Spain) for their veterinary assistance and/or support in obtaining animal samples.

Trabajo presentado en la 2nd FEMS Conference on Microbiology, celebrada en Belgrado (Serbia), del 30 de junio al 2 de julio de 2022, Animal brucellosis is characterized by abortion and bacterial shedding in
livestock. Brucella isolation is the only denite conrmation of infections. For
this, the use of adequate selective culture media able to inhibit the growth
of overcoming contaminants is an essential tool. Currently, simultaneous
culture in Farrell’s (FM) and CITA (CM) media is the recommended method.
However, attenuated mutants susceptible to polymyxins can be inhibited in
these media, providing misled safety results in the natural host., This work was funded by Agencia Estatal de Investigación of the Ministerio de Ciencia of Spain (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21) and Gobierno de Navarra (PT040-2018 and PT007-2019) projects. S.M-B. contract was granted
by an UPNA predoctoral fellowship 2018-2022. I.P-U. “Doctorados Industriales” contract was cofounded by Gobierno de Navarra and CSIC. The funders had no role in study design, data collection and analysis or decision to publish.

Tesis doctoral presentada para lograr el título de Doctor por la Universidad Pública de Navarra, Departamento de Agronomía, Biotecnología y Alimentación, Programa de Doctorado en Biotecnología, [ES] La brucelosis es una zoonosis mundialmente extendida con importantes repercusiones económicas y sanitarias tanto para la ganadería como para el ser humano. Existen doce especies distintas de Brucella con distinta preferencia de hospedador; entre ellas, B. melitensis (bovino), B. abortus (pequeños rumiantes) y B. suis (porcino) son las más frecuentes en el ganado doméstico, y a excepción de B. suis bv2, suponen un grave riesgo para el ser humano. La vacunación de los animales es una herramienta esencial para reducir la infección en los mismos y, así, evitar la transmisión al ser humano. La infección en pequeños rumiantes es ocasionada por las especies Brucella melitensis y B. ovis; la primera de ellas produce abortos a final de gestación en hembras gestantes y la segunda, orquitis y epididimitis en machos con la consecuente reducción de la fertilidad. La cepa atenuada B. melitensisRev1 es la única vacuna recomendada desde hace más de 60 años contra la brucelosis en pequeños rumiantes. Pese a su extendido empleo y demostrada eficacia, Rev1 posee ciertos inconvenientes que recomiendan la búsqueda de una vacuna alternativa más segura que, idealmente, pueda aplicarse en animales gestantes. En este contexto, en el grupo de Brucelosis del IdAB-CSIC, se han desarrollado dos candidatos de Brucella melitensis mediante deleción en fase del gen wzm (16MΔwzmy Rev1Δwzm) involucrado en el sistema de dos componentes Wzm/Wztr es responsable de la exportación del polisacárido O al periplasma bacteriano para completar la biosíntesis del LPS liso., [EN] Brucellosis is a worldwide zoonosis with important economic and health implications for both livestock and humans. There are twelve different Brucella species with different host preference; among them, B. melitensis(ovine), B. abortus(bovine) and B. suis(porcine) are the most frequent in domestic livestock,supposinga serious risk to humans. Vaccination of animals is an essential tool to reduce infection and thus, toprevent transmission to humans. Infection in small ruminants is caused by Brucella melitensisand B. ovisspecies; the former causes abortions at the end of gestation in pregnant females and the latter, orchitis and epididymitis in males with the consequent reduction of fertility. The attenuated strain B. melitensisRev1 is the only vaccine recommended for more than 60 years tocontrol of brucellosis in small ruminants. Despite its widespread use and proven efficacy, Rev1 has certain drawbacks that recommend the search for a safer alternative vaccine that, ideally, can be applied in pregnant animals. In this context, in the Brucellosis group of the IdAB-CSIC, two Brucella melitensiscandidates have been developed by phase-deletion of the wzmgene (16MΔwzmand Rev1Δwzm) involved in the two-component Wzm/Wzt system responsible for the export of the O polysaccharide to the bacterial periplasm forbiosynthesis of the smooth LPS., Contrato de formación de Doctorados Industriales de Gobierno de Navarra (convocatoria 2016), cofinanciado por CSIC y desarrollado en el IdAB. Los trabajos realizados han sido financiados por los proyectos de investigación RTI2018-098658-B-C21 del Ministerio de Ciencia e Innovación (Agencia Estatal de Investigación) y PT068-2018 y PT007 2019 del Departamento de Universidades, Innovación y Transformación Digital de Gobierno de Navarra.

Brucellosis, a worldwide zoonotic disease, is endemic in many developing countries. Besides causing significant economic losses for the livestock industry, it has severe consequences for human health. In endemic regions, small ruminants infected by Brucella melitensis are the main source of human brucellosis. Rev1, the only vaccine currently recommended to control the disease in sheep and goats, has several drawbacks. Rough lipopolysaccharide (R-LPS) mutants have been tested as alternatives, but most lack efficacy. Those in the Wzm/Wzt system responsible for O-polysaccharide export to the periplasm have been proposed as promising vaccine candidates, although to date they have been scarcely investigated in the natural host. In the present work, we studied the biological properties of a 16MΔwzm in-frame deletion mutant, including its safety in pregnant mice and sheep. In mice, 16MΔwzm prevented placental and fetal infections before parturition and protected against B. melitensis and Brucella ovis infections. In sheep, 16MΔwzm was equally safe in lambs, rams, and non-pregnant ewes, inducing some transient Rose Bengal reactions (<7 weeks). The serological reactions occurred earlier and more strongly in pregnant than in non-pregnant ewes and were significantly reduced when conjunctival rather than subcutaneous vaccination was used. In ewes vaccinated at mid-pregnancy, 16MΔwzm was not shed in vaginal discharges during the pregnancy and did not induce abortions/stillbirths. However, some ewes showed a transitory reactivation of infection in placentas and/or milk at parturition, accompanied by a seroconversion in smooth LPS (S-LPS) and/or R-LPS tests. Overall, 16MΔwzm can be considered as a safe vaccine for lambs, rams, and non-pregnant ewes, but its use at mid-pregnancy should be avoided to prevent vaccine dissemination at parturition. If the efficacy results against B. melitensis and B. ovis observed in mice are confirmed by further studies in the natural host, 16MΔwzm could constitute a useful vaccine., This work was funded by the Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades of Spain (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21) and Dirección General de Industria, Energía Proyectos Estratégicos S3 of Gobierno de Navarra, Spain (projects PT068-2018 and PT007-2019). IPU was contracted in the context of the Doctorados Industriales program of Gobierno de Navarra, co-funded by CSIC (2017-2020). SMB contracts were funded by the “Garantía Juvenil” program of CSIC-FEDER 2016-2018 and a predoctoral (2018-2019) fellowship of the Public University of Navarra (UPNA, Spain). Also, we thank the scientific advice of Professor Jean-Jacques Letesson; and the technical support of Sagrario Pérez, Elena San Miguel and Manuel Barrón (LCA-Navarra, Spain) as well as the staff of the subcontracted companies Maeva Servet-Visavet and Animalia (Spain).

Trabajo presentado para lograr el título de Doctor por la Universidad Pública de Navarra, Departamento de Agronomía, Biotecnología y Alimentación, Programa de Doctorado en Biotecnología.--Calificación: Sobresaliente cum laude, La brucelosis es una enfermedad zoonótica causada por bacterias del género Brucella que, además, conlleva grandes pérdidas económicas. La principal fuente de infección para el ser humano son los rumiantes, en los que la enfermedad cursa con aborto y otras alteraciones reproductivas. En la mayoría de las regiones afectadas, la vacunación de los animales es la única estrategia realista para controlar la infección. La cepa atenuada B. melitensis Rev1 es la única vacuna recomendada frente a la brucelosis ovina y caprina, pero posee ciertos inconvenientes, entre los que destacan la virulencia en ovejas gestantes, la interferencia serológica que generan, la posibilidad de infectar al ser humano y la resistencia a los tratamientos con estreptomicina, que instan la búsqueda de nuevas vacunas., El lipopolisacárido liso (S-LPS) de Brucella es un conocido factor de virulencia y también el antígeno inmunodominante en las pruebas de diagnóstico, por lo que sus distintas modificaciones representan una interesante aproximación en la búsqueda de una vacuna segura. Entre las rutas de biosíntesis del S-LPS, el sistema de dos componentes Wzm/Wzt es el responsable de transportar el O-PS sintetizado en la cara citoplasmática de la membrana interna hasta el espacio periplasmático, para su posterior ensamblaje al core-lípido A para formar el S-LPS. El bloqueo de esta ruta conlleva la creación de mutantes con LPS rugoso (R-LPS) capaces de acumular un O-PS dentro de las bacterias que puede ser decisivo para generar una respuesta inmune efectiva frente a la infección. Sin embargo, aún se desconocen las implicaciones biológicas de cada proteína del transportador. En particular, el bloqueo del O-PS podría acarrear modificaciones en la envuelta bacteriana, ya que su transporte requiere la participación del bactoprenol como molécula portadora, el cual también está involucrado en la biosíntesis de otras estructuras celulares. Con el objetivo de profundizar en el conocimiento de este sistema transportador de Brucella y su potencial aplicación en el desarrollo de vacunas, en el capítulo 1 de esta tesis se presenta la construcción y caracterización de mutantes Δwzm/Δwzt simples y doble derivados de Rev1. Además, con fines comparativos, se construyeron mutantes ∆wzm derivados de B. abortus 2308 y S19, y se utilizó un mutante 16M∆wzm derivado de la cepa virulenta B. melitensis 16M previamente desarrollado en el grupo de Sanidad Animal del Instituto de Agrobiotecnología (IdAB). Como resultado, los mutantes carentes de O-PS externo exhibieron cambios de expresión génica, propiedades antigénicas del O-PS interno y cambios fenotípicos asociados a modificaciones de la membrana externa y/o de la pared celular. Además, los mutantes Rev1 ∆wzm/∆wzt fueron muy susceptibles tanto a polimixinas como a los antibióticos utilizados para el tratamiento de la brucelosis humana, en particular, a la estreptomicina; y mostraron una gran atenuación en ratón. Entre ellos, Rev1∆wzm destacó por inducir un pico de esplenomegalia transitoria, ser altamente inmunogénico y generar una protección eficaz frente a la infección virulenta por B. melitensis y por B. ovis en el modelo murino. En el capítulo 2, antes de estudiar la seguridad de Rev1∆wzm en su hospedador natural, se determinó la capacidad de este candidato vacunal para crecer en los medios de cultivo selectivos Farrell (FM) y CITA (CM), actualmente recomendados para el aislamiento primario de Brucella. La fuerte susceptibilidad del mutante a las polimixinas y la sinergia observada entre colistina y vancomicina conllevaron una severa inhibición de este candidato vacunal en ambos medios selectivos. Para resolverlo, se reformuló y evaluó el denominado Brucella Selective Medium (BSM) que, tras su evaluación con más de 1.700 muestras ovinas de campo, demostró ser capaz de mejorar el rendimiento de los medios anteriores. Además, profundizando en la productividad de BSM para otras brucellae, se observó que tanto BSM como CM inhibían el crecimiento de una colección de cepas B. abortus bv1. El análisis de las posibles causas llevó al desarrollo un segundo medio selectivo denominado Brucella Selective Improved Culture (BruSIC) que resultó permisivo para todas las cepas de Brucella analizadas y tan inhibidor como CM para las bacterias contaminantes habituales en muestras veterinarias. BruSIC ofrece la ventaja adicional de no precisar suero bovino para el aislamiento de cepas de Brucella suero-dependientes, lo que simplifica la preparación del medio, abarata costes y evita el uso de compuestos de origen animal. En el capítulo 3, se analizó la seguridad y respuesta inmune inducidas en corderos inoculados con Rev1∆wzm, demostrando que se activa la formación de anticuerpos sin interferir en las pruebas serológicas estándar para el diagnóstico de la brucelosis. Estos resultados, más otros logrados por el grupo de investigación en ganado adulto y en ovejas gestantes, hacen de este mutante un prometedor candidato vacunal para el control de la infección por B. melitensis y B. ovis en pequeños rumiantes. El contenido de estos tres capítulos de tesis ha sido publicado en las revistas científicas Frontiers in Microbiology (grupo Frontiers) y Microbiology Spectrum (ASM)., La realización de la presente tesis doctoral ha sido posible gracias a la obtención de un contrato del Programa de Ayudas para la Formación de Personal Investigador Predoctoral de la UPNA 2018-2022, disfrutada en el Instituto de Agrobiotecnología (IdAB; CSIC-Gobierno de Navarra).Los trabajos realizados han sido financiados por los proyectos de investigación del Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R y RTI2018-098658-B-C21) y del Gobierno de Navarra (PT040-2018 y PT007-2019).

One of the main causes of human brucellosis is Brucella melitensis infecting small ruminants. To date, Rev1 is the only vaccine successfully used to control ovine and caprine brucellosis. However, it is pathogenic for pregnant animals, resulting in abortions and vaginal and milk shedding, as well as being infectious for humans. Therefore, there is an urgent need to develop an effective vaccine that is safer than Rev1. In efforts to further attenuate Rev1, we recently used wzm inactivation to generate a rough mutant (Rev1Δwzm) that retains a complete antigenic O-polysaccharide in the bacterial cytoplasm. The aim of the present study was to evaluate the placental pathogenicity of Rev1Δwzm in trophoblastic cells, throughout pregnancy in mice, and in ewes inoculated in different trimesters of pregnancy. This mutant was evaluated in comparison with the homologous 16M∆wzm derived from a virulent strain of B. melitensis and the naturally rough sheep pathogen B. ovis. Our results show that both wzm mutants triggered reduced cytotoxic, pro-apoptotic, and pro-inflammatory signaling in Bewo trophoblasts, as well as reduced relative expression of apoptosis genes. In mice, both wzm mutants produced infection but were rapidly cleared from the placenta, in which only Rev1Δwzm induced a low relative expression of pro-apoptotic and pro-inflammatory genes. In the 66 inoculated ewes, Rev1Δwzm was safe and immunogenic, displaying a transient serological interference in standard RBT but not CFT S-LPS tests; this serological response was minimized by conjunctival administration. In conclusion, these results support that B. melitensis Rev1Δwzm is a promising vaccine candidate for use in pregnant ewes and its efficacy against B. melitensis and B. ovis infections in sheep warrants further study., This work was funded by Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades (RTI2018-098658-B-C21 and PID2022-139200OB-C21) and Gobierno de Navarra (PT040-2018 and PT007-2019) research projects. IPU’s contract was granted by the Doctorados Industriales program of the Navarra government (2017-2019), cofounded by CSIC. SMB’s contracts were granted by the Garantía Juvenil CSIC-FEDER program (2016-2018) and by an UPNA pre-doctoral fellowship (2018–2022).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Peer reviewed

In press, Small ruminants affected by brucellosis, caused mainly by Brucella melitensis and B. ovis, suffer reproductive disorders, leading to significant economic losses worldwide. Vaccination is an essential tool to prevent the disease in ovine and caprine livestock, but the only vaccine recommended to date is B. melitensis Rev1, which in sheep is only safe for use in lambs aged 3–4 months. This restriction poses considerable practical challenges for the implementation of Rev1 in countries with endemic brucellosis and/or limited resources, where there is a need for mass vaccination with a safe vaccine to control the disease in both animals and humans. We recently developed a B. melitensis strain Rev1Δwzm showing superior vaccine properties in mice and safety in pregnant ewes. Here, we report that Rev1Δwzm (i) is safe in young and adult sheep, both male and female; (ii) induces a transient serological response in the Rose Bengal test in ≤50 % of sheep, confirmed to some extent by the complement fixation test, and a stronger, more persistent anti- rough-LPS response; and (iii) protects rams against a B. ovis challenge 25 weeks after vaccination. To resolve the problem of serological interference, the use of green fluorescent protein tagging strategy allowed us to identify vaccinated sheep with only a single inoculation. These results, together with the previously reported safety in pregnant ewes, position Rev1Δwzm as a firm vaccine candidate and a promising alternative to Rev1. Further experiments are warranted to assess its efficacy against B. melitensis in pregnant ewes., This work was funded by Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades (RTI2018-098658-B-C21 and PID2022-139200OB-C21) and Gobierno de Navarra (PT040-2018 and PT007-2019) projects. SMB contract was granted by an UPNA pre-doctoral fellowship 2018–2022., Peer reviewed