Identification of surface-exposed proteins and surface-derived structures from Streptococcus pneumoniae for vaccine and diagnostic tools purposes

Streptococcus pneumoniae (neumococo) es un patógeno humano que puede colonizar el tracto respiratorio superior o puede causar infecciones invasivas como neumonía, meningitis y empiema, y no invasivas como otitis media, sinusitis y bronquitis, principalmente en niños, ancianos e inmunodeprimidos. Es la principal causa de muertes en niños menores de 5 años a nivel mundial, sobre todo en los países en vías de desarrollo. Existen más de 90 serotipos diferentes de acuerdo a la composición de la cápsula polisacarídica y, aunque existen vacunas comerciales basadas en la cápsula, su eficacia es limitada y son caras. Así, tanto por la reciente aparición de cepas multirresistentes a antibióticos como por fenómenos derivados de la introducción de las vacunas polisacarídicas gracias a la gran capacidad de adaptación del neumococo es de urgente necesidad desarrollar nuevas, más eficientes y más baratas vacunas serotipo-independientes. Esta nueva vacuna debe ser de base proteica para reducir el coste de producción, y los candidatos, estar expuestos en superficie, antigénicamente conservados, presentes en los serotipos más prevalentes, altamente expresados, su función debe ser esencial en patogenicidad y ser capaces de estimular repuesta celular. Con el fin de descubrir nuevos candidatos proteicos, tanto con fines vacunales como para el desarrollo de herramientas de diagnóstico rápido, durante la presente tesis se ha desarrollado dos líneas de trabajo principalmente: La digestión superficial de células vivas con proteasas (1) y la producción y caracterización de vesículas extracelulares. 2. Contenido de la investigación En primer lugar se llevó a cabo la optimización de la técnica del “pelado” de células vivas de neumococo con proteasas (2), tras lo cual se aplicó a una batería de aislados clínicos de adultos (3) y de niños con el fin de describir los “pan-surfomas” (conjunto de proteínas de superficie presentes en una batería de distintas estirpes), confirmando a esta técnica como una rápida y potente aproximación para la identificación de proteínas de superficie (4, 5). Por otro lado, la reciente descripción de la producción de vesículas extracelulares en organismos Gram-positivos, nos llevó a caracterizar las vesículas de neumococo debido a que dichas estructuras están enriquecidas, presuntamente, en proteínas de membrana. Así, 40 aislados clínicos (correspondientes a 29 diferentes ST) sin relaciones evolutivas, además de las estirpes de referencia R6 y TIGR4, se analizaron mediante la aproximación de la digestión superficial de células vivas. Además, otros 4 serotipos, relacionados con diferentes capacidades de invasión (serotipos 1, 6B, 8 y 23F), se usaron para la caracterización de las vesículas derivadas de la membrana. Todo ello permitió seleccionar 95 proteínas, cuya inmunoreactividad se probó frente a sueros de pacientes enfermos y sanos (algunas de ellas, mediantes diferentes técnicas). Esto nos permitió seleccionar un conjunto de pocas proteínas (Spr0561, Spr0247, Spr1431, Spr1754 and Sph_0062) para desarrollar un test de enfermedad neumocócica rápido, fiable, barato y fácil de usar en niños de 0->5 años, que resolvería los problemas de los métodos de diagnóstico usados hasta el momento. Por otro lado, se llevaron ensayos de supervivencia usando una estirpe virulenta del serotipo 8 como agente de infección, lo cual nos permitió describir la capacidad protectora de 7 nuevas proteínas. Además, se demostró que las vesículas de membrana producidas por este mismo serotipo conferían un alto grado de protección a los ratones inmunizados...Streptococcus pneumoniae (pneumococcus) is a human pathogen that can colonize the upper respiratory tract and can cause invasive infections such as pneumonia, meningitis and empyema, and noninvasive as otitis media, sinusitis and bronchitis, especially in children, the elderly and immunocompromised. It is the leading cause of death in children under 5 years worldwide, especially in developing countries. There are more than 90 different serotypes described according to the composition of the polysaccharide capsule and, although there are commercial capsule-based vaccines, their effectiveness is limited and are expensive. Thus, both the recent emergence of multidrugresistant strains to antibiotics such as phenomena arising from the introduction of the polysaccharide vaccines, thanks to the adaptability of the pneumococcus, is an urgent need to develop new, more efficient and cheaper vaccine serotype-independent. This new vaccine should be protein based to reduce the cost of production, and candidates, surface-exposed, antigenically conserved, presents in the most prevalent serotypes, highly expressed, its function must be essential for pathogenicity and capable of stimulating cellular response. In order to discover new protein candidates, both for vaccination and for the development of rapid diagnostic tools purposes, during this thesis two lines of work were developed: The surface digestion with proteases of living (“shaving”) (1) and production and characterization of extracellular vesicles.Research content First, optimization of "shaving" of live pneumococcal cells with proteases (2) was carried out and then it was applied to a set of clinical isolates from adult (3) and children in order to describe the "pan-surfome" (set of surface proteins present in a collection of different strains). This confirmed this technique as a rapid and powerful approach to surface protein identification (4, 5). Furthermore, the recent description of the production of extracellular vesicles in a Gram-positive bacteria, prompted us to characterize pneumococcus vesicles because such structures are enriched, presumably, in membrane proteins. So, 40 clinical isolates (corresponding to 29 different ST) without evolutionary relationships, as well as reference strains R6 and TIGR4, were analyzed by the “shaving” approach. In addition, other 4 serotypes, with different capabilities of invasion (serotypes 1, 6B, 8 and 23F) were used for the characterization of the extracelluar vesicles (5, 6). All of this allowed to select 95 proteins and tested their immunoreactivity against sera of convalescent and healthy patients (some of them

Diseño e implementación de sistema fotovoltaico de bajo costo para alimentar un circuito cerrado de televisión

Práctica SocialEl presente trabajo analiza la situación que se da dentro de una institución “Rompiendo Cadenas” que ayuda a las personas con problemas de drogas que quieren rehabilitarse y poder volver a formar parte de la sociedad, es ahí donde la Universidad Católica de Colombia busca brindar a través de estas tecnologías (Circuito cerrado de televisión), realizar la supervisión de las personas internas en las instalaciones de la fundación por medio de monitoreo constante y poder de esta manera detectar situaciones o procedimientos en los cuales se observe la necesidad de reforzar, mejorar o modificar alguna de estas por parte del personal que trabaja en las instalaciones de la institución.INTRODUCCIÓN 1. GENERALIDADES 2. PROCEDIMIENTOS REALIZADOS 3. PLANO DE DISTRIBUCIÓN DE CÁMARAS 4. DESCRIPCIÓN ECONÓMICA DEL PROYECTO 5. CONCLUSIONES BIBLIOGRAFÍA ANEXOSPregradoIngeniero Electrónic

Métodos de enriquecimiento de glicoproteínas bacterianas para su posterior análisis y caracterización mediante espectrometría de masas

Comunicaciones a congreso

Approaching In Vivo Models of Pneumococcus–Host Interaction: Insights into Surface Proteins, Capsule Production, and Extracellular Vesicles

Infections caused by the Gram-positive bacterium Streptococcus pneumoniae have become a major health problem worldwide because of their high morbidity and mortality rates, especially in developing countries. This microorganism colonizes the human upper respiratory tract and becomes pathogenic under certain circumstances, which are not well known. In the interaction with the host, bacterial surface structures and proteins play major roles. To gain knowledge into gradual changes and adaptive mechanisms that this pathogen undergoes from when it enters the host, we mimicked several in vivo situations representing interaction with epithelial and macrophage cells, as well as a condition of presence in blood. Then, we analyzed, in four pneumococcal strains, two major surface structures, the capsule and extracellular vesicles produced by the pneumococci, as well as surface proteins by proteomics, using the “shaving” approach, followed by LC-MS/MS. We found important differences in both surface ultrastructures and proteins among the culture conditions and strains used. Thus, this work provides insights into physiological adaptations of the pneumococcus when it interacts with the host, which may be useful for the design of strategies to combat infections caused by this pathogen

Bioinformaticsas educational resource:Genetic engineering project

La utilización de medios informáticos para procesar los contenidos curriculares de las diferentes materias, así como para aprender su propio manejo, ha crecido exponencialmente en los últimos años. El uso de las Tecnologías de la Información y la Comunicación (TIC) pueden contribuir al acceso universal a la educación mediante microproyectos de bajo coste. Sin embargo, para ello hay que diseñar y desarrollar proyectos educativos coherentes con las necesidades educativas. Con este proyecto, programas bioinformáticos básicos necesarios para el desarrollo de un proyecto de ingeniería genética se transformarán en recursos educativos con los que el alumnado no solo adquirirá nociones básicas sobre el uso y manejo de estos programas -que les serán requeridos en su vida profesional como científicos-, sino que les servirá para afianzar y contextualizar conceptos relacionados con el propio proceso. Partiendo del objetivo de la mutación de un gen concreto, el alumnado será capaz de desarrollar una estrategia que le reporte una gran cantidad de información sobre éste usando únicamente medios informáticos.The use computational toolsto process curricular contents of the different subjects, as well as to learn their own management, has grown exponentially in recent years. The use of Information and Communication Technologies (ICT) can contribute to universal access to education, equality in education, the exercise of quality teaching andto thelearning and professional development of teachers, as well as to a moreefficient management and administration of the education system. However, it is necessary to design and develop educational projects that are consistent with educational needs. With thisproject, basedon the use of basic bioinformatics programs within the framework of a genetic engineering project, students will not only acquire basic notions about the use and management of these programs, which will be required in their professional life as scientists, but also it will serve to strengthen and contextualize concepts related to the process itself. Based on the objective of mutation of a particular gene, students will be able to develop a strategy that will report a large amount of information about it using only computational tools

Optimization of digestion of living cells of streptococcus pneumoniae for searching protein vaccine candidates

Comunicaciones a congreso

Identification of Potential New Protein Vaccine Candidates through Pan-Surfomic Analysis of Pneumococcal Clinical Isolates from Adults

Purified polysaccharide and conjugate vaccines are widely used for preventing infections in adults and in children against the Gram-positive bacterium Streptococcus pneumoniae, a pathogen responsible for high morbidity and mortality rates, especially in developing countries. However, these polysaccharide-based vaccines have some important limitations, such as being serotype-dependent, being subjected to losing efficacy because of serotype replacement and high manufacturing complexity and cost. It is expected that protein-based vaccines will overcome these issues by conferring a broad coverage independent of serotype and lowering production costs. In this study, we have applied the ‘‘shaving’’ proteomic approach, consisting of the LC/MS/MS analysis of peptides generated by protease treatment of live cells, to a collection of 16 pneumococcal clinical isolates from adults, representing the most prevalent strains circulating in Spain during the last years. The set of unique proteins identified in all the isolates, called ‘‘pan-surfome’’, consisted of 254 proteins, which included most of the protective protein antigens reported so far. In search of new candidates with vaccine potential, we identified 32 that were present in at least 50% of the clinical isolates analyzed. We selected four of them (Spr0012, Spr0328, Spr0561 and SP670_2141), whose protection capacity has not yet been tested, for assaying immunogenicity in human sera. All of them induced the production of IgM antibodies in infected patients, thus indicating that they could enter the pipeline for vaccine studies. The pan-surfomic approach shows its utility in the discovery of new proteins that can elicit protection against infectious microorganisms

Exploring anaerobic environments for cyanide and cyano-derivatives microbial degradation

Cyanide is one of the most toxic chemicals for living organisms described so far. Its toxicity is mainly based on the high affinity that cyanide presents toward metals, provoking inhibition of essential metalloenzymes. Cyanide and its cyano-derivatives are produced in a large scale by many industrial activities related to recovering of precious metals in mining and jewelry, coke production, steel hardening, synthesis of organic chemicals, and food processing industries. As consequence, cyanide-containing wastes are accumulated in the environment becoming a risk to human health and ecosystems. Cyanide and related compounds, like nitriles and thiocyanate, are degraded aerobically by numerous bacteria, and therefore, biodegradation has been offered as a clean and cheap strategy to deal with these industrial wastes. Anaerobic biological treatments are often preferred options for wastewater biodegradation. However, at present very little is known about anaerobic degradation of these hazardous compounds. This review is focused on microbial degradation of cyanide and related compounds under anaerobiosis, exploring their potential application in bioremediation of industrial cyanide-containing wastes

Assimilation of cyanide and cyano-derivatives by Pseudomonas pseudoalcaligenes CECT5344: from omic approaches to biotechnological applications

Mining, jewellery and metal-processing industries use cyanide for extracting gold and other valuable metals, generating large amounts of highly toxic wastewater. Biological treatments may be a clean alternative under the environmental point of view to the conventional physical or chemical processes used to remove cyanide and related compounds from these industrial effluents. Pseudomonas pseudoalcaligenes CECT5344 can grow under alkaline conditions using cyanide, cyanate or different nitriles as the sole nitrogen source, and is able to remove up to 12 mM total cyanide from a jewellery industry wastewater that contains cyanide free and complexed to metals. Complete genome sequencing of this bacterium has allowed the application of transcriptomic and proteomic techniques, providing a holistic view of the cyanide biodegradation process. The complex response to cyanide by the cyanotrophic bacterium P. pseudoalcaligenes CECT5344 and the potential biotechnological applications of this model organism in the bioremediation of cyanide-containing industrial residues are reviewed