Immunological heterogeneity is the norm in most free-living vertebrate populations, creating a diverse and challenging landscape for pathogens to replicate and transmit. This dissertation work sought to determine sources of immunological heterogeneity, as well as the consequences of this heterogeneity on pathogen fitness and evolution. A major source of heterogeneity in free-living host populations is the degree of exposure to a pathogen, as well as a host's history of exposure to a pathogen, which can create variation in standing immunity. We sought to determine the role of exposure heterogeneity on host susceptibility and immunity to secondary infection, and the influence of this heterogeneity on pathogen fitness and virulence evolution in a wild bird-bacterium system. We first determined that exposure level has a significant effect on host susceptibility to infection, severity of disease and infection, as well as immunity produced to secondary infection. Subsequently, we tested whether exposure history, and the immunity formed from this previous exposure, altered the within-host fitness advantage to virulent pathogens. We determined that previous low-level repeat exposure, which wild hosts likely encounter while foraging, produces a within-host environment which greatly favors more virulent pathogens. While within-host processes are vital for understanding and interpreting the evolutionary pressures on a pathogen, the ultimate metric of pathogen fitness is transmission. We therefore tested whether exposure history altered the transmission potential of a host and whether prior host exposure selected for more virulent pathogens. The transmission potential of a host significantly decreased with previous exposure, and high levels of previous exposure selected for more virulent pathogens. While we anticipated selection to be strongest at low-levels of exposure based on our previous results, we found here that high doses of prior exposure resulted in the strongest transmission advantage to virulence. This study also provided insight into the nuanced nature of transmission, which our results indicate is determined both by the degree of within-host pathogen replication as well as host disease severity. Together, our findings underscore the importance of exposure level and exposure history in natural populations in determining susceptibility, immunity and pathogen virulence evolution. / Doctor of Philosophy / Infectious diseases disrupt and threaten all life on this planet. To better anticipate and understand why some diseases are more harmful than others, it is vital that we consider the natural variability that exists in animal populations. A major source of variation in populations that experience disease is exposure level to a pathogen, as well as the history of exposure to a pathogen, which can alter an individual’s protection against future exposures. We sought to determine the role of exposure level on the likelihood of an individual contracting an infection, their protection from future infections, and the influence this has on pathogen evolution in a wild bird-bacterium system. We determined that exposure level has a significant effect on the likelihood an individual has of becoming infected, how severe the infection became, as well as how protected they were from future infections. Subsequently, we tested whether exposure history, and the immunity formed from previous exposure, altered the ability of pathogen strains that cause different levels of harm to replicate. We determined that previous low-level exposure, which hosts likely encounter in the wild, creates a level of immunity that favors more harmful strains of the pathogen. While understanding what happens within a host is important, the ultimate metric for predicting whether more or less harmful types of pathogens will persist is the ability of each pathogen type to spread from one host to another. We therefore tested whether exposure history altered the spread potential of a host and whether previous exposure preferentially favored the spread of more harmful pathogens. The spread potential of a host was much lower if that host had previously been exposed to the pathogen, and high levels of previous exposure in hosts only allowed the more harmful pathogen types to spread. We also found that a host’s spread potential was the result of both how much pathogen they had in their body, as well as how inflamed their affected tissues were. Together, our results indicate that natural variation in prior exposure to pathogens, which is common in all animal populations, including humans, can favor more harmful pathogen types.

Los alojamientos ganaderos son una fuente importante de material particulado (“particulate matter”, PM) y bioaerosoles. Estas sustancias tienen un efecto perjudicial tanto para la salud humana y animal como para el medio ambiente. Para reducir los niveles de PM y bioaerosoles en alojamientos ganaderos es necesario conocer el origen de los mismos y los factores que afectan a su generación y suspensión en el aire. Esta Tesis Doctoral aborda aspectos relacionados con la concentración, origen y propiedades físicas, químicas y biológicas del PM en el aire de granjas de conejos y aves, su relación con los bioaerosoles patógenos y técnicas para reducirlos. Este trabajo pretende contribuir a paliar los efectos negativos de estas sustancias tanto en el interior de los alojamientos ganaderos como en el exterior. Los objetivos específicos planteados en la presente Tesis Doctoral fueron: i). caracterizar la morfología y la composición química del PM de distintos tamaños así como la concentración de bacterias en el aire de granjas de conejos, ii). cuantificar la concentración y emisión del PM de distintos tamaños en el aire e identificar las principales actividades que contribuyen a la generación del PM en granjas de conejos, iii). evaluar la distribución espacial de bacterias aerobias mesófilas en el aire durante un ciclo de producción de broilers y examinar su relación con la concentración y evolución del PM, iv). evaluar y comparar diferentes técnicas para muestrear y detectar el patógeno Salmonella spp. en el aire de granjas de broilers y v). evaluar la aplicación de desinfectantes en el aire como medida de reducción de los bioaerosoles en granjas de gallinas ponedoras con especial atención al patógeno Mycoplasma gallisepticum. Los resultados de esta Tesis indican que en los alojamientos avícolas y cunícolas se generan y emiten cantidades importantes de PM y bioaerosoles, por encima de los valores límite de exposición que marca la Directiva 2008/50/CE relativa a la calidad del aire ambiente y a una atmósfera más limpia en Europa, sobre todo en granjas de aves. Estas sustancias deben ser controladas y reducidas para proteger el medio ambiente, la salud y bienestar de las personas y animales. En alojamientos cunícolas, el PM mostró una morfología y composición química compleja, siendo las partículas irregulares y angulosas, ricas en S, Ca, Mg, Na y Cl, las más abundantes. La concentración de bacterias aerobias mesófilas en el aire por metro cúbico varió entre 3,1x103 y 1,6x106 unidades formadoras de colonia (UFC). Las principales fuentes generadoras de PM fueron la piel, el pienso y las heces provenientes de las actividades de limpieza de la nave, sobre todo de barrer y de los propios animales. La concentración media de PM10 (partículas de 10 μm de diámetro o inferior) fue 0,08±0,06 mg/m3 para conejos de cebo y 0,05±0,06 mg/m3 para conejas y la concentración media de PM2,5 (partículas de 2,5 μm de diámetro o inferior) fue 0,01±0,02 mg/m3 para conejos de cebo y 0,01±0,04 mg/m3 para conejas. Las emisiones variaron entre 6 y 15 mg/plaza/día para PM10 y entre 0,2 y 3,0 mg/plaza/día para PM2,5. En alojamientos de broilers, la concentración de bacterias varió entre 3,0 y 6,5 log UFC/m3. La mayoría de bacterias se asociaron con partículas entre 3,3 y más de 7,0 μm de diámetro obteniéndose una correlación positiva entre las concentraciones de PM10 y PM2,5 y las de bacterias. Respecto a la detección de patógenos en el aire, no se detectó Salmonella spp. cultivable en una explotación de broilers infectados experimentalmente mediante el uso de borboteadores de aire y técnicas de cultivo tradicional. No obstante, se detectó este patógeno en el aire mediante impactación y técnicas moleculares. Por lo tanto, no se recomienda el uso de borboteadores y técnicas de cultivo para la detección y/o cuantificación de Salmonella spp. cultivable en el aire. En alojamientos de gallinas, la concentración media de PM10 fue 0,55±0,38 mg/m3 y 0,02±0,03 mg/m3 para PM2,5. La concentración de bacterias varió entre 4,1 y 5,7 log UFC/m3. La aplicación de un desinfectante químico de amplio espectro en el aire no fue efectiva ni para reducir los niveles de bacterias aerobias mesófilas en el aire ni de Mycoplasma spp. Es necesario estudiar diferentes productos, dosis o técnicas de aplicación. En su conjunto, los resultados presentados en esta Tesis Doctoral proporcionan una información útil sobre el PM y los bioaerosoles en el aire de alojamientos ganaderos, que permitirá diseñar e implementar medidas de reducción prácticas y eficaces que mejoren la calidad del aire en los alojamientos ganaderos y reduzcan su emisión al exterior. / Adell Sales, E. (2014). Material particulado y bioaerosoles en el aire de granjas de aves y conejos: cuantificación, caracterización y medidas de reducción [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/44231

Throughout their lives, animals are faced with numerous ecological challenges stemming from abiotic and biotic conditions of their environment. Phenotypic shifts in response to one challenge can have cascading effects on other organismal systems, with downstream implications for individual fitness. Infectious disease presents a significant ecological challenge for most organisms on earth. Additionally, how an animal responds to disease can be shifted by exposure to other ecological challenges. Thus, infectious disease can both present an ecological challenge itself or shift as a consequence of another challenge. In this work, I used experimental captive studies on wild-caught house finches (Haemorhous mexicanus) to elucidate how an animal might shift its phenotypes when presented with an ecological challenge. In the first experiment, I examined how nutritional stress during nestling development impacted the magnitude of house finch responses to the bacterial pathogen Mycoplasma gallisepticum (MG). Although nutritional stress limited mass gain in nestlings, individual responses to MG did not vary with nutritional stress, possibly indicating that the development of immune responses is resilient even in the face of suboptimal nutritional conditions. Next, I investigated infectious disease as a challenge in itself and asked how individual social preferences were shifted by MG infection. I demonstrated that MG-infected house finches showed augmented sociality relative to control birds, choosing to spend more time with a group of conspecifics than alone. Because this increased social preference was no longer present once birds recovered, this phenotypic change in sociality may have specific benefits for actively infected birds. Finally, my last experiment expands upon these results, exploring whether group-living particularly benefits infected birds by offsetting two common fitness costs of infection: reduced foraging abilities and decreased anti-predator responses. Here we found that group-living provides all individuals with improved foraging and anti-predator behaviors, with the strongest benefits of group-living apparent for infected finches. This suggests that augmented sociality in infected house finches has important implications for surviving infection, and potentially, for the spread of MG within populations. As animals continue to face increasing and novel ecological challenges, it is vitally important to understand individual responses to environmental challenges, which can have long-term effects for all levels of biological organization. In particular, my work highlights the role of social behavior as a potentially adaptive phenotypic response to infectious disease in wild animals. Taken together, my results demonstrate the importance of continuing to study infectious disease from multiple perspectives to better understand how animals will respond to a shifting world. / Doctor of Philosophy / All animals must respond to challenges in their environment, which can impact their lives in a variety of ways. Infectious disease is a significant challenge for most organisms on earth. Infection with a disease-causing pathogen must be met by the individual with behavioral, physiological, and immunological responses to increase the animal's likelihood of survival. Additionally, an animal's response to disease can be shifted by exposure to other adverse environmental conditions, such as reduced access to food. On the one hand, infectious disease can present a challenge in itself. Alternatively, how an animal responds to disease may shift as a consequence of another challenge. In this work, I brought wild-caught birds into a captive setting and performed three experiments to determine how an animal might respond to common ecological challenges. First, I studied how food shortages during early life impacted how strongly birds responded to infection with a disease-causing bacteria. In this study I found that host responses to disease did not shift, even when birds were given less food and experienced reduced mass growth during early life. Although young animals are developing rapidly and are particularly vulnerable to challenges in their environment, my results indicate that the development of responses to disease is resilient even in the face of suboptimal conditions. Next, I investigated how social behaviors were shifted due to disease. Here I demonstrated that diseased birds were more social than healthy birds, preferring to spend more time with a group of other birds than alone. In contrast, once these same birds had recovered from infection and were again healthy they became less social, which suggests that diseased birds in particular may benefit from being part of a group. My final experiment expanded upon these results, exploring whether group-living can help increase an individual's survival by compensating for two consequences of disease: reduced ability to acquire food and evade predators. Here I found that group-living provides individual benefits in terms of both acquiring food and evading predators, both of which have important implications for an individual's survival, especially while experiencing disease. As animals continue to face increasing and new challenges due to global change, it becomes vitally important to understand individual responses to environmental changes. While the work highlighted here presents an important step in understanding individual responses, future work should use observational studies in the wild to determine how the social preferences and behaviors I demonstrated here are actually occurring in a natural habitat. Taken together, my results highlight the importance of continuing to study infectious disease from multiple perspectives to better understand how animals will respond to a shifting world.

Made available in DSpace on 2015-03-04T18:51:03Z (GMT). No. of bitstreams: 1 DissertacaoOberdan.pdf: 3322063 bytes, checksum: e9c70e2132e91ffd20f094daea677edd (MD5) Previous issue date: 2008-06-04 / Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior / The advent of genome sequencing technology and complete genome analysis has provided new data on prokaryote and eukaryote metabolic pathways. The comparative analysis of metabolic pathways from different organisms can help us understand inter and intra species organizational relationships. Having this in mind, this work focused on building a system that allows for comparing the bacterial metabolic pathways, according to a set of pre-established criteria. SAMPA (System for comparative Analysis of Metabolic PAthways) comprises a database containing information on metabolic pathways in many organisms, and a set of five tools that can be used to compare these metabolic pathways and to group organisms carrying metabolic pathways that are related. As a case study to validate the tool, we the Mycoplasmataceae family of organisms was used. / Com o advento das tecnologias que propiciaram os seqüenciamentos e as análises de genomas completos em tempo relativamente curto, muitos dados sobre vias metabólicas de procariotos e eucariotos puderam ser gerados. Análises comparativas de vias metabólicas de diferentes genomas podem auxiliar no entendimento das relações organizacionais dentre e fora das espécies. Com base em tais perspectivas, este trabalho tem como finalidade implementar um sistema que permita comparar, através de diferentes critérios, vias metabólicas de bactérias. O sistema SAMPA (System for comparative Analysis of Metabolic PAthways) é composto por um banco de dados, com informações sobre vias metabólicas de diversos organismos, e um conjunto de 5 ferramentas utilizadas para comparar estas vias metabólicas e agrupar os organismos que possuam vias metabólicas relacionadas. Como estudo de caso para teste da ferramenta, foi utilizada a família Mycoplasmataceae.

Mycoplasma hyopneumoniae est l’agent causal de la pneumonie enzootique. On le retrouve dans plusieurs élevages de porcs à travers le monde. Même si ce micro-organisme est présent dans plusieurs troupeaux canadiens, peu d’informations sont présentement disponibles sur les isolats québécois. Un total de 160 poumons de porcs possédant des lésions de pneumonie ont été récupérés à l’abattoir, mis en culture et testés par PCR pour M. hyopneumoniae et Mycoplasma hyorhinis. D’autres pathogènes bactériens communs du porc et les virus du syndrome reproducteur et respiratoire porcin (VSRRP), de l’influenza et le circovirus porcin de type 2 (CVP2) ont été également testés. Quatre-vingt-dix pourcent des échantillons étaient positifs pour M. hyopneumoniae et 5.6% l’étaient seulement pour M. hyorhinis. Dans ces échantillons positifs pour M. hyopneumoniae, la concentration de ce mycoplasme variait de 1.17 x 105 à 3.37 x 109 génomes/mL. Vingt-cinq poumons positifs en culture ou par PCR en temps réel pour M. hyopneumoniae ont été sélectionnés, parmi ceux-ci 10 étaient en coinfection avec Pasteurella multocida, 12 avec Streptococcus suis, 9 avec CVP2 et 2 avec le VSRRP. Les analyses des nombres variables de répétitions en tandem à de multiples loci (MLVA) et PCR-polymorphisme de longueur de fragments de restriction (PCR-RFLP) de M. hyopneumoniae ont démontré une forte diversité des isolats de terrain. Par contre, il semble y avoir plus d’homogénéité à l’intérieur d’un même élevage. L’analyse MLVA a également démontré que près de la moitié des isolats possédaient moins de 55% d’homologie avec les souches vaccinales et de référence utilisées dans la présente étude. L’absence d’amplification du locus 1 de M. hyopneumoniae en MLVA a été significativement associée à une baisse de la concentration de bactérie et de la sévérité des lésions. Pour tous les isolats de M. hyopneumoniae, des concentrations minimales inhibitrices (CMI) de faibles à intermédiaires ont été obtenues envers tous les antimicrobiens testés. Les isolats possédant des CMI intermédiaires envers les tétracyclines, les macrolides et les lincosamides ont été testés pour la présence des gènes de résistance tetM, ermB et pour des mutations ponctuelles dans les gènes des protéines L4, L22 et de l’ARNr 23S. Aucun de ces gènes n’a été détecté mais la mutation ponctuelle G2057A a été identifiée. Cette mutation est responsable de la résistance intrinsèque de M. hyopneumoniae face aux macrolides à 14 carbones. Ces résultats indiquent qu’il ne semble pas y avoir de résistance acquise aux antimicrobiens parmi ces isolats. En conclusion, cette recherche a permis d’obtenir de nouvelles données scientifiques sur les isolats québécois de M. hyopneumoniae. / Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, is present in swine herds worldwide. However, little is known about the prevalence of this microorganism in Quebec and Canadian herds. A total of 160 swine lungs with lesions suggestive of enzootic pneumonia were recovered from two slaughterhouses. They were cultured and tested by PCR for M. hyopneumoniae and Mycoplasma hyorhinis and for the porcine reproductive and respiratory syndrome virus (PRRSV), the influenza virus, and the porcine circovirus type 2 (PCV2). Samples were also cultured for other commonly encountered swine pathogenic bacteria. Ninety percent of the samples were positive for M. hyopneumoniae (Real-time PCR) whereas 5.6% were positive only for M. hyorhinis (PCR). The concentration of M. hyopneumoniae in these positive samples varied between 1.17 x 105 and 3.37 x 109 genomes/mL Among 25 selected M. hyopneumoniae positive lungs (culture or real-time PCR), 10 demonstrated a co-infection with Pasteurella multocida, 12 with Streptococcus suis, 9 with PCV2 and 2 with the PRRS virus. Multiple loci variable number of tandem repeats analysis (MLVA) and PCR-restriction fragments length polymorphism (PCR-RFLP) analyses showed a high diversity among field M. hyopneumoniae isolates. However, there seemed to be greater homogeneity within the same herd. The MLVA analysis also demonstrated that almost half of the field isolates presented less than 55% homology with the vaccine and reference strains used in our study. The absence of amplification of one locus (locus 1) of M. hyopneumoniae was significantly associated with a lower number of bacteria and a lower severity of lung lesions. All M. hyopneumoniae isolates showed low to intermediate MICs against the antimicrobials tested. Cultures with intermediate MICs for tetracyclines, macrolides and lincosamides were tested for the presence of previously described resistance genes tetM, ermB and L4, L22 and 23S rRNA point mutations. None of these genes were found, although one point mutation, G2057A, was identified. This mutation is responsible for the intrinsic resistance of M. hyopneumoniae against 14-membered macrolides. These results indicate that there is probably no acquired antimicrobial resistance within these isolates. This research provides new scientific data on M. hyopneumoniae isolates from Canada.

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP. / QC 20100719

Aquesta tesi se centra en l’estudi de les possibles glicosiltransferases de Micoplasma genitalium involucrades en la síntesi de glicolípids. Aquests compostos formen part de la membrana plasmàtica del microorganisme, únic envolcall que el protegeix del seu entorn ja que no disposa de paret cel•lular. La hipòtesi sobre la qual s’ha desenvolupat el treball és la possibilitat que aquests glicolípids i els enzims encarregats de la seva producció, les glicosiltransferases, siguin essencials per a la viabilitat del micoplasma i per tant, la seva inhibició sigui una forma d’eradicar les infeccions causades pel patògen. De les tres seqüències classificades com a glicosiltrasferases en el genoma de Mycoplasma genitalium, mg025, mg060 i mg517, s’ha determinat que mg025 és l’únic dels tres gens que no és essencial per al bacteri, tot i que s’ha observat que els tres s’expressen tant a la fase exponencial com a la fase estacionària del seu creixement. La funció de mg025 i mg060 és encara desconeguda, mentre que mg517 és la glicosiltransferasa encarregada de la síntesi dels dos principals glicolípids del micoplasma, el monoglicosildiacilglicerol (MGlcDG) i el diglicosildiacilglicerol (DGlcDG). En aquesta tesi s’ha desenvolupat un protocol d’expressió per a la glicosiltransferasa codificada per mg517, anomenada GT-MG517, el qual fa ús d’una coexpressió amb xaperones i solubilitza la proteïna amb detergents, glicerol i una elevada força iònica. Aquesta metodologia ha estat necessària ja que GT-MG517 és una proteïna associada a membrana i la seva expressió recombinant en E.coli presenta dificultats. La proteïna s’ha purificat mitjançant cromatografia d’afinitat per Ni, tot i que el grau de puresa assolit no ha estat suficient per a intentar la seva cristal•lització. Les diverses proves realitzades usant cromatografia d’exclusió molecular han permès determinar que GT-MG517 forma oligòmers d’alt pes molecular. A partir de la proteïna purificada s’ha realitzat un estudi cinètic de la seva doble activitat glicosildiacilglicerolsintasa. D’aquest estudi s’extreu que GT-MG517 pot transferir un sucre, Glc o Gal, a una molècula principalment hidrofòbica, com és el DOG, i a una molècula molt més hidrofílica, com el MGlcDG. Ambdós compostos però posseeixen un alcohol primari sobre el qual té lloc la transferència creant l’enllaç (16). Amb qualsevol dels substrats acceptors provats, l’enzim presenta activitats específiques superiors si el substrat donador és UDP-Gal. L’acceptor preferit de GT-MG517 és el lípid DOG, però la glicosiltransferasa és capaç d’elongar, ja sigui amb una Glc o amb una Gal, glicolípids com el MGlcDG i el MGDEG, preferint en aquest cas el compost amb una Gal a l’extrem no reductor. Tot i això, l’afinitat de l’enzim és superior per a donadors amb Glc (KM inferiors a les dels donadors amb Gal). D’altra banda, s’ha demostrat que el lípid aniònic DOPG es comporta com a activador de l’activitat enzimática. GT-MG517 posseeix un teòric domini d’unió d’UDP-Glc a l’extrem N-terminal. Aquesta zona presenta similitud de seqüència amb altres glicosiltransferases i permet la classificació de GT-MG517 dins de la família GT-2. En canvi, el seu extrem C-terminal presenta una seqüència particular que no s’alinea amb altres proteïnes. En aquesta tesi es formula la hipòtesi que aquesta zona podria ser d’interacció amb la membrana i, a més, que aquesta interacció podria regular l’activitat enzimàtica. Per això es preparen diverses formes truncades de GT-MG517, en les quals s’eliminen alguns aminoàcids de l’extrem C-terminal, i una forma en la qual només es conserva l’hipotètic domini d’unió d’UDP-Glc. Aquestes noves proteïnes s’expressen, solubilitzen i purifiquen aplicant el protocol establert per a la forma completa. Amb l’anàlisi dels glicolípids sintetitzats s’estableix que els deu últims aminoàcids de GT-MG517 són prescindibles per a la seva activitat, ja que les formes truncades corresponents continuen tenint la capacitat de sintetitzar glicolípids. No obstant, l’eliminació d’un nombre superior de residus, inactiva la proteïna. La purificació de l’hipotètic domini d’unió d’UDP-Glc posa de manifest que forma oligòmers, de la mateixa forma que ho fa la proteïna completa. / Esta tesis se centra en el estudio de las posibles glicosiltransferasas de Mycoplasma genitalium involucradas en la síntesis de glicolípidos. Estas moléculas constituyen parte de la membrana plasmática del miroorganismo, única estructura de protección frente al entorno ya que los micoplasmas carecen de pared celular. La hipótesis sobre la cual se ha desarrollado el trabajo es la posibilidad que los mencionados glicolípidos y las enzimas encargadas de su producción, las glicosiltransferasas, sean esenciales para la viabilidad del micoplasma y, por tanto, su inhibición sea una forma de erradicar las infecciones causadas por el patógeno. De las tres secuencias clasificadas como glicosiltransferasas en el genoma de Mycoplasma genitalium, mg025, mg060 y mg517, se determinó que mg025 es el único de los tres genes que no es esencial para la bacteria, aunque se observó que los tres se expresan tanto en la fase exponencial como en la fase estacionaria de su crecimiento. Se desconoce todavía la función de mg025 y mg060. En cambio, se conoce que mg517 es la glicosiltransferasa responsable de la síntesis de los dos principales glicolípidos del micoplasma, el monoglicosildiacilglicerol (MGlcDG) y el diglicosildiacilglicerol (DGlcDG). En esta tesis se desarrolló un protocolo de expresión para la glicosiltransferasa codificada por mg517, llamada GT-MG517, el cual emplea una coexpresión con chaperonas y solubiliza la proteína con detergentes, glicerol y una fuerza iónica elevada. Dicha metodología fue necesaria ya que GT-MG517 es una proteína asociada a membrana y su expresión recombinante en E.coli presenta dificultades. La proteína se purificó mediante cromatografía de afinidad por Ni pero el grado de pureza obtenido no fue suficiente para intentar su cristalización. Distintas pruebas realizadas usando cromatografía d’exclusión molecular permitieron determinar que GT-MG517 forma oligómeros de alto peso molecular. Con la proteína purificada se realizó un estudio cinético de su doble actividad glicosildiacilglicerolsintasa. De este estudio se extrae que GT-MG517 puede transferir un azúcar, Glc o Gal, a una molécula principalmente hidrofóbica, como es el DOG, y a una molécula mucho más hidrofílica, como el MGlcDG. Pese a su diferencia, los dos compuestos poseen un alcohol primario sobre el que tiene lugar la transferencia creándose el enlace (16). Con cualquiera de los sustratos aceptores probados, la enzima presenta actividades específicas superiores si el sustrato dador es UDP-Gal. El aceptor preferido de GT-MG517 es el lípido DOG, aunque la glicosiltransferasa es capaz de elongar, ya sea con una Glc o con una Gal, glicolípidos como el MGlcDG y el MGDEG, prefiriendo en este caso el compuesto con una Gal en el extremo no reductor. Aún así, la afinidad de la enzima es superior para dadores con Glc (KM inferiores a las de los dadores con Gal). Por otro lado, se demostró que el lípido aniónico DOPG se comporta como activador de la actividad enzimática. GT-MG517 posee un dominio teórico de unión de UDP-Glc en su extremo N-terminal. Esta zona presenta similitud de secuencia con otras glicosiltransferasas y permite la clasificación de GT-MG517 dentro de la familia GT-2. En cambio, su extremo C-terminal presenta una secuencia particular que no se alinea con otras proteínas. En esta tesis se formula la hipótesis que dicha zona podría ser de interacción con la membrana y, además, que dicha interacción podría regular la actividad enzimática. Por eso se prepararon distintas formas trucadas de GT-MG517, en las cuales se eliminaron algunos aminoácidos del extremo C-terminal, y una forma en la cual sólo se conservó el hipotético dominio de unión de UDP-Glc. Las nuevas proteínas se expresaron, solubilizaron y purificaron aplicando el protocolo establecido para la forma completa. Con el análisis de los glicolípidos sintetizados se determinó que los diez últimos aminoácidos de GT-MG517 eran prescindibles para su actividad, ya que las correspondientes formas truncadas conservaban su capacidad de sintetizar glicolípidos. No obstante, la eliminación de un número superior de residuos inactiva la proteína. La purificación del hipotético dominio de unión de UDP puso de manifiesto que forma oligómeros, de la misma forma en que lo hace la proteína completa. / This thesis is focused on the glycolipid producing glycosyltransferases from Mycoplasma genitalium. Glycolipids are part of the microorganism plasma membrane, which is the external covering of mycoplasmas since they lack a cell wall. Our working hypothesis is that glycolipids and the enzymes responsible for their production are essential to mycoplasma. Thus, glycosyltransferase inhibition could be a way to eradicate this pathogen caused infections. There are three genes described as putative glycosyltransferases in Mycoplasma genitalium genome, mg025, mg060 and mg517. We determined that mg025 is the only one essential to the bacteria, although the three of them are expressed both during the exponential and stationary growth phases. The function of mg025 and mg060 still remains unknown, whereas mg517 codifies for the glycosyltransferase responsible for monoglycosyldiacylglycerol (MGlcDG) and diglycosyldiacylglycerol (DGlcDG) synthesis, main mycoplasma glycolipids. In this work an expression protocol for mg517 codified glycosyltransferase was designed. The protocol included chaperone coexpression and protein solubilization with detergents, glycerol and high ionic strength. This methodology was necessary since GT-MG517 is a membrane associated protein and its recombinant expression in E.coli presents some difficulties. GT-MG517 was purified by Ni affinity chromatography. However, a suitable purity degree to attempt protein crystallization was not achieved. Some experiments using size exclusion chromatography revealed that GT-MG517 forms high molecular weight oligomers. A kinetic study of the protein double glycosyldiacylglycerol synthase activity was performed. From this study we learned that GT-MG517 is able to transfer a sugar moiety, Glc or Gal, both to a hydrophobic molecule such as DOG or to a more hydrophilic compound such as MGlcDG. These acceptor substrates share a primary alcohol where the sugar transfer takes place forming a (16) bond. The enzyme presents higher specific activities when UDP-Gal acts as reaction donor, regardless of the acceptor tested. DOG is the preferred acceptor although the enzyme is able to transfer Glc or Gal moieties to glycolipids such as MGlcDG and MGDEG. In this case, the reaction is faster with acceptors with Gal in the non-reducing end. As for donor substrate, enzyme’s affinity is higher for Glc containing molecules, with lower KM values than for Gal donors. In addition, our study proved the anionic lipid DOPG to act as an enzymatic activity enhancer. GT-MG517 has a putative binding domain for UDP-Glc in the N-terminal end. This sequence is similar to other glycosyltransferases and allows its classification in Cazy’s GT-2 family. On the contrary, the C-terminal end has a particular sequence which does not match up with any other protein. Our hypothesis was that this C-terminal end of GT-MG517 could contain a membrane interaction sequence, which could at the same time modulate enzymatic activity. To test this hypothesis some truncated forms of GT-MG517, where C-terminal aminoacids had been removed, were prepared. Moreover, a form where only the putative UDP-Glc binding domain was conserved was also expressed. All these proteins were expressed, solubilized and purified with the same protocol used for the full-length form of GT-MG517. Glycolipids produced by truncated forms were analysed and results implied that the last ten aminoacids were not involved in the enzyme’s activity. Elimination of a higher number of aminoacids caused protein inactivation. When the putative UDP-binding domain was purified, it showed high molecular weight oligomers such as those developed by the complete form of the protein.

Mycoplasma hyopneumoniae est l’agent causal de la pneumonie enzootique. On le retrouve dans plusieurs élevages de porcs à travers le monde. Même si ce micro-organisme est présent dans plusieurs troupeaux canadiens, peu d’informations sont présentement disponibles sur les isolats québécois. Un total de 160 poumons de porcs possédant des lésions de pneumonie ont été récupérés à l’abattoir, mis en culture et testés par PCR pour M. hyopneumoniae et Mycoplasma hyorhinis. D’autres pathogènes bactériens communs du porc et les virus du syndrome reproducteur et respiratoire porcin (VSRRP), de l’influenza et le circovirus porcin de type 2 (CVP2) ont été également testés. Quatre-vingt-dix pourcent des échantillons étaient positifs pour M. hyopneumoniae et 5.6% l’étaient seulement pour M. hyorhinis. Dans ces échantillons positifs pour M. hyopneumoniae, la concentration de ce mycoplasme variait de 1.17 x 105 à 3.37 x 109 génomes/mL. Vingt-cinq poumons positifs en culture ou par PCR en temps réel pour M. hyopneumoniae ont été sélectionnés, parmi ceux-ci 10 étaient en coinfection avec Pasteurella multocida, 12 avec Streptococcus suis, 9 avec CVP2 et 2 avec le VSRRP. Les analyses des nombres variables de répétitions en tandem à de multiples loci (MLVA) et PCR-polymorphisme de longueur de fragments de restriction (PCR-RFLP) de M. hyopneumoniae ont démontré une forte diversité des isolats de terrain. Par contre, il semble y avoir plus d’homogénéité à l’intérieur d’un même élevage. L’analyse MLVA a également démontré que près de la moitié des isolats possédaient moins de 55% d’homologie avec les souches vaccinales et de référence utilisées dans la présente étude. L’absence d’amplification du locus 1 de M. hyopneumoniae en MLVA a été significativement associée à une baisse de la concentration de bactérie et de la sévérité des lésions. Pour tous les isolats de M. hyopneumoniae, des concentrations minimales inhibitrices (CMI) de faibles à intermédiaires ont été obtenues envers tous les antimicrobiens testés. Les isolats possédant des CMI intermédiaires envers les tétracyclines, les macrolides et les lincosamides ont été testés pour la présence des gènes de résistance tetM, ermB et pour des mutations ponctuelles dans les gènes des protéines L4, L22 et de l’ARNr 23S. Aucun de ces gènes n’a été détecté mais la mutation ponctuelle G2057A a été identifiée. Cette mutation est responsable de la résistance intrinsèque de M. hyopneumoniae face aux macrolides à 14 carbones. Ces résultats indiquent qu’il ne semble pas y avoir de résistance acquise aux antimicrobiens parmi ces isolats. En conclusion, cette recherche a permis d’obtenir de nouvelles données scientifiques sur les isolats québécois de M. hyopneumoniae. / Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, is present in swine herds worldwide. However, little is known about the prevalence of this microorganism in Quebec and Canadian herds. A total of 160 swine lungs with lesions suggestive of enzootic pneumonia were recovered from two slaughterhouses. They were cultured and tested by PCR for M. hyopneumoniae and Mycoplasma hyorhinis and for the porcine reproductive and respiratory syndrome virus (PRRSV), the influenza virus, and the porcine circovirus type 2 (PCV2). Samples were also cultured for other commonly encountered swine pathogenic bacteria. Ninety percent of the samples were positive for M. hyopneumoniae (Real-time PCR) whereas 5.6% were positive only for M. hyorhinis (PCR). The concentration of M. hyopneumoniae in these positive samples varied between 1.17 x 105 and 3.37 x 109 genomes/mL Among 25 selected M. hyopneumoniae positive lungs (culture or real-time PCR), 10 demonstrated a co-infection with Pasteurella multocida, 12 with Streptococcus suis, 9 with PCV2 and 2 with the PRRS virus. Multiple loci variable number of tandem repeats analysis (MLVA) and PCR-restriction fragments length polymorphism (PCR-RFLP) analyses showed a high diversity among field M. hyopneumoniae isolates. However, there seemed to be greater homogeneity within the same herd. The MLVA analysis also demonstrated that almost half of the field isolates presented less than 55% homology with the vaccine and reference strains used in our study. The absence of amplification of one locus (locus 1) of M. hyopneumoniae was significantly associated with a lower number of bacteria and a lower severity of lung lesions. All M. hyopneumoniae isolates showed low to intermediate MICs against the antimicrobials tested. Cultures with intermediate MICs for tetracyclines, macrolides and lincosamides were tested for the presence of previously described resistance genes tetM, ermB and L4, L22 and 23S rRNA point mutations. None of these genes were found, although one point mutation, G2057A, was identified. This mutation is responsible for the intrinsic resistance of M. hyopneumoniae against 14-membered macrolides. These results indicate that there is probably no acquired antimicrobial resistance within these isolates. This research provides new scientific data on M. hyopneumoniae isolates from Canada.

No description available.

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 130-174).