Pela utilização de métodos bioquímicos, biofísicos, de tipagem sorológica e de visualização das proteínas totais bacterianas, isolados de surubim Pseudoplatystoma corruscans, tilápia Oreochromis niloticus e pacu Piaractus mesopotamicus, foram caracterizados, identificados e sua virulência determinada. Dentre as linhagens de referência, o isolado de surubim caracterizou-se como sendo Plesiomonas shiguelloides e os demais isolados de tilápia e pacu foram identificados como Aeromonas hydrophila, todos pertencentes à família Vibrionaceae. Os isolados de tilápia e pacu caracterizaram-se como linhagens virulentas, resistentes aos antibióticos ampicilina, amoxicilina, lincomicina, novobiocina, oxacilina, penicilina, rifampicina e trimetoprim+sulfametoxazol, em ensaios de antibiograma realizados em meio YEA que evidenciaram que as linhagens isoladas de peixes são resistentes a oito das dezessete substâncias antimicrobianas testadas pelo método de difusão em disco. Essas características são compatíveis com as apresentadas pelo espécime tipo de A. hydrophila. Ambas as linhagens quando cultivadas em meio YEA compartilharam a mesma banda de aproximadamente 33,61kDa com o espécime tipo para A. hydrophila. Em meio enriquecido com glucose, a banda compartilhada entre elas teve peso molecular aproximado de 144,28kDa. Os testes de aglutinação sorológica evidenciaram nestas duas linhagens a presença de antígenos estáveis ao calor do tipo O. A técnica de dupla imunodifusão de Ouchterlony demonstrou que o antígeno preparado a partir do isolado de tilápia é a linhagem de A. hydrophila mais indicada para ser utilizada em estudos visando o desenvolvimento de uma vacina polivalente. / Bacteria isolated from surubim Pseudoplatystoma corruscans, tilapia Oreochromis niloticus e pacu Piaractus mesopotamicus, were characterized and identified by biochemical, biophysical, serology, and SDS-PAGE, and their virulence observed. The strain isolated from surubim was characterized as Plesiomonas shigelloides. The other strains isolated from tilapia and pacu were Aeromonas hydrophila. The isolated A. hydrophila strains presented virulence and resistance against the follow antibacterial substances: ampicillin, amoxicillin, lincomicin, novobiocin, oxacillin, penicillin, rifampin and trimetoprim+sulfametoxazole. Both strains when cultivated in YEA medium shared with the A. hydrophila type strain a similar protein band of 33,61kDa. In a medium supplemented with glucose, only one protein exhibiting relative molecular mass of 144.28 kDa, was shared by the type strains isolated from fish and the type strain. The serology tests revealed that all isolated strains presented heat-stable O-antigens. The Ouchterlony double-immunodiffusion showed that the antigen prepared from the tilapia strain possessed surface antigens similar to A. hydrophila type strain and the strains isolated from pacu. This suggested the possibility of development and usage of a common or polyvalent vaccine for A. hydrophila among tilapia and pacu or other freshwater fish species.

This diploma thesis focused on screening for bacterial pathogens and antagonists suppressing common scab mainly caused by Streptomyces scabiei. Common scab affects some agricultural crops causing significant economical losses. Bacterial strains, mostly streptomycetes, were isolated from potato rhizosphere because they belong to most important producents of secondary metabolites and the causative agents of the disease are also members of this genus. The isolated bacteria were characterised by PCR amplification and sequencing of 16S rRNA gene to reveal their phylogenetic relationships. The ability of isolated strains to suppress growth of Streptomyces scabiei was tested by a simple co-cultivation experiment. The strains were tested by PCR for presence of specific genes for biosynthesis of thaxtomin A, a common virulence factor found in all described pathogens causing symptoms of this disease on the surface of affected tubers. Genes for synthesis of thaxtomin belong to pathogenicity island. Standard of phytotoxin thaxtomin A was used to optimize its analysis by mass spectroscopy for further in vivo and in vitro experiments. Phylogenetic analysis of strains harboring one of the genes necessary for thaxtomin A biosynthesis supported the hypothesis of sharing the pathogenicity island by horizontal gene...

No description available.

This research addressed the effects of cultural management practices, cultivar selection, and applications of seasonal fungicides on ultradwarf bermudagrass health and playability and occurrence of foliar diseases. Additionally, novel ectotrophic root-infecting fungi were isolated from ultradwarf bermudagrass roots exhibiting symptoms of summer decline, identified via multilocus phylogenetic analyses, and characterized by morphological assessments and pathogenicity evaluations.

Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. Other species cause campylobacteriosis relatively infrequently; while this could be attributed to bias in diagnostic methods, the pathogenicity of non-jejuni/coli Campylobacter spp. such as C. upsaliensis and C. helveticus (isolated from dogs and cats) is uncertain. Galleria mellonella larvae are suitable models of the mammalian innate immune system and have been applied to C. jejuni studies. This study compared the pathogenicity of C. jejuni, C. upsaliensis, and C. helveticus isolates. Larvae inoculated with either C. upsaliensis or C. helveticus showed significantly higher survival than those inoculated with C. jejuni. All three Campylobacter species induced indistinguishable histopathological changes in the larvae. C. jejuni could be isolated from inoculated larvae up to eight days post-inoculation whereas C. upsaliensis and C. helveticus could only be isolated in the first two days. There was a significant variation in the hazard rate between batches of larvae, in Campylobacter strains, and in biological replicates as random effects, and in species and bacterial dose as fixed effects. The Galleria model is applicable to other Campylobacter spp. as well as C. jejuni, but may be subject to significant variation with all Campylobacter species. While C. upsaliensis and C. helveticus cannot be considered non-pathogenic, they are significantly less pathogenic than C. jejuni.

No description available.

Tesis por compendio / [ES] Se desarrolló un ensayo qPCR para la detección y cuantificación de inóculo aéreo de D. amygdali, incluyendo el diseño de un par de cebadores específicos para esta especie. Esta metodología se utilizó para estudiar la dinámica del inóculo de D. amygdali en trampas de esporas colocadas en dos huertos de almendros de diferentes ubicaciones, en dos temporadas de crecimiento. Con los datos climáticos registrados se construyó un modelo Hurdle de dos partes; la primera parte relacionó la presencia o ausencia de ADN de D. amygdali (Bernoulli o cualitativa) y la otra consideró la concentración promedio de ADN del hongo (Gamma o cuantitativa). El efecto de la temperatura estuvo relacionado con la amplitud térmica diaria; los rangos térmicos más amplios redujeron la concentración de ADN, mientras que los rangos térmicos más estrechos aumentaron la detección de ADN. Los días con un promedio de humedad relativa superior al 80% tuvieron un efecto negativo en la concentración de ADN de D. amygdali. Las precipitaciones tuvieron una influencia positiva en ambas partes del modelo, confirmando la contribución de la lluvia en la dispersión del inóculo. Finalmente, la variable velocidad del viento influyó positivamente en ambas partes del modelo, en ambas temporadas de crecimiento. En ensayos de laboratorio, se inocularon ramitas de 25 cultivares de almendro con cuatro aislados de D. amygdali, mientras que las inoculaciones de campo implicaron la inoculación de brotes en crecimiento de cultivares de almendro injertados en el portainjerto 'GF-677', durante cuatro años. En ambos tipos de experimentos, el inóculo consistió en discos de agar con micelio, que se insertaron debajo de la corteza y se midió la longitud de las lesiones causadas por el hongo. Todos los cultivares evaluados mostraron lesiones necróticas en respuesta a D. amygdali, confirmando su susceptibilidad. El análisis de conglomerados clasificó los cultivares como susceptibles o muy susceptibles. Las características agronómicas, como el tiempo de floración y maduración, se vincularon con la susceptibilidad de los cultivares. Los cultivares de floración tardía, muy tardía y de maduración temprana a media, mostraron una alta susceptibilidad. Se realizaron prospecciones entre 2018 y 2020 en seis provincias españolas, para recolectar e identificar oomicetos asociados con síntomas de pudrición de raíz y cuello en almendros. Se obtuvieron un total de 104 aislados de oomicetos procedentes de árboles enfermos, y la secuenciación de la región espaciadora transcrita interna (ITS) identificó especies de diferentes géneros. Phytopythium vexans y Ph. niederhauserii fueron las especies más frecuentes. Las pruebas de patogenicidad en plantas de almendro de un año de edad injertadas en el portainjerto 'Garnem' mostraron síntomas graves, como defoliación, marchitez y muerte regresiva, y algunas plantas murieron cuando se inocularon con Pp. vexans y Ph. niederhauserii. Algunos aislados de Ph. niederhauserii redujeron significativamente el peso seco de las raíces en comparación con el control, pero este efecto no se observó en plantas inoculadas con Pp. vexans. Se evaluó la patogenicidad de 12 especies de oomicetos presentes en el suelo en tres portainjertos híbridos de Prunus utilizados frecuentemente en la cuenca mediterránea. Se realizaron pruebas de patogenicidad con 15 aislados de oomicetos en plantas de portainjertos de 1 año de edad. Noventa días después de la inoculación se evaluaron los síntomas de la enfermedad con una escala de severidad y se calculó el AUDPC y la probabilidad de supervivencia de las plantas inoculadas. Todos los aislados fueron patógenos para las plántulas de los portainjertos y se volvieron a aislar de las lesiones de las raíces. Para cada portainjerto se detectaron grandes diferencias en virulencia entre las diferentes especies de oomicetos y aislados de Ph. niederhauserii. Phytophthora multivora y Pp. helicoides fueron generalmente los más virulentos. / [CA] Es va desenvolupar un assaig qPCR per a la detecció i quantificació d'inòcul aeri de D. amygdali, inclòs el disseny d'un parell d'encebadors específics per a aquesta espècie. Aquesta metodologia es va utilitzar per estudiar la dinàmica de l'inòcul de D. amygdali en trampes d'espores col·locades en dos horts d'ametllers de diferents indrets en dues temporades de creixement. Es va construir un model Hurdle de dues parts amb les dades climàtiques registrades; la primera part relacionava la presència o absència d'ADN de D. amygdali (Bernoulli o qualitatiu) i l'altra considerava la concentració mitjana d'ADN del fong (Gamma o quantitativa). L'efecte de la temperatura estava relacionat amb l'amplitud tèrmica diària; rangs tèrmics més amplis van reduir la concentració d'ADN, mentre que rangs tèrmics més estrets van augmentar la detecció d'ADN. Els dies amb una mitjana d'humitat relativa superior al 80% van tenir un efecte negatiu en la concentració d'ADN de D. amygdali. Les precipitacions van tenir una influència positiva en ambdues parts del model, confirmant la contribució de la pluja en la dispersió de l'inòcul. Finalment, la variable de velocitat del vent va influir positivament en ambdues parts del model, en ambdues temporades de creixement. En proves de laboratori, es van inocular branquetes de 25 cultivars d'ametller amb quatre aïllats de D. amygdali, mentre que les inoculacions de camp van implicar la inoculació de brots en creixement de cultivars d'ametller empeltats al portaempelt 'GF-677' durant quatre anys. En ambdós tipus d'experiments, l'inòcul consistia en discs d'agar amb miceli, que s'introduïen sota l'escorça i es mesuraven les longituds de les lesions provocades pel fong. Tots els cultivars avaluats van mostrar lesions necròtiques en resposta a D. amygdali, confirmant la seva susceptibilitat. L'anàlisi de conglomerats va categoritzar els cultivars com a susceptibles o molt susceptibles. Els trets agronòmics, com ara els temps de floració i maduració, estaven relacionats amb la susceptibilitat del conreu. Els cultivars de floració tardana, molt tardana i de maduració precoç a mitjana van mostrar una alta susceptibilitat. Entre el 2018 i el 2020 s'han realitzat prospeccions a 6 províncies d'Espanya per recollir i identificar oomicets associats a símptomes de podridura de l'arrel i del coll d'ametllers. Es van obtenir un total de 104 aïllats d'oomicets d'arbres malalts i la seqüenciació de la regió de l'espaiador transcrit intern (ITS) va identificar espècies de diferents gèneres. Phytopythium vexans i Ph. niederhauserii eren les espècies més comunes. Les proves de patogenicitat en plàntules d'ametller d'un any empeltades en el portaempelts 'Garnem' van revelar símptomes greus, com ara defoliació, marciment i mort, amb algunes plantes que van morir quan es van inocular amb Pp. vexans i Ph. niederhauserii. Alguns aïllats de Ph. niederhauserii van reduir significativament el pes sec de les arrels en comparació amb el control, però aquest efecte no es va observar en les plàntules inoculades amb Pp. vexans. Es va avaluar la patogenicitat de 12 espècies d'oomicets del sòl en tres portaempelts híbrids de Prunus d'ús habitual a la conca mediterrània. Es van realitzar proves de patogenicitat amb 15 aïllats d'oomicets en plàntules de portaempelt d'un any. Noranta dies després de la inoculació, es van avaluar els símptomes de la malaltia en una escala de gravetat i es va calcular AUDPC i la probabilitat de supervivència de les plàntules inoculades. Tots els aïllats van ser patògens per a les plàntules de portaempelt i es van tornar a aïllar de les lesions de les arrels. Per a cada portaempelt es van detectar grans diferències de virulència entre les diferents espècies d'oomicets i aïllats de Ph. niederhauserii. Phytophthora multivora i Pp. helicoides eren en general els més virulents. / [EN] A qPCR assay for the detection and quantification of aerial inoculum of D. amygdali, including the design of a pair of specific primers for this species, was developed. This methodology was used to study the dynamics of D. amygdali inoculum in spore traps placed at two almond orchards from different locations in two growing seasons. A two-part Hurdle model was built with the recorded climatic data; the first part related the presence or absence of D. amygdali DNA (Bernoulli or qualitative) and the other considered the average concentration of DNA of the fungus (Gamma or quantitative). The temperature effect was related to daily thermal amplitude; wider thermal ranges reduced DNA concentration, while narrower thermal ranges increased DNA detection. The days with average relative humidity higher than 80% had a negative effect on the concentration of D. amygdali DNA. Rainfall had a positive influence on both parts of the model, confirming the contribution of precipitation in the dispersal of inoculum. Finally, wind speed variable positively influenced both parts of the model, in both growing seasons. In laboratory tests, twigs from 25 almond cultivars were inoculated with four isolates of D. amygdali, while field inoculations involved the inoculation of growing shoots of almond cultivars grafted onto 'GF-677' rootstock over four years. In both type of experiments, inoculum consisted of agar plugs with mycelium, which were inserted underneath the bark and the lesion lengths caused by the fungus were measured. All evaluated cultivars showed necrotic lesions in response to D. amygdali, confirming their susceptibility. Cluster analysis categorized cultivars as susceptible or very susceptible. Agronomic traits, such as blooming and ripening times, were linked to cultivar susceptibility. Late blooming, very late blooming, and early to medium ripening cultivars exhibited high susceptibility. Surveys were carried out between 2018 and 2020 in 6 provinces of Spain to collect and identify oomycetes associated with root and crown rot symptoms on almond trees. A total of 104 oomycete isolates were obtained from diseased trees, and sequencing of the internal transcribed spacer (ITS) region identified species from different genera. Phytopythium vexans and Ph. niederhauserii were the most common species. Pathogenicity tests on one-year-old almond seedlings of 'Garnem' rootstock revealed severe symptoms, including defoliation, wilting, and dieback, with some plants dying when inoculated with Pp. vexans and Ph. niederhauserii. Some isolates of Ph. niederhauserii significantly reduced the dry weight of the roots compared with the control, but this effect was not observed in seedlings inoculated with Pp. vexans. Pathogenicity of 12 soil-borne oomycete species on three commonly used Prunus hybrid rootstocks in the Mediterranean Basin was evaluated. Pathogenicity tests with 15 oomycete isolates were conducted on 1-year-old rootstock seedlings. Ninety days after inoculation, disease symptoms were evaluated on a severity scale, and the AUDPC and the survival probability of the inoculated seedlings were calculated. All the isolates were pathogenic to the rootstock seedlings and were re-isolated from root lesions. For each rootstock large differences in virulence were detected among the different oomycete species and isolates of Ph. niederhauserii. Phytophthora multivora and Pp. helicoides were generally the most virulent. / Francisco Beluzán was supported by Agencia Nacional de Investigación y Desarrollo/Subdirección de Capital Humano/Doctorado Becas Chile en el Extranjero/72200145. / Beluzán Flores, FJ. (2024). Study of Diseases Caused by Diaporthe Amygdali and Oomycetes on Almond Crops [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/204445 / Compendio

La bactériose vasculaire du riz (BLB) et bactériose non-vasculaire du riz (BLS), causées respectivement par Xanthomonas oryzae pv. oryzae (Xoo) et X. oryzae pv. oryzicola (Xoc), sont les deux plus importantes maladies bactériennes du riz. Ces maladies limitent le rendement de la production de riz dans les zones rizicoles en Asie et dans certaines régions d'Afrique. L'infection et la multiplication bactérienne dans les tissus de l'hôte dépendent souvent des facteurs de virulence de ces bactéries dont le type le système de sécrétion de type III (T3SS) et ses substrats. Dans cette thèse, nous avons identifié neuf effecteurs non-TAL (transcription Transcription activator-like) sécrétés par des effecteurs de type III de la souche chinoise 13751 de Xoo en utilisant le domaine d'induction HR de la protéine avirulente AvrBs1 comme gène reporter. Parmi eux, XopAE13751 a été expérimentalement confirmé pour la première fois comme étant un effecteur non-TAL. Ensuite, par l'analyse mutationnelle de ces gènes effecteurs identifiés dans Xoo, nous avons constaté que l'effecteur non-TAL XopR13751 était nécessaire pour la virulence de la souche chinoise de Xoo sur le riz hybride Teyou63. En parallèle, nous avons démontré que le gène rsmA (repressor of secondary metabolism) - comme le gène rsmAXoo de l'espèce chinoise Xoo 13751- régule positivement l'expression des gènes associés aux facteurs de virulence, tels que le système de sécrétion de type III, les enzymes extracellulaires et le DSF (diffusible signal factor). De plus, le gène effecteur non-TAL xopO s'est avéré être peu répandu chez les Xanthomonas puisqu'il est présent uniquement chez X. euvesicatoria (Xe) et Xoc mais est absent chez Xoo. En considérant les deux pathovars de X. oryzae, avec deux modes d'infection différents, xopO a été examiné comme un facteur de la spécificité du tissu par l'inactivation mutationnelle du gène dans Xoc et par l'expression du gène dans Xoo. Les résultats ont montré que xopO n'est pas la cause déterminante de la spécificité de tissu chez Xoc. Enfin, nous avons étudié les VNTRs (Variable Number of Tandem Repeats) comme outil de typage moléculaire rapide, fiable et rentable, pour améliorer le contrôle des épidémies et pour évaluer la structure de population des souches de Xoc. 28 loci candidats VNTR ont été prédits par le criblage de trois génomes de Xoc (souche philippine BLS256, souche chinoise GX01 et souche malienne MAI10). Des paires d'amorces pour l'amplification de PCR de chacun des 28 loci ont été conçues et testées à un pannel de 20 souches de Xoc provenant de l'Asie et de l'Afrique. Le séquençage des amplicons de PCR a confirmé 25 loci VNTR robustes et polymorphes communs entre les souches Xoc asiatiques et africaines. Un dendrogramme, construit à partir de la combinaison des 25 loci de VNTR (MLVA-25), a montré que la plupart des souches asiatiques sont clairement distinguables des souches africaines. Cependant, en accord avec de précédents rapports, une souche Malienne se distingue et semble être liée aux souches asiatiques, suggérant une introduction possible de souches sur le continent africain. Ce nouvel outil de typage basé sur les VNTR sera utile pour l'étude de structures de populations et pour la surveillance épidémiologique de Xoc. / Bacterial leaf blight (BLB) and Bacterial leaf streak (BLS), caused respectively by Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), are two most important bacterial diseases on rice, constraining severely the rice yield in the rice-growing areas in Asia and in parts of Africa. Successful infection and bacterial multiplication in host tissue often depend on virulence factors from these bacteria including type Ⅲ secretion system (T3SS) and its substrates. In this thesis, we identified nine type Ⅲ secreted non-TAL (Transcription activator-like) effectors of Xoo Chinese strain 13751 using HR-inducing domain of avirulence protein AvrBs1 as the reporter, among them, XopAE13751 was first found experimentally to be non-TAL effector. Subsequently, through mutational analysis of these identified effector genes in Xoo, we showed non-TAL effector XopR13751 was found to be required for full virulence of Xoo Chinese strain in hybrid rice Teyou63. In parallel, we demonstrated that rsmA (repressor of secondary metabolism)-like gene rsmAXoo of Xoo Chinese strain 13751 positively regulated the expression of genes associated with virulence factors such as type Ⅲ secretion system, extracellular enzymes and diffusible signal factor (DSF). Furthermore, non-TAL effector gene xopO was found to be narrowly distributed in Xanthomonas, which was only present in X. euvesicatoria (Xe) and Xoc, but not in Xoo. Based on the consideration of two X. oryzae pathovars carrying two different infection ways, xopO was tested in host and tissue specificity by analysis of mutational analysis of the gene in Xoc and expression of the gene in Xoo. The results showed that xopO of Xoc did not function as a determinant in host and tissue specificity. Finally, we explored Variable Number of Tandem Repeats (VNTRs) as a fast, reliable and cost-effective molecular typing tool, to better monitoring epidemics and assess the population structure of Xoc strains. 28 candidate VNTR loci were predicted by screening of three Xoc genome sequences (Philippine strain BLS256, Chinese strain GX01 and Malian strain MAI10). Primer pairs for PCR amplification of all 28 loci were designed and applied to a panel of 20 Xoc strains originating from Asia and Africa. Sequencing of PCR amplicons revealed 25 robust and polymorphic VNTR loci which are shared among Asian and African strains of Xoc. A dendrogram was constructed from 25 VNTR loci-combinating data (MLVA-25), indicating that most Asian strains were clearly discriminated from African strains. However, in agreement with previous reports, one strain from Mali appeared to be related to Asian strains, pointing to a possible introduction of strains to the African continent. A detailed analysis of the evolutionary relationships among a larger set of Xoc strains from China will be presented, considering different spatial scales. In conclusion, a new VNTR-based tool useful for studies of population structures and epidemiological monitoring of Xoc was successfully established.

Depuis la découverte du virus de l'Immunodéficience humaine, un lentivirus, comme agent pathogène responsable de l'épidémie du SIDA en 1983, beaucoup de progrès sur le sujet ont été réalisés. Il existe deux types de virus différents pouvant infecter l'Homme, le HIV-1 et le HIV-2. Ces deux virus se regroupent en différents groupes et sous-types qui témoignent d'une grande diversité inter et intra individus (notions de quasi-espèces). La découverte de lentivirus infectant naturellement au moins quarante-cinq espèces de primates en Afrique sub-saharienne, a permis un enrichissement des connaissances sur les origines des épidémies lentivirales humaines. Aujourd'hui , il est clairement admis que l'origine des épidémies d'HIV-1 et HIV-2 sont le résultat de transmissions zoonotiques de virus de chimpanzés/gorilles et de mangabeys enfumées, respectivement. La mise en évidence de nombreux SIV circulant chez ces primates non-humains indique bien le risque potentiel de nouvelles zoonoses dans la population humaine exposée, cependant, il peut paraître surprenant que jusqu'à maintenant, deux lignées lentivirales seulement ont été capables de franchir cette barrière d'espèce. Pour pouvoir se répliquer dans les cellules d'un nouvel hôte, un lentivirus doit pouvoir contrecarrer les différents facteurs de restriction exprimés par les cellules cibles tout en exploitant au maximum la machinerie cellulaire. La famille de protéines TRIM5, APOBEC3 et les protéines Tetherin/Bst2 et SAMHD1 sont capables de bloquer une infection rétrovirale. Dans ce travail, le rôle des protéines TRIM5 a été étudié ainsi que celui d'autres protéines interagissant avec des capsides rétrovirales, dans un contexte de transmission inter-espèces de lentivirus de primates. L'étude de TRIM5α humain a montré que cette protéine n'était capable de bloquer aucune des infections par les lentivirus primates testés dans cette étude, ni par les autres SIV. Nous avons pu mettre en évidence que la dépendance de la liaison à la Cyclophiline A pour l'infection des différents SIV était variable en fonction de la capside testée. Ainsi, si cette interaction est largement répandue parmi les différentes lignées de SIV, elle n'est toutefois pas universelle. La sensibilité des SIV à la déplétion de nucléoporines qui sont connues pour affecter l'infection par HIV-1, était également variable pour différents SIV, et la même diversité a été observée concernant les déplétions de RanBP2 et Nup153. De plus, nous avons découvert une capside de SIV soumise à une forte restriction de son infection dans les cellules humaines, ce phénotype a été nommé Ref2.Il a été suggéré qu'il existait une possible corrélation entre des variations de la capside de HIV-2 et la progression vers le SIDA, nous avons donc élaboré une étude afin de déterminer si les protéines TRIM5 étaient impliquées dans ce phénotype. La conclusion est que TRIM5α humaine ne restreint fortement aucune des capsides de HIV-2 testées provenant d'une cohorte d'individus à “progression rapide“ ou “lente“ vers le SIDA. Cependant nous avons observé une capacité d'infection qui corrélait avec la pathogénicité. Il est intéressant de noter que toutes les capsides d'HIV-2 testées étaient dépendantes de la présence de Cyclophiline A pour leur infection. Toutes ces capsides étaient sensibles à la déplétion de RanBP2, et l'interaction est très probablement médiée par le motif C-ter de RanBP2 qui a une forte homologie avec la Cyclophiline A. En conclusion, il est très probable que des SIV infectant naturellement des singes puissent utiliser les mêmes protéines que HIV-1, pour un éventuel passage inter-espèces. TRIM5α ne semble pas être une barrière efficace aux différents SIV, et l'interaction avec la Cyclophiline A est probablement très conservée par les lentivirus primates / Ever since HIV has been discovered to be the pathogenic agent that causes AIDS in 1983, much progress has been made in the field. Two different viruses are now known to infect humans, HIV-1 and HIV-2. These two distinct viruses have many sub-types and clades representing a high diversity inter and intra-individuals (quasi-species). The finding of HIV simian counterparts, the Simian Immunodeficiency Viruses (SIVs), has broadened the knowledge of primate lentiviruses and to date forty-five species of non-human primates are known to be infected with SIVs in sub-saharan Africa. It is now clear that HIV-1 and HIV-2 epidemics are the result of zoonosis from chimpanzees/gorillas and sooty mangabeys, respectively. With such a big diversity of SIVs in the wild and a frequent contact of SIV infected monkey species with humans, it is interesting that so far, only two lineages breached the species barrier and infected human populations. To be able to correctly infect a cell, a lentivirus has to overcome the installed cellular barriers known as restriction factors while at the same time correctly exploiting the established host cellular machinery. Proteins such as TRIM5, APOBEC3, Tetherin/Bst2, SAMHD1 are able to restrict retroviral infections in certain conditions. In this thesis, it has been evaluated the role of TRIM5 proteins and other capsid interacting proteins with a scope to the eventuality of a cross-species transmission infection. The results showed that human TRIM5alpha does not restrict any of the primate lentiviruses tested, and so far, no primate lentivirus is known to be restricted by it. Cyclophilin A binding and dependence is variable depending on the SIV capsid; this interaction is widespread among the primate lentiviruses phylogenetic tree but not a universal phenotype. Different capsids from SIVs have been tested for the sensitivity to the depletion of nucleoporins that are known to be used by HIV-1 in its infection; it has been concluded that the same diversity applies to the interaction with RanBP2 and Nup153. Additionally, we identified a SIV capsid that is highly restricted in human cells; this phenotype was called Ref2. With the report of a possible correlation between HIV-2 capsid variations and different levels of progression to AIDS, we devised a study aiming to identify if TRIM5 proteins were involved in this phenotype. We concluded that human TRIM5alpha does not restrict any HIV-2 capsid obtained from a HIV-2 cohort, in which individuals were presenting different levels of progression to AIDS. However, we observed a different viral fitness that correlated with pathogenicity. Moreover, Cyclophilin A dependence seems ubiquitous among all of the tested HIV-2 capsids. All of these capsids are sensitive to RanBP2 depletion and the interaction is much likely mediated by RanBP2's C-terminal motif that shares a high homology with Cyclophilin A. Summing up, it is much likely that some SIVs that still circulate in the wild can hijack the same specific cellular co-factors as HIV-1 to produce a new epidemic in humans. TRIM5α does not seem to be a potent barrier to an eventual cross-species transmission from lower primates to humans, and Cyclophilin A interaction seems to play a major role to the infection of some SIVs.

Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que pode se comportar como um patógeno oportunista. A linhagem PA14 apresenta duas ilhas de patogenicidade. A maior delas, PAPI-1, contém dois grupos de genes envolvidos com virulência, transcritos de maneira oposta e que estão entre duas seqüências repetidas diretas. O primeiro grupo compreende quatro genes dispostos em dois operons, que codificam para proteínas de sistemas de dois componentes (PvrS, PvrR, RcsC e RcsB). PvrS e RcsC são proteínas sensoras híbridas, que apresentam domínios de histidina-quinase e de reguladores de resposta. PvrR é um regulador de resposta com um domínio EAL com atividade de fosfodiesterase de diGMP cíclico e RcsB apresenta um domínio de ligação a DNA, além de um domínio fosfoaceptor. O outro grupo é composto de cinco genes, cupD1 a cupD5, que codificam para uma fímbria do tipo chaperone-usher e que apresenta alta similaridade com cupA, envolvido na formação de biofilme em outras linhagens de P. aeruginosa. Trabalhos anteriores mostraram que pvrS, pvrR, rcsC, rcsB e cupD2 estão relacionados com a virulência de PA14. Como estes grupos de genes parecem ter sido inseridos na ilha em um único evento de recombinação, este trabalho investigou se os sistemas de dois componentes estão relacionados com a regulação da expressão de cupD. Foi observado que a expressão de cupD é maior a 28ºC do que que a 37ºC e é influenciada positivamente pelo regulador global de expressão, MvaT, uma proteína tipo H-NS. Ensaios de β-galactosidase a partir de uma fusão de transcrição mostraram que a atividade promotora de cupD é cerca de 50% menor numa linhagem com deleção em rcsB em relação à linhagem selvagem. Nenhuma diferença consistente foi observada entre as linhagens com deleções em pvrS, pvrR, rcsC e rcsB e PA14 em relação a motilidade dos tipos swarming, swimming ou twitching ou à formação de biofilme. A linhagem de P. aeruginosa PA14 superexpressando RcsB mostrou níveis exacerbados de mRNA de cupD1, sendo a atuação de RcsB específica em cupD, já que os outros grupos de genes cup presentes em PA14 não mostraram a mesma variação na expressão, conforme analisado por RT-PCR quantitativo. Essa linhagem mostrou também um aumento na formação de biofilme, sem que a motilidade fosse alterada. Ainda visando elucidar os mecanismos de regulação de cupD, linhagens que superexpressam pvrR também foram analisadas quanto a estes fenótipos. Nesse caso, a superexpressão de pvrR diminuiu a formação de biofilme, conforme esperado, aumentou a motilidade do tipo swarming, porém não alterou a expressão de cupD. Os dados do presente trabalho demonstraram que a cupD é regulado pelos genes do sistemas de dois componentes adjacentes a ele e que o ativador de transcrição RcsB está relacionado com a formação de biofilme em tubos de vidro, provavelmente via a fímbria CupD. / Pseudomonas aeruginosa is a γ-proteobacteria that can behave as an opportunistic pathogen. The strain PA14 carries two pathogenicity islands, the largest of them, PAPI-1, contains two gene clusters between two direct repeat sequences that are transcribed in opposite directions and are involved in virulence. The first group consists of four genes arranged in two operons encoding two-component system proteins (PvrS, PvrR, RcsC and RcsB). PvrS and RcsC are hybrid sensor proteins, which contain domains of histidine kinase and response regulator domains. PvrR is a response regulator with a phosphodiesterase EAL domain and RcsB presents a C- terminal HTH DNA biding domain, in addition to a phosphoaceptor domain. The other group is composed of five genes, cupD1-5, that encodes components and assembly factors of a putative fimbrial CupD, which has high similarity with CupA, involved in the biofilm formation in other P. aeruginosa strains. Earlier work showed that pvrS, pvrR, rcsC, rcsB and cupD2 are related to the virulence of PA14. As these groups of genes appear to have been inserted on the island in a single event of recombination, this study investigated whether the two-component systems are related to the regulation of cupD expression. It was observed that cupD promoter activity is higher at 28oC than at 37oC and it is positively influenced by the global regulator, MvaT, a H-NS like protein. A lacZ transcriptional fusion showed about 50% less promoter activity of cupD from a strain with deletion in rcsB as compared to PA14. No consistent differences were found among the strains with deletions in pvrS, pvrR, rcsC and rcsB and PA14 on swarming, swimming and twitching motilities or biofilmsformation. A strain overexpressing overexpression showed heigher levels of cupD1mRNA of, and the role of RcsB as an activator is specific to cupD, as the other groups of cup genes present in PA14 did not show the same variation in the expression, as analyzed by quantitative RT-PCR. This strain also showed an increase in biofilm formation. In further assays aiming to elucidate the mechanisms of regulation of cupD, a strains overexpressing pvrR was also analyzed. Overexpression of pvrR decreased the formation of biofilm, as expected, and increased swarming motility, but did not alter the expression of cupD. The data from this study demonstrated that cupD is regulated by RcsB, and that this transcriptional activator is involved in the formation of biofilm in glass tubes, probably via CupD fimbriae.