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Isolation, Functional Characterization and Biotechnological Applications of Glycoside Hydrolases from the Intestinal Microbiota of Breastfed InfantsMoya Gonzálvez, Eva María 27 August 2024 (has links)
Tesis por compendio / [ES] Los oligosacáridos de la leche humana (OLHs) y la parte glicana de los glicoconjugados son hidrolizados por las glicosil hidrolasas (GHs), las cuales son expresadas por la microbiota intestinal de niños lactantes promoviendo el establecimiento de una microbiota intestinal con beneficios para su salud. El objetivo de esta Tesis Doctoral consistió en la caracterización funcional de GHs de la microbiota intestinal de niños lactantes capaces de metabolizar OLHs y glicoconjugados, y el estudio de su relevancia biológica y su potencial biotecnológico.
Se aislaron cepas bacterianas a partir de heces de niños lactantes.Solo las cepas del género Bifidobacterium metabolizaron alguno de los OLHs testados. Bifidobacterium infantis Y538 consumió eficientemente todos los OLHs testados, mientras que las dos cepas de Bifidobacterium dentium Y510 y Y521 solo metabolizaron lacto-N-tetraosa (LNT) y lacto-N-neotetraosa (LNnT). Se caracterizaron dos ß-galactosidasas de B. dentium Y510; Bdg42A mostró la mayor actividad en LNT, hidrolizándolo en galactosa y lacto-N-triosa (LNTII), mientras que Bdg2A mostró actividad contra lactosa, 6'-galactopiranósil-N-acetilglucosamina, N-acetillactosamina y LNnT. También se aislaron cepas bacterianas con actividad glicosidasa extracelular de las heces de lactantes. B. infantis E17 y E18, y Enterococcus faecalis E8 y E41 exhibieron actividad de endo-ß-N-acetilglucosaminidasa, liberando N-glicanos de glicoproteínas. La endo-ß-N-acetilglucosaminidasa EndoE de E. faecalis E8 deglicosiló eficientemente la proteína S1 del coronavirus 2 del síndrome respiratorio agudo severo (SARS-CoV-2). Tanto la EndoE silvestre como un mutante catalíticamente inactive mostraron actividad lectina frente a la proteína S1 y actividad neutralizante frente a la infección de un virus pseudotipado que presenta la proteína S de SARS-CoV-2 expresada.
También se identificaron GHs putativas a través del análisis metagenómico de las heces de niños lactantes, pertenecientes a los géneros Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola y Streptococcus. Se seleccionaron diez ¿-L-fucosidasas GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358 y Fuc5372). Las ¿-L-fucosidasas Fuc18, Fuc19A, Fuc35B, Fuc39 y Fuc1584 mostraron actividad hidrolítica frente a enlaces de fucosa ¿-1,3/4 y Fuc35A, Fuc193 y Fuc2358 mostraron actividad enlaces de fucosa ¿-1,2/3/4/6. Fuc30 mostró actividad sobre la enlaces de fucosa ¿-1,6 mientras que Fuc5372 mostró preferencia por los enlaces ¿-1,2. Fuc2358 mostró actividad frente a glicoconjugados con lacto-N-fucopentaosa II, lacto-N-fucopentaosa III y contra la mucina. Fuc18, Fuc19A y Fuc39 eliminaron fucosa de neoglicoproteínas y de la glicoproteína ¿-1 ácida.
Las ¿-L-fucosidasas aisladas fueron evaluadas por su capacidad para sintetizar fucosil-oligosacáridos (FUS) a través de reacciones de transfucosilación. Fuc2358 produjo rendimientos del 35% de 2'-fucosillactosa (2'FL) y también 3'-fucosillactosa (3'FL) y 1-fucosillactosa (1FL). Fuc5372 sintetizó 2'FL, 3'FL y 1FL, con una proporción más alta de 3'FL. Se llevó a cabo mutagénesis dirigida para aumentar los rendimientos de transfucosilación. Los mutantes Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R y Fuc5372-R230K mostraron una mayor relación entre la 2'FL producida y el pNP-Fuc hidrolizado que sus respectivas enzimas silvestres. Además, se observó que los residuos F184 de Fuc2358 y W151 de Fuc5378 afectan a la regioselectividad de la transfucosilación, la fenilalanina aumentando la selectividad por los enlaces ¿-1,2 y el triptófano aumentando la selectividad por los enlaces ¿-1,3.
Los resultados presentados muestran la diversidad de GHs presentes en la microbiota intestinal de niños lactantes y expanden el conocimiento sobre su especificidad, contribuyendo al conocimiento del posible papel de las GHs en la colonización bacteriana del tracto gastrointestinal y, además, muestra su potencial biotecnológico / [CA] Els oligosacàrids de la llet humana (OLHs) i la part glicana de glicoconjugats són hidrolitzats per les glicosil hidrolases (GHs), les quals són expressades per la microbiota intestinal de xiquets lactants, promovent l'establiment d'una microbiota intestinal amb beneficis per a la seua salut. L'objectiu d'aquesta Tesi Doctoral va ser la caracterització funcional de GHs de la microbiota intestinal de xiquets lactants capaços de metabolitzar OLHs i glicoconjugats i l'estudi de la seua rellevància biològica i el seu potencial biotecnològic.
Es van aïllar soques bacterianes a partir de les femtes de xiquets lactants. Només els soques del gènere Bifidobacterium van metabolitzar algun dels OLHs testats. Bifidobacterium infantis Y538 va consumir eficientment tots els OLHs testats. Les dos soques de Bifidobacterium dentium Y510 i Y521 sol van metabolitzar lacto-N-tetraosa (LNT) i lacto-N-neotetraosa (LNnT).Es van caracteritzar dos ß-galactosidasas de B. dentium Y510; Bdg42A va exhibir la major activitat enfront de LNT, hidrolitzant-la en galactosa i lacto-N-triosa (LNTII), mentre que Bdg2A va mostrar activitat enfront de lactosa, 6'-galactopiranósil-N-acetilglucosamina, N-acetillactosamina i LNnT. També es van aïllar soques bacterianes amb activitat glicosidasa extracelul·lar. B. infantis E17 i E18, i Enterococcus faecalis E8 i E41 van exhibir activitat endo-ß-N-acetilglucosaminidasa, alliberant N-glicans de glicoproteïnes. La endo-ß-N-acetilglucosaminidasa EndoE de E. faecalis E8 va deglicosilar eficientment la proteïna S1 del coronavirus 2 del síndrome respiratori agut greu (SARS-CoV-2).Tant la EndoE salvatge com un mutant catalíticament inactiu van mostrar activitat lectina enfront de la proteïna S1 i activitat neutralitzador enfront de la infecció d'un virus pseudotipat que presenta la proteïna S de SARS-CoV-2 expressada.
També es van identificar GHs putatives a través de l'anàlisi metagenómic de la femta de xiquets lactants, pertanyents als gèneres Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola i Streptococcus. Es van seleccionar deu ¿-L-fucosidasas GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358 i Fuc5372). Les ¿-L-fucosidasas Fuc18, Fuc19A, Fuc35B, Fuc39 i Fuc1584 van mostrar activitat hidrolítica enfront d'enllaços de fucosa ¿-1,3/4 i Fuc35A, Fuc193 i Fuc2358 van mostrar activitat enllaços de fucosa ¿-1,2/3/4/6. Fuc30 va mostrar activitat enfront d'enllaços de fucosa ¿-1,6 mentre que Fuc5372 va mostrar preferència pels enllaços ¿-1,2. Fuc2358 va mostrar activitat enfront de glicoconjugats amb lacto-N-fucopentaosa II, lacto-N-fucopentaosa III i contra la glicoproteïna de la mucina. Fuc18, Fuc19A i Fuc39 van eliminar fucosa de neoglicoproteïnes i de la glicoproteïna ¿-1 àcida.
Les ¿-L-fucosidasas aïllades van ser avaluades per la seua capacitat per a sintetitzar fucosil-oligosacàrids (FUS) mediant reaccions de transfucosilació. Fuc2358 va produir rendiments del 35% de 2'-fucosillactosa (2'FL) i també 3'-fucosillactosa (3'FL) i 1-fucosillactosa (1FL). Fuc5372 va sintetitzar 2'FL, 3'FL i 1FL, amb una proporció més alta de 3'FL. Es va dur a terme mutagénesis dirigida per a augmentar els rendiments de transfucosilación. Els mutants Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R i Fuc5372-R230K van mostrar una major relació entre la 2'FL produïda i el pNP-Fuc hidrolitzat que els seus respectius enzims salvatges.A més, els residus F184 de Fuc2358 i W151 de Fuc5378 afecten la regioselectivitat de la transfucosilación; la fenilalanina augmenta la selectivitat pels enllaços ¿-1,2 i el triptòfan augmenta la selectivitat pels enllaços ¿-1,3.
Els resultats presentats mostren la diversitat de GHs presents en la microbiota intestinal de xiquets lactants i expandixen el coneixement sobre la seua especificitat, contribuint al coneixement del possible paper de les GHs en la colonització bacteriana del tracte gastrointestinal i, a més, mostra el seu potencial biotecnològic. / [EN] Human milk oligosaccharides (HMOs) and the glycan portion of glycoconjugates are hydrolyzed by glycoside hydrolases (GHs) that are expressed by the neonatal intestinal microbiota, promoting the establishment of an intestinal microbiota with health benefits for infants. The objective of this Doctoral Thesis consisted of the functional characterization of GHs from the intestinal microbiota of breastfed infants capable of metabolizing HMOs and glycoconjugates and the study of their biological relevance and their biotechnological potential.
Bacterial strains were isolated from breastfed infant faeces, showing that only Bifidobacterium genus strains metabolized any of the HMOs tested. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs, while the two strains isolated from Bifidobacterium dentium Y510 and Y521 only metabolized lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). Two ß-galactosidases from B. dentium Y510 were characterized; Bdg42A exhibited the highest activity on LNT, hydrolyzing it into galactose and lacto-N-triose (LNTII), while Bdg2A displayed activity against lactose, 6'-galactopyranosyl-N-acetylglucosamine, N-acetyllactosamine and LNnT. Bacterial strains with extracellular glycosidase activity were also isolated from breastfed infant faeces. B. infantis E17 and E18, and Enterococcus faecalis E8 and E41 exhibited endo-ß-N-acetylglucosaminidase activity, releasing N-glycans from glycoproteins. The endo-ß-N-acetylglucosaminidase EndoE from E. faecalis E8 efficiently deglycosylated the spike S1 protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Both the EndoE wild-type and a catalytically inactive mutant exhibited lectin activity towards the S1 protein and neutralizing activity against SARS-CoV-2 S pseudotyped virus infection.
Putative GHs were also identified through metagenomic analysis of breastfed infant faeces, belonging to Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola, and Streptococcus genera. Ten ¿-L-fucosidases GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358, and Fuc5372) were selected. The ¿-L-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on ¿-1,3/4-linked fucose and Fuc35A, Fuc193 and Fuc2358 showed activity on ¿-1,2/3/4/6-linked fucose. Fuc30 displayed activity only on ¿-1,6-linked fucose, and Fuc5372 showed a preference for ¿-1,2-linked fucose. Fuc2358 displayed activity against glycoconjugates carrying lacto-N-fucopentaose II, lacto-N-fucopentaose III and against the mucin glycoprotein. Fuc18, Fuc19A, and Fuc39 removed fucose from neoglycoproteins and human ¿-1 acid glycoprotein.
The isolated ¿-L-fucosidases were evaluated for their capacity to synthesize fucosyl-oligosaccharides (FUS) through transfucosylation reactions. Fuc2358 produced 35 % yields of 2'-fucosyllactose (2'FL) and also 3'-fucosyllactose (3'FL) and 1-fucosyllactose (1FL). Fuc5372 synthesized 2'FL, 3'FL and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis was conducted to increase the transglycosylation yields. Mutants Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R and Fuc5372-R230K showed a higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes. The transfucosylation activity results also showed that the residues F184 of Fuc2358 and W151 of Fuc5378 affect transfucosylation regioselectivity, with phenylalanine increasing the selectivity for ¿-1,2 linkages and tryptophan for ¿-1,3 linkages.
The results presented in this doctoral thesis illustrate the diversity of GHs in the intestinal microbiota of breastfed infants and have expanded our knowledge of their specificities, which could contribute to a better understanding of the possible role of GHs in the bacterial colonization of the infant gastrointestinal tract and presents significant biotechnological potential. / This work is part of the Grant PID2020-115403RB (C21 and C22) funded by
the Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research
Agency (AEI)/10.13039/501100011033. The study was also supported by
Valencian Government grant AICO/2021/033. EMM-G was supported by the Grant
PRE2018-085768 funded by MICIN/AEI/10.13039/501100011033 and by “ESF
Investing in your future”. / Moya Gonzálvez, EM. (2024). Isolation, Functional Characterization and Biotechnological Applications of Glycoside Hydrolases from the Intestinal Microbiota of Breastfed Infants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203171 / Compendio
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Sulphate‐reducing bacterial diversity in a calcareous sandy sediment of Mallorca and community response to hydrocarbon contaminationSuárez Suárez, Ana Belén 25 July 2012 (has links)
Aquesta tesi tracta sobre l'efecte de la contaminació per cru de petroli sobre
l'ecosistema costaner mediterrani i sobre el paper fonamental dels sediments marins
en la regulació i el manteniment dels processos biogeoquímics. L'estudi presta especial
atenció a les comunitats bacterianes reductores de sulfat i la seva implicació en la
degradació de contaminants orgànics. La diversitat, abundància i fisiologia dels bacteris
reductors de sulfat que habiten el sediment arenós del nord de Mallorca (Illes Balears),
van ser analitzades mitjançant un enfocament polifàsic, basat en la combinació
d'experiments in situ i in vitro, biologia molecular clàssica i d’última generació, cultius i
determinació d'activitats metabòliques. Els resultats obtinguts durant aquesta tesi
demostren que el sediment mediterrani alberga una microbiota autòctona que podria
prosperar després d'un vessament de cru de petroli i el paper de la qual podria ser
crucial per a la transformació i l'eliminació de compostos orgànics xenobiòtics en
aquest ambient. / Esta tesis trata sobre el efecto de la contaminación por crudo de petróleo en el
ecosistema costero mediterráneo y sobre el papel fundamental de los sedimentos
marinos en la regulación y el mantenimiento de los procesos biogeoquímicos. El
estudio presta especial atención a las comunidades bacterianas reductoras de sulfato y
a su implicación en la degradación de contaminantes orgánicos. La diversidad,
abundancia y fisiología de las bacterias reductoras de sulfato que habitan el sedimento arenoso del norte de Mallorca (Islas Baleares), fueron analizadas mediante un enfoque
polifásico, basado en la combinación de experimentos in situ e in vitro, biología
molecular clásica y de última generación, cultivos y determinación de actividades
metabólicas. Los resultados obtenidos durante esta tesis demuestran que el sedimento
mediterráneo alberga una microbiota autóctona que podría prosperar después de un
derrame de crudo de petróleo y cuyo papel podría ser crucial para la transformación y
la eliminación de compuestos orgánicos xenobióticos en este ambiente. / This thesis discusses the fate and behave of crude oil contamination in the
Mediterranean coastal ecosystem, and the essential role of the marine sediments in
the regulation and maintenance of biogeochemical processes. The study pays
particular attention to the role of sulphate reducing bacterial communities in the
degradation of organic matter and pollutants entering the Mediterranean
environment. A polyphasic approach based in the combination of in situ and in vitro
experiments, next generation and classical molecular biology, cultivation, and the
determination of metabolic activities, provided first insights into the diversity, abundance and physiology of sulphate reducing bacteria inhabiting the undisturbed
sandy sediment at the north of Mallorca (Balearic Islands). The results obtained during
the thesis demonstrate that the undisturbed Mediterranean sediment harbours an
autochthonous microbiota that could prosper after a crude oil spill and which role
might be crucial for the transformation and removal of hazardous organic compounds
in this environment.
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Dynamique spatio-temporelle des communautés virales et microbiennes des tourbières à Sphagnum / Spatio-temporal dynamic of viral and microbial communities in Sphagnum-dominated peatlandsBallaud, Flore 17 December 2015 (has links)
Les tourbières couvrent 3 % des surfaces continentales et jouent un rôle important dans le cycle du carbone en stockant le tiers du carbone des sols. L'accumulation de tourbe est liée au déséquilibre production primaire/décomposition du à une activité microbienne limitée par les conditions environnementales. L'infection et la lyse virale ont un impact sur la diversité et l'activité des communautés microbiennes, et influent sur le cycle du carbone. Cependant, le fonctionnement de ce compartiment viral n'avait jamais été pris en compte dans les études de fonctionnement des tourbières. Le but de ce travail de thèse était d'analyser et comprendre la dynamique spatiale et temporelle de l'abondance et de la diversité virale des tourbières à Sphagnum. L'analyse de l'abondance virale et procaryote et de 12 metaviromes en lien avec la physico-chimie d'une tourbière tempérée en France montre une forte variation saisonnière des communautés virales. Cette variation semble très liée aux conditions environnementales générées par la fluctuation de la nappe d'eau. Dans cette même tourbière, l'analyse de la diversité taxonomique et fonctionnelle des communautés de microorganismes présents (métagénomes) et métaboliquement actifs (métatranscriptomes) indique que la structure taxonomique est différente entre les des deux principaux stades, le fen et le bog, mais que ces communautés présentent une diversité fonctionnelle similaire, dont l'expression est liée aux changements des conditions environnementales avec la profondeur. L'abondance des particules virales étudiées dans 5 tourbières à Sphagnum réparties en Finlande, au Canada, en France, et sur l'île subantarctique d'Amsterdam varie fortement avec les sites. L'analyse de la diversité virale de la matrice et de l'eau de tourbe du Canada et de Finlande montre que la diversité virale est structurée par le site, puis le stade dynamique, puis la profondeur, avec un rôle important de la saturation en eau au niveau du site. Ces résultats valident le fonctionnement proposé du compartiment viral et de la communauté d'hôtes procaryotes. Ces connaissances ont été utilisées pour analyser le fonctionnement du compartiment microbien de tourbières à Sphagnum soumises à des perturbations d'origine anthropique. Les 31 métaviromes produits pour cette thèse constituent l'une des plus grandes bases de données sur la diversité virale des écosystèmes alors que la diversité virale des sols n'avait presque jamais été étudiée auparavant. / Peatlands cover 3 % of the continental surfaces but represent up to a third of the soil carbon stock. Peat accumulation results from the imbalance between primary production and decomposition due to the limitation of the prokaryote activity caused by the environmental conditions. Viral infection and lysis impact the diversity and the activity of the microbial communities and influence the carbon cycle. However, the functioning of the viral compartment had never been taken into account in peatlands. The aim of this thesis was to gain knowledge about the spatio-temporal dynamic of viral abundance and diversity in Sphagnum-dominated peatlands. Spatio-temporal analysis of viral and prokaryote abundance and of 12 metaviromes (viral diversity) in relation to the physico-chemical features in a temperate Sphagnum-dominated peatland in France revealed the high seasonal variability of the viral communities. This dynamic appeared mainly related to the environmental conditions shaped by the fluctuation of the water-table level. In the same peatland the taxonomic diversity of the present microorganisms (metagenomes) differed between the fen and the bog, but these communities present a similar functional diversity, which expression in selected in the same way in the two dynamic stages, in relation to depth-related environmental conditions. Viral abundance analyzed in 5 Sphagnum-dominated peatlands from Finland, Canada, France and subantarctic Amsterdam Isle presented a high geographical variability. Investigation of the diversity of the viral communities from the peat matrix and the pore-water in Finland and Canada emphasized the structuration of the viral communities by the site, then the dynamic stage, and finally depth. These results confirm the first hypotheses about the functioning of the viral compartment depending on environmental conditions and prokaryote activity. Effects of human-derived disturbances on viral ecology in peatlands were investigated based on this knowledge. While soil viral diversity was poorly documented at the start of this thesis, the collection of 31 metaviromes from Sphagnum-dominated peatlands produced for this project represents the second largest dataset representing the viral diversity from environmental samples.
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Microbiome cutané et maladie fongique émergente du syndrome du museau blanc chez les chauves-souris d’Amérique du NordLemieux-Labonté, Virginie 09 1900 (has links)
Le syndrome du museau blanc (SMB), causé par le champignon Pseudogymnoascus destructans
(Pd), a mis en péril les populations de chauves-souris hibernantes en Amérique du
Nord. Certaines espèces sont hautement vulnérables à la maladie alors que d’autres espèces
semblent être résistantes ou tolérantes à l’infection. Plusieurs facteurs physiologiques et
environnementaux peuvent expliquer ces différences. Or avant 2015, peu d’études avaient
porté sur le microbiome de la peau en relation avec cette maladie. La présente thèse vise
à caractériser le microbiome cutané de chiroptères affectés par le SMB afin d’identifier les
facteurs de vulnérabilité ou de résistance à la maladie. L’objectif principal est de déterminer
comment le microbiome est affecté par la maladie ainsi que de déterminer si celui-ci à un
rôle dans la protection face à l’infection fongique.
Au Chapitre 1, nous avons tout d’abord exploré et comparé le microbiote cutané
de petites chauves-souris brunes (Myotis lucifugus) non affectées par le SMB avec celui
de chauves-souris survivantes au SMB pour tester l’hypothèse selon laquelle le microbiote
cutané est modifié par la maladie. Nos résultats montrent que le site d’hibernation influence
fortement la composition et la diversité du microbiote cutané. Les sites d’hibernations Pd
positifs et négatifs diffèrent significativement en termes de diversité, ainsi qu’en termes de
composition du microbiote. La diversité est réduite au sein du microbiote des chauves-souris
survivantes au SMB et enrichi en taxons tels que Janthinobacterium, Micrococcaceae,
Pseudomonas, Ralstonia et Rhodococcus. Certains de ces taxons sont reconnus pour leur
potentiel antifongique et des souches spécifiques de Rhodococcus et de Pseudomonas peuvent
inhiber la croissance de Pd. Nos résultats sont cohérents avec l’hypothèse selon laquelle
l’infection par Pd modifie le microbiote cutané des chauves-souris survivantes et suggèrent
que le microbiote peut jouer un rôle de protection face au SMB.
Au Chapitre 2, nous avons étudié le microbiote d’une espèce résistante au champignon
Pd en milieu contrôlé avant et après infection afin d’établir la réponse potentielle à la maladie.
L’espèce étudiée est la grande chauve-souris brune (Eptesicus fuscus) dont le microbiote
cutané pourrait jouer un rôle de protection contre l’infection. Nos résultats montrent que la
diversité du microbiote de la grande chauve-souris brune inoculée avec Pd est plus variable
dans le temps, tandis que la diversité du microbiote des chauves-souris du groupe contrôle
demeure stable. Parmi les taxons les plus abondants, Pseudomonas et Rhodococcus, deux
taxons connus pour leur potentiel antifongique contre Pd et d’autres champignons, sont
restés stables durant l’expérience. Ainsi, bien que l’inoculation par le champignon Pd ait
déstabilisé le microbiote cutané, les bactéries aux propriétés antifongiques n’ont pas été
affectées. Cette étude est la première à démontrer le potentiel du microbiote cutané d’une
espèce de chauves-souris pour la résistance au SMB.
Au Chapitre 3, le microbiome cutané de la petite chauve-souris brune a été évalué
en milieu naturel dans le contexte du SMB, à l’aide de la métagénomique, une approche
haute résolution pour observer le potentiel fonctionnel du microbiome (métagénome
fonctionnel). Nos résultats ont permis d’établir que le temps depuis l’infection a un effet
significatif sur le métagénome fonctionnel. En effet, les chauves-souris dans la première
année suivant l’infection ont un métagénome fonctionnel perturbé qui subit une perte de
diversité fonctionnelle importante. Toutefois, le métagénome fonctionnel revient à une
structure et composition similaire d’avant infection après 10 ans. Certaines fonctions
détectées suite à l’infection sont associées à des gènes reliés au transport et à l’assimilation
de métaux, des facteurs limitants pour la croissance du champignon. Ces gènes
pourraient donc avoir un rôle à jouer dans la résistance ou la vulnérabilité à la maladie.
Globalement, l’étude du métagénome chez la petite chauve-souris brune indique une
vulnérabilité du métagénome fonctionnel au champignon, mais que celui-ci semble se rétablir
après 10 ans. Une telle réponse pourrait avoir un impact sur la résilience de M. lucifugus.
Cette thèse a permis d’acquérir des connaissances fondamentales sur le microbiome cutané
des chauves-souris en hibernation pour mieux comprendre les communautés microbiennes de
la peau dans le contexte du SMB. Le microbiome pourrait en effet jouer un rôle dans la
vulnérabilité et la résistance des chauves-souris à la maladie, et il est essentiel d’adapter
notre façon d’aborder la protection de ces espèces et de leur microbiome. Nous souhaitons
que les travaux de cette thèse permettent de sensibiliser les acteurs de la conservation à
l’existence et à l’importance potentielle du microbiome pour la santé de son hôte. Cette
thèse fait également état de l’avancement des méthodes d’analyses qui permettront d’être
de plus en plus précis et d’appliquer les connaissances du microbiome en biologie de la
conservation. / White-nose syndrome (WNS) caused by the fungus Pseudogymnoascus destructans (Pd)
has put hibernating bat populations at risk in North America. Some species are highly
vulnerable to the disease while other species appear to be resistant or tolerant. Several
physiological and environmental factors can explain these differences. However, before 2015,
few studies have focused on the skin microbiome in relation to this disease. The present
thesis aims to characterize the cutaneous microbiome of bats affected by WNS in order to
identify the factors of vulnerability or resistance to the disease. The main objective is to
determine how the microbiome can protect against the Pd fungus, or conversely how the
microbiome is altered by the fungal infection.
In Chapter 1, we first explored and compared the skin microbiota of little brown bats
(Myotis lucifugus) unaffected by WNS with that of WNS survivors to test the hypothesis
that the skin microbiota is modified by the disease. Our results show that the hibernation
site strongly influences the composition and diversity of the skin microbiota. The Pd
positive and negative sites differ significantly in terms of diversity, as well as in terms of the
composition of the microbiota. Diversity is reduced within the microbiota of bats surviving
WNS and enriched in taxa such as Janthinobacterium, Micrococcaceae, Pseudomonas,
Ralstonia, and Rhodococcus. Some of these taxa are recognized for their antifungal potential
and specific strains of Rhodococcus and Pseudomonas may inhibit the growth of Pd. Our
results are consistent with the hypothesis that Pd infection modifies the skin microbiota of
surviving bats and suggest that the microbiota may play a protective role against WNS.
In Chapter 2, we studied in a controlled environment the microbiota of a species that
exhibits evidence of resistance with mild WNS symptoms, before and after infection, to
establish the potential response to the disease. The species studied is the big brown bat
(Eptesicus fuscus), whose skin microbiota could play a protective role against infection.
Our results show that the diversity of the microbiota of big brown bats inoculated with
Pd is more variable over time, while the diversity of the microbiota of the control bats
remains stable. Among the most abundant taxa, Pseudomonas and Rhodococcus, two taxa known for their antifungal potential against Pd and other fungi, remained stable during
the experiment. Thus, although inoculation with the Pd fungus destabilized the skin
microbiota, bacteria with antifungal properties were not affected. This study is the first to
demonstrate the potential of the skin microbiota of a bat species for resistance to WNS.
In Chapter 3, the skin microbiome of the little brown bat was evaluated in the natural
environment in the context of WNS, using metagenomics, a higher-resolution approach to
observe the functional potential of the microbiome (functional metagenome). Our results established
that the time since infection has a significant effect on the functional metagenome.
Indeed, bats in the first year after infection have a disrupted functional metagenome that
undergoes a significant loss of functional diversity. However, the functional metagenome
returns to a similar structure and composition to that observed before infection after 10
years. Certain functions detected following infection are associated with genes linked to the
transport and assimilation of metals, known limiting factors for the growth of the fungus.
These genes could therefore have a role to play in resistance or vulnerability to the disease.
Overall, this metagenomics study indicates functional metagenome vulnerability to the
fungus, although the original functional metagenome is reestablished after 10 years. Such
diversified response could impact M. lucifugus resilence.
This thesis provides fundamental knowledge on the skin microbiome of hibernating bats
to better understand the microbial communities of the skin in the context of WNS. The
microbiome could indeed play a role in the vulnerability and resistance of bats to disease
and it is essential to adapt our way of approaching the protection of these species and their
microbiomes. We hope that the results of this thesis will raise awareness among conservation
stakeholders about the existence and potential importance of the microbiome for the health
of its host. This thesis also reports on the advancement of analytical methods that will
make it possible to be more and more precise and to apply knowledge of the microbiome in
conservation biology.
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Metagenomic and Metabolomic Approaches to Determine Contributors to Residual Cardiovascular Disease RiskFerrell, Marc 26 May 2023 (has links)
No description available.
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The Effect of Aluminium Industry Effluents on Sediment Bacterial CommunitiesGill, Hardeep 19 October 2012 (has links)
The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.
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The Effect of Aluminium Industry Effluents on Sediment Bacterial CommunitiesGill, Hardeep 19 October 2012 (has links)
The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.
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The Effect of Aluminium Industry Effluents on Sediment Bacterial CommunitiesGill, Hardeep January 2012 (has links)
The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.
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Identifikation von Genen und Mikroorganismen, die an der dissimilatorischen Fe(III)-Reduktion beteiligt sind / Isolation of Genes and Microorganisms Involved in Dissimilatory Fe(III)-ReductionÖzyurt, Baris 21 January 2009 (has links)
No description available.
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