Les souches humaines de rotavirus du groupe A (RVA) reconnaissent des glycannes fucosylés de la famille des Histo-Blood Group Antigens (HBGAs) et des gangliosides via la protéine de capside VP8*. L’interaction avec les gangliosides est essentielle pour l’entrée cellulaire et l’absence de ligands fucosylés dû au polymorphisme génétique des HBGAs est associée à une résistance à la gastroentérite sévère. Nos objectifs étaient de délimiter la contribution des HBGAs et du ganglioside GM1a dans le processus d’infection et d’explorer les conséquences du polymorphisme des HBGAs sur la transmission du virus et l’efficacité des vaccins vivants disponibles.génoty Ces travaux ont permis de montrer la concordance entre la spécificité glycannique des VP8* P[8], génotype le plus fréquent en France, et la sensibilité HBGA-dépendante à la gastroentérite sévère. La reconnaissance des HBGAs par les souches humaines de RVA apparaît donc essentielle pour l’infection symptomatique. Néanmoins, nos résultats suggèrent que l’attachement aux HBGAs correspond à un événement précoce puisqu’il n’est pas nécessaire pour l’infection de cellules peu différenciées par les souches P[8] adaptées à la culture. La contribution du GM1a dans l’infection reste incertaine. Enfin, nous avons montré que la reconnaissance des HBGAs est conservée entre des souches P[8] récentes et anciennes, indiquant que le polymorphisme des HBGAs pourrait contribuer à expliquer le défaut d’efficacité des vaccins dans les régions où la fréquence d’individus n’exprimant pas les ligands fucosylés est élevée. / Human strains of rotavirus A (RVAs) recognize fucosylated glycans of the histo-blood group family (HBGAs) as well as gangliosides through the VP8* protein of their capsid. Interaction with gangliosides is essential for cell entry and lack of fucosylated ligands due to HBGAs genetic polymorphism is associated with resistance to RVA gastroenteritis. Our goals are to delineate the contribution of HBGAs and gangliosides in the infection process and to explore the consequences of HBGAs polymorphisms on the virus transmission and efficacy of the available live vaccines. This study highlighted the concordance between the glycan specificity of P[8] VP8*, the most common genotype in France, and the HBGA-dependant susceptibility to RVA gastroenteritis. The recognition of HBGAs by human RVA strains therefore appears essential to the infection. Yet, our results suggest that HBGA binding corresponds to an early event since it is not required for infection of poorly differentiated cells by cell culture-adapted P[8] strains. The contribution of GM1a on infection remains unclear. Finally, we showed that HBGA recognition is conserved between recent and older P[8] strains, suggesting that HBGAs polymorphism may contribute to explain the low efficacy of vaccines in areas where the frequency of individuals who do not express fucosylated ligands is high.

No description available.

No description available.

Viruses are common causes of both endemic and epidemic gastroenteritis, infecting millions of people per year, with norovirus, rotavirus and adenovirus-F as the main causative agents, and sapovirus and astrovirus as contributing viruses. These viruses are highly infectious and most severe in the very young, old, or individuals who are immunocompromised. The viral infection usually causes self-limited gastroenteritis, although chronic infection has been observed in highly immunocompromised patients. African and South-East Asian regions are disproportionally affected by diarrhoeal disease. These regions (especially South Africa) are also more severely affected by human immunodeficiency virus (HIV) infections. It has been suggested that immunocompromised individuals may form part of a reservoir for novel virus variants and recombinants. It should be taken into account that not every person is equally susceptible to infection after pathogen exposure and that not all infected persons develop clinical symptoms (Ramani and Giri, 2019). One host genetic factor that can influence susceptibility to enteric infection is the expression of histo-blood group antigens (HBGAs). Histo-blood group antigens are a major group of complex carbohydrates and are determinants of both human and animal ABO blood groups and the Lewis blood group systems, which are distributed in abundance on the mucosal epithelia of the gastrointestinal tract. Histo-blood group antigens have been proven to influence susceptibility to rotavirus and norovirus infections. Saliva, blood and stool specimens (n=205) have previously been collected from children (≤ 5 years of age) hospitalised with gastroenteritis at Kalafong Provincial Tertiary Hospital from June 2016 to December 2017. Follow up stool specimens were then collected six weeks after enrolment when possible. A descriptive questionnaire was completed by each child’s guardian, giving information on age, residential area, HIV status etc. of the participating child. The stool specimens were screened for six gastroenteritis causing viruses (norovirus GI and –GII, rotavirus, sapovirus, astrovirus and adenovirus) by multiplex PCR. Forty-seven percent (96/205) of specimens tested positive for at least one gastroenteritis causing virus. Rotavirus predominated (46/205), followed by norovirus (32/205), adenovirus (15/205), sapovirus (9/205) and astrovirus (3/205). A total of 27/32 norovirus (GI.3, GII.2, GII.3, GII.4, GII.7, GII.12 and GII.21), 44/46 rotavirus (G1P[8], G2P[4], G2P[6], G3P[4], G3P[8], G8P[4], G8P[6], G9P[6] and G9P[8]) and 8/9 sapovirus (GI.1, GI.2, GII.1, GII.4 and GII.8) strains have been genotyped, of which norovirus GII.4 and rotavirus G3P[4] predominated. A total of 46/205 children submitted a follow up stool specimen to be tested. Of the 46 children, 9 tested positive for norovirus infection with initial stool specimen testing. Follow up screening resulted in 13/46 (28%) specimens testing positive for either norovirus GI or GII, with all patients presenting as asymptomatic. After genotyping it was observed that only one of the follow up specimens were identical to the original sequence genotyped, indicating prolonged shedding. FUT2 genotyping of 205/205 children showed a 71%:29% ratio between secretors and non-secretors. Eighty percent (77/96) of the virus-infected children were secretors whereas only 20% (19/96) were non-secretors. Rotavirus (p<0.01) and norovirus GII.4 (p<0.05) specifically were found to be more prevalent in secretors. In this study, no statistical significance was observed in terms of severity of and susceptibility to gastroenteritis viruses between HIV-infected, HIV-exposed uninfected or HIV-uninfected individuals. Histo-blood group phenotyping has resulted in various combinations, with Le(b) being the most prevalent antigen found. Next generation sequencing was unsuccessful. In future, fresh specimens should be considered for testing, with more funding and time for optimisation of this process and to give adequate results. In summary, gastroenteritis is still a leading cause of childhood morbidity and mortality, with all advancements in understanding the disease helping to decrease the impact of it. This study again reinforced the importance of these viruses, as they are circulating in such high abundance. It also reinforced the concept that susceptibility to noro- and rotavirus infection is affected by the secretor status of a person. This could in future help with better understanding the viral infection mechanisms and in turn help with vaccine development and treatment / Dissertation (MSc (Medical Virology))--University of Pretoria, 2020. / Reese Mushrooms / Discovery grant / PRF / Medical Virology / Msc / Unrestricted

Les norovirus sont l'une des causes principales de gastroentérite. Depuis 2002, des variants de norovirus GII.4 successifs ont circulé dans la population par cycle de 2-3 ans, ce qui suscite des interrogations quant au rôle de leurs ligands, les antigènes tissulaires de groupes sanguins (HBGA), dans leur évolution. Nous avons analysé l'interaction entre des variants de GII.4 représentatifs et des HBGA, et déterminé le rôle d’acides aminés (aa) clés. Par mutagénèse dirigée, nous avons montré qu’une configuration stricte des aa directement impliqués dans l’accroche est indispensable. La suppression de la thréonine 395, caractéristique des variants après 2002, confère la capacité de se lier à Lex et Si-Lex, démontrant que les aa en dehors du site de liaison peuvent modifier les propriétés d’attachement. L'analyse de l'accroche de VLP de 6 variants isolés de 1987 à 2007 à des échantillons de salive phénotypés et des HBGA synthétiques montre que tous les variants sont capables de s’attacher à la salive des sécréteurs indépendamment du phénotype ABO et aux oligosaccharides propres au phénotype sécréteur. Deux variants récents ont pu également s’accrocher aux sucres présents dans la salive des nonsécréteurs Le(+). Nos données suggèrent que la capacité de se lier à Lex et Si-Lex serait une conséquence de la variation génétique des aa situés à proximité du site de liaison. L'analyse des propriétés d’attachement par résonance plasmonique de surface a montré que seuls les variants après 2002 présentent une affinité forte pour les antigènes A et B, suggérant que l’accélération évolutive des GII.4 pourrait être liée à une affinité accrue des variants pour les HBGA après 2002. / Noroviruses are one of the leading causes of gastroenteritis worldwide. Since 2002 successive GII.4 variants have circulated in the population before being replaced every 2-3 years, which raises questions about the role of their histo-blood group antigen (HBGAs) receptors in their evolution. We analyzed the interaction between representative GII.4 variants and HBGAs and determined the role of selected amino acids (aa) in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the aa directly implicated in HBGA bindings. The ablation of the threonine 395 residue, an epidemiological feature of the post 2002 variants, allowed to gain the capacity to bind to the Lewis x and sialyl-Lewis x antigens, demonstrating that aa residues outside the HBGA binding site can modify the binding properties. The analysis of the attachment of VLPs from 6 variants isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs shows that all variants could attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, two recent variants additionally bound to carbohydrates present in the saliva of Lewis-positive non-secretors. Our data suggest that GII.4 binding to Lex and Si-Lex antigens might be a by-product of the genetic variation of the aa located in the vicinity of the binding site. Analysis of the binding properties by surface plasmon resonance showed that only post 2002 variants presented a strong affinity for A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post 2002 variants.

No description available.

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