Hacia la caracterización del epítopo del autoantígeno en el Síndrome de Goodpasture

El síndrome de Goodpasture (GP) es un desorden autoinmune que cursa con glomerulonefritis rápidamente progresiva y hemorragia pulmonar. El ataque inmunológico se lleva a cabo mediante (auto)-anticuerpos (anticuerpos GP) dirigidos contra el dominio C terminal, no colagenoso (NC1), de la cadena α3 del colágeno IV, presente en la membrana basal glomerular (GBM), así como en la membrana basal alveolar. Tres cadenas α se asocian para construir una molécula de colágeno IV, que recibe el nombre de protómero, dos protómeros de colágeno IV se unen a través de sus extremos NC1 para formar una estructura cuaternaria de naturaleza hexamérica (hexámero) en la red de colágeno IV. Estudios cristalográficos han mostrado que en la estabilización del hexámero participan tanto interacciones hidrofóbicas, como interacciones hidrofílicas que junto con enlaces covalentes tipo sulfilimina entre protómeros opuestos sellan el hexámero. Lo que se conoce del epítopo GP son dos regiones, llamadas EA y EB, en las que se identificaron los residuos Ala18, Ile19, Val27 y Pro28 conformando parte del epítopo patogénico. Así, éste parece ubicarse en la interfaz de los dominio NC1 que forman el hexámero de la red de colágeno de la GBM, resultando en un epítopo críptico e inaccesible para los anticuerpos GP. Cuando se disocia el hexámero, estos aminoácidos se tornan accesibles a los anticuerpos GP. En contraste, otros residuos que forman parte del epítopo, como los residuos hidrofílicos Ser21 y Ser 31, así como la Pro28, son accesibles a los anticuerpos GP tanto si el dominio NC1 se encuentra en forma hexamérica como monomérica. Un aspecto relevante en la enfermedad GP, es el mecanismo mediante el cual los autoanticuerpos tienen acceso al epítopo. En este sentido, la proteína de unión al antígeno GP (GPBP) podría jugar un papel clave en el plegamiento de las cadenas de colágeno IV condicionando la diferente accesibilidad del antígeno GP. Objetivos El objetivo general de este trabajo persigue esclarecer la etiología de la enfermedad Goodpasture y profundizar en el conocimiento del epítopo de los autoanticuerpos y en la estructura 3D y ensamblaje de los componentes proteicos que forman el heterohexámero (α3,4,5)2(IV)NC1 y albergan el epítopo. 1 Caracterización mediante tecnología phage display y bioinformática del epítopo de los autoanticuerpos GP, ya localizado en el dominio α3(IV)NC1 del colágeno tipo IV. Mientras el estudio del epítopo GP se había abordado clásicamente a partir del análisis del antígeno, aquí obtenemos información del epítopo GP a partir del análisis de los autoanticuerpos GP patogénicos. Para ello, (1) ponemos a punto un sistema de expresión que usa células de insecto y baculovirus que permite la producción recombinante de α3(IV)NC1 y del resto de cadenas proteicas del colágeno tipo IV humano en forma abundante, funcional y estructuralmente competente; y (2) aplicamos la tecnología phage display y procedimientos bioinformáticos para cartografiar el epítopo para los anticuerpos circulantes de un paciente Goodpasture sobre un modelo de la estructura 3D del heterohexámero (α3,4,5)2(IV)NC1. 2 Establecer las bases estructurales de los dominios NC1 del colágeno IV para comprender la biología y patología asociadas al colágeno IV en las membranas basales. Cristalizamos los seis α(IV)NC1 y varias asociaciones de ellos para, mediante la resolución de sus respectivas estructuras a nivel atómico, entender el proceso de ensamblaje y formación del protómero en el colágeno IV, así como de las interacciones que lo mantienen y que lo preservan del ataque de los anticuerpos GP. 3 Implicación de GPBP-1 en el ensamblaje del colágeno tipo IV y consecuencias inmunopatogénicas. Mostramos mediante la coexpresión simultánea de α3(IV)NC1 y GPBP-1 y estudios de resonancia de plasmón de superficie, de estructura de proteínas por cristalografía de rayos X y de caracterización inmunoquímica y de mutagénesis dirigida, que GPBP-1 modifica el plegamiento de α3(IV)NC1 y la exposición del epítopo de anticuerpos patogénicos. Resultados y conclusiones. Objetivo 1. Hemos aplicado la tecnología phage display por primera vez en la caracterización del epítopo de los anticuerpos GP. Hemos seleccionado péptidos específicos (fagotopos) para los autoanticuerpos GP a partir de dos librerías phage display-M13 de péptidos combinatoriales comerciales que hipotéticamente mimetizan las propiedades físico-químicas y la organización espacial de los auto-epítopos. En base a esta información, hemos generado quimeras, preparadas a partir de la proteína α2(IV)NC1, que son reconocidas por los anticuerpos GP con similar intensidad a como lo hacen con la diana natural α3(IV)NC1. Nuestros resultados, no sólo corroboraron hallazgos previos en la definición del epítopo sino que identificaron nuevos residuos (Thr26, Tyr30, Thr127, Pro131, His134, Lys141, Thr99 y Ala196) involucrados en la unión de los autoanticuerpos GP. Además de proporcionar nueva información estructural sobre los epítopos GP, proponemos el uso de nuevos compuestos (péptidos) con capacidad de bloquear la unión de los autoanticuerpos GP a su autoantígeno “in vivo” y aportamos datos cinéticos de la interacción antígeno-anticuerpo que, eventualmente, podrían resultar críticos en la generación de agentes bloqueantes terapéuticos. Objetivo 2. Pretendíamos establecer la estructura del heterohexámero (α3,4,5)2(IV)NC1, que es la diana de los autoanticuerpos en el síndrome de Goodpasture, y trasladar los hallazgos sobre el epítopo obtenidos en el objetivo 1 a una estructura hasta entonces desconocida. No tuvimos éxito en cristalizar tal heterohexámero probablemente por la asombrosa facilidad de uno de sus componentes (α5(IV)NC1) para cristalizar de forma aislada. Sí resolvimos, sin embargo, la estructura atómica, mediante cristalografía de rayos X, de homohexámeros de (α1(IV)NC1), (α3(IV)NC1), (α4(IV)NC1) y α5(IV)NC1, así como de heterohexámeros de ((α1)2(α2))2(IV)NC1 y de (α5,6,6)2(IV)NC1. La estructura de cada uno de los homohexámeros, excepto el de (α4(IV)NC1), junto con aquella de ambos heterohexámeros, resultó ser extremadamente similar, con un valor RMSD de superposición inferior a 0,8 Å. Todas tienen una estructura hexamérica cuaternaria típica para los α(IV)NC1, ya observada en el heterohexámero ((α1)2(α2))2(IV)NC1 encontrado en la placenta humana (PDB id:1LI1) y bovina (PDB id:1T61) y en la cápsula de cristalino bovino (PDB id:1M3D, 1T60). Esto demostraba que las nuevas estructuras aquí obtenidas poseen estructura cuaternaria fisiológica, validando, por tanto, el sistema de expresión que hemos usado, así como la idoneidad de los procesos de purificación y cristalización seguidos. El cristal del heterohexámero (α5,6,6)2(IV)NC1 reveló que este protómero no está constituido por dos moléculas de α5(IV)NC1 y una de α6(IV)NC1, como se creía, sino por dos moléculas de α6(IV)NC1 y una de α5(IV)NC1. La estructura de α4(IV)NC1 mostró claras diferencias respecto al resto de homohexámeros, evidenciando que las proteínas α(IV)NC1 pares exhiben menor plasticidad, lo que probablemente sea la causa de y limite su tendencia a la autoasociación. Las diferencias estructurales en α4(IV)NC1 impiden la formación del protómero y del hexámero al cambiar y limitar las interacciones que se establecen entre diferentes regiones en el resto de proteínas α(IV)NC1. El análisis de los heterohexámeros ((α1)2(α2))2(IV)NC1 y (α5,6,6)2(IV)NC1 reveló unas estructuras muy similares al resto de monómeros α(IV)NC1 permitiendo comprender que, una vez los α(IV)NC1 pares interaccionan con α(IV)NC1 impares, modifican su conformación para favorecer la formación del protómero, así como para proveerle estabilidad. La información estructural nos permitió concluir que los anticuerpos patogénicos GP se dirigen a dos regiones en α3(IV)NC1 que comprenden los residuos 15-TAIPSCPEGTVPLYS-29 (región EA) y 125-TDIPPCPHGWISLWK-139 (región EB), dos regiones estructuralmente rígidas situadas en el ápice de los dos dominios estructurales C4. Objetivo 3. Presentamos evidencias de que GPBP-1 influencia la estructura de α3(IV)NC1, como se observa, tras la co-expresión de ambas proteínas, en la estructura cristalina a nivel atómico de α3(IV)NC1, en la caracterización termodinámica de la interacción de α3(IV)NC1 con anticuerpos conformacionales y en su reconocimiento inmunoquímico. Mientras que los resultados inmunoquímicos y mediante SPR son indicativos de que GPBP-1 influencia la conformación de α3(IV)NC1, el estudio estructural de la proteína α3(IV)NC1 expresada y no expresada junto a GPBP-1, claramente revela la naturaleza de los cambios mediados por la presencia de la cinasa. GPBP-1 promueve la generación de cristales diferentes (en términos de grupo espacial y moléculas en la unidad asimétrica) a aquellos constituidos por un solo tipo de moléculas de α3(IV)NC1 (obtenidas en ausencia de GPBP-1 y que son competentes para ensamblarse en hexámeros) por contener también un segundo tipo de molécula de α3(IV)NC1 modificada (α3(IV)NC1c), sólo encontrada cuando su expresión ocurre junto a GPBP-1, y cuya capacidad de interacción se restringe a trímeros. Los cambios estructurales observados se localizan esencialmente en dos lazos de las regiones I y VI del dominio C4 condicionando la capacidad de ensamblaje de los trímeros en hexámeros. Este hecho se evidencia mejor en el ensanchamiento general de los trímeros originados por α3(IV)NC1c que impide el ensamblaje con el trímero vecino para formar el hexámero. Además, uno de estos lazos alberga la Lys211 involucrada en un enlace sulfilimina que da estabilidad al hexámero y restringe la accesibilidad de los autoanticuerpos GP por lo que se le atribuye un papel substancial en la patogenia del síndrome GP. La predicción para un heterohexámero (α3,4,5)2(IV)NC1 que contuviese una molécula de α3(IV)NC1c apuntaría a que se produjese una perturbación de las interacciones con las moléculas de α4(IV)NC1 y α5(IV)NC1 vecinas dentro del heterohexámero y una abolición de los enlaces sulfilimina, convirtiéndolo, de este modo, más susceptible al acceso de los anticuerpos GPc. La observación de que la reactividad de los autoanticuerpos GP sea mucho mayor hacia homohexámeros de α3(IV)NC1 que se han expresado simultáneamente con GPBP-1 que frente a homohexámeros de α3(IV)NC1 preparados en ausencia de GPBP o frente a formas monoméricas de α3(IV)NC1 bajo cualquier circunstancia, es consistente con el conocimiento presente del autoepítopo y refuerza la propuesta del papel de la cinasa en la etiología de la enfermedad Goodpasture, quizás facilitando la exposición del epítopo, la activación del sistema inmune y la producción de autoanticuerpos patogénicos. Los resultados de mutagénesis dirigida tanto en α3(IV)NC1 como en GPBP-1 se complementan y confirman que las modificaciones estructurales observadas en α3(IV)NC1 cuando se co-expresa junto a GPBP-1 se deben a la presencia de la cinasa. Estos resultados son consistentes con la observación de que la expresión elevada de GPBP-1 in vivo desorganiza la MBG, lo que podría permitir la exposición de epítopos crípticos y desencadenar un proceso autoinmune en el síndrome de Goodpasture y/o un proceso inflamatorio que culminaría en una Glomerulonefritis

Structural investigations on novel non-nucleoside inhibitors of human norovirus polymerase

Human norovirus is the first cause of foodborne disease worldwide, leading to extensive outbreaks of acute gastroenteritis, and causing around 200,000 children to die annually in developing countries. No specific vaccines or antiviral agents are currently available, with therapeutic options limited to supportive care to prevent dehydration. The infection can become severe and lead to life-threatening complications in young children, the elderly and immunocompromised individuals, leading to a clear need for antiviral agents, to be used as treatments and as prophylactic measures in case of outbreaks. Due to the key role played by the viral RNA-dependent RNA polymerase (RdRp) in the virus life cycle, this enzyme is a promising target for antiviral drug discovery. In previous studies, following in silico investigations, we identified different small-molecule inhibitors of this enzyme. In this study, we rationally modified five identified scaffolds, to further explore structure-activity relationships, and to enhance binding to the RdRp. The newly designed compounds were synthesized according to multiple-step synthetic routes and evaluated for their inhibition of the enzyme in vitro. New inhibitors with low micromolar inhibitory activity of the RdRp were identified, which provide a promising basis for further hit-to-lead optimization

The rotavirus vaccine landscape, an update

Rotavirus is the leading cause of severe acute childhood gastroenteritis, responsible for more than 128,500 deaths per year, mainly in low-income countries. Although the mortality rate has dropped significantly since the introduction of the first vaccines around 2006, an estimated 83,158 deaths are still preventable. The two main vaccines currently deployed, Rotarix and RotaTeq, both live oral vaccines, have been shown to be less effective in developing countries. In addition, they have been associated with a slight risk of intussusception, and the need for cold chain maintenance limits the accessibility of these vaccines to certain areas, leaving 65% of children worldwide unvaccinated and therefore unprotected. Against this backdrop, here we review the main vaccines under development and the state of the art on potential alternatives

Relevance of secretor status genotype and microbiota composition in susceptibility to rotavirus and norovirus infections in humans

Host genetic factors, such as histo-blood group antigens (HBGAs), are associated with susceptibility to norovirus (NoV) and rotavirus (RV) infections. Recent advances point to the gut microbiome as a key player necessary for a viral pathogen to cause infection. In vitro NoV attachment to host cells and resulting infections have been linked to interactions with certain bacterial types in the gut microbiota. We investigated the relationship between host genotype, gut microbiota, and viral infections. Saliva and fecal samples from 35 adult volunteers were analysed for secretor status genotype, the gut microbiota composition by 16S rRNA gene sequencing, and salivary IgA titers to NoV and RV. Higher levels of IgA against NoV and RV were related to secretor-positive status. No significant differences were found between the FUT2 genotype groups, although the multivariate analysis showed a significant impact of host genotype on specific viral susceptibilities in the microbiome composition. A specific link was found between the abundance of certain bacterial groups, such as Faecalibacterium and Ruminococcus spp., and lower IgA titers against NoV and RV. As a conclusion, we can state that there is a link between host genetics, gut microbiota, and susceptibility to viral infections in humans

Recombinant Noroviruses Circulating in Spain from 2016 to 2020 and Proposal of Two Novel Genotypes within Genogroup I

Noroviruses are the leading cause of sporadic cases and outbreaks of viral gastroenteritis. For more than 20 years, most norovirus infections have been caused by the pandemic genotype GII.4, yet recent studies have reported the emergence of recombinant strains in many countries. In the present study, 4,950 stool samples collected between January 2016 and April 2020 in Valencia, Spain, from patients with acute gastroenteritis were analyzed to investigate the etiological agent. Norovirus was the most frequently detected enteric virus, with a positivity rate of 9.5% (471/4,950). Among 224 norovirus strains characterized, 175 belonged to genogroup II (GII) and 49 belonged to GI. Using dual genotyping based on sequencing of the open reading frame 1 (ORF1)/ORF2 junction region, we detected 25 different capsid-polymerase-type associations. The most common GII capsid genotype was GII.4 Sydney 2012, followed by GII.2, GII.3, GII.6, and GII.17. A high prevalence of recombinant strains (90.4%) was observed among GII infections between 2018 and 2020. GII.4 Sydney[P16] was the predominant genotype from 2019 to 2020. In addition, GII.P16 polymerase was found harbored within six different capsid genes. GI.4 and GI.3 were the predominant genotypes in genogroup I, in which recombinant strains were also found, such as GI.3[P10], GI.3[P13], and GI.5[P4]. Interestingly, applying the criterion of 2 times the standard deviation, we found that 12 sequences initially classified as GI.3 may represent two new tentative genotypes in genogroup I, designated GI.10 and GI.11. This study shows the extensive diversity of recombinant noroviruses circulating in Spain and highlights the role of recombination events in the spread of noroviruses

The role of histo-blood group antigens and microbiota in human norovirus replication in zebrafish larvae

Human norovirus (HuNoV) is the major agent for viral gastroenteritis, causing >700 million infections yearly. Fucose-containing carbohydrates named histo-blood group antigens (HBGAs) are known (co)receptors for HuNoV. Moreover, bacteria of the gut microbiota expressing HBGA-like structures have shown an enhancing effect on HuNoV replication in an in vitro model. Here, we studied the role of HBGAs and the host microbiota during HuNoV infection in zebrafish larvae. Using whole-mount immunohistochemistry, we visualized the fucose expression in the zebrafish gut for the HBGA Lewis X [LeX, α(1,3)-fucose] and core fucose [α(1,6)-fucose]. Costaining of HuNoV-infected larvae proved colocalization of LeX and to a lower extent core fucose with the viral capsid protein VP1, indicating the presence of fucose residues on infected cells. Upon blocking of fucose expression by a fluorinated fucose analogue, HuNoV replication was strongly reduced. Furthermore, by comparing HuNoV replication in conventional and germfree zebrafish larvae, we found that the natural zebrafish microbiome does not have an effect on HuNoV replication, contrary to earlier reports about the human gut microbiome. Interestingly, monoassociation with the HBGA-expressing Enterobacter cloacae resulted in a minor decrease in HuNoV replication, which was not triggered by a stronger innate immune response. Overall, we show here that fucose has an essential role for HuNoV infection in zebrafish larvae, as in the human host, but their natural gut microbiome does not affect viral replication. IMPORTANCE Despite causing over 700 million infections yearly, many gaps remain in the knowledge of human norovirus (HuNoV) biology due to an historical lack of efficient cultivation systems. Fucose-containing carbohydrate structures, named histo-blood group antigens, are known to be important (co)receptors for viral entry in humans, while the natural gut microbiota is suggested to enhance viral replication. This study shows a conserved mechanism of entry for HuNoV in the novel zebrafish infection model, highlighting the pivotal opportunity this model represents to study entry mechanisms and identify the cellular receptor of HuNoV. Our results shed light on the interaction of HuNoV with the zebrafish microbiota, contributing to the understanding of the interplay between gut microbiota and enteric viruses. The ease of generating germfree animals that can be colonized with human gut bacteria is an additional advantage of using zebrafish larvae in virology. This small animal model constitutes an innovative alternative to high-severity animal models

In vitro assessment of the combined effect of letermovir and sirolimus on cytomegalovirus replication

Introduction. Letermovir (LMV) is used for prophylaxis of cytomegalovirus (CMV) reactivation and end-organ disease in adult CMV-seropositive allogeneic hematopoietic stem cell transplant recipients (allo-HSCT). In turn, sirolimus (SLM) which displays in vitro anti-CMV activity, is frequently employed for prophylaxis of Graft vs. Host disease in allo-HSCT. Here, we aimed at assessing whether LMV and SLM used in combination may act synergistically in vitro on inhibiting CMV replication. Material and methods. The antiviral activity of LMV and SLM alone or in combination was evaluated by a checkerboard assay, using ARPE-19 cells infected with CMV strain BADrUL131-Y. LMV and SLM were used at concentrations ranging from 24 nM to 0.38 nM and 16 nM to 0.06 nM, respectively. Results. The mean EC50 for LMV and SLM was 2.44 nM (95% CI, 1.66-3.60) and 1.40 nM (95% CI, 0.41-4.74), respective. LMV and SLM interaction yielded mainly additive effects over the range of concentrations tested. Conclusion. The additive nature of the combination of LMV and SLM against CMV may have relevant clinical implications in management of CMV infection in allo-HSCT recipients undergoing prophylaxis with LMV

Infant gut microbiota modulation by human milk disaccharides in humanized microbiome mice

Human milk glycans present a unique diversity of structures that suggest different mechanisms by which they may affect the infant microbiome development. A humanized mouse model generated by infant fecal transplantation was utilized here to evaluate the impact of fucosyl-α1,3-GlcNAc (3FN), fucosyl-α1,6-GlcNAc, lacto-N-biose (LNB) and galacto-N-biose on the fecal microbiota and host-microbiota interactions. 16S rRNA amplicon sequencing showed that certain bacterial genera significantly increased (Ruminococcus and Oscillospira) or decreased (Eubacterium and Clostridium) in all disaccharide-supplemented groups. Interestingly, cluster analysis differentiates the consumption of fucosyl-oligosaccharides from galactosyl-oligosaccharides, highlighting the disappearance of Akkermansia genus in both fucosyl-oligosaccharides. An increment of the relative abundance of Coprococcus genus was only observed with 3FN. As well, LNB significantly increased the relative abundance of Bifidobacterium, whereas the absolute levels of this genus, as measured by quantitative real-time PCR, did not significantly increase. OTUs corresponding to the species Bifidobacterium longum, Bifidobacterium adolescentis and Ruminococcus gnavus were not present in the control after the 3-week intervention, but were shared among the donor and specific disaccharide groups, indicating that their survival is dependent on disaccharide supplementation. The 3FN-feeding group showed increased levels of butyrate and acetate in the colon, and decreased levels of serum HDL-cholesterol. 3FN also down-regulated the pro-inflammatory cytokine TNF-α and up-regulated the anti-inflammatory cytokines IL-10 and IL-13, and the Toll-like receptor 2 in the large intestine tissue. The present study revealed that the four disaccharides show efficacy in producing beneficial compositional shifts of the gut microbiota and in addition, the 3FN demonstrated physiological and immunomodulatory roles

Replication of Human Norovirus in Mice after Antibiotic-Mediated Intestinal Bacteria Depletion

Human noroviruses (HuNoVs) are the main cause of acute gastroenteritis causing more than 50,000 deaths per year. Recent evidence shows that the gut microbiota plays a key role in enteric virus infectivity. In this context, we tested whether microbiota depletion or microbiota replacement with that of human individuals susceptible to HuNoVs infection could favor viral replication in mice. Four groups of mice (n = 5) were used, including a control group and three groups that were treated with antibiotics to eliminate the autochthonous intestinal microbiota. Two of the antibiotic-treated groups received fecal microbiota transplantation from a pool of feces from infants (age 1-3 months) or an auto-transplantation with mouse feces that obtained prior antibiotic treatment. The inoculation of the different mouse groups with a HuNoVs strain (GII.4 Sydney [P16] genotype) showed that the virus replicated more efficiently in animals only treated with antibiotics but not subject to microbiota transplantation. Viral replication in animals receiving fecal microbiota from newborn infants was intermediate, whereas virus excretion in feces from auto-transplanted mice was as low as in the control mice. The analysis of the fecal microbiota by 16S rDNA NGS showed deep variations in the composition in the different mice groups. Furthermore, differences were observed in the gene expression of relevant immunological mediators, such as IL4, CXCL15, IL13, TNFα and TLR2, at the small intestine. Our results suggest that microbiota depletion eliminates bacteria that restrict HuNoVs infectivity and that the mechanism(s) could involve immune mediators

Infant Gut Microbial Metagenome Mining of α-l-Fucosidases with Activity on Fucosylated Human Milk Oligosaccharides and Glycoconjugates

The gastrointestinal microbiota members produce α-l-fucosidases that play key roles in mucosal, human milk, and dietary oligosaccharide assimilation. Here, 36 open reading frames (ORFs) coding for putative α-l-fucosidases belonging to glycosyl hydrolase family 29 (GH29) were identified through metagenome analysis of breast-fed infant fecal microbiome. Twenty-two of those ORFs showed a complete coding sequence with deduced amino acid sequences displaying the highest degree of identity with α-l-fucosidases from Bacteroides thetaiotaomicron, Bacteroides caccae, Phocaeicola vulgatus, Phocaeicola dorei, Ruminococcus gnavus, and Streptococcus parasanguinis. Based on sequence homology, 10 α-l-fucosidase genes were selected for substrate specificity characterization. The α-l-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on α1,3/4-linked fucose present in Lewis blood antigens and the human milk oligosaccharide (HMO) 3-fucosyllactose. In addition, Fuc1584 also hydrolyzed fucosyl-α-1,6-N-acetylglucosamine (6FN), a component of the core fucosylation of N-glycans. Fuc35A and Fuc193 showed activity on α1,2/3/4/6 linkages from H type-2, Lewis blood antigens, HMOs and 6FN. Fuc30 displayed activity only on α1,6-linked l-fucose, and Fuc5372 showed a preference for α1,2 linkages. Fuc2358 exhibited a broad substrate specificity releasing l-fucose from all the tested free histo-blood group antigens, HMOs, and 6FN. This latest enzyme also displayed activity in glycoconjugates carrying lacto-N-fucopentaose II (Lea) and lacto-N-fucopentaose III (Lex) and in the glycoprotein mucin. Fuc18, Fuc19A, and Fuc39 also removed l-fucose from neoglycoproteins and human α-1 acid glycoprotein. These results give insight into the great diversity of α-l-fucosidases from the infant gut microbiota, thus supporting the hypothesis that fucosylated glycans are crucial for shaping the newborn microbiota composition