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The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.

Background This study aims to investigate the difference in vaginal microecology, local immunity and HPV infection among childbearing-age women with different degrees of cervical lesions. Methods A total of 432 patients were included in this study. Among these patients, 136 patients had LSIL, 263 patients had HSIL and 33 patients had CSCC. These patients were assigned as the research groups. In addition, 100 healthy females were enrolled and assigned as the control group. Results The microbiological indexes of vaginal secretions were evaluated. Furthermore, the concentrations of SIgA, IgG, IL-2 and IL-10 in vaginal lavage fluid, as well as the presence of HPV, mycoplasma and Chlamydia in cervical secretions, were detected. The results is that: (1) Differences in evaluation indexes of vaginal microecology among all research groups and the control group were statistically significant ( P  < 0.0001). As the degree of cervical lesions increased, the number of Lactobacillus decreased, and there was an increase in prevalence of bacterial imbalance, and the diversity, density and normal proportion of bacteria was reduced. Furthermore, the incidence of HPV, trichomonads, clue cell and Chlamydia infection increased. Moreover, the positive rate of H 2 O 2 decreased, while the positive rates of SNa and GADP increased. (2) Differences in the ratio of IL-2 and IL-10 in the female genital tract among all research groups and the control group were statistically significant ( P  < 0.0001). Conclusions As the degree of cervical lesions increased, IL-2 decreased, IL-10 increased and IL-2/IL-10 decreased, while SIgA and IgG were elevated. The reduction of dominant Lactobacillus in the vagina, impairment of H 2 O 2 function, flora ratio imbalance, pathogen infections, reduction in IL-2/IL-10 ratio, and changes in SIgA and IgG levels could all be potential factors that influenced the pathogenicity of HPV infection and the occurrence and development of cervical lesions.

The hemagglutinin (HA) and neuraminidase (NA) glycoproteins of influenza A virus are responsible for the surface interactions of the virion with the host. Entry of the virus is mediated by functions of the HA: binding to cellular receptors and facilitating fusion of the virion membrane with the endosomal membrane. The HA structure contains receptor binding sites in the globular membrane distal head domains of the trimer, and the fusion machinery resides in the stem region. These sites have specific characteristics associated with subtype and host, and the differences often define species barriers. For example, avian viruses preferentially recognize α2,3-Sialic acid terminating glycans as receptors and mammalian viruses recognize α2,6-Sialic acid. The neuraminidase, or the receptor-destroying protein, cleaves the sialic acid from cellular membrane constituents and viral glycoproteins allowing for egress of nascent virions. A functional balance of activity has been demonstrated between the two glycoproteins, resulting in an optimum level of HA affinity and NA enzymatic cleavage to allow for productive infection. As more is understood about both HA and NA, the relevance for functional balance between HA and NA continues to expand, with potential implications for interspecies transmission, host adaptation, and pathogenicity.

The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those on double-sulfated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the sialyl T-antigen specificity of a sialidase of a previously unknown family. Cell-attached sialidases and fucosidases displayed mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, neither the sialic acid nor fucose contributed to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unprecedented mechanistic insight into the initiation of mucin O -glycan degradation by A. muciniphila and nutrient sharing between mucus-associated bacteria. This study offers molecular insight into the sialidase and fucosidase decapping apparatus that initiates growth on mucin and promotes nutrient sharing by the dedicated mucolytic symbiont Akkermansia muciniphila with the mucus-associated microbiota.

Antibiotic treatment disturbs the commensal microbiota and is often followed by infection with enteric pathogens such as Salmonella typhimurium and Clostridium difficile; pathogen expansion is fuelled by antibiotic-driven accumulation of commensal-liberated host mucosal carbohydrates. Gut microbes support pathogen proliferation Intestinal microbiota can provide protection against invading pathogens through competition for resources and production of specific antimicrobial products. But disruption of the microbiota with antibiotics can contribute to the emergence of several enteric pathogens. Justin Sonnenburg and colleagues show here that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium and Clostridium difficile , catabolize microbiota-liberated host sugars to fuel their growth in the mouse gut. In particular, the ability to use sialic acid cleaved from host polysaccharides by Bacteroides thetaiotaomicron is important for pathogen expansion. These findings identify a role for the gut microbiota in facilitating enteric pathogen infection and provide new options for developing therapeutics. The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens 1 , 2 , 3 , 4 . How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium ( S. typhimurium ) and Clostridium difficile , use a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo . Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo . Colonization of gnotobiotic mice with a sialidase-deficient mutant of Bacteroides thetaiotaomicron , a model gut symbiont, reduces free sialic acid levels resulting in C. difficile downregulating its sialic acid catabolic pathway and exhibiting impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

The His²⁷⁴[rightward arrow]Tyr²⁷⁴ (H274Y) mutation confers oseltamivir resistance on N1 influenza neuraminidase but had long been thought to compromise viral fitness. However, beginning in 2007-2008, viruses containing H274Y rapidly became predominant among human seasonal H1N1 isolates. We show that H274Y decreases the amount of neuraminidase that reaches the cell surface and that this defect can be counteracted by secondary mutations that also restore viral fitness. Two such mutations occurred in seasonal H1N1 shortly before the widespread appearance of H274Y. The evolution of oseltamivir resistance was therefore enabled by \"permissive\" mutations that allowed the virus to tolerate subsequent occurrences of H274Y. An understanding of this process may provide a basis for predicting the evolution of oseltamivir resistance in other influenza strains.

Ruminococcus gnavus is a human gut symbiont wherein the ability to degrade mucins is mediated by an intramolecular trans -sialidase ( Rg NanH). Rg NanH comprises a GH33 catalytic domain and a sialic acid-binding carbohydrate-binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis and binding assays to determine the structure and function of Rg NanH_CBM40 ( Rg CBM40). Rg CBM40 displays the canonical CBM40 β-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. Rg CBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that Rg CBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut. The mucus layer is an important physical niche within the gut which harbours a distinct microbial community. Here the authors show that specific carbohydrate-binding modules associated with bacterial carbohydrate-active enzymes are mucus adhesins that target regions of the distal colon rich in sialomucins.

The intestinal anaerobic bacterium Akkermansia muciniphila is specialized in the degradation of mucins, which are heavily O -glycosylated proteins that constitute the major components of the mucus lining the intestine. Despite that adhesion to mucins is considered critical for the persistence of A. muciniphila in the human intestinal tract, our knowledge of how this intestinal symbiont recognizes and binds to mucins is still limited. Here, we first show that the mucin-binding properties of A. muciniphila are independent of environmental oxygen concentrations and not abolished by pasteurization. We then dissected the mucin-binding properties of pasteurized A. muciniphila by use of a recently developed cell-based mucin array that enables display of the tandem repeats of human mucins with distinct O -glycan patterns and structures. We found that A. muciniphila recognizes the unsialylated LacNAc (Galβ1-4GlcNAcβ1-R) disaccharide selectively on core2 and core3 O -glycans. This disaccharide epitope is abundantly found on human colonic mucins capped by sialic acids, and we demonstrated that endogenous A. muciniphila neuraminidase activity can uncover the epitope and promote binding. In summary, our study provides insights into the mucin-binding properties important for colonization of a key mucin-foraging bacterium. Intestinal mucus consists of densely O-glycosylated mucins, serving as a nutrient source for bacteria. Elzinga et al. show that mucin-degrading Akkermansia muciniphila selectively binds to O-glycan structures found on human colonic mucins.

The functions of the influenza virus neuraminidase has been well documented but those of the mammalian neuraminidases remain less explored. Here, we characterize the role of neuraminidase 1 (NEU1) in unilateral ureteral obstruction (UUO) and folic acid (FA)-induced renal fibrosis mouse models. We find that NEU1 is significantly upregulated in the fibrotic kidneys of patients and mice. Functionally, tubular epithelial cell-specific NEU1 knockout inhibits epithelial-to-mesenchymal transition, inflammatory cytokines production, and collagen deposition in mice. Conversely, NEU1 overexpression exacerbates progressive renal fibrosis. Mechanistically, NEU1 interacts with TGFβ type I receptor ALK5 at the 160-200aa region and stabilizes ALK5 leading to SMAD2/3 activation. Salvianolic acid B, a component of Salvia miltiorrhiza , is found to strongly bind to NEU1 and effectively protect mice from renal fibrosis in a NEU1-dependent manner. Collectively, this study characterizes a promotor role for NEU1 in renal fibrosis and suggests a potential avenue of targeting NEU1 to treat kidney diseases. The influenza virus neuraminidase has been well documented, yet the functions of mammalian neuraminidases remain less explored. Here, the authors show that neuraminidase 1 promotes renal fibrosis development by interacting with ALK5 to activate SMAD2/3.

Four human sialidases (hNEUs, E.C 3.2.1.18) have been identified. Each is an exosialidase identified as either NEU1, NEU2, NEU3, or NEU4. They exhibit differences in structure, subcellular distribution, substrate specificity, and the diseases with which they are associated. Similarly, microbial sialidases (NAs) may catalyze the release of sialyl residues from the same sialoglycoconjugates as hNEUs, even though they have low sequence homology with human NEUs. Use of sequence homology, plus the crystalline structure of human NEU2, has provided researchers with the basis for developing inhibitors that may differentiate between them. While microbial-induced diseases that use sialidase to complete their infectious cycle have been the driving force behind interrogation of possible NA inhibitors, errors affecting expression of functional hNEUs and their correlation with clinical problems has led to study of the sialidases per se. Information gained about sialidase structure, function, mechanism of action, mutations affecting expression, and their role(s) in disease, has provided the information about the different sialidases needed for development of specific therapies.