NanI Sialidase Enhances the Action of Clostridium perfringens Enterotoxin in the Presence of Mucus.

Clostridium perfringens enterotoxin (CPE) is the main virulence factor for C. perfringens type F strains to cause human gastrointestinal diseases, which can involve lethal enterotoxemia. During type F disease, CPE encounters an adherent mucus layer overlying the intestines, so the current study evaluated if NanI potentiates CPE activity in the presence of adherent mucus. CPE alone caused more cytotoxicity transepithelial electrical resistance (TEER) and permeability to fluorescent dextran (FD) for minimal mucus-producing HT29 cells versus that in their derivative HT29-MTX-E12 cells, which produce abundant adherent mucus. However, for HT29-MTX-E12 cells, the presence of NanI significantly increased CPE binding and pore formation, which enhanced their sensitivity to CPE effects on cytotoxicity, TEER, and FD permeability. When the ability of NanI to potentiate CPE-induced enterotoxemia was then tested in a mouse small intestinal loop enterotoxemia model, a pathophysiologically relevant 50 µg/mL dose of CPE did not kill mice. However, the copresence of purified NanI resulted in significant CPE-induced lethality. More CPE was detected in the sera of mice challenged with 50 µg/mL of CPE when NanI was copresent during challenge. The copresence of NanI and CPE during challenge also significantly increased intestinal histologic damage compared to that after challenge with CPE alone, suggesting that NanI enhancement of CPE-induced intestinal damage may increase CPE absorption into blood. Overall, these results indicate that (i) mucus inhibits CPE action and (ii) NanI can potentiate CPE action in the presence of mucus, which may help explain why type F strains that produce relatively low levels of CPE are still pathogenic. IMPORTANCE NanI is a sialidase produced by some Clostridium perfringens type F strains. Here, we found that NanI can significantly increase the action of C. perfringens enterotoxin (CPE), which is the main toxin responsible for severe human enteric disease caused by type F strains. This effect likely helps to explain why even some type F strains that produce small amounts of CPE are pathogenic.

NanI Sialidase Contributes to the Growth and Adherence of Clostridium perfringens Type F Strain F4969 in the Presence of Adherent Mucus.

Clostridium perfringens type F strains causing nonfoodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence and growth using Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI-carrying versus non-nanI-carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, versus their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells versus HT29 cells. Those effects involved mucus production by HT29-MTX-E12 cells since mucus reduction using N-acetyl cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.

Complement Decay-Accelerating Factor is a modulator of influenza A virus lung immunopathology.

Clearance of viral infections, such as SARS-CoV-2 and influenza A virus (IAV), must be fine-tuned to eliminate the pathogen without causing immunopathology. As such, an aggressive initial innate immune response favors the host in contrast to a detrimental prolonged inflammation. The complement pathway bridges innate and adaptive immune system and contributes to the response by directly clearing pathogens or infected cells, as well as recruiting proinflammatory immune cells and regulating inflammation. However, the impact of modulating complement activation in viral infections is still unclear. In this work, we targeted the complement decay-accelerating factor (DAF/CD55), a surface protein that protects cells from non-specific complement attack, and analyzed its role in IAV infections. We found that DAF modulates IAV infection in vivo, via an interplay with the antigenic viral proteins hemagglutinin (HA) and neuraminidase (NA), in a strain specific manner. Our results reveal that, contrary to what could be expected, DAF potentiates complement activation, increasing the recruitment of neutrophils, monocytes and T cells. We also show that viral NA acts on the heavily sialylated DAF and propose that the NA-dependent DAF removal of sialic acids exacerbates complement activation, leading to lung immunopathology. Remarkably, this mechanism has no impact on viral loads, but rather on the host resilience to infection, and may have direct implications in zoonotic influenza transmissions.

[Enzyme activity of neuraminidase 1 correlated with the biological behavior of prostate cancer PC3 and DU145 cell lines].

OBJECTIVE: To investigate the effects of the enzyme activity of neuraminidase 1 (Neu1) on the biological behavior of prostate cancer PC3 and DU145 cell lines. METHODS: We detected the expression of Neul in the prostate cancer PC3 and DU145 cell lines by Western blot. Using sialidase inhibitors and antibody blocking, we suppressed the enzyme activity of Neu1 and then measured the proliferation and invasiveness of the two cell lines by CCK-8 and Transwell assay, respectively. RESULTS: No statistically significant difference was found in the Neu1 expression between the PC3 and DU145 cell lines. The proliferation and invasiveness of the two types of cells were both increased after inhibition of the Neu1 enzyme activity. CONCLUSIONS: The enzyme activity of Neu1 is correlated with the biological behavior of prostate cancer PC3 and DU145 cells and capable of inhibiting the proliferation and invasiveness of the two types of cells.

The sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid is a glucose-dependent potentiator of insulin secretion.

Sialidase cleaves sialic acid residues from a sialoglycoconjugate: oligosaccharides, glycolipids and glycoproteins that contain sialic acid. Histochemical imaging of the mouse pancreas using a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe used to assess sialidase activity, showed that pancreatic islets have intense sialidase activity. The sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) remarkably enhances glutamate release from hippocampal neurons. Since there are many similar processes between synaptic vesicle exocytosis and secretory granule exocytosis, we investigated the effect of DANA on insulin release from ß-cells. Insulin release was induced in INS-1D cells by treatment with 8.3 mM glucose, and the release was enhanced by treatment with DANA. In a mouse intraperitoneal glucose tolerance test, the increase in serum insulin levels was enhanced by intravenous injection with DANA. However, under fasting conditions, insulin release was not enhanced by treatment with DANA. Calcium oscillations induced by 8.3 mM glucose treatment of INS-1D cells were not affected by DANA. Blood insulin levels in sialidase isozyme Neu3-deficient mice were significantly higher than those in WT mice under ad libitum feeding conditions, but the levels were not different under fasting conditions. These results indicate that DANA is a glucose-dependent potentiator of insulin secretion. The sialidase inhibitor may be useful for anti-diabetic treatment with a low risk of hypoglycemia.

Sialidase NEU1 suppresses progression of human bladder cancer cells by inhibiting fibronectin-integrin α5ß1 interaction and Akt signaling pathway.

BACKGROUND: Sialic acids are widely distributed in animal tissues, and aberrantly expressed in a variety of cancer types. High expression of sialic acid contributes to tumor aggressiveness by promoting cell proliferation, migration, angiogenesis, and metastasis. Sialidases are responsible for removal of sialic acids from glycoproteins and glycolipids. METHODS: N-glycomics of bladder cancer cells were detected by MALDI-TOF mass spectrometry. Sialic acid modification in bladder cancer tissue was determined by lectin blot. The down-regulation of NEU1 in bladder cancer cells was determined by high resolution liquid chromatography mass spectrometry (HR LC-MS). The effects of sialidase NEU1 expression on proliferation and apoptosis of human bladder cancer cells were examined by western blot, RT-PCR, confocal imaging and flow cytometry. Moreover, the function of sialic acids on fibronectin-integrin α5ß1 interaction were assayed by immunoprecipitation and ELISA. The importance of NEU1 in tumor formation in vivo was performed using BALB/c-nu mice. Expression of NEU1 in primary human bladder cancer tissue samples was estimated using bladder cancer tissue microarray. RESULTS: (1) Downregulation of NEU1 was primarily responsible for aberrant expression of sialic acids in bladder cancer cells. (2) Decreased NEU1 expression was correlated with bladder cancer progression. (3) NEU1 overexpression enhanced apoptosis and reduced proliferation of bladder cancer cells. (4) NEU1 disrupted FN-integrin α5ß1 interaction and deactivated the Akt signaling pathway. (5) NEU1 significantly suppressed in vivo tumor formation in BALB/c-nu mice. CONCLUSIONS: Our data showed that NEU1 inhibited cancer cell proliferation, induced apoptosis, and suppressed tumor formation both in vitro and in vivo, by disrupting interaction of FN and integrin ß1 and inhibiting the Akt signaling pathway. Our observations indicate that NEU1 is an important modulator of the malignant properties of bladder cancer cells, and is a potential therapeutic target for prognosis and treatment of bladder cancer. Video Abstract.

Aberrant sialylation in ovarian cancers.

Sialylation (the covalent addition of sialic acid to the terminal end of glycoproteins or glycans), tightly regulated cell- and microenvironment-specific process and orchestrated by sialyltransferases and sialidases (neuraminidases) family, is one of the posttranslational modifications, which plays an important biological role in the maintenance of normal physiology and involves many pathological dysfunctions. Glycans have roles in all the cancer hallmarks, referring to capabilities acquired during all steps of cancer development to initiate malignant transformation (a driver of a malignant genotype), enable cancer cells to survive, proliferate, and metastasize (a consequence of a malignant phenotype), which includes sustaining proliferative signaling, evading growth suppressor, resisting cell apoptosis, enabling replicative immortality, inducing angiogenesis, reprogramming of energy metabolism, evading tumor destruction, accumulating inflammatory microenvironment, and activating invasion and accelerating metastases. Regarding the important role of altered sialylation of cancers, further knowledge about the initiation and the consequences of altered sialylation pattern in tumor cells is needed, because all may offer a better chance for developing novel therapeutic strategy. In this review, we would like to update alteration of sialylation in ovarian cancers.

Non-small cell lung cancer (NSCLC), EGFR downstream pathway activation and TKI targeted therapies sensitivity: Effect of the plasma membrane-associated NEU3.

Adenocarcinoma of Non-Small Cell Lung Cancer (NSCLC) is a severe disease. Patients carrying EGFR mutations may benefit from EGFR targeted therapies (e.g.: gefitinib). Recently, it has been shown that sialidase NEU3 directly interacts and regulates EGFR. In this work, we investigate the effect of sialidase NEU3 overexpression on EGFR pathways activation and EGFR targeted therapies sensitivity, in a series of lung cancer cell lines. NEU3 overexpression, forced after transfection, does not affect NSCLC cell viability. We demonstrate that NEU3 overexpression stimulates the ERK pathway but this activation is completely abolished by gefitinib treatment. The Akt pathway is also hyper-activated upon NEU3 overexpression, but gefitinib is able only to decrease, and not to abolish, such activation. These findings indicate that NEU3 can act directly on the ERK pathway through EGFR and both directly and indirectly with respect to EGFR on the Akt pathway. Furthermore, we provide evidence that a healthy mucosa cell line (with EGFR wild-type gene sequence) is slightly sensitive to gefitinib, especially in the presence of NEU3 overexpression, thus hypothesizing that NEU3 overexpressing patients may benefit from EGFR targeted therapies also in absence of EGFR point mutations. Overall, the expression of NEU3 may be a novel diagnostic marker in NSCLC because, by its ability to stimulate EGFR downstream pathways with direct and indirect mechanisms, it may help in the identification of patients who can profit from EGFR targeted therapies in absence of EGFR activating mutations or from new combinations of EGFR and Akt inhibitors.

Differences in survival, virulence and biofilm formation between sialidase-deficient and W83 wild-type Porphyromonas gingivalis strains under stressful environmental conditions.

BACKGROUND: Porphyromonas gingivalis is a major causative pathogen of chronic periodontitis. Within the inflammatory microenvironment, there exists extreme pH values, elevated temperatures and oxidative stress. Pathogens adapt to these stressful environmental conditions by regulating the transcription of virulence genes, modifying themselves with macromolecules and by aggregating and entering into a biofilm growth phase. Our previous study showed that the P. gingivalis sialidase can help cells obtain sialic acid from the environment, which is used to modify macromolecules on the surface of P. gingivalis cells. In this study, we compared the survival, virulence factors and biofilm formation of a sialidase-deficient strain (ΔPG0352) and the wild-type P. gingivalis W83 strain under various pH values, temperatures and oxidative stress conditions to identify the roles of sialidase in the adaptation of P. gingivalis to stressful conditions. RESULTS: Compared to the growth of the P. gingivalis W83 strain, the growth of the △PG0352 was more inhibited by oxidative stress (0.25 and 0.5 mM H2O2) and exhibited greater cell structure damage when treated with H2O2 as assessed by transmission electron microscopy. Both Lys-gingipain (Kgp) and Arg-gingipain (Rgp) activities were lower in the ΔPG0352 than those in the P. gingivalis W83 strain under all the assayed culture conditions. The lipopolysaccharide (LPS) activity of the W83 strain was higher than that of the ΔPG0352 under acidic conditions (pH 5.0), but no differences between the strains were observed under other conditions. Compared to the biofilms formed by P. gingivalis W83, those formed by the ΔPG0352 were decreased and discontinuous under acidic, alkaline and oxidative stress conditions. CONCLUSION: Compared to the P. gingivalis W83 strain, the survival, virulence and biofilm formation of the ΔPG0352 were decreased under stressful environmental conditions.

Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization.

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and ß-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role.

Enhanced replication of avian-origin H3N2 canine influenza virus in eggs, cell cultures and mice by a two-amino acid insertion in neuraminidase stalk.

Canine influenza virus (CIV) is a newly identified, highly contagious respiratory pathogen in dogs. Recent studies indicate that avian-origin H3N2 CIV are circulating in Chinese dogs. To investigate the effects of a two-amino acid (2-aa) insertion naturally occurring at the distal end of the neuraminidase (NA) stalk found in Chinese isolates since 2010 on virus replication and virulence, we rescued the CIV strain, A/canine/Jiangsu/06/2011(H3N2) and its NA mutant without the 2-aa insertion using reverse genetics. The NA stalk length affected virus growth in cell culture. Compared to the short stalk strain (without 2-aa insertion), the long stalk strain (with 2-aa insertion) exhibited higher peak titers and greater yields in Madin-Darby canine kidney (MDCK) cells, chicken embryo fibroblasts and canine bronchiolar epithelial cells, as well as much larger plaques in MDCK cell monolayers. Furthermore, mice inoculated with the long stalk strain showed more severe pathologic damage in lung and higher proportion of detectable viral RNA in tissues. The long stalk strain induced local IFN-γ production with faster kinetics and higher levels in mice. However, in chickens, the two viral strains showed no significant difference with nearly the same proportion of detectable viral RNA loads in tissues. These observations suggest that the 2-aa insertion in the NA stalk acquired by avian-origin H3N2 CIV helps to enhance viral replication and is likely a result of adaptive evolution in canine hosts.

[Multidimensional Analysis of Hippocampal Excitatory Neurotransmission and Development of Analytical Tools for Glycans].

Sialidase removes sialic acid residues from sialoglycoconjugates such as glycoproteins and glycolipids. Since sialic acid plays crucial roles in synaptic plasticity and memory in the hippocampus, the regulation of sialyl signaling by sialidase is also necessary for neural functions. However, since mammalian sialidase activity is remarkably weak, it has been difficult to detect sialidase activity in mammalian tissues. Determination of the distribution of sialidase activity in living mammalian tissues would provide much valuable information for understanding the roles of sialidase in physiological functions. Therefore, we synthesized a novel benzothiazolylphenol-based sialic acid derivative (BTP-Neu5Ac) as a fluorescent sialidase substrate. After cleavage of BTP-Neu5Ac, which is water soluble and shows little fluorescence, with sialidase, the water-insoluble fluorophore benzothiazolylphenol (BTP) released from BTP-Neu5Ac stains tissue and shows bright fluorescence. BTP-Neu5Ac can visualize sialidase activity in brain tissue with high levels of sensitivity and specificity. The sialidase expression level is markedly high in various human cancers such as colon, renal, prostate, and ovarian cancers. BTP-Neu5Ac can detect human colon cancers sensitively. Thus, BTP-Neu5Ac is useful not only for physiological research but also as a cancer probe. BTP-Neu5Ac is now being used in virology research. In this review, methods for histochemical imaging of sialidase activity and the role of sialidase in hippocampal memory are described based on the author's study of multidimensional analysis of hippocampal excitatory neurotransmission and development of analytical tools for glycans, which was awarded a prize by the Tokai branch of the Pharmaceutical Society of Japan.

Role of plasma-membrane-bound sialidase NEU3 in clathrin-mediated endocytosis.

Gangliosides are sialic acid-containing glycosphingolipids mainly expressed at the outer leaflet of the plasma membrane. Sialidase NEU3 is a key enzyme in the catabolism of gangliosides with its up-regulation having been observed in human cancer cells. In the case of CME (clathrin-mediated endocytosis), although this has been widely studied, the role of NEU3 and gangliosides in this cellular process has not yet been established. In the present study, we found an increased internalization of Tf (transferrin), the archetypical cargo for CME, in cells expressing complex gangliosides with high levels of sialylation. The ectopic expression of NEU3 led to a drastic decrease in Tf endocytosis, suggesting the participation of gangliosides in this process. However, the reduction in Tf endocytosis caused by NEU3 was still observed in glycosphingolipid-depleted cells, indicating that NEU3 could operate in a way that is independent of its action on gangliosides. Additionally, internalization of α2-macroglobulin and low-density lipoprotein, other typical ligands in CME, was also decreased in NEU3-expressing cells. In contrast, internalization of cholera toxin ß-subunit, which is endocytosed by both clathrin-dependent and clathrin-independent mechanisms, remained unaltered. Kinetic assays revealed that NEU3 caused a reduction in the sorting of endocytosed Tf to early and recycling endosomes, with the Tf binding at the cell surface being also reduced. NEU3-expressing cells showed an altered subcellular distribution of clathrin adaptor AP-2 (adaptor protein 2), but did not reveal any changes in the membrane distribution of clathrin, PtdIns(4,5)P2 or caveolin-1. Overall, these results suggest a specific and novel role of NEU3 in CME.

Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo.

Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.

Neuraminidase inhibition primes short-term depression and suppresses long-term potentiation of synaptic transmission in the rat hippocampus.

Neuraminidase (NEU) is a key enzyme that cleaves negatively charged sialic acid residues from membrane proteins and lipids. Clinical and basic science studies have shown that an imbalance in NEU metabolism or changes in NEU activity due to various pathological conditions parallel with behavior and cognitive impairment. It has been suggested that the decreases of NEU activity could cause serious neurological consequences. However, there is a lack of direct evidences that modulation of endogenous NEU activity can impair neuronal function. Using combined rat entorhinal cortex/hippocampal slices and a specific inhibitor of NEU, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NADNA), we examined the effect of downregulation of NEU activity on different forms of synaptic plasticity in the hippocampal CA3-to-CA1 network. We show that NEU inhibition results in a significant decrease in long-term potentiation (LTP) and an increase in short-term depression. Synaptic depotentiation restores LTP in NADNA-pretreated slices to the control level. These data suggest that short-term NEU inhibition produces the LTP-like effect on neuronal network, which results in damping of further LTP induction. Our findings demonstrate that downregulation of NEU activity could have a major impact on synaptic plasticity and provide a new insight into the cellular mechanism underlying behavioral and cognitive impairment associated with abnormal metabolism of NEU.

Influenza neuraminidase as a vaccine antigen.

Neuraminidase (NA) is the second most abundant influenza surface glycoprotein and contributes to virus replication in several ways, most notably by removing sialic acids from the host and viral glycoproteins, releasing newly formed virus particles from infected cells. Antibodies that block this enzyme activity restrict virus replication in vitro. This chapter describes foundational epidemiologic and human influenza challenge studies that provide evidence of an association between NA inhibiting antibodies and resistance to disease. Mouse challenge studies show that while NA immunity is infection-permissive, NA-specific antibodies attenuate infection and prevent severe disease. NA immunity is most effective against homologous viruses but there is substantial protection against viruses with a heterologous NA (different lineage within a NA subtype). Monoclonal antibodies specific for conserved antigenic domains of subtype N1 protect against seasonal and pandemic H1N1 as well as H5N1 virus challenge. Clinical studies demonstrate that licensed seasonal vaccines contain immunogenic amounts of NA, but the contribution of this immunity to vaccine efficacy is currently not known. New types of influenza vaccines could be designed to elicit NA immunity. Because NA induces heterologous immunity, it could be an important constituent of universal influenza vaccines that aim to protect against unexpected emerging viruses.

Sialylation of prion protein controls the rate of prion amplification, the cross-species barrier, the ratio of PrPSc glycoform and prion infectivity.

The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP(C)) into the disease-associated, transmissible form (PrP(Sc)). Pr(PC) is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP(C) and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrP(C) glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP(Sc) glycoform ratio. The current study demonstrates that undersialylated PrP(C) is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrP(C). As a result, PMCAb-derived PrP(Sc) was less sialylated than brain-derived PrP(Sc). A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP(C) using sialidase was found to increase the rate of PrP(Sc) amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP(C) reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP(C) was also found to control PrP(Sc) glycoform ratio. A decrease in Pr(PC) sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP(Sc). 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP(C) differed from that of spleen-derived PrP(C). Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP(C), suggesting that Neu1 is not responsible for desialylation of PrP(C). The current work highlights previously unappreciated role of PrP(C) sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.

Functional balance between the hemagglutinin and neuraminidase of influenza A(H1N1)pdm09 HA D222 variants.

D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/10(6)) was higher than that of G222 (1.7 nmol/h/10(6) viruses) and E/N222 variants (4.4 nmol/h/10(6) viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2,6- preference, nor estimate the hemagglutinin-neuraminidase balance in A(H1N1)pdm09 strains.

A 20-amino-acid deletion in the neuraminidase stalk and a five-amino-acid deletion in the NS1 protein both contribute to the pathogenicity of H5N1 avian influenza viruses in mallard ducks.

Since 2003, H5N1-subtype avian influenza viruses (AIVs) with both a deletion of 20 amino acids in the stalk of the neuraminidase (NA) glycoprotein (A-) and a deletion of five amino acids at positions 80 to 84 in the non-structural protein NS1 (S-) have become predominant. To understand the influence of these double deletions in the NA and NS1 proteins on the pathogenicity of H5N1-subtype AIVs, we selected A/mallard/Huadong/S/2005 as a parental strain to generate rescued wild-type A-S- and three variants (A-S+ with a five-amino-acid insertion in the NS1 protein, A+S- with a 20-amino-acid insertion in the NA stalk, and A+S+ with insertions in both NA and NS1 proteins) and evaluated their biological characteristics and virulence. The titers of the AIVs with A- and/or S- replicated in DEF cells were higher than that of A+S+, and the A-S- virus exhibited a replication predominance when co-infected with the other variants in DEF cells. In addition, A-S- induced a more significant increase in the expression of immune-related genes in peripheral blood mononuclear cells of mallard ducks in vitro compared with the other variants. Furthermore, an insertion in the NA and/or NS1 proteins of AIVs resulted in a notable decrease in virulence in ducks, as determined by intravenous pathogenicity index, and the two insertions exerted a synergistic effect on the attenuation of pathogenicity in ducks. In addition, compared with A+S+ and A+S-, the A-S+ and A-S- viruses that were introduced via the intranasal inoculation route exhibited a faster replication ability in the lungs of ducks. These data indicate that both the deletions in the NA stalk and the NS1 protein contribute to the high pathogenicity of H5N1 AIVs in ducks.

Viral determinants of influenza A virus host range.

Typical avian influenza A viruses are restricted from replicating efficiently and causing disease in humans. However, an avian virus can become adapted to humans by mutating or recombining with currently circulating human viruses. These viruses have the potential to cause pandemics in an immunologically naïve human population. It is critical that we understand the molecular basis of host-range restriction and how this can be overcome. Here, we review our current understanding of the mechanisms by which influenza viruses adapt to replicate efficiently in a new host. We predominantly focus on the influenza polymerase, which remains one of the least understood host-range barriers.