Organic Electrochemical Transistors (OECTs): Advancements and Exciting Prospects for Future Biosensing Applications

Over the past few decades, the field of organic electronics has depicted proliferated growth, due to the advantageous characteristics of organic semiconductors, such as tunability through synthetic chemistry, simplicity in processing, cost-effectiveness, and low-voltage operation, to cite a few. Organic electrochemical transistors (OECTs) have recently emerged as a highly promising technology in the area of biosensing and flexible electronics. OECT-based biosensors are capable of sensing brain activities, tissues, monitoring cells, hormones, DNAs, and glucose. Sensitivity, selectivity, and detection limit are the key parameters adopted for measuring the performance of OECT-based biosensors. This article highlights the advancements and exciting prospects of OECTs for future biosensing applications, such as cell-based biosensing, chemical sensing, DNA/ribonucleic acid (RNA) sensing, glucose sensing, immune sensing, ion sensing, and pH sensing. OECT-based biosensors outperform other conventional biosensors because of their excellent biocompatibility, high transconductance, and mixed electronic-ionic conductivity. At present, OECTs are fabricated and characterized in millimeter and micrometer dimensions, and miniaturizing their dimensions to nanoscale is the key challenge for utilizing them in the field of nanobioelectronics, nanomedicine, and nanobiosensing.

SIGLEC-9 RESTRAINS ANTIBODY-DEPENDENT NK CELL CYTOTOXICITY AGAINST SARS-CoV-2

Background: SARS CoV 2 infection alters the immunological profiles of natural killer (NK) cells. However, whether NK anti-viral functions (direct cytotoxicity and/or antibody-dependent cell cytotoxicity (ADCC)) are impaired during severe COVID-19 and what host factors modulate these functions remain unclear. Method(s): Using functional assays, we examined the ability of NK cells from SARS-CoV-2 negative controls (n=12), mild COVID-19 patients (n=26), and hospitalized COVID-19 patients (n=41) to elicit direct cytotoxicity and ADCC [NK degranulation by flow] against cells expressing SARS-CoV-2 antigens. SARS-CoV- 2 N antigen plasma load was measured using an ultra-sensitive Simoa assay. We also phenotypically characterized the baseline expression of NK activating (CD16 and NKG2C), maturation (CD57), and inhibitory (NKG2A and the glyco-immune negative checkpoint Siglec-9) by flow cytometry. Finally, an anti-Siglec-9 blocking antibody was used to examine the impact of Siglec-9 expression on anti-SARS-CoV-2-specific ADCC [degranulation and target cell lysis]. Result(s): NK cells from hospitalized COVID-19 patients degranulate less against SARS-CoV-2-antigen-expressing cells (in direct cytolytic and ADCC assays) than did cells from mild COVID-19 patients or negative controls (Fig. 1A). The lower NK degranulation was associated with higher plasma levels of SARS-CoV-2 N-antigen (P<=0.02). Phenotypic and functional analyses showed that NK cells expressing Siglec-9 elicited higher ADCC than Siglec-9- NK cells (P<0.05;Fig. 1B). Consistently, Siglec-9+ NK cells expressed an activated and mature phenotype with higher expression of CD16, CD57, and NKG2C, and lower expression of NKG2A, than Siglec-9- NK cells (P<=0.03). These data are consistent with the concept that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic. However, the Siglec-9 molecule itself is an inhibitory receptor that restrains NK cytotoxicity during cancer and other infections. Indeed, blocking Siglec-9 significantly enhanced the ADCC-mediated NK degranulation and lysis of SARS-CoV-2-antigen-positive target cells (P<=0.05;Fig. 1C). Conclusion(s): These data support a model (Fig. 1D) in which the Siglec-9+ CD56dim NK subpopulation is cytotoxic even while being restrained by the inhibitory effects of Siglec-9. However, alleviating the Siglec-9-mediated restriction on NK cytotoxicity can further improve NK immune surveillance and presents an opportunity to develop novel immunotherapeutic tools against SARS-CoV-2 infected cells. (Figure Presented).

SARS-CoV-2 Binding to Terminal Sialic Acid of Gangliosides Embedded in Lipid Membranes.

Multiple recent reports indicate that the S protein of SARS-CoV-2 specifically interacts with membrane receptors and attachment factors other than ACE2. They likely have an active role in cellular attachment and entry of the virus. In this article, we examined the binding of SARS-CoV-2 particles to gangliosides embedded in supported lipid bilayers (SLBs), mimicking the cell membrane-like environment. We show that the virus specifically binds to sialylated (sialic acid (SIA)) gangliosides, i.e., GD1a, GM3, and GM1, as determined from the acquired single-particle fluorescence images using a time-lapse total internal reflection fluorescence (TIRF) microscope. The data of virus binding events, the apparent binding rate constant, and the maximum virus coverage on the ganglioside-rich SLBs show that the virus particles have a higher binding affinity toward the GD1a and GM3 compared to the GM1 ganglioside. Enzymatic hydrolysis of the SIA-Gal bond of the gangliosides confirms that the SIA sugar unit of GD1a and GM3 is essential for virus attachment to the SLBs and even the cell surface sialic acid is critical for the cellular attachment of the virus. The structural difference between GM3/GD1a and GM1 is the presence of SIA at the main or branched chain. We conclude that the number of SIA per ganglioside can weakly influence the initial binding rate of SARS-CoV-2 particles, whereas the terminal or more exposed SIA is critical for the virus binding to the gangliosides in SLBs.

Neuraminidase is a host-directed approach to regulate neutrophil responses in sepsis and COVID-19.

BACKGROUND AND PURPOSE: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. EXPERIMENTAL APPROACH: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. KEY RESULTS: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. CONCLUSION AND IMPLICATIONS: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

Derivatization of sialylated glycopeptides plus based sialoglycopeptides enrichment using cation exchange media.

Glycosylation is one of the most important post-translational modifications. However, the characterizations of glycopeptides, especially the negatively charged sialoglycopeptides that are associated with various diseases, remain challenging, due to the co-existence with high abundant peptides and the low ionization efficiency of sialoglycopeptides resulting from the carboxyl groups. Therefore, it is essential to develop an efficient enrichment method for sialoglycopeptides. Here, we present a novel derivatization-based enrichment method that can (i) identify linkage isomers of sialic acids by generating mass difference, (ii) unify the net charge of peptides into zero, and (iii) introduce positive charges to sialoglycopeptides by conjugating quaternary ammonium with sialic acid. The derivatization, termed derivatization of sialylated glycopeptides plus (DOSG+), enables efficient enrichment through electrostatic interaction using weak cation exchange (WCX) media. DOSG+ -based WCX enrichment was validated and optimized with samples derived from bovine fetuin. Peptides were removed efficiently (recovery rate <1%). The signal intensity of a selected model sialoglycopeptide was increased by ∼30% (suggesting recovery rate >100%). The method was employed on human alpha-1 acid glycoprotein (AGP), and recombinant human erythropoietin (EPO), demonstrating the application of DOSG+ -based WCX enrichment on complexed N-linked and O-linked sialoglycopeptides. The method is simple, efficient, and targets small-scale sialoglycopeptide enrichment.

Mechanisms of Atrial Fibrillation in Covid-19

Atrial fibrillation (AF) is the most frequent form of cardiac arrhythmia in COVID-19 infected patients. The occurrence of AF paroxysms is often associated with the acute period of infection in time. At the same time, the pathophysiological mechanisms of the occurrence of AF associated with COVID-19 remain insufficiently studied. The review considers the available literature data on the influence of factors such as reduced availability of angiotensin-converting enzyme 2 receptors, interaction of the virus with the cluster of differentiation 147 and sialic acid, increased inflammatory signaling, "cytokine storm", direct viral damage to the endothelium, electrolyte and acid-alkaline balance in the acute phase of severe illness and increased sympathetic activity.Copyright © Autors 2023.

Identification and Comparison of the Sialic Acid-Binding Domain Characteristics of Avian Coronavirus Infectious Bronchitis Virus Spike Protein.

Infectious bronchitis virus (IBV) infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host factors and fuses the viral and cell membranes. The N-terminal domain of the S1 subunit of IBV S protein binds to sialic acids, but the precise location of the sialic acid binding domain (SABD) and the role of the SABD in IBV-infected chickens remain unclear. Here, we identify the S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19) and GD (GI-7), and the corresponding region of M41 (GI-1), as the minimal SABD using truncated protein histochemistry and neuraminidase assays. Both α-2,3- and α-2,6-linked sialic acids on the surfaces of CEK cells can be used as attachment receptors by IBV, leading to increased infection efficiency. However, 9-O acetylation of the sialic acid glycerol side chain inhibits IBV S1 and SABD protein binding. We further constructed recombinant strains in which the S1 gene or the SABD in the GD and SD genomes were replaced with the corresponding region from M41 by reverse genetics. Infecting chickens with these viruses revealed that the virulence and nephrotropism of rSDM41-S1, rSDM41-206, rGDM41-S1, and rGDM41-206 strains were decreased to various degrees compared to their parental strains. A positive sera cross-neutralization test showed that the serotypes were changed for the recombinant viruses. Our results provide insight into IBV infection of host cells that may aid vaccine design. IMPORTANCE To date, only α-2,3-linked sialic acid has been identified as a potential host binding receptor for IBV. Here, we show the minimum region constituting the sialic acid binding domain (SABD) and the binding characteristics of the S1 subunit of spike (S) protein of IBV strains SD (GI-19), GD (GI-7), and M41 (GI-1) to various sialic acids. The 9-O acetylation modification partially inhibits IBV from binding to sialic acid, while the virus can also bind to sialic acid molecules linked to host cells through an α-2,6 linkage, serving as another receptor determinant. Substitution of the putative SABD from strain M41 into strains SD and GD resulted in reduced virulence, nephrotropism, and a serotype switch. These findings suggest that sialic acid binding has diversified during the evolution of γ-coronaviruses, impacting the biological properties of IBV strains. Our results offer insight into the mechanisms by which IBV invades host cells.

Sialylation status in placentas from pregnancies with SARS-CoV-2 infection.

INTRODUCTION: Recent investigations suggest the potential negative impact of SARS-CoV-2 infection on pregnant women and pregnancy outcome. In addition, some studies have described pathological changes in the placental tissue of SARS-CoV-2-positive mothers, which are related or not to the infection severity and/or infection trimester. Among the various molecules involved in the normal structure and functionality of the placenta, sialic acids (Sias) seem to play an important role. Hence, we aimed to investigate possible changes in the distribution and content of Sias with different glycosidic linkages, namely α2,3 and α2,6 Galactose- or N-acetyl-Galactosamine-linked Sias and polymeric Sia (PolySia), in placentas from pregnant women infected by SARS-CoV-2 during the three different pregnancy trimesters. METHODS: α2,3 and α2,6 Galactose-linked Sias were evaluated by lectin histochemistry (Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA), respectively), while immunohistochemistry was used for PolySia detection. RESULTS: Data showed lower levels of α2,3 Galactose-linked Sias in the trophoblast and underlying basement membrane/basal plasma membrane in placentas from women infected during the second and third infection trimester compared with uninfected cases and those infected during first trimester. On the other hand, higher levels of PolySia were detected in the trophoblast during the second and third infection trimester. CONCLUSIONS: Our findings suggest that changes in the sialylation status of trophoblast and its basement membrane/basal plasma membrane, together with other concomitant factors, could be at the basis of the most common placental histopathological alterations and gestational complications found especially in pregnancies with SARS-CoV-2 infection during the second and third trimester.

A new approach for extracting information from protein dynamics.

Increased ability to predict protein structures is moving research focus towards understanding protein dynamics. A promising approach is to represent protein dynamics through networks and take advantage of well-developed methods from network science. Most studies build protein dynamics networks from correlation measures, an approach that only works under very specific conditions, instead of the more robust inverse approach. Thus, we apply the inverse approach to the dynamics of protein dihedral angles, a system of internal coordinates, to avoid structural alignment. Using the well-characterized adhesion protein, FimH, we show that our method identifies networks that are physically interpretable, robust, and relevant to the allosteric pathway sites. We further use our approach to detect dynamical differences, despite structural similarity, for Siglec-8 in the immune system, and the SARS-CoV-2 spike protein. Our study demonstrates that using the inverse approach to extract a network from protein dynamics yields important biophysical insights.

α2,6-Sialylation Is Upregulated in Severe COVID-19, Implicating the Complement Cascade.

Better understanding of the molecular mechanisms underlying COVID-19 severity is desperately needed in current times. Although hyper-inflammation drives severe COVID-19, precise mechanisms triggering this cascade and what role glycosylation might play therein are unknown. Here we report the first high-throughput glycomic analysis of COVID-19 plasma samples and autopsy tissues. We find that α2,6-sialylation is upregulated in the plasma of patients with severe COVID-19 and in autopsied lung tissue. This glycan motif is enriched on members of the complement cascade (e.g., C5, C9), which show higher levels of sialylation in severe COVID-19. In the lung tissue, we observe increased complement deposition, associated with elevated α2,6-sialylation levels, corresponding to elevated markers of poor prognosis (IL-6) and fibrotic response. We also observe upregulation of the α2,6-sialylation enzyme ST6GAL1 in patients who succumbed to COVID-19. Our work identifies a heretofore undescribed relationship between sialylation and complement in severe COVID-19, potentially informing future therapeutic development.

SARS-CoV-2 Spike Protein Induces Hemagglutination: Implications for COVID-19 Morbidities and Therapeutics and for Vaccine Adverse Effects.

Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID-19. To provide further insight into these glycan attachments and their potential clinical relevance, the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The electrostatic potential of the central region of spike protein from these four lineages was studied through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 spike protein glycan sites. The results of these experiments were, first, that spike protein from these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower threshold concentration of spike protein than the three prior lineages and was much more electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to spike protein and reversed HA when added afterward. These results validate and extend prior findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore suggest therapeutic options using competitive glycan-binding agents such as IVM and may help elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines, which use spike protein as the generated antigen.

Genome-Wide CRISPR/Cas9 Screen Reveals a Role for SLC35A1 in the Adsorption of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, not only causes diarrhea in piglets but also possesses the potential to infect humans. To better understand host-virus genetic dependencies and find potential therapeutic targets for PDCoV, we used a porcine single-guide RNA (sgRNA) lentivirus library to screen host factors related to PDCoV infection in LLC-PK1 cells. The solute carrier family 35 member A1 (SLC35A1), a key molecule in the sialic acid (SA) synthesis pathway, was identified as a host factor required for PDCoV infection. A knockout of SLC35A1 caused decreases in the amounts of cell surface sialic acid (SA) and viral adsorption; meanwhile, trypsin promoted the use of SA in PDCoV infection. By constructing and assessing a series of recombinant PDCoV strains with the deletion or mutation of possible critical domain or amino acid residues for SA binding in the S1 N-terminal domain, we found that S T182 might be a PDCoV SA-binding site. However, the double knockout of SLC35A1 and amino peptidase N (APN) could not block PDCoV infection completely. Additionally, we found that different swine enteric coronaviruses, including transmissible gastroenteritis coronavirus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome coronavirus, are differentially dependent on SA. Overall, our study uncovered a collection of host factors that can be exploited as drug targets against PDCoV infection and deepened our understanding of the relationship between PDCoV and SA. IMPORTANCE Identifying the host factors required for replication will be helpful to uncover the pathogenesis mechanisms and develop antivirals against the emerging coronavirus porcine deltacoronavirus (PDCoV). Herein, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout screen, the results of which revealed that the solute carrier family 35 member A1 (SLC35A1) is a host factor required for PDCoV infection that acts by regulating cell surface sialic acid (SA). We also identified the T182 site in the N-terminal domain of PDCoV S1 subunit as being associated with the SA-binding site and found that trypsin promotes the use of cell surface SA by PDCoV. Furthermore, different swine enteric coronaviruses use SLC35A1 differently for infection. This is the first study to screen host factors required for PDCoV replication using a genome-wide CRISPR-Cas9 functional knockout, thereby providing clues for developing antiviral drugs against PDCoV infection.

Structural and energetic analyses of SARS-CoV-2 N-terminal domain characterise sugar binding pockets and suggest putative impacts of variants on COVID-19 transmission.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is an ongoing pandemic that causes significant health/socioeconomic burden. Variants of concern (VOCs) have emerged affecting transmissibility, disease severity and re-infection risk. Studies suggest that the - N-terminal domain (NTD) of the spike protein may have a role in facilitating virus entry via sialic-acid receptor binding. Furthermore, most VOCs include novel NTD variants. Despite global sequence and structure similarity, most sialic-acid binding pockets in NTD vary across coronaviruses. Our work suggests ongoing evolutionary tuning of the sugar-binding pockets and recent analyses have shown that NTD insertions in VOCs tend to lie close to loops. We extended the structural characterisation of these sugar-binding pockets and explored whether variants could enhance sialic acid-binding. We found that recent NTD insertions in VOCs (i.e., Gamma, Delta and Omicron variants) and emerging variants of interest (VOIs) (i.e., Iota, Lambda and Theta variants) frequently lie close to sugar-binding pockets. For some variants, including the recent Omicron VOC, we find increases in predicted sialic acid-binding energy, compared to the original SARS-CoV-2, which may contribute to increased transmission. These binding observations are supported by molecular dynamics simulations (MD). We examined the similarity of NTD across Betacoronaviruses to determine whether the sugar-binding pockets are sufficiently similar to be exploited in drug design. Whilst most pockets are too structurally variable, we detected a previously unknown highly structurally conserved pocket which can be investigated in pursuit of a generic pan-Betacoronavirus drug. Our structure-based analyses help rationalise the effects of VOCs and provide hypotheses for experiments. Our findings suggest a strong need for experimental monitoring of changes in NTD of VOCs.

Roles of Sialyl Glycans in HCoV-OC43, HCoV-HKU1, MERS-CoV and SARS-CoV-2 Infections.

Ongoing seasonal HCoV-OC43 and HCoV-HKU1 (common cold), an ongoing zoonotic infection of highly lethal MERS-CoV in humans (MERS disease), and an ongoing pandemic SARS-CoV-2 (COVID-19) with high mutability giving some variants causing severe illness and death have been reported to attach to sialyl receptors via their spike (S) glycoproteins and via additional short spikes, hemagglutinin-esterase (HE) glycoproteins, for HCoV-OC43 and HCoV-HKU1. There is lack of zoonotic viruses that are origins of HCoV-HKU1 and the first recorded pandemic CoV (SARS-CoV-2) for studies. In this chapter, we review current knowledge of the roles of sialyl glycans in infections with these viruses in distinct infection stages. Determination of the similarities and differences in roles of sialyl glycans in infections with these viruses could lead to a better understanding of the pathogenesis and transmission that is essential for combating infections with CoVs that recognize sialyl glycans.

COVID-19 patient fibrinogen produces dense clots with altered polymerization kinetics, partially explained by increased sialic acid.

BACKGROUND: Thrombogenicity is a known complication of COVID-19, resulting from SARS-CoV-2 infection, with significant effects on morbidity and mortality. OBJECTIVE: We aimed to better understand the effects of COVID-19 on fibrinogen and the resulting effects on clot structure, formation, and degradation. METHODS: Fibrinogen isolated from COVID-19 patients and uninfected subjects was used to form uniformly concentrated clots (2 mg/ml), which were characterized using confocal microscopy, scanning electron microscopy, atomic force microscopy, and endogenous and exogenous fibrinolysis assays. Neuraminidase digestion and subsequent NANA assay were used to quantify sialic acid residue presence; clots made from the desialylated fibrinogen were then assayed similarly to the original fibrinogen clots. RESULTS: Clots made from purified fibrinogen from COVID-19 patients were shown to be significantly stiffer and denser than clots made using fibrinogen from noninfected subjects. Endogenous and exogenous fibrinolysis assays demonstrated that clot polymerization and degradation dynamics were different for purified fibrinogen from COVID-19 patients compared with fibrinogen from noninfected subjects. Quantification of sialic acid residues via the NANA assay demonstrated that SARS-CoV-2-positive fibrinogen samples contained significantly more sialic acid. Desialylation via neuraminidase digestion resolved differences in clot density. Desialylation did not normalize differences in polymerization, but did affect rate of exogenous fibrinolysis. DISCUSSION: These differences noted in purified SARS-CoV-2-positive clots demonstrate that structural differences in fibrinogen, and not just differences in gross fibrinogen concentration, contribute to clinical differences in thrombotic features associated with COVID-19. These structural differences are at least in part mediated by differential sialylation.

Heparan Sulfate and Sialic Acid in Viral Attachment: Two Sides of the Same Coin?

Sialic acids and heparan sulfates make up the outermost part of the cell membrane and the extracellular matrix. Both structures are characterized by being negatively charged, serving as receptors for various pathogens, and are highly expressed in the respiratory and digestive tracts. Numerous viruses use heparan sulfates as receptors to infect cells; in this group are HSV, HPV, and SARS-CoV-2. Other viruses require the cell to express sialic acids, as is the case in influenza A viruses and adenoviruses. This review aims to present, in a general way, the participation of glycoconjugates in viral entry, and therapeutic strategies focused on inhibiting the interaction between the virus and the glycoconjugates. Interestingly, there are few studies that suggest the participation of both glycoconjugates in the viruses addressed here. Considering the biological redundancy that exists between heparan sulfates and sialic acids, we propose that it is important to jointly evaluate and design strategies that contemplate inhibiting the interactions of both glycoconjugates. This approach will allow identifying new receptors and lead to a deeper understanding of interspecies transmission.

SARS-CoV-2 and MERS-CoV Spike Protein Binding Studies Support Stable Mimic of Bound 9-O-Acetylated Sialic Acids.

Many disease-causing viruses target sialic acids (Sias), a class of nine-carbon sugars known to coat the surface of many cells, including those in the lungs. Human beta coronaviridae, known for causing respiratory tract diseases, often bind Sias, and some preferentially bind to those with 9-O-Ac-modification. Currently, co-binding of SARS-CoV-2, a beta coronavirus responsible for the COVID-19 pandemic, to human Sias has been reported and its preference towards α2-3-linked Neu5Ac has been shown. Nevertheless, O-acetylated Sias-protein binding studies are difficult to perform, due to the ester lability. We studied the binding free energy differences between Neu5,9Ac2α2-3GalßpNP and its more stable 9-NAc mimic binding to SARS-CoV-2 spike protein using molecular dynamics and alchemical free energy simulations. We identified multiple Sia-binding pockets, including two novel sites, with similar binding affinities to those of MERS-CoV, a known co-binder of sialic acid. In our binding poses, 9-NAc and 9-OAc Sias bind similarly, suggesting an experimentally reasonable mimic to probe viral mechanisms.

Wild and domestic animals variably display Neu5Ac and Neu5Gc sialic acids.

Sialic acids are used as a receptor by several viruses and variations in the linkage type or C-5 modifications affect the binding properties. A species barrier for multiple viruses is present due to α2,3- or α2,6-linked sialic acids. The C-5 position of the sialic acid can be modified to form N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), which acts as a determinant for host susceptibility for pathogens such as influenza A virus, rotavirus, and transmissible gastroenteritis coronavirus. Neu5Gc is present in most mammals such as pigs and horses but is absent in humans, ferrets, and dogs. However, little is known about C-5 content in wildlife species or how many C-5 modified sialic acids are present on N-linked glycans or glycolipids. Using our previously developed tissue microarray system, we investigated how 2 different lectins specific for Neu5Gc can result in varying detection levels of Neu5Gc glycans. We used these lectins to map Neu5Gc content in wild Suidae, Cervidae, tigers, and European hedgehogs. We show that Neu5Gc content is highly variable among different species. Furthermore, the removal of N-linked glycans reduces the binding of both Neu5Gc lectins while retention of glycolipids by omitting methanol treatment of tissues increases lectin binding. These findings highlight the importance of using multiple Neu5Gc lectins as the rich variety in which Neu5Gc is displayed can hardly be detected by a single lectin.

The structure of Phocaeicola vulgatus sialic acid acetylesterase.

Sialic acids terminate many N- and O-glycans and are widely distributed on cell surfaces. There are a diverse range of enzymes which interact with these sugars throughout the tree of life. They can act as receptors for influenza and specific betacoronaviruses in viral binding and their cleavage is important in virion release. Sialic acids are also exploited by both commensal and pathogenic bacteria for nutrient acquisition. A common modification of sialic acid is 9-O-acetylation, which can limit the action of sialidases. Some bacteria, including human endosymbionts, employ esterases to overcome this modification. However, few bacterial sialic acid 9-O-acetylesterases (9-O-SAEs) have been structurally characterized. Here, the crystal structure of a 9-O-SAE from Phocaeicola vulgatus (PvSAE) is reported. The structure of PvSAE was determined to resolutions of 1.44 and 2.06 Šusing crystals from two different crystallization conditions. Structural characterization revealed PvSAE to be a dimer with an SGNH fold, named after the conserved sequence motif of this family, and a Ser-His-Asp catalytic triad. These structures also reveal flexibility in the most N-terminal α-helix, which provides a barrier to active-site accessibility. Biochemical assays also show that PvSAE deacetylates both mucin and the acetylated chromophore para-nitrophenyl acetate. This structural and biochemical characterization of PvSAE furthers the understanding of 9-O-SAEs and may aid in the discovery of small molecules targeting this class of enzyme.

Histones released by NETosis enhance the infectivity of SARS-CoV-2 by bridging the spike protein subunit 2 and sialic acid on host cells.

Neutrophil extracellular traps (NETs) can capture and kill viruses, such as influenza viruses, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV), thus contributing to host defense. Contrary to our expectation, we show here that the histones released by NETosis enhance the infectivity of SARS-CoV-2, as found by using live SARS-CoV-2 and two pseudovirus systems as well as a mouse model. The histone H3 or H4 selectively binds to subunit 2 of the spike (S) protein, as shown by a biochemical binding assay, surface plasmon resonance and binding energy calculation as well as the construction of a mutant S protein by replacing four acidic amino acids. Sialic acid on the host cell surface is the key molecule to which histones bridge subunit 2 of the S protein. Moreover, histones enhance cell-cell fusion. Finally, treatment with an inhibitor of NETosis, histone H3 or H4, or sialic acid notably affected the levels of sgRNA copies and the number of apoptotic cells in a mouse model. These findings suggest that SARS-CoV-2 could hijack histones from neutrophil NETosis to promote its host cell attachment and entry process and may be important in exploring pathogenesis and possible strategies to develop new effective therapies for COVID-19.