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At the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel human coronavirus, emerged and rapidly caused a global pandemic. SARS-CoV-2 is the causative agent of coronavirus disease 2019 (COVID-19), which affects the respiratory tract and lungs of infected individuals. Due to the increased transmissibility of the SARS-CoV-2 virus compared to its previous versions, determining as fully as possible the various structural aspects of the virus became critical for the development of therapeutics and vaccines to combat this virus. Knowing the structures of viral proteins and their glycosylation is an essential foundation for the understanding of the mechanism of the disease. Glycopeptide analysis has been used to map the glycosylation of viral glycoproteins, including those of influenza and HIV. Thanks to the developments in the field over the last few decades, scientists were able to quickly develop therapeutics against SARS-CoV-2. This chapter discusses the four structural proteins of SARS-CoV-2, their glycosylation and modifications, and the techniques used to map SARS-CoV-2 glycosylation. 

ABSTRACT Pseudomonas aeruginosais considered one of the most challenging, drug-resistant, opportunistic pathogens partly due to its ability to synthesize robust biofilms. Biofilm is a mixture of extracellular polymeric substances (EPS) that encapsulates microbial cells, leading to immune evasion, antibiotic resistance, and thus higher risk of infection. In the cystic fibrosis lung environment,P. aeruginosaundergoes a mucoid transition, defined by overproduction of the exopolysaccharide alginate. Alginate encapsulation results in bacterial resistance to antibiotics and the host immune system. Given its role in airway inflammation and chronic infection, alginate is an obvious target to improve treatment forP. aeruginosainfection. Previously, we demonstrated polysaccharide lyase Smlt1473 fromStenotrophomonas maltophiliastrain k279a can catalyze the degradation of multiple polyuronidesin vitro, including D-mannuronic acid (poly-ManA). Poly-ManA is a major constituent ofP. aeruginosaalginate, suggesting that Smlt1473 could have potential application against multidrug-resistantP. aeruginosaand perhaps other microbes with related biofilm composition. In this study, we demonstrate that Smlt1473 can inhibit and degrade alginate fromP. aeruginosa. Additionally, we show that testedP. aeruginosastrains are dominant in acetylated alginate and that all but one have similar M-to-G ratios. These results indicate that variation in enzyme efficacy among the isolates is not primarily due to differences in total EPS or alginate chemical composition. Overall, these results demonstrate Smlt1473 can inhibit and degradeP. aeruginosaalginate and suggest that other factors including rate of EPS production, alginate sequence/chain length, or non-EPS components may explain differences in enzyme efficacy. IMPORTANCEPseudomonas aeruginosais a major opportunistic human pathogen in part due to its ability to synthesize biofilms that confer antibiotic resistance. Biofilm is a mixture of polysaccharides, DNA, and proteins that encapsulate cells, protecting them from antibiotics, disinfectants, and other cleaning agents. Due to its ability to increase antibiotic and immune resistance, the exopolysaccharide alginate plays a large role in airway inflammation and chronicP. aeruginosainfection. As a result, colonization withP. aeruginosais the leading cause of morbidity and mortality in CF patients. Thus, it is an obvious target to improve the treatment regimen forP. aeruginosainfection. In this study, we demonstrate that polysaccharide lyase, Smlt1473, inhibits alginate secretion and degrades established alginate from a variety of mucoidP. aeruginosaclinical isolates. Additionally, Smlt1473 differs from other alginate lyases in that it is active against acetylated alginate, which is secreted during chronic lung infection. These results suggest that Smlt1473 may be useful in treating infections associated with alginate-producingP. aeruginosa, as well as have the potential to reduceP. aeruginosaEPS in non-clinical settings. 

ABSTRACT The development of vaccines for fungal diseases, including cryptococcosis, is an emergent line of research and development. In previous studies, we showed that aCryptococcusmutant lacking theSGL1gene (∆sgl1) accumulates certain glycolipids called steryl glucosides (SGs) on the fungal capsule, promoting an effective immunostimulation that totally protects the host from a secondary cryptococcal infection. However, this protection is lost when the cryptococcal capsule is absent in the∆sgl1background. The cryptococcal capsule is mainly composed of glucuronoxylomannan (GXM), a polysaccharide microfiber consisting of glucuronic acid, xylose, and mannose linked by glycosidic bonds forming specific triads. In this study, we engineered cells to lack each of the GXM components and tested the effect of these deletions on protection under the condition of SG accumulation. We found that glucuronic acid and xylose are required for protection, and their absence abrogates the production of IFNγ and IL-17A by γδ T cells, which are necessary stimulants for the protective phenotype of the∆sgl1. We analyzed the structure of the GXM microfibers and found that although the deletion ofSGL1only slightly affects the size and distribution of these microfibers, it significantly changes the ratio of mannose to other components. In conclusion, this study identifies the structural modifications that the deletion ofSGL1and the consequent accumulation of SGs impart to the GXM structure ofC. neoformans. This provides significant insights into the protective mechanisms mediated by SG accumulation on the capsule, with important implications for the future development of an efficacious cryptococcal vaccine.IMPORTANCECryptococcus neoformansis an encapsulated fungus that causes invasive fungal infections with high morbidity and mortality in susceptible patients. With increasing drug resistance and high toxicity of current antifungal drugs, there is a need for alternative therapeutic strategies, such as a cryptococcal vaccine. In this study, we identify the necessary capsular components and their structural organization required for a cryptococcal vaccine to protect the host against challenge with a virulent strain. These capsular components are glucuronic acid, xylose, and mannose, and they work together with certain glycolipids called steryl glucosides (SGs) to stimulate host immunity. Interestingly, SGs on the capsule may favor the formation of small capsular microfibers organized in specific mannose triads. Thus, the results of this paper are important because they identify a mechanism by which SGs affect the structure of the cryptococcal capsule, with important implications for the future development of a cryptococcal vaccine using capsular components and SGs. 

The Neurospora crassa genome has a gene cluster for the synthesis of galactosaminogalactan (GAG). The gene cluster includes the following: (1) UDP-glucose-4-epimerase to convert UDP-glucose and UDP-N-acetylglucosamine to UDP-galactose and UDP-N-acetylgalactosamine (NCU05133), (2) GAG synthase for the synthesis of an acetylated GAG (NCU05132), (3) GAG deacetylase (/NCW-1/NCU05137), (4) GH135-1, a GAG hydrolase with specificity for N-acetylgalactosamine-containing GAG (NCU05135), and (5) GH114-1, a galactosaminidase with specificity for galactosamine-containing GAG (NCU05136). The deacetylase was previously shown to be a major cell wall glycoprotein and given the name of NCW-1 (non-GPI anchored cell wall protein-1). Characterization of the polysaccharides found in the growth medium from the wild type and the GAG synthase mutant demonstrates that there is a major reduction in the levels of polysaccharides containing galactosamine and N-acetylgalactosamine in the mutant growth medium, providing evidence that the synthase is responsible for the production of a GAG. The analysis also indicates that there are other galactose-containing polysaccharides produced by the fungus. Phenotypic characterization of wild-type and mutant isolates showed that deacetylated GAG from the wild type can function as an adhesin to a glass surface and provides the fungal mat with tensile strength, demonstrating that the deacetylated GAG functions as an intercellular adhesive. The acetylated GAG produced by the deacetylase mutant was found to function as an adhesive for chitin, alumina, celite (diatomaceous earth), activated charcoal, and wheat leaf particulates. 

ABSTRACT Whole genome sequencing has revealed that the genome ofStaphylococcus aureuspossesses an uncharacterized 5-gene operon (SAOUHSC_00088–00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus assscfor staphylococcal surface carbohydrate. We found that thesscgenes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed inEscherichia coliand extracted by heat treatment. Ssc was also conjugated to AcrA fromCampylobacter jejuniinE. coliusing protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting ofN-acetylgalactosamine. We further demonstrated that the expression of thesscgenes inS. aureusaffected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed. IMPORTANCESurface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens.Staphylococcus aureusproduces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide inS. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide. 

Galactofuranose is a constituent of the cell walls of filamentous fungi. The galactofuranose can be found as a component of N-linked oligosaccharides, in O-linked oligosaccharides, in GPI-anchored galactomannan, and in free galactomannan. The Neurospora genome contains a single UDP-galactose mutase gene (ugm-1/NCU01824) and two UDP-galactofuranose translocases used to import UDP-galactofuranose into the lumen of the Golgi apparatus (ugt-1/NCU01826 and ugt-2/NCU01456). Our results demonstrate that loss of galactofuranose synthesis or its translocation into the lumen of the secretory pathway affects the morphology and growth rate of the vegetative hyphae, the production of conidia (asexual spores), and dramatically affects the sexual stages of the life cycle. In mutants that are unable to make galactofuranose or transport it into the lumen of the Golgi apparatus, ascospore development is aborted soon after fertilization and perithecium maturation is aborted prior to the formation of the neck and ostiole. The Neurospora genome contains three genes encoding possible galactofuranosyltransferases from the GT31 family of glycosyltransferases (gfs-1/NCU05878, gfs-2/NCU07762, and gfs-3/NCU02213) which might be involved in generating galactofuranose-containing oligosaccharide structures. Analysis of triple KO mutants in GT31 glycosyltransferases shows that these mutants have normal morphology, suggesting that these genes do not encode vital galactofuranosyltransferases. 

Fungal glycosphingolipids (GSLs) are important membrane components which play a key role in vesicle trafficking. To assess the importance of GSLs in the fungal life cycle, we performed a mutant phenotypic study of the acidic and neutral GSL biosynthetic pathways in Neurospora crassa. GSL biosynthesis begins with two reactions leading up to the formation of dihydrosphingosine. The first of these reactions is catalyzed by serine palmitoyltransferase and generates 3-keto dihydrosphinganine. In N. crassa, this reaction is catalyzed by GSL-1 and GSL-2 and is required for viability. The second reaction is carried out by GSL-3, a 3-keto dihydrosphinoganine reductase to generate dihydrosphingosine, which is used for the synthesis of neutral and acidic GSLs. We found that deletion mutations in the acidic GSL pathway leading up to the formation of mannosylinositol-phosphoceramide are lethal, indicating that acidic GSLs are essential for viability in N. crassa. Once mannosylinositol-phosphoceramide is made, it is further modified by GSL-5, an inositol-phosphoceramide-B C26 hydroxylase, which adds a hydroxyl group to the amide-linked fatty acid. GSL-5 is not required for viability but gives a clear mutant phenotype affecting all stages of the life cycle. Our results show that the synthesis of mannosylinositol-phosphoceramide is required for viability and that the modification of the amide-linked fatty acid is important for acidic GSL functionality. We also examined the neutral GSL biosynthetic pathway and identified the presence of glucosylceramide. The deletion of neutral GSL biosynthetic genes affected hyphal morphology, vegetative growth rate, conidiation, and female development. Our results indicate that the synthesis of neutral GSLs is essential for normal growth and development of N. crassa. 

Abstract The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both theN-terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved bothN- andO- glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation.