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ABSTRACT K2-capsular Klebsiella pneumoniae is a hypervirulent pathogen that causes fatal infections. Here, we describe a phage tailspike protein, named K2-2, that specifically depolymerizes the K2 capsular polysaccharide (CPS) of K. pneumoniae into tetrasaccharide repeating units. Nearly half of the products contained O-acetylation, which was thought crucial to the immunogenicity of CPS. The product-bound structures of this trimeric enzyme revealed intersubunit carbohydrate-binding grooves, each accommodating three tetrasaccharide units of K2 CPS. The catalytic residues and the key interactions responsible for K2 CPS recognition were identified and verified by site-directed mutagenesis. Further biophysical and functional characterization, along with the structure of a tetrameric form of K2-2, demonstrated that the formation of intersubunit catalytic center does not require trimerization, which could be nearly completely disrupted by a single-residue mutation in the C-terminal domain. Our findings regarding the assembly and catalysis of K2-2 provide cues for the development of glycoconjugate vaccines against K. pneumoniae infection.IMPORTANCEGenerating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of K. pneumoniae into tetrasaccharide fragments while retaining the vital O-acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2 K. pneumoniae, we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center.

O-Acetyl esterification is an important structural and functional feature of pectins present in the cell walls of all land plants. The amount and positions of pectin acetyl substituents varies across plant tissues and stages of development. Plant growth and response to biotic and abiotic stress are known to be significantly influenced by pectin O-acetylation. Gel formation is a key characteristic of pectins, and many studies have shown that gel formation is dependent upon the degree of acetylation. Previous studies have indicated that members of the TRICHOME BIREFRINGENCE-LIKE (TBL) family may play a role in the O-acetylation of pectin, however, biochemical evidence for acceptor specific pectin acetyltransferase activity remains to be confirmed and the exact mechanism(s) for catalysis must be determined. Pectin acetylesterases (PAEs) affect pectin acetylation as they hydrolyze acetylester bonds and have a role in the amount and distribution of O-acetylation. Several mutant studies suggest the critical role of pectin O-acetylation; however, additional research is required to fully understand this. This review aims to discuss the importance, role, and putative mechanism of pectin O-acetylation.

The serine-rich repeat (SRR) glycoproteins of gram-positive bacteria are a family of adhesins that bind to a wide range of host ligands, and expression of SRR glycoproteins is linked with enhanced bacterial virulence. The biogenesis of these surface glycoproteins involves their intracellular glycosylation and export via the accessory Sec system. Although all accessory Sec components are required for SRR glycoprotein export, Asp2 of Streptococcus gordonii also functions as an O-acetyltransferase that modifies GlcNAc residues on the SRR adhesin gordonii surface protein B (GspB). Because these GlcNAc residues can also be modified by the glycosyltransferases Nss and Gly, it has been unclear whether the post-translational modification of GspB is coordinated. We now report that acetylation modulates the glycosylation of exported GspB. Loss of O-acetylation due to aps2 mutagenesis led to the export of GspB glycoforms with increased glucosylation of the GlcNAc moieties. Linkage analysis of the GspB glycan revealed that both O-acetylation and glucosylation occurred at the same C6 position on GlcNAc residues and that O-acetylation prevented Glc deposition. Whereas streptococci expressing nonacetylated GspB with increased glucosylation were significantly reduced in their ability to bind human platelets in vitro, deletion of the glycosyltransferases nss and gly in the asp2 mutant restored platelet binding to WT levels. These findings demonstrate that GlcNAc O-acetylation controls GspB glycosylation, such that binding via this adhesin is optimized. Moreover, because O-acetylation has comparable effects on the glycosylation of other SRR adhesins, acetylation may represent a conserved regulatory mechanism for the post-translational modification of the SRR glycoprotein family.

Caloric restriction (CR) can prolong life and ameliorate age‐related diseases; thus, its molecular basis might provide new insights for finding biomarker and intervention for aging and age‐related disease. Glycosylation is an important post‐translational modification, which can timely reflect the changes of intracellular state. Serum N‐glycosylation was found changed with aging in humans and mice. CR is widely accepted as an effective anti‐aging intervention in mice and could affect mouse serum fucosylated N‐glycans. However, the effect of CR on the level of global N‐glycans remains unknown. In order to explore whether CR affect the level of global N‐glycans, we performed a comprehensive serum glycome profiling in mice of 30% calorie restriction group and ad libitum group at 7 time points across 60 weeks by MALDI‐TOF‐MS. At each time point, the majority of glycans, including galactosylated and high mannose glycans, showed a consistent low level in CR group. Interestingly, O‐acetylated sialoglycans presented an upward change different from other derived traits, which is mainly reflected in two biantennary α2,6‐linked sialoglycans (H5N4Ge2Ac1, H5N4Ge2Ac2). Liver transcriptome analysis further revealed a decreased transcriptional level of genes involved in N‐glycan biosynthesis while increased level of acetyl‐CoA production. This finding is consistent with changes in serum N‐glycans and O‐acetylated sialic acids. Therefore, we provided one possible molecular basis for the beneficial effect of CR from N‐glycosylation perspective. [ABSTRACT FROM AUTHOR]

O-Acetylated pectins are abundant in the primary cell wall of plants and growing evidence suggests they have important roles in plant cell growth and interaction with the environment. Despite their importance, genes required for O-acetylation of pectins are still largely unknown. In this study, we showed that TRICHOME BIREFRINGENCE LIKE 10 (AT3G06080) is involved in O-acetylation of pectins in Arabidopsis (Arabidopsis thaliana). The activity of the TBL10 promoter was strong in tissues where pectins are highly abundant (e.g. leaves). Two homozygous knock-out mutants of Arabidopsis, tbl10-1 and tbl10-2, were isolated and shown to exhibit reduced levels of wall-bound acetyl esters, equivalent of ~50% of the wild-type level in pectin-enriched fractions derived from leaves. Further fractionation revealed that the degree of acetylation of the pectin rhamnogalacturonan-I (RG-I) was reduced in the tbl10 mutant compared to the wild type, whereas the pectin homogalacturonan (HG) was unaffected. The degrees of acetylation in hemicelluloses (i.e. xyloglucan, xylan and mannan) were indistinguishable between the tbl10 mutants and the wild type. The mutant plants contained normal trichomes in leaves and exhibited a similar level of susceptibility to the phytopathogenic microorganisms Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea; while they displayed enhanced tolerance to drought. These results indicate that TBL10 is required for O-acetylation of RG-I, possibly as an acetyltransferase, and suggest that O-acetylated RG-I plays a role in abiotic stress responses in Arabidopsis.

Bacterial cell envelope polymers are commonly modified with acyl groups that provide fitness advantages. Many polymer acylation pathways involve pairs of membrane-bound O -acyltransferase (MBOAT) and SGNH family proteins. As an example, the MBOAT protein PatA and the SGNH protein PatB are required in Gram-negative bacteria for peptidoglycan O-acetylation. The mechanism for how MBOAT-SGNH transferases move acyl groups from acyl-CoA donors made in the cytoplasm to extracellular polymers is unclear. Using the peptidoglycan O -acetyltransferase proteins PatAB, we explore the mechanism of MBOAT-SGNH pairs. We find that the MBOAT protein PatA catalyzes auto-acetylation of an invariant Tyr residue in its conserved C-terminal hexapeptide motif. We also show that PatB can use a synthetic hexapeptide containing an acetylated tyrosine to donate an acetyl group to a peptidoglycan mimetic. Finally, we report the structure of PatB, finding that it has structural features that shape its activity as an O -acetyltransferase and distinguish it from other SGNH esterases and hydrolases. Taken together, our results support a model for peptidoglycan acylation in which a tyrosine-containing peptide at the MBOAT's C-terminus shuttles an acyl group from the MBOAT active site to the SGNH active site, where it is transferred to peptidoglycan. This model likely applies to other systems containing MBOAT-SGNH pairs, such as those that O -acetylate alginate, cellulose, and secondary cell wall polysaccharides. The use of an acyl-tyrosine intermediate for MBOAT-SGNH acyl transfer is also shared with AT3-SGNH proteins, a second major group of acyltransferases that modify cell envelope polymers., Competing Interests: Competing Interest Statement: The authors declare no competing interest.

To understand the mechanisms of immunomodulatory effect, Dendrobium Officinale polysaccharides (DOP) were treated by ultrasound and mild base separately to generate fractions of various weight-average molecular weight (Mw) and degrees of acetylation (DA). The structural features, conformational properties, functional properties and immunomodulatory activities of original and modified DOPs were investigated. Ultrasonic treatment decreased the Mw and apparent viscosity and improved the water solubility of DOP. Mild base treatment remarkably reduced the DA and the water solubility, while the overall apparent viscosity was increased. Conformational analysis by triple-detector high performance size-exclusion chromatography showed that the molecular chain of DOP turned more compact coil conformation with decreased DA. Results from the macrophages RAW 264.7 analysis showed that samples sonicated for 200 min (Mw 34.2 kDa) showed the highest immune-regulation effects. However, the immunomodulatory effects of the samples after de-acetylation were all compromised compared to the original DOP. This study inspires further research to establish the structural-immunomodulatory relationships, which promote the application of DOP in both the food and medicine fields.

SUMMARY: Modification of the O‐acetylation level of xyloglucan (XyG) appears to affect aluminum (Al) sensitivity in Arabidopsis by modulating its binding capacity to Al. However, the transcriptional regulation of this process remains largely unknown. In our previous studies, we found that the expression of TRICHOME BIREFRINGENCE‐LIKE27 (TBL27), which is responsible for the O‐acetylation of XyG, was downregulated under Al stress. In the present study, we showed that the expression of an R2R3‐type transcription factor‐encoding gene, MYB103, was also inhibited by Al exposure and exhibited a co‐expression pattern with TBL27 in roots and siliques, suggesting a potential link between MYB103 and TBL27. The loss of function of MYB103 resulted in increased Al sensitivity, as indicated by more inhibited root growth and elevated root Al content compared with the wild type. Moreover, we also detected increased Al accumulation in the root cell wall and the hemicellulose fraction, which was attributed to the changes in the O‐acetylation level of XyG rather than the XyG content itself. In addition, further analysis revealed that MYB103 positively activated TBL27 expression by directly binding to the TBL27 promoter region, and TBL27 overexpression in the myb103 mutant rescued the Al‐sensitive phenotype of the mutant to the wild‐type level. Taken together, we conclude that MYB103 acts upstream of TBL27 to positively regulate Al resistance by modulating the O‐acetylation of the cell wall XyG. Significance Statement: In this study, a member of the MYB family that is involved in the regulation of aluminum (Al) sensitivity in Arabidopsis, MYB103, was identified. Further analysis indicated that MYB103 acts upstream of TRICHOME BIREFRINGENCE‐LIKE27 in regulating Al sensitivity by modulating the O‐acetylation level of the cell wall xyloglucan. [ABSTRACT FROM AUTHOR]

Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of arylamine carcinogens such as 4-aminobiphenyl (ABP), and following N-hydroxylation, the O-acetylation of N-hydroxy-arylamine carcinogens such as N-hydroxy-ABP (N-OH-ABP). Genetic polymorphisms in NAT1 are linked to cancer susceptibility following exposures. The effects of individual single nucleotide polymorphisms (SNPs) in the NAT1 coding exon on Michaelis-Menten kinetic constants was assessed for ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activity following transfection of human NAT1 into COS-1 cells (SV40-transformed African green monkey kidney cells). NAT1 coding region SNPs 97C > T (rs56318881) (R33stop), 190C > T (rs56379106) (R64W), 559C > T (rs5030839) (R187stop) and 752A > T (rs56172717) (D251V) reduced ABP N- acetyltransferase and N-OH-ABP O-acetyltransferase activity below detection. 21T > G (rs4986992) (synonymous), 402T > C (rs146727732) (synonymous), 445G > A (rs4987076) (V149I), 613A > G (rs72554609) (M205V) and 640T > G (rs4986783) (S241A) did not significantly affect Vmax for ABP N-acetyltransferase or N-OH-ABP O-acetyltransferase. 781G > A (rs72554610) (E261K), and 787A > G (rs72554611) (I263V) slightly reduced ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activities whereas 560G > A (rs4986782) (R187Q) substantially and significantly reduced them. 560G > A (rs4986782) (R187Q) significantly reduced the apparent Km for ABP and N-OH-ABP a finding that was not observed with any of the other NAT1 SNPs tested. These findings suggest that the role of the 560G > A (rs4986782) (R187Q) SNP cancer risk assessment may be modified by exposure level to aromatic amine carcinogens such as ABP. [ABSTRACT FROM AUTHOR]

Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of arylamine carcinogens such as 4-aminobiphenyl (ABP), and following N-hydroxylation, the O-acetylation of N-hydroxy-arylamine carcinogens such as N-hydroxy-ABP (N-OH-ABP). Genetic polymorphisms in NAT1 are linked to cancer susceptibility following exposures. The effects of individual single nucleotide polymorphisms (SNPs) in the NAT1 coding exon on Michaelis-Menten kinetic constants was assessed for ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activity following transfection of human NAT1 into COS-1 cells (SV40-transformed African green monkey kidney cells). NAT1 coding region SNPs 97C > T (rs56318881) (R33stop), 190C > T (rs56379106) (R64W), 559C > T (rs5030839) (R187stop) and 752A > T (rs56172717) (D251V) reduced ABP N- acetyltransferase and N-OH-ABP O-acetyltransferase activity below detection. 21T > G (rs4986992) (synonymous), 402T > C (rs146727732) (synonymous), 445G > A (rs4987076) (V149I), 613A > G (rs72554609) (M205V) and 640T > G (rs4986783) (S241A) did not significantly affect Vmax for ABP N-acetyltransferase or N-OH-ABP O-acetyltransferase. 781G > A (rs72554610) (E261K), and 787A > G (rs72554611) (I263V) slightly reduced ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activities whereas 560G > A (rs4986782) (R187Q) substantially and significantly reduced them. 560G > A (rs4986782) (R187Q) significantly reduced the apparent Km for ABP and N-OH-ABP a finding that was not observed with any of the other NAT1 SNPs tested. These findings suggest that the role of the 560G > A (rs4986782) (R187Q) SNP cancer risk assessment may be modified by exposure level to aromatic amine carcinogens such as ABP.

Arylamine N-acetyltransferases catalyze the transfer of acetyl groups from the endogenous cofactor acetyl coenzyme A (AcCoA) to arylamine (N-acetylation) and N-hydroxy-arylamine (O-acetylation) acceptors. Humans express two arylamine N-acetyltransferase isozymes (NAT1 and NAT2) which catalyze both N- and O-acetylation but differ in genetic regulation, substrate selectivity, and expression in human tissues. We investigated recombinant human NAT1 and NAT2 expressed in an Escherichia coli JM105 and Schizosaccharomyces pombe expression systems as well as in Chinese hamster ovary (CHO) cells to assess the relative affinity of AcCoA for human NAT1 and NAT2. NAT1 and NAT2 affinity for AcCoA was higher for recombinant human NAT1 than NAT2 when catalyzing N-acetylation of aromatic amine carcinogens 2-aminofluroene (AF), 4-aminobiphenyl (ABP), and β-naphthylamine (BNA) and the metabolic activation of N-hydroxy-2-aminofluorene (N-OH-AF) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) via O-acetylation. These results suggest that AcCoA level may influence differential rates of arylamine carcinogen metabolism catalyzed by NAT1 and NAT2 in human tissues. Affinity was higher for NAT2 than for NAT1 using N-OH-AF and N-OH-ABP as substrate consistent with a larger active site for NAT2. In conclusion, following recombinant expression in bacteria, yeast, and CHO cells, we report significant differences in affinity between human NAT1 and NAT2 for its required co-factor AcCoA, as well as for N-hydroxy-arylamines activated via O-acetylation. The findings provide important information to understand the relative contribution of human NAT1 vs NAT2 towards N-acetylation and O-acetylation reactions in human hepatic and extrahepatic tissues.

ABSTRACT Escherichia coli K1 causes bacteremia and meningitis in human neonates. The K1 capsule, an α2,8-linked polysialic acid (PSA) homopolymer, is its essential virulence factor. PSA is usually partially modified by O-acetyl groups. It is known that O-acetylation alters the antigenicity of PSA, but its impact on the interactions between E. coli K1 and host cells is unclear. In this study, a phase variant was obtained by passage of E. coli K1 parent strain, which expressed a capsule with 44% O-acetylation whereas the capsule of the parent strain has only 3%. The variant strain showed significantly reduced adherence and invasion to macrophage-like cells in comparison to the parent strain. Furthermore, we found that O-acetylation of PSA enhanced the modulation of trafficking of E. coli-containing vacuoles (ECV), enabling them to avoid fusing with lysosomes in these cells. Intriguingly, by using quartz crystal microbalance, we demonstrated that the PSA purified from the parent strain interacted with human sialic acid-binding immunoglobulin-like lectins (Siglecs), including Siglec-5, Siglec-7, Siglec-11, and Siglec-14. However, O-acetylated PSA from the variant interacted much less and also suppressed the production of Siglec-mediated proinflammatory cytokines. The adherence of the parent strain to human macrophage-like cells was significantly blocked by monoclonal antibodies against Siglec-11 and Siglec-14. Furthermore, the variant strain caused increased bacteremia and higher lethality in neonatal mice compared to the parent strain. These data elucidate that O-acetylation of K1 capsule enables E. coli to escape from Siglec-mediated innate immunity and lysosomal degradation; therefore, it is a strategy used by E. coli K1 to regulate its virulence. IMPORTANCE Escherichia coli K1 is a leading cause of neonatal meningitis. The mortality and morbidity of this disease remain significantly high despite antibiotic therapy. One major limitation on advances in prevention and therapy for meningitis is an incomplete understanding of its pathogenesis. E. coli K1 is surrounded by PSA, which is observed to have high-frequency variation of O-acetyl modification. Here, we present an in-depth study of the function of O-acetylation in PSA at each stage of host-pathogen interaction. We found that a high level of O-acetylation significantly interfered with Siglec-mediated bacterial adherence to macrophage-like cells, and blunted the proinflammatory response. Furthermore, the O-acetylation of PSA modulated the trafficking of ECVs to prevent them from fusing with lysosomes, enabling them to escape degradation by lysozymes within these cells. Elucidating how subtle modification of the capsule enhances bacterial defenses against host innate immunity will enable the future development of effective drugs or vaccines against infection by E. coli K1.

The O-acetylation of exopolysaccharides, including the essential bacterial cell wall polymer peptidoglycan, confers resistance to their lysis by exogenous hydrolases. Like the enzymes catalyzing the O-acetylation of exopolysaccharides in the Golgi of animals and fungi, peptidoglycan O-acetyltransferase A (OatA) is predicted to be an integral membrane protein comprised of a membrane-spanning acyltransferase-3 (AT-3) domain and an extracytoplasmic domain; for OatA, these domains are located in the N- and C-terminal regions of the enzyme, respectively. The recombinant C-terminal domain (OatAC) has been characterized as an SGNH acetyltransferase, but nothing was known about the function of the N-terminal AT-3 domain (OatAN) or its homologs associated with other acyltransferases. We report herein the experimental determination of the topology of Staphylococcus aureus OatAN, which differs markedly from that predicted in silico. We present the biochemical characterization of OatAN as part of recombinant OatA and demonstrate that acetyl-CoA serves as the substrate for OatAN. Using in situ and in vitro assays, we characterized 35 engineered OatA variants which identified a catalytic triad of Tyr-His-Glu residues. We trapped an acetyl group from acetyl-CoA on the catalytic Tyr residue that is located on an extracytoplasmic loop of OatAN. Further enzymatic characterization revealed that O-acetyl-Tyr represents the substrate for OatAC. We propose a model for OatA action involving the translocation of acetyl groups from acetyl-CoA across the cytoplasmic membrane by OatAN and their subsequent intramolecular transfer to OatAC for the O-acetylation of peptidoglycan via the concerted action of catalytic Tyr and Ser residues. [ABSTRACT FROM AUTHOR]

The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O -acetylated sialyl residues susceptible to the action of its sialidase and sialic acid uptake system, Tannerella produces NanS, an O -acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine–histidine–aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl- O -acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7- O -acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl- O -acetylesterases and fills the current gap in tools targeting 7- O -acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di- O -acetylated sialoglycans by monitoring the gain in 9- O -acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9- O -acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9- O -acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9- O -acetylation proved advantageous over de-9- O -acetylation as the additional removal of the 7- O -acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di- O -acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O -acetylation. [ABSTRACT FROM AUTHOR]

Shigella are gram-negative bacteria that cause severe diarrhea and dysentery, with a high level of antimicrobial resistance. Disease-induced protection against reinfection in Shigella-endemic areas provides convincing evidence on the feasibility of a vaccine and on the importance of Shigella lipopolysaccharides as targets of the host humoral protective immune response against disease. This article provides an overview of the original and current strategies toward the development of a Shigella glycan-protein conjugate vaccine that would cover the most commonly detected strains. Going beyond pioneering “lattice”-type polysaccharide-protein conjugates, progress, and challenges are addressed with focus on promising alternatives, which have reached phases I and II clinical trial. Glycoengineered bioconjugates and “sun”-type conjugates featuring well-defined synthetic carbohydrate antigens are discussed with insights on the molecular parameters governing the rational design of a cost-effective glycoconjugate vaccine efficacious in preventing diseases caused by Shigella in the most at risk populations, young children living in endemic areas.

K2-capsular Klebsiella pneumoniae is a hypervirulent pathogen that causes fatal infections. Here, we describe a phage tailspike protein, named K2-2, that specifically depolymerizes the K2 capsular polysaccharide (CPS) of K. pneumoniae into tetrasaccharide repeating units. Nearly half of the products contained O -acetylation, which was thought crucial to the immunogenicity of CPS. The product-bound structures of this trimeric enzyme revealed intersubunit carbohydrate-binding grooves, each accommodating three tetrasaccharide units of K2 CPS. The catalytic residues and the key interactions responsible for K2 CPS recognition were identified and verified by site-directed mutagenesis. Further biophysical and functional characterization, along with the structure of a tetrameric form of K2-2, demonstrated that the formation of intersubunit catalytic center does not require trimerization, which could be nearly completely disrupted by a single-residue mutation in the C-terminal domain. Our findings regarding the assembly and catalysis of K2-2 provide cues for the development of glycoconjugate vaccines against K. pneumoniae infection., Importance: Generating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of K. pneumoniae into tetrasaccharide fragments while retaining the vital O -acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2 K. pneumoniae , we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center., Competing Interests: The authors declare no conflict of interest.

Shigella flexneri is a major causative agent of bacillary dysentery in developing countries, where serotype 2a2 is the prevalent strain. To date, approximately 30 serotypes have been identified for S. flexneri, and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to S. flexneri. LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of S. flexneri serotypes (Y and X). It uses its tailspike protein (Sf6TSP) to establish initial interaction with LPS Oags that it then hydrolyzes. Currently, there is a lack of comprehensive study on the parent and serotype variant strains from the same genetic background and an understanding of the importance of LPS Oag O-acetylations. Therefore, a set of isogenic strains (based on S. flexneri 2457T [2a2]) with deletions of different Oag modification genes (oacB, oacD, and gtrII) that resemble different naturally occurring serotype Y and 2a strains was created. The impacts of these Oag modifications on S. flexneri sensitivity to Sf6 and the pathogenesis-related properties were then compared. We found that Sf6TSP can hydrolyze serotype 2a LPS Oag, identified that 3/4-O-acetylation is essential for resistance of serotype 2a strains to Sf6, and showed that serotype 2a strains have better invasion ability. Lastly, we revealed two new serotype conversions for S. flexneri, thereby contributing to understanding the evolution of this important human pathogen. IMPORTANCE The emergence of antibiotic-resistant strains and lack of efficient vaccines have made Shigella a priority organism for the World Health Organization (1). Therefore, bacteriophage therapy has received increasing attention as an alternative therapeutic approach. LPS Oag is the most variable part of LPS due to chemical modifications and is the target of bacteriophage Sf6 (S. flexneri specific). We dissected the evolution of S. flexneri serotype Y to 2a2, which revealed a new role for a gene acquired during serotype conversion and furthermore identified new specific forms of LPS receptor for Sf6. Collectively, these results unfold the importance of the acquisition of those Oag modification genes and further our understanding of the relationship between Sf6 and S. flexneri. [ABSTRACT FROM AUTHOR]

ABSTRACT Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection. IMPORTANCE Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.

Staphylococcus aureus produces an antiphagocytic polysaccharide capsule to evade neutrophil-mediated killing. Many vaccines against encapsulated bacterial pathogens require generation of functional anti-capsular antibodies to mediate protection against infection and disease. Here it is shown that the generation of such antibody responses to S. aureus in vivo and in vitro requires the presence of O-acetyl modifications on the capsular polysaccharides. O-acetylation of S. aureus capsular polysaccharide therefore should be monitored carefully during vaccine development and production. This finding may provide additional insight into the previous failure of a S. aureus capsular polysaccharide conjugate vaccine.

Shigellosis is a diarrheal disease caused prevalently by Shigella flexneri and S. sonnei and representing a major global health risk, particularly in developing countries. Bacterial O-antigen (OAg) is the primary target of the host immune response and modifications of its oligosaccharide units, including O-acetylation, are responsible for the variability among the circulating S. flexneri serotypes. No vaccines are widely available against shigellosis and the understanding of the immunogenicity induced by the OAg is fundamental for the design of a vaccine that could cover the most prevalent Shigella serotypes. To understand whether a different O-acetylation pattern could influence the immune response elicited by S. flexneri OAg, we employed as a vaccine technology GMMA purified from S. flexneri 2a and 1b strains that were easily engineered to obtain differently O-acetylated OAg. Resulting GMMA were tested in mice, demonstrating not only no major impact of O-acetyl decorations on the immune response elicited by the two OAg against the homologous strains, but also that the O-acetylation of the Rhamnose III residue (O-factor 9), shared among serotypes 1b, 2a and 6, does not induce cross-reactive antibodies against these serotypes. This work contributes to the optimization of vaccine design against Shigella, providing indication about the ability of shared epitopes to elicit broad protection against S. flexneri serotypes and supporting the identification of critical quality attributes of OAg-based vaccines.

The solid acids such as ZrO 2 , Al 2 O 3 and ZrO 2 -Al 2 O 3 containing different ZrO 2 loadings (10–80 mol%) were prepared by solution combustion method (SCM) and characterized for their total surface acidity by NH 3 -TPD/ n -butylamine back titration method and crystallinity by powder X-ray diffraction (PXRD) technique. These solid acids were evaluated for their catalytic activity in the synthesis of novel O -acetylated products from substituted phenols, pyridine alcohols and aryl alcohols with acetic anhydride (AA) as an acetylating agent. The reaction conditions were optimized by varying the catalyst, molar ratio of the reactants, reaction temperature and amount of the catalyst. All the solid acids used in this study exhibited good catalytic activity in the reaction. In particular, ZrO 2 -Al 2 O 3 containing 80 mol% of ZrO 2 was found to be highly active in the acetylation reaction with high yield of acetylated products. Triangular correlation between the surface acidity, crystallinity and catalytic activity of solid acids was observed. These solid acids were found to be reactivable and reusable. [ABSTRACT FROM AUTHOR]

This report discloses a mild and efficient O-acetylation using easily accessible TMSOAc as a novel acetyl reagent and O-trimethylsilylation using HMDS for various alcohols catalyzed by tunable Brønsted acidic ionic liquids (TBAILs). Imidazolium-based TBAILs were prepared by a two-step atom-economic reaction and acidities measured by using UV-visible spectroscopy. Both protections for alcohols were accomplished at room temperature with good to excellent yields, while the products and TBAILs were separated by simple work-up for O-silylation and column chromatography for O-acetylation. Notably, with the simple post-process, TBAILs catalyst in this solvent free method easily recovered and recycled several times without significant degradation.

The O-acetylated form of GD2, almost exclusively expressed in cancerous tissues, is considered to be a promising therapeutic target for neuroectoderm-derived tumors, especially for breast cancer. Our recent data have shown that 9-O-acetylated GD2 (9-OAcGD2) is the major O-acetylated ganglioside species in breast cancer cells. In 2015, Baumann et al. proposed that Cas 1 domain containing 1 (CASD1), which is the only known human sialyl-O-acetyltransferase, plays a role in GD3 O-acetylation. However, the mechanisms of ganglioside O-acetylation remain poorly understood. The aim of this study was to determine the involvement of CASD1 in GD2 O-acetylation in breast cancer. The role of CASD1 in OAcGD2 synthesis was first demonstrated using wild type CHO and CHOΔCasd1 cells as cellular models. Overexpression using plasmid transfection and siRNA strategies was used to modulate CASD1 expression in SUM159PT breast cancer cell line. Our results showed that OAcGD2 expression was reduced in SUM159PT that was transiently depleted for CASD1 expression. Additionally, OAcGD2 expression was increased in SUM159PT cells transiently overexpressing CASD1. The modulation of CASD1 expression using transient transfection strategies provided interesting insights into the role of CASD1 in OAcGD2 and OAcGD3 biosynthesis, and it highlights the importance of further studies on O-acetylation mechanisms.

Abstract Defects in the biosynthesis and/or deposition of secondary plant cell wall polymers result in the collapse of xylem vessels causing a dwarfed plant stature and an altered plant architecture termed irregular xylem (irx) syndrome. For example, reduced xylan O‐acetylation causes strong developmental defects and increased freezing tolerance. Recently, we demonstrated that the irx syndrome in the trichome birefringence‐like 29/eskimo1 (tbl29/esk1) mutant is dependent on MORE AXILLARY GROWTH 4 (MAX4), a key enzyme in the biosynthesis of the phytohormone strigolactone (SL). In this report, we show that other xylan‐ and cellulose‐deficient secondary wall mutants exhibit increased freezing tolerance correlated with the irx syndrome. In addition, these phenotypes are also dependent on MAX4, suggesting a more general interaction between secondary wall defects and SL biosynthesis. In contrast, MAX4 does not play a role in developmental defects triggered by primary wall deficiencies, suggesting that the interaction is restricted to vascular tissue. Through a reverse genetics approach, the requirement of different components of the SL pathway impacting the irx syndrome in tbl29 was evaluated. Our results show that the tbl29‐associated irx phenotypes are dependent on the MAX3 and MAX4 enzymes, involved in the early steps of SL biosynthesis. In contrast, this signaling is independent on downstream enzymes in the biosynthesis and perception of SL such as MAX1 and MAX2.

Neisseria gonorrhoeae and Neisseria meningitidis release peptidoglycan (PG) fragments from the cell as the bacteria grow. For N. gonorrhoeae these PG fragments are known to cause damage to human Fallopian tube tissue in organ culture that mimics the damage seen in patients with pelvic inflammatory disease. N. meningitidis also releases pro-inflammatory PG fragments, but in smaller amounts than those from N. gonorrhoeae. It is not yet known if PG fragment release contributes to the highly inflammatory conditions of meningitis and meningococcemia caused by N. meningitidis. Examination of the mechanisms of PG degradation and recycling identified proteins required for these processes. In comparison to the model organism E. coli, the pathogenic Neisseria have far fewer PG degradation proteins, and some of these proteins show differences in subcellular localization compared to their E. coli homologs. In particular, some N. gonorrhoeae PG degradation proteins were demonstrated to be in the outer membrane while their homologs in E. coli were found free in the periplasm or in the cytoplasm. The localization of two of these proteins was demonstrated to affect PG fragment release. Another major factor for PG fragment release is the allele of ampG. Gonococcal AmpG was found to be slightly defective compared to related PG fragment permeases, thus leading to increased release of PG. A number of additional PG-related factors affect other virulence functions in Neisseria. Endopeptidases and carboxypeptidases were found to be required for type IV pilus production and resistance to hydrogen peroxide. Also, deacetylation of PG was required for virulence of N. meningitidis as well as normal cell size. Overall, we describe the processes involved in PG degradation and recycling and how certain characteristics of these proteins influence the interactions of these pathogens with their host.

The pathogenic bacterium Shigella is a leading cause of diarrheal disease and mortality, disproportionately affecting young children in low-income countries. The increasing prevalence of antibiotic resistance in Shigella necessitates an effective vaccine, for which the bacterial lipopolysaccharide O-antigen is the primary target. S. flexneri serotype 6 has been proposed as a multivalent vaccine component to ensure broad protection against Shigella. We have previously explored the conformations of S. flexneri O-antigens from serogroups Y, 2, 3, and 5 that share a common saccharide backbone (serotype Y). Here we consider serogroup 6, which is of particular interest because of an altered backbone repeat unit with non-stoichiometric O-acetylation, the antigenic and immunogenic importance of which have yet to be established. Our simulations show significant conformational changes in serogroup 6 relative to the serotype Y backbone. We further find that O-acetylation has little effect on conformation and hence may not be essential for the antigenicity of serotype 6. This is corroborated by an in vivo study in mice, using Generalized Modules for Membrane Antigens (GMMA) as O-antigen delivery systems, that shows that O-acetylation does not have an impact on the immune response elicited by the S. flexneri serotype 6 O-antigen.

O-acetylation of sialic acid residues is one of the main modifications of gangliosides, and modulates ganglioside functions. O-acetylation of gangliosides is dependent on sialyl-O-acetyltransferases and sialyl-O-acetyl-esterase activities. CAS1 Domain-Containing Protein 1 (CASD1) is the only human sialyl-O-acetyltransferases (SOAT) described until now. O-acetylated ganglioside species are mainly expressed during embryonic development and in the central nervous system in healthy adults, but are re-expressed during cancer development and are considered as markers of cancers of neuroectodermal origin. However, the specific biological roles of O-acetylated gangliosides in developing and malignant tissues have not been extensively studied, mostly because of the requirement of specific approaches and tools for sample preparation and analysis. In this review, we summarize our current knowledge of ganglioside biosynthesis and expression in normal and pathological conditions, of ganglioside O-acetylation analysis and expression in cancers, and of the possible use of O-acetylated gangliosides as targets for cancer immunotherapy.

The expression and biological functions of oncofetal markers GD2 and GD3 were extensively studied in neuroectoderm-derived cancers in order to characterize their potential as therapeutic targets. Using immunological approaches, we previously identified GD3, GD2, and OAcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for O-acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of O-acetylated ganglioside species. Consequently, the aim of the present study was to use structural approaches in order to apprehend ganglioside diversity in melanoma, neuroblastoma, and breast cancer cells, focusing on O-acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the O-acetyl modification for the analysis of native gangliosides by MALDI-TOF. We identified the expression of GM1, GM2, GM3, GD2, GD3, GT2, and GT3 in SK-Mel28 (melanoma), LAN-1 (neuroblastoma), Hs 578T, SUM 159PT, MDA-MB-231, MCF-7 (BC), and BC cell lines over-expressing GD3 synthase. Among O-acetylated gangliosides, we characterized the expression of OAcGM1, OAcGD3, OAcGD2, OAcGT2, and OAcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the O-acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside O-acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different O-acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of O-acetylated ganglioside profiles in cancer cells.

The post-synthetic O-acetylation of the essential component of bacterial cell walls, peptidoglycan (PG), is performed by many pathogenic bacteria to help them evade the lytic action of innate immunity responses. Occurring at the C-6 hydroxyl of N-acetylmuramoyl residues, this modification to the glycan backbone of PG sterically blocks the activity of lysozymes. As such, the enzyme responsible for this modification in Gram-positive bacteria is recognized as a virulence factor. With Gram-negative bacteria, the O-acetylation of PG provides a means of control of their autolysins at the substrate level. In this review, we discuss the pathways for PG O-acetylation and de-O-acetylation and the structure and function relationship of the O-acetyltransferases and O-acetylesterases that catalyze these reactions. The current understanding of their mechanisms of action is presented and the prospects of targeting these systems for the development of novel therapeutics are explored.

The extracellular matrix of plants, algae, bacteria, fungi, and some archaea consist of a semipermeable composite containing polysaccharides. Many of these polysaccharides are O-acetylated imparting important physiochemical properties to the polymers. The position and degree of O-acetylation is genetically determined and varies between organisms, cell types, and developmental stages. Despite the importance of wall polysaccharide O-acetylation, only recently progress has been made to elucidate the molecular mechanism of O-acetylation. In plants, three protein families are involved in the transfer of the acetyl substituents to the various polysaccharides. In other organisms, this mechanism seems to be conserved, although the number of required components varies. In this review, we provide an update on the latest advances on plant polysaccharide O-acetylation and related information from other wall polysaccharide O-acetylating organisms such as bacteria and fungi. The biotechnological impact of understanding wall polysaccharide O-acetylation ranges from the design of novel drugs against human pathogenic bacteria to the development of improved lignocellulosic feedstocks for biofuel production.

Sialic acids are a family of nine carbon keto-aldononulosonic acids presented at the terminal ends of glycans on cellular membranes. α-Linked sialoglycoconjugates often undergo post-glycosylation modifications, among which O-acetylation of N-acetyl neuraminic acid (Neu5Ac) is the most common in mammalian cells. Isoforms of sialic acid are critical determinants of virus pathogenesis. To date, the focus of viral receptor-mediated attachment has been on Neu5Ac. O-Acetylated Neu5Acs have been largely ignored as receptor determinants of virus pathogenesis, although it is ubiquitous across species. Significantly, the array of structures resulting from site-specific O-acetylation by sialic acid O-acetyltransferases (SOATs) provides a means to examine specificity of viral binding to host cells. Specifically, C4 O-acetylated Neu5Ac can influence virus pathogenicity. However, the biological implications of only O-acetylated Neu5Ac at C7−9 have been explored extensively. This review will highlight the biological significance, extraction methods, and synthetic modifications of C4 O-acetylated Neu5Ac that may provide value in therapeutic developments and targets to prevent virus related diseases.

The post-synthetic O-acetylation of the essential component of bacterial cell walls, peptidoglycan (PG), is performed by many pathogenic bacteria to help them evade the lytic action of innate immunity responses. Occurring at the C-6 hydroxyl of N -acetylmuramoyl residues, this modification to the glycan backbone of PG sterically blocks the activity of lysozymes. As such, the enzyme responsible for this modification in Gram-positive bacteria is recognized as a virulence factor. With Gram-negative bacteria, the O-acetylation of PG provides a means of control of their autolysins at the substrate level. In this review, we discuss the pathways for PG O-acetylation and de-O-acetylation and the structure and function relationship of the O -acetyltransferases and O -acetylesterases that catalyze these reactions. The current understanding of their mechanisms of action is presented and the prospects of targeting these systems for the development of novel therapeutics are explored. [ABSTRACT FROM AUTHOR]

The high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR) analysis of Plesiomonas shigelloides 78/89 lipopolysaccharide directly on bacteria revealed the characteristic structural features of the O-acetylated polysaccharide in the NMR spectra. The O-antigen profiles were unique, yet the pattern of signals in the, spectra along with their ¹H,13C chemical shift values, resembled these of d-galactan I of Klebsiella pneumoniae. The isolated O-specific polysaccharide (O-PS) of P. shigelloides strain CNCTC 78/89 was investigated by ¹H and 13C NMR spectroscopy, mass spectrometry and chemical methods. The analyses demonstrated that the P. shigelloides 78/89 O-PS is composed of →3)-α-d-Galp-(1→3)-β-d-Galf2OAc-(1→ disaccharide repeating units. The O-acetylation was incomplete and resulted in a microheterogeneity of the O-antigen. This O-acetylation generates additional antigenic determinants within the O-antigen, forms a new chemotype, and contributes to the epitopes recognized by the O-serotype specific antibodies. The serological cross-reactivities further confirmed the inter-specific structural similarity of these O-antigens. [ABSTRACT FROM AUTHOR]

The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria.

The peptidoglycan sacculus of both Gram-positive and Gram-negative bacteria acts as a protective mesh and provides structural support around the entirety of the cell. The integrity of this structure is of utmost importance for cell viability and so naturally is the first target for attack by the host immune system during bacterial infection. Lysozyme, a muramidase and the first line of defense of the innate immune system, targets the peptidoglycan sacculus hydrolyzing the β-(1→4) linkage between repeating glycan units, causing lysis and the death of the invading bacterium. The O-acetylation of N-acetylmuramoyl residues within peptidoglycan precludes the productive binding of lysozyme, and in doing so renders it inactive. This modification has been shown to be an important virulence factor in pathogens such as Staphylococcus aureus and Neisseria gonorrhoeae and is currently being investigated as a novel target for anti-virulence therapies. This article reviews interactions made between peptidoglycan and the host immune system, specifically with respect to lysozyme, and how the O-acetylation of the peptidoglycan interrupts these interactions, leading to increased pathogenicity.

O-acetylation is a common modification of bacterial glycoconjugates. By modifying oligosaccharide structure and chemistry, O-acetylation has important consequences for biotic and abiotic recognition events and thus bacterial fitness in general. Previous studies of the broad-spectrum O-linked protein glycosylation in pathogenic Neisseria species (including N. gonorrhoeae and N. meningitidis) have revealed O-acetylation of some of their diverse glycoforms and identified the committed acetylase, PglI. Herein, we extend these observations by using mass spectrometry to examine a complete set of all glycan variants identified to date. Regardless of composition, all glycoforms and all sugars in the oligosaccharide are subject to acetylation in a PglI-dependent fashion with the only exception of di-N-acetyl-bacillosamine. Moreover, multiple sugars in a single oligosaccharide could be simultaneously modified. Interestingly, O-acetylation status was found to be correlated with altered chain lengths of oligosaccharides expressed in otherwise isogenic backgrounds. Models for how this unprecedented phenomenon might arise are discussed with some having potentially important implications for the membrane topology of glycan O-acetylation. Together, the findings provide better insight into how O-acetylation can both directly and indirectly govern glycoform structure and diversity. [ABSTRACT FROM AUTHOR]

The incidence of infectious diseases caused by several bacterial pathogens such as Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis, has been dramatically reduced over the last 25 years through the use of glycoconjugate vaccines. The structures of the bacterial capsular polysaccharide (CPS) antigens, extracted and purified from microbial cultures and obtained with very high purity, show that many of them are decorated by O-acetyl groups. While these groups are often considered important for the structural identity of the polysaccharides, they play a major role in the functional immune response to some vaccines such as meningococcal serogroup A and Salmonella typhi Vi, but do not seem to be important for many others, such as meningococcal serogroups C, W, Y, and type III Group B Streptococcus. This review discusses the O-acetylation status of CPSs and its role in the immunological responses of these antigens.

The high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR) analysis of Plesiomonas shigelloides 78/89 lipopolysaccharide directly on bacteria revealed the characteristic structural features of the O-acetylated polysaccharide in the NMR spectra. The O-antigen profiles were unique, yet the pattern of signals in the, spectra along with their 1H,13C chemical shift values, resembled these of d-galactan I of Klebsiella pneumoniae. The isolated O-specific polysaccharide (O-PS) of P. shigelloides strain CNCTC 78/89 was investigated by 1H and 13C NMR spectroscopy, mass spectrometry and chemical methods. The analyses demonstrated that the P. shigelloides 78/89 O-PS is composed of →3)-α-d-Galp-(1→3)-β-d-Galf2OAc-(1→ disaccharide repeating units. The O-acetylation was incomplete and resulted in a microheterogeneity of the O-antigen. This O-acetylation generates additional antigenic determinants within the O-antigen, forms a new chemotype, and contributes to the epitopes recognized by the O-serotype specific antibodies. The serological cross-reactivities further confirmed the inter-specific structural similarity of these O-antigens.

Cryptococcus neoformans causes life-threatening diseases mainly in immunosuppressed hosts such as AIDS patients; C. gattii causes disseminated infections even in healthy hosts. To identify the possible molecular mechanisms underlying this difference in virulence, we investigated the survival and histopathology of lung tissue in wild-type and CD4-depleted mice infected with C. neoformans H99 and C. gattii JP02 (the highly virulent strain isolated in Japan); we then compared dendritic cell (DC) cytokine release responses to different cell fractions from these two strains. JP02-infected mice exhibited shorter survival and fewer inflammatory cells in the lung than H99-infected control mice. Depletion of CD4-related cellular immunity reduced survival of H99-infected mice but had no effect on the survival or inflammatory cell infiltration in JP02-infected mice, suggesting that JP02 evades immune detection. To identify the molecule(s) conferring this difference, we measured cytokine production from murine DCs co-cultured with H99 and JP02 in vitro. The levels of inflammatory cytokines from DCs treated with intact JP02 cells, the extracted capsule, secreted extracellular polysaccharides, and purified glucuronoxylomannan (GXM) were markedly lower than those induced by intact H99 cells and corresponding H99 fractions. Structural analysis of GXM indicated that JP02 altered one of two O-acetyl groups detected in the H99 GXM. Deacetylated GXM lost the ability to induce inflammatory cytokine release from DCs, implicating these O-acetyl groups in immune recognition. We conclude that the highly virulent C. gattii processes a structural alteration in GXM that allows this pathogen to evade the immune response and therefore elimination. [ABSTRACT FROM AUTHOR]

Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ∼70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1- expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production. [ABSTRACT FROM AUTHOR]

O -Acetyl esterification is an important structural and functional feature of pectins present in the cell walls of all land plants. The amount and positions of pectin acetyl substituents varies across plant tissues and stages of development. Plant growth and response to biotic and abiotic stress are known to be significantly influenced by pectin O -acetylation. Gel formation is a key characteristic of pectins, and many studies have shown that gel formation is dependent upon the degree of acetylation. Previous studies have indicated that members of the TRICHOME BIREFRINGENCE-LIKE (TBL) family may play a role in the O -acetylation of pectin, however, biochemical evidence for acceptor specific pectin acetyltransferase activity remains to be confirmed and the exact mechanism(s) for catalysis must be determined. Pectin acetylesterases (PAEs) affect pectin acetylation as they hydrolyze acetylester bonds and have a role in the amount and distribution of O -acetylation. Several mutant studies suggest the critical role of pectin O -acetylation; however, additional research is required to fully understand this. This review aims to discuss the importance, role, and putative mechanism of pectin O -acetylation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V.)

Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently, a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties, we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase, is detected. Using the homology modeling approach, we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site-directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum corresponding to 8.4-8.5. Moreover, glycosylation influences the biological activity of the enzyme and is essential for release of SIAE into the culture medium. According to these findings, co-localization experiments demonstrated the presence of SIAE in membranous structures corresponding to endoplasmic reticulum and Golgi complex. Thus, at least in COS7 cells, SIAE behaves as a typical secreted enzyme, subjected to glycosylation and located along the classical secretory route or in the extracellular space. In these environments, the enzyme could act on 9-O-acetylated sialic acid residues, contributing to the fine-tuning of the various functions played by this acidic sugar. [ABSTRACT FROM AUTHOR]