O-linked Glycosylation System Research Articles

One-step preparation of a self-assembled bioconjugate nanovaccine against Brucella

ABSTRACT Brucellosis, caused by Brucella, is a severe zoonosis, and the current Brucella live attenuated vaccine cannot be used in humans due to major safety risks. Although polysaccharide antigens can be used to prepare the Brucella vaccine, their lower immunogenicity limits them from producing efficient and broad protection. In this study, we produced a high-performance bioconjugate nanovaccine against different species of Brucella by introducing a self-assembly nanoparticle platform and an O-linked glycosylation system into Yersinia enterocolitica serotype O:9, which has an O-polysaccharide composed of the same unit as Brucella. After successfully preparing the vaccine and confirming its stability, we subsequently demonstrated the safety of the vaccine in mice by high-dose immunization. Then, by a series of mouse experiments, we found that the nanovaccine greatly promoted antibody responses. In particular, the increase of IgG2a was more obvious than that of IgG1. Most importantly, this nanovaccine could provide cross-protection against B. abortus, B. melitensis, and B. suis strains by lethal dose challenged models, and could improve the clearance of B. melitensis, the most common pathogenic species in human brucellosis, by non-lethal dose infection. Overall, for the first time, we biocoupled polysaccharide antigens with nano carriers to prepare a Brucella vaccine, which showed pronounced and extensive protective effects in mice. Thus, we provided a potential candidate vaccine and a new direction for Brucella vaccine design.

Specificity in glycosylation of multiple flagellins by the modular and cell cycle regulated glycosyltransferase FlmG.

How specificity is programmed into post-translational modification of proteins by glycosylation is poorly understood, especially for O-linked glycosylation systems. Here we reconstitute and dissect the substrate specificity underpinning the cytoplasmic O-glycosylation pathway that modifies all six flagellins, five structural and one regulatory paralog, in Caulobacter crescentus, a monopolarly flagellated alpha-proteobacterium. We characterize the biosynthetic pathway for the sialic acid-like sugar pseudaminic acid and show its requirement for flagellation, flagellin modification and efficient export. The cognate NeuB enzyme that condenses phosphoenolpyruvate with a hexose into pseudaminic acid is functionally interchangeable with other pseudaminic acid synthases. The previously unknown and cell cycle-regulated FlmG protein, a defining member of a new class of cytoplasmic O-glycosyltransferases, is required and sufficient for flagellin modification. The substrate specificity of FlmG is conferred by its N-terminal flagellin-binding domain. FlmG accumulates before the FlaF secretion chaperone, potentially timing flagellin modification, export, and assembly during the cell division cycle.

2020 FASEB Science Research Conference on Microbial Glycobiology, July 13-14, 2020.

2020 FASEB Science Research Conference on Microbial Glycobiology, July 13-14, 2020.

Application of an O-Linked Glycosylation System in Yersinia enterocolitica Serotype O:9 to Generate a New Candidate Vaccine against Brucella abortus.

Brucellosis is a major zoonotic public health threat worldwide, causing veterinary morbidity and major economic losses in endemic regions. However, no efficacious brucellosis vaccine is yet available, and live attenuated vaccines commonly used in animals can cause human infection. N- and O-linked glycosylation systems have been successfully developed and exploited for the production of successful bioconjugate vaccines. Here, we applied an O-linked glycosylation system to a low-pathogenicity bacterium, Yersinia enterocolitica serotype O:9 (Y. enterocolitica O:9), which has repeating units of O-antigen polysaccharide (OPS) identical to that of Brucella abortus (B. abortus), to develop a bioconjugate vaccine against Brucella. The glycoprotein we produced was recognized by both anti-B. abortus and anti-Y. enterocolitica O:9 monoclonal antibodies. Three doses of bioconjugate vaccine-elicited B. abortus OPS-specific serum IgG in mice, significantly reducing bacterial loads in the spleen following infection with the B. abortus hypovirulent smooth strain A19. This candidate vaccine mitigated B. abortus infection and prevented severe tissue damage, thereby protecting against lethal challenge with A19. Overall, the results indicated that the bioconjugate vaccine elicited a strong immune response and provided significant protection against brucellosis. The described vaccine preparation strategy is safe and avoids large-scale culture of the highly pathogenic B. abortus.

Glycoengineering bioconjugate vaccines, therapeutics, and diagnostics in E. coli

The first, general glycosylation pathway in bacteria, the N-linked glycosylation system of Campylobacter jejuni, was discovered two decades ago. Since then, many diverse prokaryotic glycosylation systems have been characterized, including O-linked glycosylation systems that have no homologous counterparts in eukaryotic organisms. Shortly after these discoveries, glycosylation pathways were recombinantly introduced into E. coli creating the field of bacterial glycoengineering. Bacterial glycoengineering is an emerging biotechnological tool that harnesses prokaryotic glycosylation systems for the generation of recombinantly glycosylated proteins using E. coli as a host. Over the last decade, as our understanding of prokaryotic glycosylation systems has advanced, so too has the glycoengineering toolbox. Currently, glycoengineering utilizes two broad approaches to recombinantly glycosylate proteins, both of which can generate N- or O-linkages: oligosaccharyltransferase (OTase)-dependent and OTase-independent. This review discusses the applications of these bacterial glycoengineering techniques as they relate to the development of glycoconjugate vaccines, therapeutic proteins, and diagnostics.

Design and production of conjugate vaccines against S. Paratyphi A using an O-linked glycosylation system in vivo

Enteric fever, mainly caused by Salmonella enterica serovar Paratyphi A, remains a common and serious infectious disease worldwide. As yet, there are no licensed vaccines against S. Paratyphi A. Biosynthesis of conjugate vaccines has become a promising approach against bacterial infection. However, the popular biosynthetic strategy using N-linked glycosylation systems does not recognize the specialized O-polysaccharide structure of S. Paratyphi A. Here, we describe an O-linked glycosylation approach, the only currently available glycosylation system suitable for an S. Paratyphi A conjugate vaccine. We successfully generated a recombinant S. Paratyphi A strain with a longer O-polysaccharide chain and transformed the O-linked glycosylation system into the strain. Thus, we avoided the need for construction of an O-polysaccharide expression vector. In vivo assays indicated that this conjugate vaccine could evoke IgG1 antibody to O-antigen of S. Paratyphi A strain CMCC 50973 and elicit bactericidal activity against S. Paratyphi A strain CMCC 50973 and five other epidemic strains. Furthermore, we replaced the peptides after the glycosylation site (Ser) with an antigenic peptide (P2). The results showed that the anti-lipopolysaccharide antibody titer, bactericidal activity of serum, and protective effect during animal challenge could be improved, indicating a potential strategy for further vaccine design. Our system provides an easier and more economical method for the production of S. Paratyphi A conjugate vaccines. Modification of the glycosylation site sequon provides a potential approach for the development of next-generation “precise conjugate vaccines.”

Role of Glycosyltransferases Modifying Type B Flagellin of Emerging Hypervirulent Clostridium difficile Lineages and Their Impact on Motility and Biofilm Formation

Clostridium difficile is the principal cause of nosocomial infectious diarrhea worldwide. The pathogen modifies its flagellin with either a type A or type B O-linked glycosylation system, which has a contributory role in pathogenesis. We study the functional role of glycosyltransferases modifying type B flagellin in the 023 and 027 hypervirulent C. difficile lineages by mutagenesis of five putative glycosyltransferases and biosynthetic genes. We reveal their roles in the biosynthesis of the flagellin glycan chain and demonstrate that flagellar post-translational modification affects motility and adhesion-related bacterial properties of these strains. We show that the glycosyltransferases 1 and 2 (GT1 and GT2) are responsible for the sequential addition of a GlcNAc and two rhamnoses, respectively, and that GT3 is associated with the incorporation of a novel sulfonated peptidyl-amido sugar moiety whose structure is reported in our accompanying paper (Bouché, L., Panico, M., Hitchen, P., Binet, D., Sastre, F., Faulds-Pain, A., Valiente, E., Vinogradov, E., Aubry, A., Fulton, K., Twine, S., Logan, S. M., Wren, B. W., Dell, A., and Morris, H. R. (2016) J. Biol. Chem. 291, 25439–25449). GT2 is also responsible for methylation of the rhamnoses. Whereas type B modification is not required for flagellar assembly, some mutations that result in truncation or abolition of the glycan reduce bacterial motility and promote autoaggregation and biofilm formation. The complete lack of flagellin modification also significantly reduces adhesion of C. difficile to Caco-2 intestinal epithelial cells but does not affect activation of human TLR5. Our study advances our understanding of the genes involved in flagellar glycosylation and their biological roles in emerging hypervirulent C. difficile strains.

Biosynthesis of Conjugate Vaccines Using an O-Linked Glycosylation System.

ABSTRACT Conjugate vaccines are known to be one of the most effective and safest types of vaccines against bacterial pathogens. Previously, vaccine biosynthesis has been performed by using N-linked glycosylation systems. However, the structural specificity of these systems for sugar substrates has hindered their application. Here, we report a novel protein glycosylation system (O-linked glycosylation via Neisseria meningitidis) that can transfer virtually any glycan to produce a conjugate vaccine. We successfully established this system in Shigella spp., avoiding the construction of an expression vector for polysaccharide synthesis. We further found that different protein substrates can be glycosylated using this system and that the O-linked glycosylation system can also effectively function in other Gram-negative bacteria, including some strains whose polysaccharide structure was not suitable for conjugation using the N-linked glycosylation system. The results from a series of animal experiments show that the conjugate vaccine produced by this O-linked glycosylation system offered a potentially protective antibody response. Furthermore, we elucidated and optimized the recognition motif, named MOOR, for the O-glycosyltransferase PglL. Finally, we demonstrated that the fusion of other peptides recognized by major histocompatibility complex class II around MOOR had no adverse effects on substrate glycosylation, suggesting that this optimized system will be useful for future vaccine development. Our results expand the glycoengineering toolbox and provide a simpler and more robust strategy for producing bioconjugate vaccines against a variety of pathogens.

Insights into type IV pilus biogenesis and dynamics from genetic analysis of a C‐terminally tagged pilin: a role for O‐linked glycosylation

Type IV pili are surface organelles essential for pathogenicity of many Gram-negative bacteria. In Neisseria gonorrhoeae, the major subunit of type IV pili, PilE, is a target of its general O-linked glycosylation system. This system modifies a diverse set of periplasmic and extracellular gonococcal proteins with a variable set of glycans. Here we show that expression of a particular hexa-histidine-tagged PilE was associated with growth arrest. By studying intra- and extragenic suppressors, we found that this phenotype was dependent on pilus assembly and retraction. Based on these results, we developed a sensitive tool to identify factors with subtle effects on pilus dynamics. Using this approach, we found that glycan chain length has differential effects on the growth arrest that appears to be mediated at the level of pilin subunit-subunit interactions and bidirectional remodelling of pilin between its membrane-associated and assembled states. Gonococcal pilin glycosylation thus plays both an intracellular role in pilus dynamics and potential extracellular roles mediated through type IV pili. In addition to demonstrating the effect of glycosylation on pilus dynamics, the study provides a new way of identifying factors with less dramatic effects on processes involved in type IV pilus biogenesis.

Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae

Protein glycosylation is an important element of biologic systems because of its significant effects on protein properties and functions. Although prominent within all domains of life, O-linked glycosylation systems modifying serine and threonine residues within bacteria and eukaryotes differ substantially in target protein selectivity. In particular, well-characterized bacterial systems have been invariably dedicated to modification of individual proteins or related subsets thereof. Here we characterize a general O-linked glycosylation system that targets structurally and functionally diverse groups of membrane-associated proteins in the gram-negative bacterium Neisseria gonorrhoeae, the etiologic agent of the human disease gonorrhea. The 11 glycoproteins identified here are implicated in activities as varied as protein folding, disulfide bond formation, and solute uptake, as well as both aerobic and anaerobic respiration. Along with their common trafficking within the periplasmic compartment, the protein substrates share quasi-related domains bearing signatures of low complexity that were demonstrated to encompass sites of glycan occupancy. Thus, as in eukaryotes, the broad scope of this system is dictated by the relaxed specificity of the glycan transferase as well as the bulk properties and context of the protein-targeting signal rather than by a strict amino acid consensus sequence. Together, these findings reveal previously unrecognized commonalities linking O-linked protein glycosylation in distantly related life forms.