How bacterial carbohydrates influence the adaptive immune system.

The capsular polysaccharides (CPSs) of most pathogenic bacteria are T cell-independent antigens whose conjugation to carrier proteins evokes a carbohydrate-specific response eliciting T cell help. However, certain bacterial CPSs, known as zwitterionic polysaccharides (ZPSs), activate the adaptive immune system through processing by antigen-presenting cells and presentation by the major histocompatibility complex class II pathway to CD4(+) T cells. This discovery was the first mechanistic insight into how carbohydrates-a class of biological molecules previously thought to be T cell independent-can in fact activate T cells. Through their ability to activate CD4(+) T cells, ZPSs direct the cellular and physical maturation of the developing immune system. In this review, we explore the still-enigmatic relations between CPSs and the adaptive immune machinery at the cellular and molecular levels, and we discuss how new insights into the biological impact of ZPSs expand our concepts of the role of carbohydrates in microbial interactions with the adaptive immune system.

Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells.

Coevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host's endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood, and hosts are protected against experimental iNKT cell-mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids-exemplified by an isolated peak (MW = 717.6) called GSL-Bf717-reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive.

The immune interactions of gut glycans and microbiota in health and disease.

The human digestive system harbors a vast diversity of commensal bacteria and maintains a symbiotic relationship with them. However, imbalances in the gut microbiota accompany various diseases, such as inflammatory bowel diseases (IBDs) and colorectal cancers (CRCs), which significantly impact the well-being of populations globally. Glycosylation of the mucus layer is a crucial factor that plays a critical role in maintaining the homeostatic environment in the gut. This review delves into how the gut microbiota, immune cells, and gut mucus layer work together to establish a balanced gut environment. Specifically, the role of glycosylation in regulating immune cell responses and mucus metabolism in this process is examined.

Serine-rich repeat proteins: well-known yet little-understood bacterial adhesins.

Serine-rich-repeat proteins (SRRPs) are large mucin-like glycoprotein adhesins expressed by a plethora of pathogenic and symbiotic Gram-positive bacteria. SRRPs play major functional roles in bacterial-host interactions, like adhesion, aggregation, biofilm formation, virulence, and pathogenesis. Through their functional roles, SRRPs aid in the development of host microbiomes but also diseases like infective endocarditis, otitis media, meningitis, and pneumonia. SRRPs comprise shared domains across different species, including two or more heavily O-glycosylated long stretches of serine-rich repeat regions. With loci that can be as large as ~40 kb and can encode up to 10 distinct glycosyltransferases that specifically facilitate SRRP glycosylation, the SRRP loci makes up a significant portion of the bacterial genome. The significance of SRRPs and their glycans in host-microbe communications is becoming increasingly evident. Studies are beginning to reveal the glycosylation pathways and mature O-glycans presented by SRRPs. Here we review the glycosylation machinery of SRRPs across species and discuss the functional roles and clinical manifestations of SRRP glycosylation.

Harnessing galactose oxidase in the development of a chemoenzymatic platform for glycoconjugate vaccine design.

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.

Tn antigen interactions of macrophage galactose-type lectin (MGL) in immune function and disease.

Protein-carbohydrate interactions are essential in maintaining immune homeostasis and orchestrating inflammatory and regulatory immune processes. This review elucidates the immune interactions of macrophage galactose-type lectin (MGL, CD301) and Tn carbohydrate antigen. MGL is a C-type lectin receptor (CLR) primarily expressed by myeloid cells such as macrophages and immature dendritic cells. MGL recognizes terminal O-linked N-acetylgalactosamine (GalNAc) residue on the surface proteins, also known as Tn antigen (Tn). Tn is a truncated form of the elongated cell surface O-glycan. The hypoglycosylation leading to Tn may occur when the enzyme responsible for O-glycan elongation-T-synthase-or its associated chaperone-Cosmc-becomes functionally inhibited. As reviewed here, Tn expression is observed in many different neoplastic and non-neoplastic diseases, and the recognition of Tn by MGL plays an important role in regulating effector T cells, immune suppression, and the recognition of pathogens.

Host protein glycosylation in nucleic acid vaccines as a potential hurdle in vaccine design for nonviral pathogens.

Nucleic acid vaccines introduce the genetic materials encoding antigenic proteins into host cells. If these proteins are directed into the secretory pathway with a signal/leader sequence, they will be exposed to the host's glycosylation machinery, and, if their amino acid sequences contain consensus sequons for N-linked glycosylation, they may become glycosylated. The presence of host glycans on the proteins of microbial origin may prevent a strong protective immune response either through hindering access to key epitopes by lymphocytes or through altering immune responses by binding to immunoregulatory glycan-binding receptors on immune cells. Ag85A expressed by Mycobacterium tuberculosis (Mtb) is a bacterial surface protein that is commonly used in nucleic acid vaccines in multiple clinical trials. Here we show that, when Ag85A is expressed in mammalian cells, it is glycosylated, does not induce a strong humoral immune response in mice, and does not activate Ag85A-specific lymphocytes as highly as Ag85A natively expressed by the bacterium. Our study indicates that host glycosylation of the vaccine target can impede its antigenicity and immunogenicity. Glycosylation of the antigenic protein targets therefore must be carefully evaluated in designing nucleic acid vaccines.

Glycosyltransferases within the psrP Locus Facilitate Pneumococcal Virulence.

The pneumococcal serine-rich repeat protein (PsrP) is a high-molecular-weight, glycosylated adhesin that promotes the attachment of Streptococcus pneumoniae to host cells. PsrP, its associated glycosyltransferases (GTs), and dedicated secretion machinery are encoded in a 37-kb genomic island that is present in many invasive clinical isolates of S. pneumoniae PsrP has been implicated in establishment of lung infection in murine models, although specific roles of the PsrP glycans in disease progression or bacterial physiology have not been elucidated. Moreover, enzymatic specificities of associated glycosyltransferases are yet to be fully characterized. We hypothesized that the glycosyltransferases that modify PsrP are critical for the adhesion properties and infectivity of S. pneumoniae Here, we characterize the putative S. pneumoniaepsrP locus glycosyltransferases responsible for PsrP glycosylation. We also begin to elucidate their roles in S. pneumoniae virulence. We show that four glycosyltransferases within the psrP locus are indispensable for S. pneumoniae biofilm formation, lung epithelial cell adherence, and establishment of lung infection in a mouse model of pneumococcal pneumonia.IMPORTANCE PsrP has previously been identified as a necessary virulence factor for many serotypes of S. pneumoniae and studied as a surface glycoprotein. Thus, studying the effects on virulence of each glycosyltransferase (GT) that builds the PsrP glycan is of high importance. Our work elucidates the influence of GTs in vivo We have identified at least four GTs that are required for lung infection, an indication that it is worthwhile to consider glycosylated PsrP as a candidate for serotype-independent pneumococcal vaccine design.

Broadly Reactive Human Monoclonal Antibodies Targeting the Pneumococcal Histidine Triad Protein Protect against Fatal Pneumococcal Infection.

Streptococcus pneumoniae remains a leading cause of bacterial pneumonia despite the widespread use of vaccines. While vaccines are effective at reducing the incidence of most serotypes included in vaccines, a rise in infection due to nonvaccine serotypes and moderate efficacy against some vaccine serotypes have contributed to high disease incidence. Additionally, numerous isolates of S. pneumoniae are antibiotic or multidrug resistant. Several conserved pneumococcal proteins prevalent in the majority of serotypes have been examined for their potential as vaccines in preclinical and clinical trials. An additional, yet-unexplored tool for disease prevention and treatment is the use of human monoclonal antibodies (MAbs) targeting conserved pneumococcal proteins. Here, we isolated the first human MAbs (PhtD3, PhtD6, PhtD7, PhtD8, and PspA16) against the pneumococcal histidine triad protein (PhtD) and the pneumococcal surface protein A (PspA), two conserved and protective antigens. MAbs to PhtD target diverse epitopes on PhtD, and MAb PspA16 targets the N-terminal segment of PspA. The PhtD-specific MAbs bind to multiple serotypes, while MAb PspA16 serotype breadth is limited. MAbs PhtD3 and PhtD8 prolong the survival of mice infected with pneumococcal serotype 3. Furthermore, MAb PhtD3 prolongs the survival of mice in intranasal and intravenous infection models with pneumococcal serotype 4 and in mice infected with pneumococcal serotype 3 when administered 24 h after pneumococcal infection. All PhtD and PspA MAbs demonstrate opsonophagocytic activity, suggesting a potential mechanism of protection. Our results identify new human MAbs for pneumococcal disease prevention and treatment and identify epitopes on PhtD and PspA recognized by human B cells.

Characterization of the ß-glucuronidase Pn3Pase as the founding member of glycoside hydrolase family GH169.

Paenibacillus sp. 32352 is a soil-dwelling bacterium capable of producing an enzyme, Pn3Pase that degrades the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (Pn3P). Recent reports on Pn3Pase have demonstrated its initial characterization and potential for protection against highly virulent S. pneumoniae serotype 3 infections. Initial experiments revealed this enzyme functions as an exo-ß1,4-glucuronidase cleaving the ß(1,4) linkage between glucuronic acid and glucose. However, the catalytic mechanism of this enzyme is still unknown. Here, we report the detailed biochemical analysis of Pn3Pase. Pn3Pase shows no significant sequence similarity to known glycoside hydrolase (GH) families, thus this novel enzyme establishes a new carbohydrate-active enzyme (CAZy) GH family. Site-directed mutagenesis studies revealed two catalytic residues along with truncation mutants defining essential domains for function. Pn3Pase and its mutants were screened for activity, substrate binding and kinetics. Additionally, nuclear magnetic resonance spectroscopy analysis revealed that Pn3Pase acts through a retaining mechanism. This study exhibits Pn3Pase activity at the structural and mechanistic level to establish the new CAZy GH family GH169 belonging to the large GH-A clan. This study will also serve toward generating Pn3Pase derivatives with optimal activity and pharmacokinetics aiding in the use of Pn3Pase as a novel therapeutic approach against type 3 S. pneumoniae infections.

FAP-Targeted Photodynamic Therapy Mediated by Ferritin Nanoparticles Elicits an Immune Response against Cancer Cells and Cancer Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) are present in many types of tumors and play a pivotal role in tumor progression and immunosuppression. Fibroblast-activation protein (FAP), which is overexpressed on CAFs, has been indicated as a universal tumor target. However, FAP expression is not restricted to tumors, and systemic treatment against FAP often causes severe side effects. To solve this problem, a photodynamic therapy (PDT) approach was developed based on ZnF16Pc (a photosensitizer)-loaded and FAP-specific single chain variable fragment (scFv)-conjugated apoferritin nanoparticles, or αFAP-Z@FRT. αFAP-Z@FRT PDT efficiently eradicates CAFs in tumors without inducing systemic toxicity. When tested in murine 4T1 models, the PDT treatment elicits anti-cancer immunity, causing suppression of both primary and distant tumors, i.e. abscopal effect. Treatment efficacy is enhanced when αFAP-Z@FRT PDT is used in combination with anti-PD1 antibodies. Interestingly, it is found that the PDT treatment not only elicits a cellular immunity against cancer cells, but also stimulates an anti-CAFs immunity. This is supported by an adoptive cell transfer study, where T cells taken from 4T1-tumor-bearing animals treated with αFAP PDT retard the growth of A549 tumors established on nude mice. Overall, our approach is unique for permitting site-specific eradication of CAFs and inducing a broad spectrum anti-cancer immunity.

Therapeutic Activity of Type 3 Streptococcus pneumoniae Capsule Degrading Enzyme Pn3Pase.

PURPOSE: Streptococcus pneumoniae (Spn) serotype 3 (Spn3) is considered one of the most virulent serotypes with resistance to conventional vaccine and treatment regimens. Pn3Pase is a glycoside hydrolase that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn, sensitizing it to host immune clearance. To begin assessing the value and safety of this enzyme for future clinical studies, we investigated the effects of high doses of Pn3Pase on host cells and immune system. METHODS: We assessed the enzyme's catalytic activity following administration in mice, and performed septic infection models to determine if prior administration of the enzyme inhibited repeat treatments of Spn3-challenged mice. We assessed immune populations in mouse tissues following administration of the enzyme, and tested Pn3Pase toxicity on other mammalian cell types in vitro. RESULTS: Repeated administration of the enzyme in vivo does not prevent efficacy of the enzyme in promoting bacterial clearance following bacterial challenge, with insignificant antibody response generated against the enzyme. Immune homeostasis is maintained following high-dose treatment with Pn3Pase, and no cytotoxic effects were observed against mammalian cells. CONCLUSIONS: These data indicate that Pn3Pase has potential as a therapy against Spn3. Further development as a drug product could overcome a great hurdle of pneumococcal infections.

Metabolic labeling of HIV-1 envelope glycoprotein gp120 to elucidate the effect of gp120 glycosylation on antigen uptake.

The glycan shield on the envelope glycoprotein gp120 of HIV-1 has drawn immense attention as a vulnerable site for broadly neutralizing antibodies and for its significant impact on host adaptive immune response to HIV-1. Glycosylation sites and glycan composition/structure at each site on gp120 along with the interactions of gp120 glycan shield with broadly neutralizing antibodies have been extensively studied. However, a method for directly and selectively tracking gp120 glycans has been lacking. Here, we integrate metabolic labeling and click chemistry technology with recombinant gp120 expression to demonstrate that gp120 glycans could be specifically labeled and directly detected. Selective labeling of gp120 by N-azidoacetylmannosamine (ManNAz) and N-azidoacetylgalactosamine (GalNAz) incorporation into the gp120 glycan shield was characterized by MS of tryptic glycopeptides. By using metabolically labeled gp120, we investigated the impact of gp120 glycosylation on its interaction with host cells and demonstrated that oligomannose enrichment and sialic acid deficiency drastically enhanced gp120 uptake by bone marrow-derived dendritic cells. Collectively, our data reveal an effective labeling and detection method for gp120, serving as a tool for functional characterization of the gp120 glycans and potentially other glycosylated proteins.

Glycoconjugate synthesis using chemoselective ligation.

Chemoselective ligation of carbohydrates and polypeptides was achieved using an adipic acid dihydrazide cross-linker. The reducing end of a carbohydrate is efficiently attached to peptides in two steps, constructing a glycoconjugate in high yield and with high regioselectivity, enabling the production of homogeneous glycoconjugates.

T Cell-Mediated Humoral Immune Responses to Type 3 Capsular Polysaccharide of Streptococcus pneumoniae.

Most pathogenic bacteria express surface carbohydrates called capsular polysaccharides (CPSs). CPSs are important vaccine targets because they are easily accessible and recognizable by the immune system. However, CPS-specific adaptive humoral immune responses can only be achieved by the covalent conjugation of CPSs with carrier proteins to produce glycoconjugate vaccines. We previously described a mechanism by which a model glycoconjugate vaccine can activate the adaptive immune system and demonstrated that the mammalian CD4+ T cell repertoire contains a population of carbohydrate-specific T cells. In this study, we use glycoconjugates of type 3 Streptococcus pneumoniae CPS (Pn3P) to assess whether the carbohydrate-specific adaptive immune response exemplified in our previous study can be applied to the conjugates of this lethal pathogen. In this article, we provide evidence for the functional roles of Pn3P-specific CD4+ T cells utilizing mouse immunization schemes that induce Pn3P-specific IgG responses in a carbohydrate-specific T cell-dependent manner.

Enzymatic Hydrolysis of Pneumococcal Capsular Polysaccharide Renders the Bacterium Vulnerable to Host Defense.

Despite a century of investigation, Streptococcus pneumoniae remains a major human pathogen, causing a number of diseases, such as pneumonia, meningitis, and otitis media. Like many encapsulated pathogens, the capsular polysaccharide (CPS) of S. pneumoniae is a critical component for colonization and virulence in mammalian hosts. This study aimed to evaluate the protective role of a glycoside hydrolase, Pn3Pase, targeting the CPS of type 3 S. pneumoniae, which is one of the most virulent serotypes. We have assessed the ability of Pn3Pase to degrade the capsule on a live type 3 strain. Through in vitro assays, we observed that Pn3Pase treatment increases the bacterium's susceptibility to phagocytosis by macrophages and complement-mediated killing by neutrophils. We have demonstrated that in vivo Pn3Pase treatment reduces nasopharyngeal colonization and protects mice from sepsis caused by type 3 S. pneumoniae Due to the increasing shifts in serotype distribution, the rise in drug-resistant strains, and poor immune responses to vaccine-included serotypes, it is necessary to investigate approaches to combat pneumococcal infections. This study evaluates the interaction of pneumococcal CPS with the host at molecular, cellular, and systemic levels and offers an alternative therapeutic approach for diseases caused by S. pneumoniae through enzymatic hydrolysis of the CPS.

Identification and characterization of the Streptococcus pneumoniae type 3 capsule-specific glycoside hydrolase of Paenibacillus species 32352.

Bacillus circulans Jordan 32352 was isolated from decaying organic matter in the New Jersey soil in the early 1930s. This soil-dwelling bacterium produced an enzyme capable of degrading the type 3 capsular polysaccharide (Pn3P) of Streptococcus pneumoniae (Spn). Early reports of this enzyme, Pn3Pase, demonstrated its inducibility by, and specificity for Pn3P. We set out to identify and clone this enzyme for its recombinant expression and characterization. We first sequenced the genome of this bacterial species, and reclassified the Pn3Pase producing bacterium as Paenibacillus species 32352. We identified the putative protein of Pn3Pase through mass spectrometry-based proteomics and cloned the gene for recombinant expression. We then characterized the oligosaccharide products generated upon the enzymatic depolymerization of Pn3P. Sequence analysis suggests that this glycoside hydrolase belongs to a new carbohydrate-active enzyme GH family. To our knowledge, this is the only enzyme to demonstrate Pn3P depolymerization activity.

Rapid screening of sugar-nucleotide donor specificities of putative glycosyltransferases.

Determining the correct enzymatic activity of putative glycosyltransferases (GTs) can be challenging as these enzymes can utilize multiple donor and acceptor substrates. Upon initial determination of the donor-sugar nucleotide(s), a GT utilizes various acceptor molecules that can then be tested. Here, we describe a quick method to screen sugar-nucleotide donor specificities of GTs utilizing a sensitive, nonradioactive, commercially available bioluminescent uridine diphosphate detection kit. This in vitro method allowed us to validate the sugar-nucleotide donor-substrate specificities of recombinantly expressed human, bovine, bacterial and protozoan GTs. Our approach, which is less time consuming than many traditional assays that utilize radiolabeled sugars and chromatographic separations, should facilitate discovery of novel GTs that participate in diverse biological processes.

Carbohydrates and T cells: a sweet twosome.

Carbohydrates as T cell-activating antigens have been generating significant interest. For many years, carbohydrates were thought of as T-independent antigens, however, more recent research had demonstrated that mono- or oligosaccharides glycosidically linked to peptides can be recognized by T cells. T cell recognition of these glycopeptides depends on the structure of both peptide and glycan portions of the antigen. Subsequently, it was discovered that natural killer T cells recognized glycolipids when presented by the antigen presenting molecule CD1d. A transformative insight into glycan-recognition by T cells occurred when zwitterionic polysaccharides were discovered to bind to and be presented by MHCII to CD4+ T cells. Based on this latter observation, the role that carbohydrate epitopes generated from glycoconjugate vaccines had in activating helper T cells was explored and it was found that these epitopes are presented to specific carbohydrate recognizing T cells through a unique mechanism. Here we review the key interactions between carbohydrate antigens and the adaptive immune system at the molecular, cellular and systems levels exploring the significant biological implications in health and disease.