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Introduction: The pathogenic bacterium Helicobacter pylori has evolved glycan-mediated mechanisms to evade host immune defenses. This study tests the hypothesis that genetic disruption of H. pylori glycan biosynthesis alters immune recognition and response by human gastric epithelial cells and monocyte-derived dendritic cells., Methods: To test this hypothesis, human cell lines were challenged with wildtype H. pylori alongside an array of H. pylori glycosylation mutants. The relative levels of immune response were measured via immature dendritic cell maturation and cytokine secretion., Results: Our findings indicate that disruption of lipopolysaccharide biosynthesis diminishes gastric cytokine production, without disrupting dendritic cell recognition and activation. In contrast, variable immune responses were observed in protein glycosylation mutants which prompted us to test the hypothesis that phase variation plays a role in regulating bacterial cell surface glycosylation and subsequent immune recognition. Lewis antigen presentation does not correlate with extent of immune response, while the extent of lipopolysaccharide O-antigen elaboration does., Discussion: The outcomes of this study demonstrate that H. pylori glycans modulate the host immune response. This work provides a foundation to pursue immune-based tailoring of bacterial glycans towards modulating immunogenicity of microbial pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Barrett, Kassama, Surks, Mulholland, Moulton and Dube.)

Glycans that coat the surface of bacteria are compelling antibiotic targets because they contain distinct monosaccharides that are linked to pathogenesis and are absent in human cells. Disrupting glycan biosynthesis presents a path to inhibiting the ability of a bacterium to infect the host. We previously demonstrated that O-glycosides act as metabolic inhibitors and disrupt bacterial glycan biosynthesis. Inspired by a recent study which showed that thioglycosides (S-glycosides) are 10 times more effective than O-glycosides at inhibiting glycan biosynthesis in mammalian cells, we crafted a panel of S-glycosides based on rare bacterial monosaccharides. The novel thioglycosides altered glycan biosynthesis and fitness in pathogenic bacteria but had no notable effect on glycosylation or growth in beneficial bacteria or mammalian cells. In contrast to findings in mammalian cells, S-glycosides and O-glycosides exhibited comparable potency in bacteria. However, S-glycosides exhibited enhanced selectivity relative to O-glycosides. These novel metabolic inhibitors will allow selective perturbation of the bacterial glycocalyx for functional studies and set the stage to expand our antibiotic arsenal.

Bacterial cell envelope glycans are compelling antibiotic targets as they are critical for strain fitness and pathogenesis yet are virtually absent from human cells. However, systematic study and perturbation of bacterial glycans remains challenging due to their utilization of rare deoxy amino l-sugars, which impede traditional glycan analysis and are not readily available from natural sources. The development of chemical tools to study bacterial glycans is a crucial step toward understanding and altering these biomolecules. Here we report an expedient methodology to access azide-containing analogues of a variety of unusual deoxy amino l-sugars starting from readily available l-rhamnose and l-fucose. Azide-containing l-sugar analogues facilitated metabolic profiling of bacterial glycans in a range of Gram-negative bacteria and revealed differential utilization of l-sugars in symbiotic versus pathogenic bacteria. Further application of these probes will refine our knowledge of the glycan repertoire in diverse bacteria and aid in the design of novel antibiotics.

Bacterial cell surface glycans are quintessential drug targets due to their critical role in colonization of the host, pathogen survival, and immune evasion. The dense cell envelope glycocalyx contains distinctive monosaccharides that are stitched together into higher order glycans to yield exclusively bacterial structures that are critical for strain fitness and pathogenesis. However, the systematic study and inhibition of bacterial glycosylation enzymes remains challenging. Bacteria produce glycans containing rare sugars refractory to traditional glycan analysis, complicating the study of bacterial glycans and the identification of their biosynthesis machinery. To ease the study of bacterial glycans in the absence of detailed structural information, we used metabolic glycan labeling to detect changes in glycan biosynthesis. Here, we screened wild-type versus mutant strains of the gastric pathogen Helicobacter pylori , ultimately permitting the identification of genes involved in glycoprotein and lipopolysaccharide biosynthesis. Our findings provide the first evidence that H. pylori protein glycosylation proceeds via a lipid carrier-mediated pathway that overlaps with lipopolysaccharide biosynthesis. Protein glycosylation mutants displayed fitness defects consistent with those induced by small molecule glycosylation inhibitors. Broadly, our results suggest a facile approach to screen for bacterial glycosylation genes and gain insight into their biosynthesis and functional importance, even in the absence of glycan structural information.

The bacterial cell wall is a quintessential drug target due to its critical role in colonization of the host, pathogen survival, and immune evasion. The dense cell wall glycocalyx contains distinctive monosaccharides that are absent from human cells, and proper assembly of monosaccharides into higher-order glycans is critical for bacterial fitness and pathogenesis. However, the systematic study and inhibition of bacterial glycosylation enzymes remains challenging. Bacteria produce glycans containing rare deoxy amino sugars refractory to traditional glycan analysis, complicating the study of bacterial glycans and the creation of glycosylation inhibitors. To ease the study of bacterial glycan function in the absence of detailed structural or enzyme information, we crafted metabolic inhibitors based on rare bacterial monosaccharide scaffolds. Metabolic inhibitors were assessed for their ability to interfere with glycan biosynthesis and fitness in pathogenic and symbiotic bacterial species. Three metabolic inhibitors led to dramatic structural and functional defects in Helicobacter pylori . Strikingly, these inhibitors acted in a bacteria-selective manner. These metabolic inhibitors will provide a platform for systematic study of bacterial glycosylation enzymes not currently possible with existing tools. Moreover, their selectivity will provide a pathway for the development of novel, narrow-spectrum antibiotics to treat infectious disease. Our inhibition approach is general and will expedite the identification of bacterial glycan biosynthesis inhibitors in a range of systems, expanding the glycochemistry toolkit., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Helicobacter pylori ( H. pylori ) infection poses a worldwide public health crisis, as chronic infection is rampant and can lead to gastric ulcers, gastritis, and gastric cancer. Unfortunately, frontline therapies cause harmful side effects and are often ineffective due to antibiotic resistance. The FDA-approved drug auranofin is a gold complex with a Au(I) core coordinated with triethylphosphine and peracetylated thioglucose as the ligands. Auranofin is used for the treatment of rheumatoid arthritis and also displays potent activity against H. pylori . One of auranofin's modes of action involves cell death by disrupting cellular thiol-redox balance maintained by thioredoxin reductase (TrxR), but this disruption leads to unwanted side effects due to mammalian cell toxicity. Here, we developed and tested sugar-modified analogs of auranofin as potential antibiotics against H. pylori , with the rationale that modulating the sugar moiety would bias uptake by targeting bacterial cells and mitigating mammalian cell toxicity. Sugar-modified auranofin analogs displayed micromolar minimum inhibitory concentrations against H. pylori , maintained nanomolar inhibitory activity against the target enzyme TrxR, and caused reduced toxicity to mammalian cells. Taken together, our results suggest that structurally modifying the sugar component of auranofin has the potential to yield superior antibiotics for the treatment of H. pylori infection. Broadly, glyco-tailoring is an attractive approach for repurposing approved drugs.

A structurally conserved antibody combining site, encoded by the IGH V3-23 and kappa A2 variable (V) region gene segments, predominates the adult immune response to the Haemophilus influenzae type b (Hib) capsular polysaccharide (PS). This site has been elevated to canonical status based upon its relative molecular uniformity and prevalence in adults. To date, no studies have examined the primary structure of Hib PS-specific antibodies in young infants, who are the primary targets of Hib vaccination. In this study we show that canonical Hib PS-specific heavy (H) and light (L) chain V regions are present in 4-month-old infants following two vaccinations with Hib PS-protein conjugates. The infant V regions contain sequence polymorphisms that resemble those found in adult antibodies, as well as polymorphisms at position 95a of the A2 L chain not previously observed in adults. In vitro studies of Fab fragments and recombinant IgG2 antibodies using these V regions identify sequence polymorphisms that impact Hib PS binding affinity and bactericidal activity. These results demonstrate the establishment of canonical V regions in early ontogeny and provide a structural explanation of how canonical antibodies in the infant can vary in their affinity and protective activity against Hib.

Antibodies specific for capsular polysaccharides play a central role in immunity to encapsulated Streptococcus pneumoniae, but little is known about their genetics or the variable (V) region polymorphisms that affect their protective function. To begin to address these issues, we used combinatorial library cloning to isolate pneumococcal polysaccharide (PPS)-specific Fab fragments from two vaccinated adults. We determined complete V region primary structures and performed antigen binding analyses of seven Fab fragments specific for PPS serotype 6B, 14, or 23F. Fabs were of the immunoglobulin G2 or A isotype. Several V(H)III gene segments (HV 3-7, 3-15, 3-23, and 3-11) were identified. V(L) regions were encoded by several kappa genes (KV 4-1, 3-15, 2-24, and 2D-29) and a lambda gene (LV 1-51). Deviation of the V(H) and V(L) regions from their assigned germ line counterparts indicated that they were somatically mutated. Fabs of the same serotype specificity isolated from a single individual differed in affinity, and these differences could be accounted for either by the extent of mutation among clonal relatives or by usage of different V-region genes. Thus, functionally disparate anti-PPS antibodies can arise within individuals both by activation of independent clones and by intraclonal somatic mutation. For one pair of clonally related Fabs, the more extensively mutated V(H) was associated with lower affinity for PPS 14, a result suggesting that somatic mutation could lead to diminished protective efficacy. These findings indicate that the PPS repertoire in the adult derives from memory B-cell populations that have class switched and undergone extensive hypermutation.

Antibodies having light (L) chains encoded by the kappaII-A2 variable region gene segment predominate in the human response to the Haemophilus influenzae type b polysaccharide (Hib PS). To determine whether the closely related homologue of the A2 gene, the kappaII-A18 gene, has the potential to contribute to the repertoire, we examined Hib PS binding to a series of recombinant Fab fragments having either A2 or A18 L chains isolated from a Hib PS-vaccinated adult. The ability to bind Hib PS resided exclusively with those Fab fragments having A2 and containing an insertional arginine at the variable-joining junction. Thus, despite the sequence similarity between A2 and A18, only A2 contributes to the canonical Hib PS paratope.

Abs using the kappaII-A2 V gene segment predominate the human Ab repertoire to the Haemophilus influenzae b (Hib) polysaccharide (PS). All A2 anti-Hib PS Abs sequenced to date possess a 10-amino acid L chain complementarity-determining region-3 (CDR-3) having an insertional arginine (Arg) at position 95a, the V-J junction. These findings suggest an essential requirement for this conserved Arg residue in determining Hib PS-binding affinity. We examined this requirement by performing chain recombination experiments in which a series of A2 L chains, differing at position 95a, were combined individually with an Fd region known to generate a Hib PS-combining site when paired with an A2-Arg(95a)-Jkappa1 V region. Hib PS binding of the recombinant Fabs was evaluated quantitatively using a radioantigen-binding assay. Fabs having A2 L chains with either Arg or lysine in position 95a in combination with Jkappa1 gave equivalent and strongest binding to Hib PS. Fabs having A2-Jkappa1 L chains with either tyrosine, glycine, alanine, leucine, serine, or threonine in position 95a, or having an A2-Arg(95a)-Jkappa3 L chain, gave intermediate binding. Fabs having A2-Jkappa1 L chains with glutamate or aspartate at 95a or with no junctional residue showed little or no Hib PS binding. These results demonstrate the importance of L chain junctional residue, as well as Jkappa usage and CDR-3 length, in determining Hib PS-binding affinity. Contrary to expectation, an Arg junctional residue is not essential for generating either high or intermediate affinity-binding sites.