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Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae. Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse–chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

Bacterial zwitterionic capsular polysaccharides (ZPS), such as polysaccharide A (PSA) of the intestinal commensal Bacteroides fragilis , have been shown to modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). We previously used a genomic screen to identify diverse host-associated bacteria with the predicted genetic capacity to produce ZPSs related to PSA of B. fragilis and hypothesized that genetic disruption (KO) of a key functional gene within these operons would reduce the anti-inflammatory activity of these bacteria. We found that ZPS-KO bacteria in two common gut commensals, Bacteroides uniformis and Bacteroides cellulosilyticus , had a reduced ability to induce Tregs and IL-10 in stimulations of human peripheral blood mononuclear cells (PBMCs). Additionally, we found that macrophage stimulated with either wildtype B. fragilis or B. uniformis produced significantly more IL-10 than KOs, indicating a potentially novel function of ZPS of shifting the cytokine response in macrophages to a more anti-inflammatory state. These findings support the hypothesis that these related ZPS may represent a shared strategy to modulate host immune responses.

Some marine and extremophilic microorganisms are capable of synthesizing sulfated polysaccharides with a unique structure. A number of studies indicate significant biological properties of individual sulfated polysaccharides, such as antiproliferative activity, which makes them a promising area for further research. In this study, the capsular polysaccharide (CPS) was obtained from the bacterium Cobetia marina KMM 1449, isolated from a marine sediment sample collected along the shore of the Sea of Japan. The CPS was isolated by saline solution, purified by a series of chromatographic procedures, and studied by chemical methods along with 1D and 2D 1H and 13C NMR spectroscopy. The following new structure of the CPS from C. marina KMM 1449 was established and consisted of sulfated and simultaneously phosphorylated disaccharide repeating units: →4)-α-L-Rhap2S-(1→3)-β-D-Manp6PGro-(1→. To elucidate the genetic basis of the CPS biosynthesis, the whole genomic sequence of C. marina KMM 1449 was obtained. The CPS biosynthetic gene cluster (BGC) of about 70 genes composes four regions encoding nucleotide sugar biosynthesis (dTDP-Rha and GDP-Man), assembly (GTs genes), translocation (ABC transporter genes), sulfation (PAPS biosynthesis and sulfotransferase genes) and lipid carrier biosynthesis (wcb operon). Comparative analysis of the CPS BGCs from available Cobetia genomes showed the presence of KMM 1449-like CPS BGC among strains of all three Cobetia species. The study of new natural sulfated polysaccharides, as well as the elucidation of the pathways of their biosynthesis, provides the basis for the development of potential anticancer drugs.

The structural integrity of Haemophilus influenzae type b (Hib) capsular polysaccharide (polyribosylribitol phosphate, PRP) is a critical determinant of glycoconjugate vaccine quality, yet conventional pharmacopoeial methods lack the resolution to monitor atomic-level alterations during manufacturing. This study employed an orthogonal analytical strategy integrating solution-phase 1H NMR spectroscopy and HPSEC-MALLS/RI to rigorously evaluate the preservation of PRP's structure across three consecutive production batches, encompassing purified polysaccharide, activated intermediate, ADH-derivatized polymer, and the final tetanus toxoid (TT) conjugate. 1H NMR analysis demonstrated high-degree of batch-to-batch consistency, with invariant spectral fingerprints in the diagnostic saccharide region (δ 3.4–4.4 ppm) confirming the core ribose and ribitol moieties remained entirely undisturbed through chemical activation and conjugation. HPSEC-MALLS/RI revealed consistent weight-average molecular weight (Mw) through activation and derivatization, ruling out depolymerization or aggregation, while an expected Mw increase post-conjugation quantified the successful removal of unconjugated species and enrichment of the high-molecular-weight glycoconjugate. The final lyophilized product maintained molecular size stability. These results demonstrate the preservation of PRP's structural and macromolecular integrity throughout the manufacturing process. We propose 1H NMR and HPSEC-MALLS/RI as essential quality control tools for in-process monitoring and lot-release, ensuring the structural consistency, safety, and efficacy of Hib conjugate vaccines. •Atomic-level Hib PRP integrity confirmed across manufacturing by 1H NMR.•HPSEC-MALLS/RI shows conjugation maintains polysaccharide structural stability.•Batch-to-batch consistency proven for intermediates and final conjugate vaccine.•Orthogonal analytics proposed for enhanced glycoconjugate vaccine quality control.

Haemophilus influenzae type a (Hia) disease is associated with fatality and morbidity, predominantly in children from Northern indigenous communities in Canada and Alaska. Symptoms include but are not limited to meningitis, sepsis, pneumonia, otitis media and bacteremia. A key virulence factor associated with Hia pathogenesis is the bacterial capsular polysaccharides. Prophylactic vaccines targeting the capsular polysaccharide (CPS) antigens is considered a promising intervention strategy given the effectiveness of polysaccharide-protein conjugate vaccines against diverse bacterial pathogens. In the same vein, we in this study have presented the development of Hia conjugate vaccine involving hydrazide-polyethylene glycol-hydrazide linker with rCRM197 as a carrier protein. Preclinical immunogenicity data indicated induction of robust serum bactericidal antibody responses at a 10-μg CPS antigen dose in rabbits like the PedvaxHIB® comparator, leading to evaluation of the Hia vaccine candidate in Phase I clinical trials.

Klebsiella pneumoniae has become one of the most important clinical bacterial pathogens due to its evolution into hyperresistant and hypervirulent phenotypes. The mechanism of virulence of this pathogen is not well understood, particularly because it differs from other Enterobacteriaceae pathogens such as Escherichia coli and Salmonella . The capsule polysaccharide (CPS) of this pathogen is well recognized for contributing to the virulence of K. pneumoniae , but the exact mechanisms underlying its contribution are unclear. In this study, we demonstrated that CPS does not directly contribute to the host response; rather, it forms an external coat that blocks host recognition and prevents immune cells from binding to receptor proteins on K. pneumoniae , thus inhibiting phagocytosis, which makes it more challenging for the body to fight off infections. Understanding these mechanisms is vital for developing new treatments against K. pneumoniae infections, ultimately improving patient outcomes and public health.

Streptococcus pneumoniae ( Spn ) is the leading cause of invasive disease. Importantly, only a subset of the 100 capsule types carried by Spn cause the majority of serious infections, suggesting that the biochemical properties of capsular polysaccharide are directly tied to virulence. The polysaccharide capsule that surrounds Streptococcus pneumoniae ( Spn ) is one of its most important virulence determinants, serving to protect against phagocytosis. To date, 100 biochemical and antigenically distinct capsule types, i.e., serotypes, of Spn have been identified. Yet how capsule influences pneumococcal translocation across vascular endothelial cells (VEC), a key step in the progression of invasive disease, was unknown. Here, we show that despite capsule being inhibitory of Spn uptake by VEC, capsule enhances the escape rate of internalized pneumococci and thereby promotes translocation. Upon investigation, we determined that capsule protected Spn against intracellular killing by VEC and H 2 O 2 -mediated killing in vitro . Using a nitroblue tetrazolium reduction assay and nuclear magnetic resonance (NMR) analyses, purified capsule was confirmed as having antioxidant properties which varied according to serotype. Using an 11-member panel of isogenic capsule-switch mutants, we determined that serotype affected levels of Spn resistance to H 2 O 2 -mediated killing in vitro , with killing resistance correlated positively with survival duration within VEC, rate of transcytosis to the basolateral surface, and human attack rates. Experiments with mice supported our in vitro findings, with Spn producing oxidative-stress-resistant type 4 capsule being more organ-invasive than that producing oxidative-stress-sensitive type 2 capsule during bacteremia. Capsule-mediated protection against intracellular killing was also observed for Streptococcus pyogenes and Staphylococcus aureus . We conclude that capsular polysaccharide plays an important role within VEC, serving as an intracellular antioxidant, and that serotype-dependent differences in antioxidant capabilities impact the efficiency of VEC translocation and a serotype’s potential for invasive disease. IMPORTANCE Streptococcus pneumoniae ( Spn ) is the leading cause of invasive disease. Importantly, only a subset of the 100 capsule types carried by Spn cause the majority of serious infections, suggesting that the biochemical properties of capsular polysaccharide are directly tied to virulence. Here, we describe a new function for Spn ’s capsule—conferring resistance to oxidative stress. Moreover, we demonstrate that capsule promotes intracellular survival of pneumococci within vascular endothelial cells and thereby enhances bacterial translocation across the vasculature and into organs. Using isogenic capsule-switch mutants, we show that different capsule types, i.e., serotypes, vary in their resistance to oxidative stress-mediated killing and that resistance is positively correlated with intracellular survival in an in vitro model, organ invasion during bacteremia in vivo , and epidemiologically established pneumococcal attack rates in humans. Our findings define a new role of capsule and provide an explanation for why certain serotypes of Spn more frequently cause invasive pneumococcal disease.

Background Staphylococcus aureus is the most commonly isolated bacterial species from ovine mastitis. Previous studies have mainly focused on dairy sheep, whereas knowledge about S. aureus in sheep kept for mutton and wool production remains limited. Furthermore, data on the occurrence of antimicrobial resistance genes and molecular epidemiology of S. aureus are scarce. In Norway, most sheep are kept for meat or wool, and mastitis caused by S. aureus results in substantial economic losses. This study aimed to determine the persistence, genetic relatedness and diversity, and antimicrobial resistance (AMR) of S. aureus in ewes at four farms in Norway using whole genome sequencing. The potential effect of vaccination on the occurrence of S. aureus with capsular polysaccharide type 8 ( cap 8 ) was also determined. Results A total of 101 isolates from 70 ewes across four farms were characterized. Persistent colonization was observed in 24% of ewes. Among the isolates, 65% carried the capsular polysaccharide type 8 ( cap 8 ) gene and belonged to sequence types (STs) 8, 9, 49, and 8875. The remaining isolates were cap5 -positive and belonged to STs 30, 130, 133, and 1640. No statistically significant differences were seen between isolates from vaccinated and unvaccinated ewes regarding the cap gene. Single-nucleotide polymorphism (SNP) analysis showed no evidence of inter-farm transmission but supported within-farm transmission. AMR genes were rare; the only clinically relevant gene identified was bla Z, present solely in ST8 isolates. Virulence gene profiling showed that the toxin genes tst -1, sel , and sec were common in ST133 and ST49 isolates and that no specific human-associated lineages could be detected. Conclusions Despite the limited number of isolates and farms studied, the findings revealed high genetic diversity between farms, while one or two genotypes tended to dominate within a farm. AMR genes were uncommon, consistent with Norway’s low antibiotic usage and low prevalence of AMR. Most genotypes detected in this study have previously been described in ovine populations, indicating host-adapted strains. Vaccination appeared to have limited effect on the occurrence or distribution of S. aureus with cap 8 .

Streptococcus parasuis ( S. parasuis ) is a close relative of Streptococcus suis ( S. suis ), composed of former members of S. suis serotypes 20, 22 and 26. S. parasuis could infect pigs and cows, and recently, human infection cases have been reported, making S. parasuis a potential opportunistic zoonotic pathogen. In this study, we analysed the genomic characteristics of S. parasuis , using pan-genome analysis, and compare some phenotypic determinants such as capsular polysaccharide, integrative conjugative elements, CRISPR-Cas system and pili, and predicted the potential virulence genes by associated analysis of the clinical condition of isolated source animals and genotypes. Furthermore, to discuss the relationship with S. suis , we compared these characteristics of S. parasuis with those of S. suis . We found that the characteristics of S. parasuis are similar to those of S. suis , both of them have “open” pan-genome, their antimicrobial resistance gene profiles are similar and a srtF pilus cluster of S. suis was identified in S. parasuis genome. But S. parasuis still have its unique characteristics, two novel pilus clusters are and three different type CRISPR-Cas system were found. Therefore, this study provides novel insights into the interspecific and intraspecific genetic characteristics of S. parasuis , which can be useful for further study of this opportunistic pathogen, such as serotyping, diagnostics, vaccine development, and study of the pathogenesis mechanism.