O-antigen Polysaccharide Research Articles

Synthesis of the pentasaccharide repeating unit with a conjugation-ready linker corresponding to the O-antigenic polysaccharide of Acinetobacter junii strain 65

A straightforward synthesis of the pentasaccharide with a readily available linker arm corresponding to the O-antigenic polysaccharide of Acinetobacter junii strain 65 has been achieved in good yield. The synthesis has been carried out using thioglycosides as glycosyl donor in the presence of a combination of N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) as thiophilic activator. The yields of the glycosylation steps were very good with satisfactory stereochemistry at the glycosidic linkages. The pentasaccharide derivative has also been obtained using a one-pot iterative glycosylation strategy.

Shuanghuanglian volatile oil exerts antipyretic, anti-inflammatory, and antibacterial synergistic effects through multiple pathways

Ethnopharmacological relevanceTraditional Chinese Medicine (TCM) has a rich history spanning 2000 years. Shuanghuanglian, a traditional Chinese herbal formula composed of three botanicals, is primarily used to treat colds, respiratory infections (including bacterial pneumonia), and pharyngitis. Previous research has found that the volatile oil of Shuanghuanglian is crucial for its efficacy. However, there is a lack of studies investigating its mechanisms. Aim of the studyThis study aims to explore the antibacterial and anti-inflammatory mechanisms of Shuanghuanglian volatile oil and its potential to enhance the antibacterial effects when used in conjunction with antibiotics. MethodsDetermination of the GC-MS fingerprint of SVO using Gas Chromatography-Mass Spectrometry (GC-MS), The antibacterial effects of SVO on multidrug-resistant Klebsiella pneumoniae (MDR-KP) were assessed by detecting MIC, checkerboard method assay, time-kill curves, resistance growth curves, transcriptome sequencing analysis, scanning electron microscopy(SEM), purification, and quantitative analysis of extracellular polysaccharides(EPS). In vivo part, an MDR-KP induced mouse pneumonia model was established to evaluate the mitigating effects of SVO on mouse pneumonia, using comprehensive network pharmacology and bioinformatics to identify genes related to bacterial pneumonia and potential targets of SVO. Validation was performed through molecular docking, qPCR, and ELISA tests. ResultsSVO modulates the expression of MDR-KP mRNA for wecB, wecC, murA, murD, murE, murF, inhibiting the synthesis of O-antigen polysaccharides and peptidoglycans, thereby compromising bacterial cell wall integrity and affecting the synthesis of biofilms. These actions not only exhibit antibacterial effects but also enhance antibacterial activity, restoring the sensitivity of CEF to MDR-KP. SVO suppresses the biological activity of PTGS2, reducing the production of Prostaglandin E2 (PGE2), thereby exerting antipyretic and anti-inflammatory effects, providing new insights for the development of natural non-steroidal anti-inflammatory drugs (NSAIDs). ConclusionsOur research indicates that SVO exerts antipyretic, anti-inflammatory, and antibacterial synergistic effects through multiple pathways.

Klebsiella LPS O1-antigen prevents complement-mediated killing by inhibiting C9 polymerization

The Gram-negative bacterium Klebsiella pneumoniae is an important human pathogen. Its treatment has been complicated by the emergence of multi-drug resistant strains. The human complement system is an important part of our innate immune response that can directly kill Gram-negative bacteria by assembling membrane attack complex (MAC) pores into the bacterial outer membrane. To resist this attack, Gram-negative bacteria can modify their lipopolysaccharide (LPS). Especially the decoration of the LPS outer core with the O-antigen polysaccharide has been linked to increased bacterial survival in serum, but not studied in detail. In this study, we characterized various clinical Klebsiella pneumoniae isolates and show that expression of the LPS O1-antigen correlates with resistance to complement-mediated killing. Mechanistic data reveal that the O1-antigen does not inhibit C3b deposition and C5 conversion. In contrast, we see more efficient formation of C5a, and deposition of C6 and C9 when an O-antigen is present. Further downstream analyses revealed that the O1-antigen prevents correct insertion and polymerization of the final MAC component C9 into the bacterial membrane. Altogether, we show that the LPS O1-antigen is a key determining factor for complement resistance by K. pneumoniae and provide insights into the molecular basis of O1-mediated MAC evasion.

Multiplexed real-time PCR for the detection and differentiation of Klebsiella pneumoniae O-antigen serotypes.

Klebsiella pneumoniae has emerged as a global health threat due to its role in the spread of antimicrobial resistance and because it is a frequent cause of hospital-acquired infections and neonatal sepsis. Capsular and lipopolysaccharide (LPS) O-antigen polysaccharide surface antigens are major immunogens that are useful for strain classification and are candidates for vaccine development. We have developed real-time PCR reagents for molecular serotyping, subtyping, and quantitation of the most prevalent LPS O-antigen types (i.e., O1, O2, O3, and O5) of Klebsiella pneumoniae. We describe two applications for this O-typing assay: for screening culture isolates and for direct typing of Klebsiella pneumoniae present in stool samples. We find 100% concordance between the results of the O-typing assay and whole-genome sequencing of 81 culture isolates, and >90% agreement in O-typing performed directly on specimens of human stool, with disagreement arising primarily from a lack of sensitivity of the culture-based comparator method. Additionally, we find evidence for mixed O-type populations at varying levels of abundance in direct tests of stool from a hospitalized patient population. Taken together, these results demonstrate that this novel O-typing assay can be a useful tool for K. pneumoniae epidemiologic and vaccine studies.IMPORTANCEKlebsiella pneumoniae is an important opportunistic pathogen. The gastrointestinal (GI) tract is the primary reservoir of K. pneumoniae in humans, and GI carriage is believed to be a prerequisite for invasive infection. Knowledge about the dynamics and duration of GI carriage has been hampered by the lack of tools suitable for detection and strain discrimination. Real-time PCR is particularly suited to the higher-throughput workflows used in population-based studies, which are needed to improve our understanding of carriage dynamics and the factors influencing K. pneumoniae colonization.

Characterising virulence in a nontoxigenic non-O1/non-O139 Vibrio cholerae isolate imported from Vietnam

Vibrio cholerae is a major human pathogen that can cause life-threatening acute diarrhea. V. cholerae are classified according to O-antigen polysaccharide outer membrane properties, where the serotypes O1 and O139 are strains that cause pandemics and epidemics while non-O1/non-O139 usually cause mild disease. The dynamic evolution of V. cholerae involves acquisition of new virulence factors through horizontal gene transfer and formerly nontoxigenic serogroups are increasingly being reported to cause severe forms of human disease.In this study we have serotyped one isolate (ST588-CPH) of imported V. cholerae from Vietnam to Denmark and performed whole genome sequencing to identify known virulence genes and furthermore studied the pattern of virulence in closely related pathogenic strains of V. cholerae.ST558-CPH was found to be a non-O1/non-O139 strain. Initial analysis from the whole genome sequencing gave a 96,6 % match to the O139-specific wbfZ gene, but in a second analysis with a higher identification threshold, the wbfZ gene was absent. We suggest a “de novo” display of a database misannotation, which explains the conflicting results. The MLST analysis revealed that the isolate belongs to the nontoxigenic non-O1/non-O139 sequence type ST558. ST558 has recently been reported as a sequence type forming a cluster of ST's that should be monitored, as it has shown to have virulence causing moderate to severe illness. Our analysis of virulence genes identified MakA, a recently discovered toxin, which seems to be generally present in both toxigenic and nontoxigenic strains.

MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

Non-O1/non-O139 Vibrio cholerae bacteraemia and cholangitis: An unusual case in an oncological patient in Lecco Hospital, Italy

Vibrio species are curved, motile, aerobic and facultative anaerobic, Gram-negative bacilli that are widely distributed in aquatic environments, especially marine and estuarine waters. Vibrios of primary concern in human pathology are essentially represented by the species Vibrio cholerae. Classification of Vibrio cholerae is based on the O-antigen polysaccharide type, with over 200 serogroups described. Historically, only toxigenic serogroups O1 and O139 have been associated with widespread cholera epidemics. Other serogroups can cause small outbreaks that spread via contaminated raw seafood (primarily shellfish and molluscs) and seawater exposure, and they are collectively termed Non-O1/Non-O139 Vibrio Cholerae (NOVC). NOVC, occasionally detected also in the Mediterranean Sea, most often causes sporadic gastrointestinal manifestations requiring supportive therapy. However, rarely the V. cholerae non-O1/ non-O139 serotype can result in extraintestinal manifestations such as severe wound infections, soft tissue infections and sepsis in immunocompromised patients that represent a therapeutic challenge. In particular, NOVC bacteraemia is a rare event but has the highest mortality rates among NOVC infections (almost 33%) and usually occurs in certain immunosuppressed populations, such as patients with haematologic and solid malignancies, and those with underlying liver disease.

O26 Polysaccharides as Key Players in Enteropathogenic E. coli Immune Evasion and Vaccine Development.

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

Genome-wide association study reveals serovar-associated genetic loci in Riemerella anatipestifer

BackgroundThe disease caused by Riemerella anatipestifer (R. anatipestifer, RA) results in large economic losses to the global duck industry every year. Serovar-related genomic variation, such as the O-antigen and capsular polysaccharide (CPS) gene clusters, has been widely used for serotyping in many gram-negative bacteria. RA has been classified into at least 21 serovars based on slide agglutination, but the molecular basis of serotyping is unknown. In this study, we performed a pan-genome-wide association study (Pan-GWAS) to identify the genetic loci associated with RA serovars.ResultsThe results revealed a significant association between the putative CPS synthesis gene locus and the serological phenotype. Further characterization of the CPS gene clusters in 11 representative serovar strains indicated that they were highly diverse and serovar-specific. The CPS gene cluster contained the key genes wzx and wzy, which are involved in the Wzx/Wzy-dependent pathway of CPS synthesis. Similar CPS loci have been found in some other species within the family Weeksellaceae. We have also shown that deletion of the wzy gene in RA results in capsular defects and cross-agglutination.ConclusionsThis study indicates that the CPS synthesis gene cluster of R. anatipestifer is a serotype-specific genetic locus. Importantly, our finding provides a new perspective for the systematic analysis of the genetic basis of the R anatipestifer serovars and a potential target for establishing a complete molecular serotyping scheme.

Total Synthesis of a Conjugation-Ready Tetrasaccharide Repeating Unit of Vibrio cholerae O:3 O-antigen Polysaccharide.

Herein, we report the first total synthesis of the tetrasaccharide repeating unit of Vibrio cholerae O:3 O-antigen polysaccharide. The highly complex tetrasaccharide contains rare amino sugars such as d-bacillosamine and l-fucosamine, highly labile sugar ascarylose, and higher carbon sugar d-d-heptose. Stereoselective glycosylation of the notoriously reactive ascarylose with d-d-heptose, poor nucleophilicity of the axial C4-OH of l-fucosamine, and amide coupling are the key challenges encountered in the total synthesis, which was completed via a longest linear sequence of 23 steps in 4.2% overall yield.

Effect of O antigen glycosyl isomerase gene mutation on biological property and pathogenicity of Burkholderia pseudomallei strain BPC006

Burkholderia pseudomallei, an intracellular pathogen, is responsible for melioidosis, a zoonotic disease. Its pathogenesis involves several virulence factors, among which lipopolysaccharide (LPS) plays a crucial role. Our research reveals that the O antigen present within the LPS significantly regulates the host immune response. In a previous study, we obtained a B. pseudomallei mutant strain ΔwbiI. Here, the purification of LPS from ΔwbiI and a gas chromatography–mass spectrometry (GC–MS) analysis were conducted. The results confirmed the absence of specific sugar 6-deoxy-Talp, which is a typical component of the O antigen in the wild type B. pseudomallei. Our findings underscore the potent impact the O antigen exerts on the virulence of B. pseudomallei. The ΔwbiI strain displayed significantly increased invasiveness and cytotoxicity in vitro. This enhanced cytotoxicity seems to be related to the exposure of lipid A and an increased cell membrane hydrophobicity resulting from the deletion of the O antigen. Additionally, in mouse models, the ΔwbiI strain resulted in a heightened host lethality and an excessive inflammatory response in mice. These findings indicate that the O-antigenic polysaccharide moiety of B. pseudomallei plays a role in its pathogenicity in vitro and in vivo.

Dual function of the O-antigen WaaL ligase of Aggregatibacter actinomycetemcomitans.

Protein glycosylation is critical to the quaternary structure and collagen-binding activity of the extracellular matrix protein adhesin A (EmaA) associated with Aggregatibacter actinomycetemcomitans. The glycosylation of this large, trimeric autotransporter adhesin is postulated to be mediated by WaaL, an enzyme with the canonical function to ligate the O-polysaccharide (O-PS) antigen with a terminal sugar of the lipid A-core oligosaccharide of lipopolysaccharide (LPS). In this study, we have determined that the Escherichia coli waaL ortholog (rflA) does not restore collagen binding of a waaL mutant strain of A. actinomycetemcomitans but does restore O-PS ligase activity following transformation of a plasmid expressing waaL. Therefore, a heterologous E. coli expression system was developed constituted of two independently replicating plasmids expressing either waaL or emaA of A. actinomycetemcomitans to directly demonstrate the necessity of ligase activity for EmaA collagen binding. Proper expression of the protein encoded by each plasmid was characterized, and the individually transformed strains did not promote collagen binding. However, coexpression of the two plasmids resulted in a strain with a significant increase in collagen binding activity and a change in the biochemical properties of the protein. These results provide additional data supporting the novel hypothesis that the WaaL ligase of A. actinomycetemcomitans shares a dual role as a ligase in LPS biosynthesis and is required for collagen binding activity of EmaA.

Molecular and evolutionary basis of O-antigenic polysaccharide-driven phage sensitivity in environmental pseudomonads.

Pseudomonas protegens CHA0, a bacterial strain able to suppress plant pathogens as well as efficiently kill lepidopteran pest insects, has been studied as a biocontrol agent to prevent ensuing agricultural damage. However, the success of this method is dependent on efficient plant colonization by the bacterial inoculant, while it faces competition from the resident microbiota as well as predators such as bacteriophages. One of these naturally occurring phages, ΦGP100, was found to drastically reduce the abundance of CHA0 once inoculated into plant microcosms, resulting in the loss of plant protection effect against a phytopathogen. Here, we investigated the molecular determinants implicated in the interaction between CHA0 and the phage ΦGP100 using a high-density transposon-sequencing approach. We show that lipopolysaccharide cell surface decorations, specifically the longer OBC3-type O-antigenic polysaccharide (O-PS, O-antigen) of the two dominant O-PS of CHA0, are essential for the attachment and infection of ΦGP100. Moreover, when exploring the distribution of the OBC3 cluster in bacterial genomes, we identified several parts of this gene cluster that are conserved in phylogenetically distant bacteria. Through heterologous complementation, we integrated an OBC3-type gene copy from a phylogenetically distant bacterium and were able to restore the phage sensitivity of a CHA0 mutant which lacked the ability to form long O-PS. Finally, we evidence that the OBC3 gene cluster of CHA0 displays a high genomic plasticity and likely underwent several horizontal acquisitions and genomic rearrangements. Collectively, this study underlines the complexity of phage-bacteria interactions and the multifunctional aspect of bacterial cell surface decorations. IMPORTANCE The application of plant-beneficial microorganisms to protect crop plants is a promising alternative to the usage of chemicals. However, biocontrol research often faces difficulties in implementing this approach due to the inconsistency of the bacterial inoculant to establish itself within the root microbiome. Beneficial bacterial inoculants can be decimated by the presence of their natural predators, notably bacteriophages (also called phages). Thus, it is important to gain knowledge regarding the mechanisms behind phage-bacteria interactions to overcome this challenge. Here, we evidence that the major long O-antigenic polysaccharide (O-PS, O-antigen) of the widely used model plant-beneficial bacterium Pseudomonas protegens CHA0 is the receptor of its natural predator, the phage ΦGP100. We examined the distribution of the gene cluster directing the synthesis of this O-PS and identified signatures of horizontal gene acquisitions. Altogether, our study highlights the importance of bacterial cell surface structure variation in the complex interplay between phages and their Pseudomonas hosts.

Glycoengineering directs de novo biomanufacturing of UPEC O21 O-antigen polysaccharide based glycoprotein

Glycoproteins, in which polysaccharides are usually attached to proteins, are an important class of biomolecules that are widely used as therapeutic agents in clinical treatments for decades. Uropathogenic Escherichia coli (UPEC) O21 has been identified as a serogroup that induces urinary tract infections, with a global increasing number among women and young children. Therefore, there is an urgent need to establish protective vaccines against UPEC infection. Herein, we engineered non-pathogenic E. coli MG1655 to achieve robust, cost-effective de novo biosynthesis of O21 O-antigen polysaccharide-based glycoprotein against UPEC O21. Specifically, this glycoengineered E. coli MG1655 was manipulated for high-efficient glucose-glycerol co-utilization and for the gene cluster installation and O-glycosylation machinery assembly. The key pathways of UDP-sugar precursors were also strengthened to enforce more carbon flux towards the glycosyl donors, which enhanced the glycoprotein titer by 5.6-fold. Further optimization of culture conditions yielded glycoproteins of up to 35.34 mg/L. Glycopeptide MS confirmed the preciset biosynthesis of glycoprotein. This glycoprotein elicited antigen-specific IgG immune responses and significantly reduced kidney and bladder colonization. This bacterial cell-based glyco-platform and optimized strategies can provide a guideline for the biosynthesis of other value-added glycoproteins.

NMR chemical shift prediction and structural elucidation of linker-containing oligo- and polysaccharides using the computer program CASPER

Carbohydrate structures containing alkyl groups as aglycones are useful for investigating enzyme activity and glycan-protein interactions. Moreover, linker-containing oligosaccharides with a spacer group are commonly used to print glycan microarrays or to prepare protein-conjugates as vaccine candidates. The structural accuracy of these synthesized glycans are essential for interpretation of results from biological experiments in which the compounds have been used and NMR spectroscopy can unravel and confirm their structures. An approach for efficient 1H and 13C NMR chemical shift assignments employed a parallel NOAH-10 measurement followed by NMR spin-simulation to refine the 1H NMR chemical shifts, as exemplified for a disaccharide with an azidoethyl group as an aglycone, the NMR chemical shifts of which have been used to enhance the quality of CASPER (http://www.casper.organ.su.se/casper/). The CASPER program has been further developed to aid characterization of linker-containing oligo- and polysaccharides, either by chemical shift prediction for comparison to experimental NMR data or as structural investigation of synthesized glycans based on acquired unassigned NMR data. The ability of CASPER to elucidate structures of linker-containing oligosaccharides is demonstrated and comparisons to assigned or unassigned NMR data show the utility of CASPER in supporting a proposed oligosaccharide structure. Prediction of NMR chemical shifts of an oligosaccharide, corresponding to the repeating unit of an O-antigen polysaccharide, having a linker as an aglycone and a non-natural substituent derivative thereof are presented to exemplify the diversity of structures handled. Furthermore, NMR chemical shift predictions of synthesized polysaccharides, corresponding to bacterial polysaccharides, containing a linker are described showing that in addition to oligosaccharide structures also polysaccharide structures having an aglycone spacer group can be analyzed by CASPER.

Safety, Reactogenicity, Immunogenicity, and Dose Selection of 10-Valent Extraintestinal Pathogenic Escherichia coli Bioconjugate Vaccine (VAC52416) in Adults Aged 60-85 Years in a Randomized, Multicenter, Interventional, First-in-Human, Phase 1/2a Study.

ExPEC10V is a bioconjugate vaccine containing O-antigen polysaccharides of 10 extraintestinal pathogenic Escherichia coli (ExPEC) serotypes. This phase 1/2a study (NCT03819049) assessed the safety, reactogenicity, and immunogenicity of ExPEC10V (VAC52416) to prevent invasive E coli disease in elderly adults. The observer-blind, active-controlled design included a 28-day screening, vaccination, 181-day follow-up, and 1-year follow-up. Participants (60-85 years of age) were randomized to ExPEC10V low dose (antigen dose range, 4-8 µg), ExPEC10V medium dose (4-16 µg), or ExPEC10V high dose (8-16 µg); 4-valent ExPEC vaccine (ExPEC4V); or 13-valent pneumococcal conjugate vaccine (PCV13). The incidence of adverse events (AEs; solicited, day 15; unsolicited, day 30; serious AEs, day 181) and immunogenicity (electrochemiluminescent-based assay [ECL] and multiplex opsonophagocytic assay [MOPA]) were assessed. Optimal ExPEC10V dose was determined from safety data through day 30 and an immunogenicity dose selection algorithm based on day 15 ECL and MOPA results. A total of 416 participants were included (median age, 64.0 years; 54.8% female). The incidences of solicited local and systemic AEs were, respectively, 44.2% and 39.4% for low-dose, 52.9% and 46.1% for medium-dose, 57.7% and 45.2% for high-dose ExPEC10V, and 74.1% and 48.1% for PCV13. Five serious AEs, not vaccine related, were reported. The ECL revealed a robust antibody response to ExPEC10V through year 1. Opsonophagocytic killing activity was detected against all but serotype O8; this lack of response against serotype O8 was linked to low assay sensitivity. Based on the totality of data, high-dose ExPEC10V was considered optimal. ExPEC10V was well tolerated and immunogenic in elderly adults against all but serotype O8.

Preference of Bacterial Rhamnosyltransferases for 6-Deoxysugars Reveals a Strategy To Deplete O-Antigens.

Bacteria synthesize hundreds of bacteria-specific or "rare" sugars that are absent in mammalian cells and enriched in 6-deoxy monosaccharides such as l-rhamnose (l-Rha). Across bacteria, l-Rha is incorporated into glycans by rhamnosyltransferases (RTs) that couple nucleotide sugar substrates (donors) to target biomolecules (acceptors). Since l-Rha is required for the biosynthesis of bacterial glycans involved in survival or host infection, RTs represent potential antibiotic or antivirulence targets. However, purified RTs and their unique bacterial sugar substrates have been difficult to obtain. Here, we use synthetic nucleotide rare sugar and glycolipid analogs to examine substrate recognition by three RTs that produce cell envelope components in diverse species, including a known pathogen. We find that bacterial RTs prefer pyrimidine nucleotide-linked 6-deoxysugars, not those containing a C6-hydroxyl, as donors. While glycolipid acceptors must contain a lipid, isoprenoid chain length, and stereochemistry can vary. Based on these observations, we demonstrate that a 6-deoxysugar transition state analog inhibits an RT in vitro and reduces levels of RT-dependent O-antigen polysaccharides in Gram-negative cells. As O-antigens are virulence factors, bacteria-specific sugar transferase inhibition represents a novel strategy to prevent bacterial infections.

Intrinsic B cell TLR-BCR linked coengagement induces class-switched, hypermutated, neutralizing antibody responses in absence of T cells.

Maturation of antibody responses entails somatic hypermutation (SHM), class-switch DNA recombination (CSR), plasma cell differentiation, and generation of memory B cells, and it is thought to require T cell help. We showed that B cell Toll-like receptor 4 (TLR4)-B cell receptor (BCR) (receptor for antigen) coengagement by 4-hydroxy-3-nitrophenyl acetyl (NP)-lipopolysaccharide (LPS) (Escherichia coli lipid A polysaccharide O-antigen) or TLR5-BCR coengagement by Salmonella flagellin induces mature antibody responses to NP and flagellin in Tcrβ-/-Tcrδ-/- and NSG/B mice. TLR-BCR coengagement required linkage of TLR and BCR ligands, "linked coengagement." This induced B cell CSR/SHM, germinal center-like differentiation, clonal expansion, intraconal diversification, plasma cell differentiation, and an anamnestic antibody response. In Tcrβ-/-Tcrδ-/- mice, linked coengagement of TLR4-BCR by LPS or TLR5-BCR by flagellin induced protective antibodies against E. coli or Salmonella Typhimurium. Our findings unveiled a critical role of B cell TLRs in inducing neutralizing antibody responses, including those to microbial pathogens, without T cell help.

A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice.

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler's diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.

Lipidome modulation by dietary omega-3 polyunsaturated fatty acid supplementation or selective soluble epoxide hydrolase inhibition suppresses rough LPS-accelerated glomerulonephritis in lupus-prone mice.

Lipopolysaccharide (LPS)-accelerated autoimmune glomerulonephritis (GN) in NZBWF1 mice is a preclinical model potentially applicable for investigating lipidome-modulating interventions against lupus. LPS can be expressed as one of two chemotypes: smooth LPS (S-LPS) or rough LPS (R-LPS) which is devoid of O-antigen polysaccharide sidechain. Since these chemotypes differentially affect toll-like receptor 4 (TLR4)-mediated immune cell responses, these differences may influence GN induction. We initially compared the effects of subchronic intraperitoneal (i.p.) injection for 5 wk with 1) Salmonella S-LPS, 2) Salmonella R-LPS, or 3) saline vehicle (VEH) (Study 1) in female NZBWF1 mice. Based on the efficacy of R-LPS in inducing GN, we next used it to compare the impact of two lipidome-modulating interventions, ω-3 polyunsaturated fatty acid (PUFA) supplementation and soluble epoxide hydrolase (sEH) inhibition, on GN (Study 2). Specifically, effects of consuming ω-3 docosahexaenoic acid (DHA) (10 g/kg diet) and/or the sEH inhibitor 1-(4-trifluoro-methoxy-phenyl)-3-(1-propionylpiperidin-4-yl) urea (TPPU) (22.5 mg/kg diet ≈ 3 mg/kg/day) on R-LPS triggering were compared. In Study 1, R-LPS induced robust elevations in blood urea nitrogen, proteinuria, and hematuria that were not evident in VEH- or S-LPS-treated mice. R-LPS-treated mice further exhibited kidney histopathology including robust hypertrophy, hyperplasia, thickened membranes, lymphocytic accumulation containing B and T cells, and glomerular IgG deposition consistent with GN that was not evident in VEH- or SLPS-treated groups. R-LPS but not S-LPS induced spleen enlargement with lymphoid hyperplasia and inflammatory cell recruitment in the liver. In Study 2, resultant blood fatty acid profiles and epoxy fatty acid concentrations reflected the anticipated DHA- and TPPU-mediated lipidome changes, respectively. The relative rank order of R-LPS-induced GN severity among groups fed experimental diets based on proteinuria, hematuria, histopathologic scoring, and glomerular IgG deposition was: VEH/CON< R-LPS/DHA ≈ R-LPS/TPPU<<< R-LPS/TPPU+DHA ≈ R-LPS/CON. In contrast, these interventions had modest-to- negligible effects on R-LPS-induced splenomegaly, plasma antibody responses, liver inflammation, and inflammation-associated kidney gene expression. We show for the first time that absence of O-antigenic polysaccharide in R-LPS is critical to accelerated GN in lupus-prone mice. Furthermore, intervention by lipidome modulation through DHA feeding or sEH inhibition suppressed R-LPS-induced GN; however, these ameliorative effects were greatly diminished upon combining the treatments.