Top-Down Characterization of Bacterial Lipopolysaccharides and Lipooligosaccharides Using Activated-Electron Photodetachment Mass Spectrometry.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activated-electron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Escherichia coli Nissle and Bacteroides fragilis. a-EPD is a hybrid activation method that uses ultraviolet photoirradiation to generate charge-reduced radical ions followed by collisional activation to produce informative fragmentation patterns. We benchmark the a-EPD method for top-down characterization of triacyl LOS from E. coli R2, then focus on characterization of LPS from E. coli Nissle and B. fragilis. Notably, a-EPD affords extensive fragmentation throughout the backbone of the core OS and O-antigen regions of LPS from E. coli Nissle. This hybrid approach facilitated the elucidation of structural details for LPS from B. fragilis, revealing a putative hexuronic acid (HexA) conjugated to lipid A.

Total Synthesis and Structural Studies of Zwitterionic Bacteroides fragilis Polysaccharide A1 Fragments.

Zwitterionic polysaccharides (ZPSs) are exceptional carbohydrates, carrying both positively charged amine groups and negatively charged carboxylates, that can be loaded onto MHC-II molecules to activate T cells. It remains enigmatic, however, how these polysaccharides bind to these receptors, and to understand the structural features responsible for this "peptide-like" behavior, well-defined ZPS fragments are required in sufficient quantity and quality. We here present the first total synthesis of Bacteroides fragilis PS A1 fragments encompassing up to 12 monosaccharides, representing three repeating units. Key to our successful syntheses has been the incorporation of a C-3,C-6-silylidene-bridged "ring-inverted" galactosamine building block that was designed to act as an apt nucleophile as well as a stereoselective glycosyl donor. Our stereoselective synthesis route is further characterized by a unique protecting group strategy, built on base-labile protecting groups, which has allowed the incorporation of an orthogonal alkyne functionalization handle. Detailed structural studies have revealed that the assembled oligosaccharides take up a bent structure, which translates into a left-handed helix for larger PS A1 polysaccharides, presenting the key positively charged amino groups to the outside of the helix. The availability of the fragments and the insight into their secondary structure will enable detailed interaction studies with binding proteins to unravel the mode of action of these unique oligosaccharides at the atomic level.

Bacteroides fragilis Enterotoxin Upregulates Matrix Metalloproteinase-7 Expression through MAPK and AP-1 Activation in Intestinal Epithelial Cells, Leading to Syndecan-2 Release.

Bacteroides fragilis enterotoxin (BFT) produced by enterotoxigenic B. fragilis (ETBF) causes colonic inflammation. BFT initially contacts intestinal epithelial cells (IECs) and affects the intestinal barrier. Although molecular components of the gut epithelial barrier such as metalloproteinase-7 (MMP-7) and syndecan-2 are known to be associated with inflammation, little has been reported about MMP-7 expression and syndecan-2 shedding in response to ETBF infection. This study explores the role of BFT in MMP-7 induction and syndecan-2 release in IECs. Stimulating IECs with BFT led to the induction of MMP-7 and the activation of transcription factors such as NF-κB and AP-1. MMP-7 upregulation was not affected by NF-κB, but it was related to AP-1 activation. In BFT-exposed IECs, syndecan-2 release was observed in a time- and concentration-dependent manner. MMP-7 suppression was associated with a reduction in syndecan-2 release. In addition, suppression of ERK, one of the mitogen-activated protein kinases (MAPKs), inhibited AP-1 activity and MMP-7 expression. Furthermore, the suppression of AP-1 and ERK activity was related to the attenuation of syndecan-2 release. These results suggest that a signaling cascade comprising ERK and AP-1 activation in IECs is involved in MMP-7 upregulation and syndecan-2 release during exposure to BFT.

A Large Insertion Domain in the Rho Factor From a Low G + C, Gram-negative Bacterium is Critical for RNA Binding and Transcription Termination Activity.

Rho-dependent termination of transcription (RDTT) is a critical regulatory mechanism specific to bacteria. In a subset of species including most Actinobacteria and Bacteroidetes, the Rho factor contains a large, poorly conserved N-terminal insertion domain (NID) of cryptic function. To date, only two NID-bearing Rho factors from high G + C Actinobacteria have been thoroughly characterized. Both can trigger RDTT at promoter-proximal sites or with structurally constrained transcripts that are unsuitable for the archetypal, NID-less Rho factor of Escherichia coli (EcRho). Here, we provide the first biochemical characterization of a NID-bearing Rho factor from a low G + C bacterium. We show that Bacteroides fragilis Rho (BfRho) is a bona fide RNA-dependent NTPase motor able to unwind long RNA:DNA duplexes and to disrupt transcription complexes. The large NID (~40% of total mass) strongly increases BfRho affinity for RNA, is strictly required for RDTT, but does not promote RDTT at promoter-proximal sites or with a structurally constrained transcript. Furthermore, the NID does not preclude modulation of RDTT by transcription factors NusA and NusG or by the Rho inhibitor bicyclomycin. Although the NID contains a prion-like Q/N-rich motif, it does not spontaneously trigger formation of ß-amyloids. Thus, despite its unusually large RNA binding domain, BfRho behaves more like the NID-less EcRho than NID-bearing counterparts from high G + C Actinobacteria. Our data highlight the evolutionary plasticity of Rho's N-terminal region and illustrate how RDTT is adapted to distinct genomic contents.

Bacteroides fragilis Enterotoxin Induces Sulfiredoxin-1 Expression in Intestinal Epithelial Cell Lines Through a Mitogen-Activated Protein Kinases- and Nrf2-Dependent Pathway, Leading to the Suppression of Apoptosis.

Enterotoxigenic Bacteroides fragilis is a causative agent of colitis and secrets enterotoxin (BFT), leading to the disease. Sulfiredoxin (Srx)-1 serves to protect from oxidative damages. Although BFT can generate reactive oxygen species in intestinal epithelial cells (IECs), no Srx-1 expression has been reported in ETBF infection. In this study, we explored the effects of ETBF-produced BFT on Srx-1 induction in IECs. Treatment of IECs with BFT resulted in increased expression of Srx-1 in a time-dependent manner. BFT treatment also activated transcriptional signals including Nrf2, AP-1 and NF-κB, and the Srx-1 induction was dependent on the activation of Nrf2 signals. Nrf2 activation was assessed using immunoblot and Nrf2-DNA binding activity and the specificity was confirmed by supershift and competition assays. Suppression of NF-κB or AP-1 signals did not affect the upregulation of Srx-1 expression. Nrf2-dependent Srx-1 expression was associated with the activation of p38 mitogen-activated protein kinases (MAPKs) in IECs. Furthermore, suppression of Srx-1 significantly enhanced apoptosis while overexpression of Srx-1 significantly attenuated apoptosis during exposure to BFT. These results imply that a signaling cascade involving p38 and Nrf2 is essential for Srx-1 upregulation in IECs stimulated with BFT. Following this upregulation, Srx-1 may control the apoptosis in BFT-exposed IECs.

Designing a multi-epitopic vaccine against the enterotoxigenic Bacteroides fragilis based on immunoinformatics approach.

Enterotoxigenic Bacteroides fragilis is an enteric pathogen which is described as a causative agent of various intestinal infections and inflammatory diseases. Moreover, various research studies have reported it to be a leading factor in the development of colorectal cancer. As a part of the normal human microbiome, its treatment has become quite a challenge due to the alarming resistance against the available antibiotics. Although, this particular strain of B. fragilis shows susceptibility to few antibiotics, it is pertinent to devise an effective vaccine strategy for its elimination. There is no vaccine available against this pathogen up to date; therefore, we systematically ventured the outer membrane toxin producing proteins found exclusively in the toxigenic B. fragilis through the in-silico approaches to predict a multi-epitopic chimeric vaccine construct. The designed protein constitutes of epitopes which are predicted for linear B cells, Helper and T cells of outer membrane proteins expected to be putative vaccine candidates. The finalized proteins are only expressed in the enterotoxigenic B. fragilis, thus proving them to be exclusive. The 3D structure of the protein was first predicted followed by its refinement and validation via utilizing the bioinformatic approaches. Docking of the designed protein with the TLR2 receptor forecasted apt binding. Upon immune simulation, notable levels were observed in the expression of the immune cells.

Bacterial immunogenic α-galactosylceramide identified in the murine large intestine: dependency on diet and inflammation.

The glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of αGalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS2, we screened murine intestinal tracts to identify and quantify αGalCers, and we investigated the αGalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of αGalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor αGalCer. The identified αGalCer contained a ß(R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis Unlike ß-anomeric structures, but similar to αGalCers from B. fragilis, the synthetic form of the murine αGalCer induced iNKT cell activation in vitro. Last, we observed a decrease in αGalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that αGalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future studies.

The developmentally regulated fetal enterocyte gene, ZP4, mediates anti-inflammation by the symbiotic bacterial surface factor polysaccharide A on Bacteroides fragilis.

Initial colonizing bacteria play a critical role in completing the development of the immune system in the gastrointestinal tract of infants. Yet, the interaction of colonizing bacterial organisms with the developing human intestine favors inflammation over immune homeostasis. This characteristic of bacterial-intestinal interaction partially contributes to the pathogenesis of necrotizing enterocolitis (NEC), a devastating premature infant intestinal inflammatory disease. However, paradoxically some unique pioneer bacteria (initial colonizing species) have been shown to have a beneficial effect on the homeostasis of the immature intestine and the prevention of inflammation. We have reported that one such pioneer bacterium, Bacteroides fragilis (B. fragilis), and its surface component polysaccharide A (PSA) inhibit IL-1ß-induced inflammation in a human primary fetal small intestinal cell line (H4 cells). In this study, using transcription profiling of H4 cellular RNA after pretreatment with or without PSA before an inflammatory stimulation of IL-1ß, we have begun to further determine the cellular mechanism for anti-inflammation. We show that a developmentally regulated gene, zona pellucida protein 4 (ZP4), is uniquely elevated after IL-1ß stimulation and reduced with PSA exposure. ZP4 was known as a sperm receptor-mediating species-specific binding protein in the initial life of mammals. However, its intestinal epithelial function is unclear. We found that ZP4 is a developmentally regulated gene involved with immune function and regulated by both Toll-like receptor 2 and 4. Knockdown of ZP4-affected PSA inhibited IL-8 mRNA expression in response to IL-1ß. This represents an initial study of ZP4 innate immune function in immature enterocytes. This study may lead to new opportunity for efficient treatment of NEC.NEW & NOTEWORTHY This study extends previous observations to define the cellular mechanisms of polysaccharide A-induced anti-inflammation in immature enterocytes using transcription profiling of enterocyte genes after preexposure to polysaccharide A before an inflammatory stimulus with IL-1ß.

Purification of Capsular Polysaccharide Complex from Gram-Negative Bacteria.

Capsular polysaccharides are a dominant class of antigens from bacteria, both pathogenic and symbiotic or commensal. With the rise of awareness for the influence of the microbiota over immune system development and immune homeostasis, analysis of the antigens is more important than ever. Here we describe a method for the isolation of capsular polysaccharide from gram-negative bacteria, with the purification of polysaccharide from the commensal bacterium Bacteroides fragilis serving as an example. The method efficiently removes all detectable endotoxins and other lipid components, proteins, and nucleic acids, providing a source of capsular polysaccharide for immunologic study.

Promoter orientation of the immunomodulatory Bacteroides fragilis capsular polysaccharide A (PSA) is off in individuals with inflammatory bowel disease (IBD).

Bacteroides fragilis is a member of the normal microbiota of the lower gastrointestinal tract, but some strains produce the putative tumourigenic B. fragilis toxin (BFT). In addition, B. fragilis can produce multiple capsular polysaccharides that comprise a microcapsule layer, including an immunomodulatory, zwitterionic, polysaccharide A (PSA) capable of stimulating anti-inflammatory interleukin-10 (IL-10) production. It is known that the PSA promoter can undergo inversion, thereby regulating the expression of PSA. A PCR digestion technique was used to investigate B. fragilis capsular PSA promoter orientation using human samples for the first time. It was found that approximately half of the B. fragilis population in a healthy patient population had PSA orientated in the 'ON' position. However, individuals with inflammatory bowel disease (IBD) had a significantly lower percentage of the B. fragilis population with PSA orientated 'ON' in comparison with the other patient cohorts studied. Similarly, the putative tumourigenic bft-positive B. fragilis populations were significantly associated with a lower proportion of the PSA promoter orientated 'ON'. These results suggest that the proportion of the B. fragilis population with the PSA promoter 'ON' may be an indicator of gastrointestinal health.

High prevalence of division II (cfiA positive) isolates among blood stream Bacteroides fragilis in Slovenia as determined by MALDI-TOF MS.

Bacteroides fragilis can be classified into division I (cfiA negative) and division II (cfiA positive) isolates. Division II isolates have a silent chromosomal carbapenemase gene (cfiA) that can become overexpressed by an insertion of a mobile genetic element and thus develop a phenotypic resistance to carbapenems. Aims of our study were (i) to determine the prevalence of B. fragilis division II (cfiA positive) isolates among blood stream and non-blood stream isolates from two major Slovenian tertiary-care hospitals and (ii) to assess its influence on phenotypic resistance to imipenem. Consecutive non-duplicate B. fragilis isolates from blood stream and non-blood stream specimens were included in the analysis from 2015 to 2017 period. Data from laboratory information system were matched with mass spectra obtained with Microflex LT instrument and MALDI Biotyper 3.1 software (Bruker Daltonik, Bremen, Germany). All mass spectra were reanalyzed using Bruker taxonomy library. Spectra with a log(score) > 2.0 were further analyzed with cfiA library that separates B. fragilis division I and II isolates based on a log(score) value difference of >0.3. Minimal inhibitory concentrations (MICs) for imipenem were determined with Etest (bioMérieux, Marcy l'Étoile, France), using supplemented Brucella agar and EUCAST breakpoints (S ≤ 2 mg/L, R > 8 mg/L). Altogether 623 consecutive B. fragilis isolates were included in the analysis; 47 (7.5%) were isolated from blood stream and 576 (92.5%) from non-blood stream specimens. Among all study isolates, 51 (8.2%) proved to belong to division II (cfiA positive). The proportions of division II isolates among blood stream and non-blood stream isolates were 14.9% and 7.6%, respectively (p = 0.081, ns). In total, 1.3% (n = 8) were non-susceptible to imipenem (MIC >2 mg/L); 4.3% (n = 2) among blood stream and 1% (n = 6) among non-blood stream isolates. All imipenem resistant isolates belonged to division II. Modal MICs (MIC range) were 0.064 mg/L (0.016 mg/L-2 mg/L) and 0.125 mg/L (0.064 mg/L-≥32 mg/L) for division I and II isolates, respectively.

Surface-Enhanced Raman Spectroscopy for the Detection of a Metabolic Product in the Headspace Above Live Bacterial Cultures.

In situ surface-enhanced Raman spectra of the headspace above cultures of six bacterial species showed strong characteristic bands from chemisorbed methyl sulfide. This marker compound is created by dissociation of dimethyl disulfide (DMDS), a fermentative metabolite of bacteria, on the surface of the enhancing Au or Ag nanoparticle films. Kinetic binding plots of media spiked with DMDS and of live cultures showed that the Au-based substrates were more suitable for the rapid detection of bacteria than Ag-based substrates. For E. coli DH5α, the sensitivity limit for headspace SERS detection was 1.5×107  CFU mL-1 , which corresponded to detection 15 min after inoculation of the growth medium. Since the metabolites are only produced by viable bacteria, antibiotic (gentamicin) treatment stopped the normal signal growth of the marker peak. This work is a promising step towards rapid bedside detection of bacterial infections and rapid screening of antibiotics against bacteria.

Total Synthesis of Zwitterionic Tetrasaccharide Repeating Unit from Bacteroides fragilis ATCC 25285/NCTC 9343 Capsular Polysaccharide PS A1 with Alternating Charges on Adjacent Monosaccharides.

The tetrasaccharide repeating unit of zwitterionic polysaccharide A1 (PS A1) from Bacteroides fragilis ATCC 25285/NCTC 9343 has been synthesized using a linear glycosylation approach. One key step includes an α(1,4)-stereoselective [2 + 1] glycosylation of a 2,4,6-trideoxy-2-acetamido-4-amino-d-Gal p (AAT) donor with a poorly reactive axial C4-OH disaccharide acceptor. Mild acid-mediated deacetylation and a challenging [3 + 1] glycosylation are also highlighted. The strategy is inclusive of a single-pot, three-step deprotection affording PS A1 with alternating charges on adjacent monosaccharide units.

Bacteroides fragilis: A whole MALDI-based workflow from identification to confirmation of carbapenemase production for routine laboratories.

Bacteroides fragilis is a frequent anaerobic pathogen and can cause severe infections. Resistance to carbapenems, associated with the cfiA gene encoded carbapenemase, represents an emerging problem. To date, no rapid methods are available to detect and confirm this resistance mechanism in routine laboratories, and the missed recognition of carbapenemase-producing strains can lead to therapeutic failures. In this study we have investigated a whole MALDI-TOF MS-based workflow to detect carbapenemase-producing B. fragilis, using the largest set of B. fragilis clinical isolates ever tested. The presence of the cfiA gene was predicted by MALDI subtyping into Division I (cfiA-negative) or Division II (cfiA-positive). The carbapenemase activity in cfiA-positive strains was confirmed by a MALDI-TOF MS imipenem hydrolysis assay (MBT STAR-Carba, Bruker Daltonik, Germany), that was further used for a characterization of the strains in terms of cfiA expression level. The validity of MALDI subtyping was verified by PCR for the cfiA gene, while results of MALDI hydrolysis assay were compared to conventional methods for susceptibility testing and carbapenemase detection (Carba-NP and disk diffusion synergy test). A genetic analysis of the IS elements upstream cfiA was performed, for the evaluations regarding the expression level of cfiA. A total of 5300 B. fragilis isolates (406 from Bologna, Italy, and 4894 from Dortmund, Germany) were identified and subtyped by MALDI-TOF MS, yielding 41/406 (10.1%) strains from Bologna and 374/4894 (7.6%) from Dortmund to belong to Division II. Molecular verification by PCR for the cfiA gene on a subset of strains confirmed the MALDI typing results in all cases (sensitivity and specificity of 100%). MBT STAR-Carba assay detected the carbapenemase activity in all of the 70 cfiA-carrying strains tested. Moreover, it allowed distinct separation into slow (59) and fast (11) imipenem hydrolyzers corresponding to cfiA expression levels as well as to low or high MICs for carbapenems, respectively. Among the 11 cfiA-positive strains with high carbapenem MIC, only 7 harboured IS elements upstream the carbapenemase gene showing low expression level as well. The MALDI-TOF MS-based workflow was superior to the currently available phenotypic methods for carbapenemase detection as it proved to be more sensitive and accurate than Carba NP and disk diffusion synergy test. The whole MALDI-TOF MS-based workflow allows an accurate identification of B. fragilis clinical strains with reliable classification into Division I/II, and confirmation of the carbapenemase-production, together with estimation of carbapenemase activity, within less than 2 h. This may be of particular interest for early therapeutical decisions in life-threatening infections.

Finding a needle in a haystack: Bacteroides fragilis polysaccharide A as the archetypical symbiosis factor.

Starting from birth, all animals develop a symbiotic relationship with their resident microorganisms that benefits both the microbe and the host. Recent advances in technology have substantially improved our ability to direct research toward the identification of important microbial species that affect host physiology. The identification of specific commensal molecules from these microbes and their mechanisms of action is still in its early stages. Polysaccharide A (PSA) of Bacteroides fragilis is the archetypical example of a commensal molecule that can modulate the host immune system in health and disease. This zwitterionic polysaccharide has a critical impact on the development of the mammalian immune system and also on the stimulation of interleukin 10-producing CD4+ T cells; consequently, PSA confers benefits to the host with regard to experimental autoimmune, inflammatory, and infectious diseases. In this review, we summarize the current understanding of the immunomodulatory effects of B. fragilis PSA and discuss these effects as a novel immunological paradigm. In particular, we discuss recent advances in our understanding of the unique functional mechanisms of this molecule and its therapeutic potential, and we review the recent literature in the field of microbiome research aimed at discovering new commensal products and their immunomodulatory potential.

Antimicrobial susceptibility testing of Bacteroides fragilis using the MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA).

This study evaluated the MBT-ASTRA for antimicrobial susceptibility testing of Bacteroides fragilis with different classes of antibiotics. MALDI-TOF MS peak AUCs from suspensions with B. fragilis with and without an antibiotic were used to calculate the relative growth (AUC "with antibiotic" divided by "without antibiotic"). Antimicrobial susceptibility testing of B. fragilis ATCC 25285 (susceptible) and B. fragilis O18 (resistant) was demonstrated with a clear difference of the relative growth between susceptible and resistant. The MBT-ASTRA needs further development and assessment but could be a relatively easy and inexpensive method for rapid antimicrobial susceptibility testing in specific cases of infection with B. fragilis.

Effect of hyperbaric air on endotoxin from Bacteroides fragilis strains.

The aim of the project was to determine any effect of hyperbaric air on Bacteroides fragilis strains cultivated under hyperbaric conditions. Previously, it was hypothesized that there was a correlation between the presence of Bacteroides bacteria in patients preferring a meaty diet and cancer of the small intestine, and particularly of the large intestine and rectum. With respect to the fact that Bacteroides fragilis (BAFR) group are important producers of endotoxins, measurement and statistical evaluation of endotoxin production by individual strains of isolated Bacteroides species were used to compare bacteria isolated from various clinical samples from patients with colon and rectum cancer in comparison with strains isolated from other non-cancer diagnoses. Endotoxin production was proven by quantitative detection using the limulus amebocyte lysate (LAL) test in EU/mL. Production of endotoxins in these bacteria cultured under hyperbaric air conditions was higher than those strains cultured under normobaric anaerobic conditions. But these differences in endotoxin production were not statistically significant (t test with log-transformed data, p value = 0.0910). Based on a two-tier t test for lognormal data, it is possible to cautiously conclude that a statistically significant difference was found between endotoxin production by Bacteroides fragilis strains isolated from non-carcinoma diagnoses (strains (1-6) and strains isolated from colorectal carcinoma diagnoses (strains 7-8; Wilcoxon non-parametric test p = 0.0132; t test = 0.1110; t test with log-transformed data, p value = 0.0294).

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A.

Capsular polysaccharide A (CPSA) is a four-sugar repeating unit polymer found on the surface of the gut symbiont Bacteroides fragilis that has therapeutic potential in animal models of autoimmune disorders. This therapeutic potential has been credited to its zwitterionic character derived from a positively charged N-acetyl-4-aminogalactosamine (AADGal) and a negatively charged 4,6-O-pyruvylated galactose (PyrGal). In this report, using a fluorescent polyisoprenoid chemical probe, the complete enzymatic assembly of the CPSA tetrasaccharide repeat unit is achieved. The proposed pyruvyltransferase, WcfO; galactopyranose mutase, WcfM; and glycosyltransferases, WcfP and WcfN, encoded by the CPSA biosynthesis gene cluster were heterologously expressed and functionally characterized. Pyruvate modification, catalyzed by WcfO, was found to occur on galactose of the polyisoprenoid-linked disaccharide (AADGal-Gal), and did not occur on galactose linked to uridine diphosphate (UDP) or a set of nitrophenyl-galactose analogues. This pyruvate modification was also found to be required for the incorporation of the next sugar in the pathway N-acetylgalactosamine (GalNAc) by the glycosyltransferase WcfP. The pyruvate acetal modification of a galactose has not been previously explored in the context of a polysaccharide biosynthesis pathway, and this work demonstrates the importance of this modification to repeat unit assembly. Upon production of the polyisoprenoid-linked AADGal-PyrGal-GalNAc, the proteins WcfM and WcfN were found to work in concert to form the final tetrasaccharide, where WcfM formed UDP-galactofuranose (Galf) and WcfN transfers Galf to the AADGal-PyrGal-GalNAc. This work demonstrates the first enzymatic assembly of the tetrasaccharide repeat unit of CPSA in a sequential single pot reaction.

Dendritic cell-specific Mgat2 knockout mice show antigen presentation defects but reveal an unexpected CD11c expression pattern.

Zwitterionic polysaccharide antigens such as polysaccharide A (PSA) from Bacteroides fragilis have been shown to activate CD4+ T cells upon presentation by class II major histocompatibility complex (MHCII) on professional antigen presenting cells. For T cell recognition and activation, high affinity binding between MHCII and PSA is required, and complex N-glycans on conserved MHCII asparagine residues play a central role in controlling this interaction. By truncating these glycans in a myeloid-specific knockout of Mgat2, created using the LyzM-CRE mouse (M-cKO), we previously reported defects in PSA responses in vivo. Unfortunately, the M-cKO also showed a propensity to develop common variable immunodeficiency with autoimmune hemolytic anemia features. Here, we describe a novel murine model in which Mgat2 was targeted for ablation using the dendritic cell (DC)-specific CD11c-CRE-GFP strain in order to develop a more specific and robust in vivo model of PSA presentation defects (DC-cKO). This study shows that Mgat2 deficient DCs from DC-cKO mice show ablation of PSA presentation and downstream T cell activation in vitro. However, the CD11c promoter was unexpectedly active and triggered Mgat2 deletion within multiple hematopoietic lineages, showed remarkably poor penetrance within native DC populations, and produced almost undetectable levels of green fluorescent protein signal. These findings show that the CD11c promoter is not DC-specific, and extreme care should be taken in the interpretation of data using any mouse created using the CD11c-CRE model.

Immunological evaluation of the entirely carbohydrate-based Thomsen-Friedenreich - PS B conjugate.

PS B, a naturally occurring CD4(+) T-cell simulating zwitterionic polysaccharide from Bacteroides fragilis ATCC 25285/NCTC 9343, was conjugated with aminooxy Thomsen Friedenreich (TF or T) [α-d-Gal-(1,3)-ß-d-GalNAc-ONH2] tumor antigen. Immunization in Jax C57BL/6, followed by ELISA revealed IgM and IgG antibody TF specificity. FACS data noted preferential binding to TF-laced MCF-7 cells but not to HCT-116 cells.