Zwitterionic Motif Research Articles

αMI-domain of integrin Mac-1 binds the cytokine pleiotrophin using multiple mechanisms

The integrin Mac-1 (αMβ2, CD11b/CD18, CR3) is an adhesion receptor expressed on macrophages and neutrophils. Mac-1 is also a promiscuous integrin that binds a diverse set of ligands through its αMI-domain. However, the binding mechanism of most ligands remains unclear. We have characterized the interaction of αMI-domain with the cytokine pleiotrophin (PTN), a protein known to bind αMI-domain and induce Mac-1-mediated cell adhesion and migration. Our data show that PTN's N-terminal domain binds a unique site near the N- and C-termini of the αMI-domain using a metal-independent mechanism. However, a stronger interaction is achieved when an acidic amino acid in a zwitterionic motif in PTN's C-terminal domain chelates the divalent cation in the metal ion-dependent adhesion site of active αMI-domain. These results indicate that αMI-domain can bind ligands using multiple mechanisms and that the active αMI-domain has a preference for motifs containing both positively and negatively charged amino acids.

Milk exosomes anchored with hydrophilic and zwitterionic motifs enhance mucus permeability for applications in oral gene delivery.

Exosomes have emerged as a promising tool for the delivery of drugs and genetic materials, owing to their biocompatibility and non-immunogenic nature. However, challenges persist in achieving successful oral delivery due to their susceptibility to degradation in the harsh gastrointestinal (GI) environment and impeded transport across the mucus-epithelium barrier. To overcome these challenges, we have developed high-purity bovine milk exosomes (mExo) as a scalable and efficient oral drug delivery system, which can be customized by incorporating hydrophilic and zwitterionic motifs on their surface. In our study, we observed significantly improved transport rates by 2.5-4.5-fold in native porcine intestinal mucus after the introduction of hydrophilic and zwitterionic surface modifications, as demonstrated by transwell setup and fluorescence recovery after photobleaching (FRAP) analysis. Remarkably, mExo functionalized by a block peptide (BP), consisting of cationic and anionic amino acids arranged in blocks at the two ends, demonstrated superior tolerability in the acidic gastric environment (with a protein recovery rate of 84.8 ± 7.7%) and exhibited a 2.5-fold increase in uptake by intestinal epithelial cells. Furthermore, both mExo and mExo-BP demonstrated successful intracellular delivery of functional siRNA, resulting in up to 65% suppression of the target green fluorescence protein (GFP) gene expression at a low dose of siRNA (5 pmol) without causing significant toxicity. These findings highlight the immense potential of modifying mExo with hydrophilic and zwitterionic motifs for effective oral delivery of siRNA therapies.

Mobile Protons Limit the Stability of Salt Bridges in the Gas Phase: Implications for the Structures of Electrosprayed Protein Ions.

Electrosprayed protein ions can retain native-like conformations. The intramolecular contacts that stabilize these compact gas-phase structures remain poorly understood. Recent work has uncovered abundant salt bridges in electrosprayed proteins. Salt bridges are zwitterionic BH+/A- contacts. The low dielectric constant in the vacuum strengthens electrostatic interactions, suggesting that salt bridges could be a key contributor to the retention of compact protein structures. A problem with this assertion is that H+ are mobile, such that H+ transfer can convert salt bridges into neutral B0/HA0 contacts. This possible salt bridge annihilation puts into question the role of zwitterionic motifs in the gas phase, and it calls for a detailed analysis of BH+/A- versus B0/HA0 interactions. Here, we investigate this issue using molecular dynamics (MD) simulations and electrospray experiments. MD data for short model peptides revealed that salt bridges with static H+ have dissociation energies around 700 kJ mol-1. The corresponding B0/HA0 contacts are 1 order of magnitude weaker. When considering the effects of mobile H+, BH+/A- bond energies were found to be between these two extremes, confirming that H+ migration can significantly weaken salt bridges. Next, we examined the protein ubiquitin under collision-induced unfolding (CIU) conditions. CIU simulations were conducted using three different MD models: (i) Positive-only runs with static H+ did not allow for salt bridge formation and produced highly expanded CIU structures. (ii) Zwitterionic runs with static H+ resulted in abundant salt bridges, culminating in much more compact CIU structures. (iii) Mobile H+ simulations allowed for the dynamic formation/annihilation of salt bridges, generating CIU structures intermediate between scenarios (i) and (ii). Our results uncover that mobile H+ limit the stabilizing effects of salt bridges in the gas phase. Failure to consider the effects of mobile H+ in MD simulations will result in unrealistic outcomes under CIU conditions.

Recent Advances in the Generation of Nitrilium Betaine 1,3-Dipoles

AbstractNitrilium betaine 1,3-dipoles are ubiquitous reagents in organic chemistry, with applications ranging from natural product synthesis to materials science. Given the high reactivity of these zwitterionic motifs, they are invariably generated in situ from a suitable precursor, prior to use. This short review summarises the recent progress in the development of modern approaches towards the formation of these 1,3-dipoles, and their applications within a diverse range of fields.1 Introduction2 Nitrile Ylides2.1 2H-Azirine Rearrangement2.2 Addition of Nitriles to Carbenes3 Nitrile Imines3.1 2,5-Tetrazole Thermolysis3.2 2,5-Tetrazole Photolysis3.3 Diaryl Sydnone Photolysis4 Nitrile Oxides4.1 Hypervalent Iodine4.2 The Nitroso Radical4.3 Green Chemistry Approaches4.4 Other Approaches5 Conclusions

Photoinduced Proton-Transfer Polymerization: A Practical Synthetic Tool for Soft Lithography Applications.

Proton-transfer photopolymerization through the thiol-epoxy "click" reaction is shown to be a versatile new method for the fabrication of micro- and nanosized polymeric patterns. In this approach, complexation of a guanidine base, diazabicycloundecene (DBU), with benzoylphenylpropionic acid (ketoprofen) generates a photolabile salt. Under illumination at a wavelength of 365 nm, the salt undergoes a photodecarboxylation reaction to release DBU as a base. The base-catalyzed ring opening reaction then creates cross-linked poly(β-hydroxyl thio-ether) patterns. The surface chemistry of these patterns can be altered through alkylation of the thio-ether linkages. For example, a reaction with bromoacetic acid produces a hitherto unknown sulfonium/carboxylate-based zwitterionic motif that endows antibiofouling capacity to the micropatterns.

Synthesis of di- and tri-saccharide fragments of Salmonella typhi Vi capsular polysaccharide and their zwitterionic analogues

Zwitterionic polysaccharides (ZPS) behave like traditional T cell-dependent antigens, suggesting the design of new classes of vaccines alternative to currently used glycoconjugates and based on the artificial introduction of a zwitterionic charge motif onto the carbohydrate structure of pathogen antigens. Here we report the new synthesis and antigenic evaluation of di-/tri-saccharide fragments of Salmonella typhi Vi polysaccharide, as well as of their corresponding zwitterionic analogues. Our strategy is based on versatile intermediates enabling chain elongation either by iterative single monomer attachment or by faster and more flexible approach using disaccharide donors.The effect of structural modifications of the synthetic compounds on antigenic properties was evaluated by competitive ELISA. All the oligosaccharides were recognized by specific anti-Vi polyclonal antibodies in a concentration-dependent manner, and the introduction of a zwitterionic motif into the synthetic molecules did not prevent the binding.

Understanding the hygiene hypothesis mechanism (HYP2P.331)

Abstract As the western world becomes increasingly sterilized, the rate of allergic diseases, such as asthma, have increased dramatically. Alternatively, children born vaginally, raised on a farm, or with older siblings have a reduced risk of developing a multitude of allergic conditions. This phenomenon, known as the hygiene hypothesis, has become well established; however, the cellular mechanism by which this exposure protects the individual remains unclear. We have found that the commensal bacterium B. fragilis, in particular the capsular polysaccharide PSA, can protect against airway inflammation characteristic of asthma. The population of CD4+ T cells responding to PSA treatment was found to be clonal in nature, with a CD4+Foxp3-CD45RblowCD44+CD62L- (TEM) phenotype. These cells show potent inhibition of asthma in an IL-10-dependent fashion upon transfer to naïve recipients. Additionally, analysis of top clones revealed increased prevalence of zwitterionic motifs in the CDR3 loop sequences of PSA experienced T cells, which corresponds to a need for a similar motif in PSA itself for MHCII presentation. Collectively these data show that a polysaccharide antigen induces clonal expansion of effector T cells which act to suppress asthmatic inflammation, thereby adding further evidence to the beneficial effects of commensal flora on immune homeostasis as predicted by the hygiene hypothesis.

Polysaccharide A from the Capsule of Bacteroides fragilis Induces Clonal CD4+ T Cell Expansion

For 3 decades, the view of MHCII-dependent antigen presentation has been completely dominated by peptide antigens despite our 2004 discovery in which MHCII was shown to present processed fragments of zwitterionic capsular polysaccharides to T cells. Published findings further demonstrate that polysaccharide A (PSA) from the capsule of Bacteroides fragilis is a potent activator of CD4(+) T cells and that these T cells have important biological functions, especially in the maintenance of immunological homeostasis. However, little is known about the nature of T cell recognition of the polysaccharide-MHCII complex or the phenotype of the resulting activated cells. Here, we use next-generation sequencing of the αβT cell receptor of CD4(+) T cells from mice stimulated with PSA in comparison with protein antigen simulation and non-immunized controls and found that PSA immunization induced clonal expansion of a small subset of suppressive CD4(+)CD45RB(low) effector/memory T cells. Moreover, the sequences of the complementarity-determining region 3 (CDR3) loop from top clones indicate a lack of specific variable β and joining region use and average CDR3 loop length. There was also a preference for a zwitterionic motif within the CDR3 loop sequences, aligning well with the known requirement for a similar motif within PSA to enable T cell activation. These data support a model in which PSA, and possibly other T cell-dependent polysaccharide antigens, elicits a clonal and therefore specific CD4(+) T cell response often characterized by pairing dual-charged CDR3 loop sequences with dual-charged PSA.

Alteration in immune responses toward N-deacetylation of hyaluronic acid.

Hyaluronic acid (HA) is an ubiquitous nonsulfated glycosaminoglycan of the extracellular matrix in all mammalian connective tissues. Along with the age growth, HA will lose its N-acetyl groups in vivo; however, the significance of this physiological process remains largely unknown. Herein, three highly N-deacetylated HAs (dHAs), dHA-5 kDa (Mw: 5 kDa, DD: 100%), dHA-16 kDa (Mw: 16 kDa, DD: 94%) and dHA-110 kDa (Mw: 110 kDa, DD: 72%), were generated after hydrazinolysis. Their capability in the activation of antigen-presenting cells (APCs) was compared with that of their respective HAs. Our results demonstrated that both HAs and dHAs could activate the nuclear factor-kappa B (NF-κB) transcription factor in APCs and induced cytokine production through the Toll-like receptor (TLR)/MyD88 pathway. Notably, the capacity of dHAs in cytokine induction was much lower than that of HAs. In addition, the TLR-2 pathway was much involved following the appearance of zwitterionic motifs in dHAs. Thus, our findings highlight that N-deacetylation renders HA divergences in immune response, which might be implicated in age-induced functional change in endogenous glycosaminoglycans due to the structural modification in vivo.

Three-dimensionally presented anti-fouling zwitterionic motifs sequester and enable high-efficiency delivery of therapeutic proteins

Zwitterions are well known for their anti-biofouling properties. Past investigations of zwitterionic materials for biomedical uses have been centered on exploiting their ability to inhibit non-specific adsorption of proteins. Here, we report that zwitterionic motifs, when presented in three dimensions (e.g. in crosslinked hydrogels), could effectively sequester osteogenic bone morphogenetic protein-2 (rhBMP-2). The ionic interactions between rhBMP-2 and the 3-D zwitterionic network enabled dynamic sequestering and sustained release of the protein with preserved bioactivity. We further demonstrated that the zwitterionic hydrogel confers high-efficiency in vivo local delivery of rhBMP-2. It can template the functional healing of critical-size femoral segmental defects in rats with rhBMP-2 at a loading dose substantially lower than those required for current natural or synthetic polymeric carriers. These findings reveal a novel function of zwitterionic materials beyond their commonly perceived anti-biofouling property, and may establish 3-D zwitterionic matrices as novel high-efficiency vehicles for protein/ionic drug therapeutic delivery.

Modification of the Coordination Behaviour of 4,6-Dimethylpyrimidine-2-thiol with Divalent Cadmium at pH 5.5: A Supramolecular Assembly Generated by Axially Directed Zigzag Weaving of Tripolar Zwitterionic Motifs Through Alternate Pairs of Charge-Assisted C(sp2)–H···Cl and N(sp2)–H···Cl Interactions in Solid State

At pH 5.5, coordination between Cd2+ and 4,6-dimethylpyrimidine-2-thiol was studied using CdCl2 and Cd(NO3)2. For both of these, the same complex, bis-4,6-dimethylpyrimidinium-2-thiolato cadmium (II) chloride was invariably formed. It crystallized from water in the space group P21/c, Z = 4 (a = 12.955(2) A, b = 8.429(1) A, c = 15.727(2) A, β = 97.19(0)°), displaying a distorted tetrahedral molecular geometry characterized by a [CdCl2S2]2(−) chromophore. The negative charge on the coordination zone is intramolecularly compensated by protonation of one azomethine N in each of the two thiolpyrimidine ligands, resulting in a tripolar zwitterion; its aqueous solution is consequently acidic and highly conducting. The crystal structure is mainly characterized by two kinds of charge-driven H-bonding interactions generated in pairs by the inversion symmetry of the space group and translation symmetry of its periodic lattice. This ultimately results in extensive intermolecular interactions, forming left handed zigzag H-bond networks and a consequent supramolecular growth along b. Spectroscopic studies agree well with the proposed molecular structure. The aqueous solubility of the complex and a high 50% lethal dose (mice) of its ligand seem to indicate development of the pyrimidinethiol moiety into a prospective antidote to Cd2+ toxicity. (DMPT)2Cl2Cd(II)—a tripolar zwitterion generating zigzag supramolecules through charge-augmented H-bonds in solid state.

Molecular dynamics simulations of the Salmonella typhi Vi antigenic polysaccharide and effects of the introduction of a zwitterionic motif

A series of hexasaccharides corresponding to the Vi capsular polysaccharide, a polymer of α-(1→4)-galacturonic acid, and analogs containing a zwitterionic motif with various degrees of acetylation at positions 3 have been modeled. When submitted to molecular dynamics simulations in a water box, all the structures visited only two quite restricted regions of the φ/ψ conformational space both corresponding to extended geometries without any tendency towards supercoiling. The most stable conformation showed a clockwise helix arrangement of substituents on the molecular surface whereas the opposite arrangement was observed for the other conformation. The flexibility of the system and the hydrophobic character of the molecular surface are modulated by the 3-O-acetyl groups that confer rigidity to the system. In the zwitterionic analogs, the introduction of positive charges in the place of the acetamido groups alters the hydrophobicity that can be regained by methylation of the amino groups. The needed molecular flexibility can be obtained by the complete deacetylation at positions 3.

Polysaccharides with zwitterionic charge motifs of Bacteroides fragili can affect both the development and homeostasis of the mammalian immune system

Bacterial colonization of the intestine is critical for the normal function of the mammalian immune system. Polysaccharides are characterized as T-cell-independent antigens that are not typically recognized by the full array of immune cells-specifically T cells. However， Bacteroides fragilis zwitterionic polysaccharides charge motifs are processed and presented by the MHC class ll pathway and activate T cells. Namely， Polysaccharide A (PSA) is capable of activating T cell-dependent immune responses that can affect both the development and homeostasis of the host immune system. This molecule also stimulates the innate immune system as a ligand for Toll-like receptor 2 and as such promotes interactions with the adaptive immune system that are required for T cell activation. The study of PSA reveal that polysaccharides of the bacterial microbiota can affect both the development and homeostasis of the mammalian immune system.

Delayed Etiologic Diagnosis of Meningitis in an Extremely Low Birth Weight Newborn

Delayed Etiologic Diagnosis of Meningitis in an Extremely Low Birth Weight Newborn

In vitro immunogenic and immunostimulatory effects of zwitterionized 23-valent pneumococcal polysaccharide vaccine compared with nonzwitterionized vaccine

Background: It was hypothesized that the observed slight immunostimulatory effect of the 23-valent pneumococcal polysaccharide (pneumo-23) vaccine might be due to the presence of low levels of zwitterionic motifs. Therefore, it was hypothesized further that introducing zwitterionic motifs experimentally into polysaccharides of pneumo-23 vaccine might render it an effective immunostimulatory agent. Objective: This study was conducted to assess the in vitro immunostimulatory effect of zwitterionized pneumo-23 (Z-P23) vaccine compared with the nonzwitterionized commercial pneumo-23 (C-P23) vaccine. Methods: In vitro proliferation, ELISA-based in vitro cytokine synthesis (interleukin [IL]-2, interferon [IFN]-γ, and IL-10), and immunofluorescence microscopy-based immune cell profiling (CD4 +, CD8 +, and CD21 + cells) assays were used to evaluate the immunostimulatory effect of Z-P23 on peripheral blood mononuclear cells (PBMC) of immunosuppressed cancer (IC) patients and healthy control subjects in comparison with PBMC exposed to C-P23, concanavalin A (positive control), and phosphate-buffered saline (PBS) (negative control). Results: Z-P23 induced proliferation of PBMC in the IC (81.1%) and control (75.1%) groups significantly higher than that achieved with concanavalin A in the IC group (51.0%; P = 0.01) but not in the control group (89.2%; P = NS). This was also significantly higher than that achieved with C-P23 in the IC (4.8%; P < 0.001) and control (6.2%; P < 0.001) groups. Z-P23 induced IL-2 and IFN-γ synthesis in the IC group (0.61 and 0.45 ng/mL, respectively) significantly more than that with C-P23 (0.4 and 0.45 ng/mL; P = 0.002 and P <0.001), concanavalin A (0.45 and 0.31 ng/mL; P = 0.021 and P = 0.03), and PBS (0.41 and 0.29 ng/mL; P = 0.005 and P = 0.04) but not the control group. Z-P23 induced expansion of CD4 +, CD8 +, and CD21 + lymphocytes (39.3%, 42.7%, and 8.1%, respectively) in the IC group higher than that with C-P23 (28.3%, 30.1%, and 5.5%; P = 0.01, P = 0.003, and P = NS), concanavalin A (27.2%, 35.8%, and 4.1%; P = 0.02, P = 0.048, and P = 0.035), and PBS (25.6%, 31.9%, and 4.2%; P = 0.018, P = 0.02, and P = 0.045). Conclusion: The in vitro immunostimulatory potential of Z-P23 was clearly observed on PBMC of IC patients as well as, to a lesser extent, healthy control subjects, stimulating the synthesis of core cytokines of T-helper 1, and primarily inducing CD4 + and CD8 +T cells.

Interactions between Sulfobetaine-Based Polyzwitterions and Polyelectrolytes

We investigate the interactions between sulfobetaine-based polyzwitterions and polyelectrolytes, either positive or negative ones, i.e., poly(DADMAC)s and poly(AA)s. Three different sulfobetaine motifs denoted SPE, SPP, and SHPP have been considered, presenting slight chemical changes either in the function carrying the zwitterionic group or in the zwitterionic motif itself. All three poly(sulfobetaine)s normally present critical temperatures (T(c)) above which they become fully soluble. The association with polyelectrolytes directly affects the critical temperature in a highly nonmonotonic fashion as the mixture composition is varied. Thanks to layer-by-layer deposition in a reflectometric cell, we demonstrate that a selective attraction exists between polyzwitterions and polyelectrolytes, from which an association follows at a nanoscopic scale as demonstrated by small-angle X-ray scattering and atomic force microscopy. The association of polyzwitterions with polyelectrolytes, however, is site-specific since it exists only between positive polyelectrolytes (i.e., polycations) and polyzwitterions based on SPE or SPP motifs. The range in which the association affects the critical temperature, T(c), is found to largely depend on the molecular weights of both zwitterionic and cationic species. As a result, the complexation and the creation of a hybrid object, referred to as a complex, also depend on the same parameters. By varying the latter from a few thousands to several millions, we define rules for the existence of this complex. In particular, a minimum polyzwitterion molecular weight is needed to observe alterations of the critical temperatures and closure of the complexation cone. Finally, within a Flory-like approach, we consider the polyzwitterion/polyelectrolyte complex as an effective statistical copolymer, whose composition comprises a fraction phi(A) of excess zwitterionic motifs as the majority species and a fraction 1 - phi(A) of complex motifs. We thereby reduce a polymer/polymer/solvent ternary system to a copolymer/solvent binary one, an assumption valid within the limit of small additions of cationic species. The approach predicts the reciprocal critical temperature 1/T(c) to be quadratic in phi(A), which agrees very well with all experimental results, even for a large mismatch between the molecular weights of both species, and regardless of the zwitterionic motif, SPE or SPP.

Bacterial Polysaccharides with Zwitterionic Charge Motifs: Toll-Like Receptor 2 Agonists,T Cell Antigens, or Both?

Bacterial capsular polysaccharides (PS) which naturally contain zwitterionic charge motifs (ZPS) possess specific immunostimulatory activity, leading to direct activation of antigen-presenting cells (APCs) through Toll-like receptor 2 (TLR2) and of T cells in co-culture systems. When administered intraperitoneally, ZPS and bacteria expressing them are involved in the induction or regulation of T-cell dependent inflammatory processes such as intra-abdominal abscess formation. To generate vaccine candidates with antigen and adjuvant properties in one molecule we have chemically introduced zwitterionic motifs into naturally anionic PS and find that the resulting ZPS are TLR2 agonists, able to activate human and mouse APCs. Since T-regulatory cells and other T-cell subsets express TLR2, and TLR2 engagement modifies functionality and activation state of these cells, we speculate that most effects induced by natural and chemically derived ZPS may be explained by their TLR2 agonist properties, presumably through the combined action on TLR2-expressing APCs and T cells.

Introduction of Zwitterionic Motifs into Bacterial Polysaccharides Generates TLR2 Agonists Able to Activate APCs

It was shown previously that bacterial polysaccharides (PS), which naturally contain both positive and negative charges, are able to activate T cells and APCs. However, the vast majority of bacterial PS are anionic and do not have these properties. In this study, we show that chemical introduction of positive charges into naturally anionic bacterial PS confers to the resulting zwitterionic PS (ZPS) the ability to activate pure human monocytes, monocyte-derived dendritic cells, and mouse bone marrow-derived dendritic cells, as do natural bacterial ZPS. Cells are induced to up-regulate MHC class II and costimulatory molecules and to produce cytokines. In mixed monocyte-T cell cocultures, ZPS induce MHC II-dependent T cell proliferation and up-regulation of activation markers. These stimulatory qualities of ZPS disappear when the positive charge is chemically removed from the molecules and thus the zwitterionic motif is destroyed. The ability of natural and chemically derived ZPS to activate APCs can be blocked by anti-TLR2 mAbs, and TLR2 transfectants show reporter gene transcription upon incubation with ZPS. In conclusion, the generation of a zwitterionic motif in bacterial PS confers the ability to activate both APCs and T cells. This finding has important implications for the design of novel polysaccharide vaccines.

Antibody-Independent, Interleukin-17A-Mediated, Cross-Serotype Immunity to Pneumococci in Mice Immunized Intranasally with the Cell Wall Polysaccharide

Serotype-specific immunity to Streptococcus pneumoniae is conferred by antibodies to the capsular polysaccharides, which define the 90 known serotypes. Whether antibody to the species-common cell wall polysaccharide (C-Ps) is protective has been a matter of controversy. Here we show that C-Ps given intranasally with mucosal adjuvant increased the resistance of mice to experimental nasopharyngeal colonization by capsulated S. pneumoniae of serotype 6B. This immunity could be induced in mice congenitally lacking immunoglobulin but was dependent upon CD4+ T cells. Elimination of the charged amino group on the polymer backbone by N acetylation of C-Ps reduced the immunity, as did treatment of the mice with antibody to the cytokine interleukin-17A at the time of challenge, both consistent with the hypothesis of T-cell activation due to the zwitterionic motif of the polymer. C-Ps also protected in a model of fatal aspiration pneumonia by heavily capsulated serotype 3. These findings suggest a novel immunization strategy against S. pneumoniae.

Immunochemical and biological characterization of three capsular polysaccharides from a single Bacteroides fragilis strain.

Although Bacteroides fragilis accounts for only 0.5% of the normal human colonic flora, it is the anaerobic species most frequently isolated from intra-abdominal and other infections with an intestinal source. The capsular polysaccharides of B. fragilis are part of a complex of surface polysaccharides and are the organism's most important virulence factors in the formation of intra-abdominal abscesses. Two capsular polysaccharides from strain NCTC 9343, PS A1 and PS B1, have been characterized structurally. Their most striking feature is a zwitterionic charge motif consisting of both positively and negatively charged substituent groups on each repeating unit. This zwitterionic motif is essential for abscess formation. In this study, we sought to elucidate structural features of the capsular polysaccharide complex of a commonly studied B. fragilis strain, 638R, that is distinct from strain 9343. We sought a more general picture of the species to establish basic structure-activity and structure-biosynthesis relationships among abscess-inducing polysaccharides. Strain 638R was found to have a capsular polysaccharide complex from which three distinct carbohydrates could be isolated by a complex purification procedure. Compositional and immunochemical studies demonstrated a zwitterionic charge motif common to all of the capsular polysaccharides that correlated with their ability to induce experimental intra-abdominal abscesses. Of interest is the range of net charges of the isolated polysaccharides-from positive (PS C2) to balanced (PS A2) to negative (PS 3). Relationships among structural components of the zwitterionic polysaccharides and their molecular biosynthesis loci were identified.