A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1ß production in tissue-resident macrophages.

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1ß (IL-1ß), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1ß production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1ß production, indicating that IRE1α is required for CT- or CTB-induced IL-1ß production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

Mg2+ supplementation treats secretory diarrhea in mice by activating calcium-sensing receptor in intestinal epithelial cells.

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

Application of phosphorylcholine derivative as mucosal adjuvant enhancing mucosal immune responses in the upper respiratory tract.

OBJECTIVE: A phosphorylcholine (PC)-derivative with high binding ability (PCDB) was intranasally administered to mice with ovalbumin (OVA), and immune responses were investigated to determine whether PCDB has antigenicity and adjuvanticity. METHODS: BALB/c mice were intranasally immunized with PCDB coupled with OVA, unbound PCDB plus OVA, cholera toxin (CT) plus OVA, OVA alone, and PCDB alone. Then, the production of OVA- and PC-specific antibodies in external secretions and serum, and the secretion of cytokines such as IL-4 and IFN-γ from splenic mononuclear cells by stimulation with PCDB and OVA were examined. Furthermore, the secretion of IL-12p40 from CD11c+ cells following stimulation with PCDB was observed to clarify the adjuvant effect of PCDB through TLR4. RESULTS: Intranasal immunization with PCDB plus OVA increased OVA- and PC-specific IgA in external secretions and OVA- and PC-specific antibodies in the serum. The analysis of IgG subclasses specific to OVA and PC showed a higher production of IgG1 than IgG2, and the secretion of both IL-4 and IFN-γ was enhanced. However, IL-12p40 secretion from CD11c+ cells was increased and OVA-specific IgE production was not promoted by PCDB stimulation. CONCLUSION: Intranasal administration of the protein antigen with PCDB enhanced immune responses specific to the mixed antigen and PC. Although PCDB acted to bias the immune response toward the Th2-type, antigen-specific IgE production did not increase. These findings suggest that PCDB has the potential to be a mucosal vaccine with both adjuvanticity and antigenicity without causing side effects due to type I allergy.

Calcium-sensing receptor activator cinacalcet for treatment of cyclic nucleotide-mediated secretory diarrheas.

Cyclic nucleotide elevation in intestinal epithelial cells is the key pathology causing intestinal fluid loss in secretory diarrheas such as cholera. Current secretory diarrhea treatment is primarily supportive, and oral rehydration solution is the mainstay of cholera treatment. There is an unmet need for safe, simple and effective diarrhea treatments. By promoting cAMP hydrolysis, extracellular calcium-sensing receptor (CaSR) is a regulator of intestinal fluid transport. We studied the antidiarrheal mechanisms of FDA-approved CaSR activator cinacalcet and tested its efficacy in clinically relevant human cell, mouse and intestinal organoid models of secretory diarrhea. By using selective inhibitors, we found that cAMP agonists-induced secretory short-circuit currents (Isc) in human intestinal T84 cells are mediated by collective actions of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) and Clc-2 Cl- channels, and basolateral membrane K+ channels. 30 µM cinacalcet pretreatment inhibited all 3 components of forskolin and cholera toxin-induced secretory Isc by ∼75%. In mouse jejunal mucosa, cinacalcet inhibited forskolin-induced secretory Isc by ∼60% in wild type mice, with no antisecretory effect in intestinal epithelia-specific Casr knockout mice (Casr-flox; Vil1-cre). In suckling mouse model of cholera induced by oral cholera toxin, single dose (30 mg/kg) oral cinacalcet treatment reduced intestinal fluid accumulation by ∼55% at 20 hours. Lastly, cinacalcet inhibited forskolin-induced secretory Isc by ∼75% in human colonic and ileal organoids. Our findings suggest that CaSR activator cinacalcet has antidiarrheal efficacy in distinct human cell, organoid and mouse models of secretory diarrhea. Considering its excellent clinical safety profile, cinacalcet can be repurposed as a treatment for cyclic nucleotide-mediated secretory diarrheas including cholera.

Pharmacoinformatics and molecular dynamics simulation approach to identify anti-diarrheal potentials of Centella asiatica (L.) Urb. against Vibrio cholerae.

Vibrio cholerae, the etiological agent of cholera, causes dehydration and severe diarrhea with the production of cholera toxin. Due to the acquired antibiotic resistance, V. cholerae has drawn attention to the establishment of novel medications to counteract the virulence and viability of the pathogen. Centella asiatica is a medicinal herb native to Bangladesh that has a wide range of medicinal and ethnobotanical applications including anti-bacterial properties. In the present investigation, a total of 25 bioactive phytochemicals of C. asiatica have been screened virtually through molecular docking, ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analyses, and molecular dynamics simulation. Our results revealed four lead compounds as Viridiflorol (-8.7 Kcal/mol), Luteolin (-8.1 Kcal/mol), Quercetin (-8.0 Kcal/mol) and, Geranyl acetate (-7.1 Kcal/mol) against V. cholerae Toxin co-regulated pilus virulence regulatory protein (ToxT). All the lead compounds have been found to possess favorable pharmacokinetic, pharmacodynamics, and molecular dynamics properties. Toxicity analysis revealed satisfactory results with no major side effects. Molecular dynamics simulation was performed for 100 ns that revealed noteworthy conformational stability and structural compactness for all the lead compounds, especially for Quercetin. Target class prediction unveiled enzymes in most of the cases and some experimental and investigational drugs were found as structurally similar analogs of the lead compounds. These findings could aid in the development of novel therapeutics targeting Cholera disease and we strongly recommend in vitro trials of our experimental findings.Communicated by Ramaswamy H. Sarma.

The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin.

After binding to the surface of a target cell, cholera toxin (CT) moves to the endoplasmic reticulum (ER) by retrograde transport. In the ER, the catalytic CTA1 subunit dissociates from the rest of the toxin and is transferred to the cytosol where it is degraded by a ubiquitin-independent proteasomal mechanism. However, CTA1 persists long enough to induce excessive cAMP production through the activation of Gsα. It is generally believed that only one or a few molecules of cytosolic CTA1 are necessary to elicit a cytopathic effect, yet no study has directly correlated the levels of cytosolic toxin to the extent of intoxication. Here, we used the technology of surface plasmon resonance to quantify the cytosolic pool of CTA1. Our data demonstrate that only 4% of surface-bound CTA1 is found in the cytosol after 2 h of intoxication. This represented around 2600 molecules of cytosolic toxin per cell, and it was sufficient to produce a robust cAMP response. However, we did not detect elevated cAMP levels in cells containing less than 700 molecules of cytosolic toxin. Thus, a threshold quantity of cytosolic CTA1 is required to elicit a cytopathic effect. When translocation to the cytosol was blocked soon after toxin exposure, the pool of CTA1 already in the cytosol was degraded and was not replenished. The cytosolic pool of CTA1 thus remained below its functional threshold, preventing the initiation of a cAMP response. These observations challenge the paradigm that extremely low levels of cytosolic toxin are sufficient for toxicity, and they provide experimental support for the development of post-intoxication therapeutic strategies.

The manipulation of cell signaling and host cell biology by cholera toxin.

Vibrio cholerae colonizes the small intestine and releases cholera toxin into the extracellular space. The toxin binds to the apical surface of the epithelium, is internalized into the host endomembrane system, and escapes into the cytosol where it activates the stimulatory alpha subunit of the heterotrimeric G protein by ADP-ribosylation. This initiates a cAMP-dependent signaling pathway that stimulates chloride efflux into the gut, with diarrhea resulting from the accompanying osmotic movement of water into the intestinal lumen. G protein signaling is not the only host system manipulated by cholera toxin, however. Other cellular mechanisms and signaling pathways active in the intoxication process include endocytosis through lipid rafts, retrograde transport to the endoplasmic reticulum, the endoplasmic reticulum-associated degradation system for protein delivery to the cytosol, the unfolded protein response, and G protein de-activation through degradation or the function of ADP-ribosyl hydrolases. Although toxin-induced chloride efflux is thought to be an irreversible event, alterations to these processes could facilitate cellular recovery from intoxication. This review will highlight how cholera toxin exploits signaling pathways and other cell biology events to elicit a diarrheal response from the host.

Recent advances in microbial toxin-related strategies to combat cancer.

It is a major concern to treat cancer successfully, due to the distinctive pathophysiology of cancer cells and the gradual manifestation of resistance. Specific action, adverse effects and development of resistance has prompted the urgent requirement of exploring alternative anti-tumour treatment therapies. The naturally derived microbial toxins as a therapy against cancer cells are a promisingly new dimension. Various important microbial toxins such as Diphtheria toxin, Vibrio cholera toxin, Aflatoxin, Patulin, Cryptophycin-55, Chlorella are derived from several bacterial, fungal and algal species. These agents act on different biotargets such as inhibition of protein synthesis, reduction in cell growth, regulation of cell cycle and many cellular processes. Bacterial toxins produce actions primarily by targeting protein moieties and some immunomodulation and few acts through DNA. Fungal toxins appear to have more DNA damaging activity and affect the cell cycle. Algal toxins produce alteration in mitochondrial phosphorylation. In conclusion, microbial toxins and their metabolites appear to have a great potential to provide a promising option for the treatment and management to combat cancer.

Nedd4-2-dependent Ubiquitination Potentiates the Inhibition of Human NHE3 by Cholera Toxin and Enteropathogenic Escherichia coli.

BACKGROUND & AIMS: Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Na+/H+ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. METHODS: We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Na+/H+ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. RESULTS: The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2-hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. CONCLUSIONS: The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans.

KDP, a Lactobacilli Product from Kimchi, Enhances Mucosal Immunity by Increasing Secretory IgA in Mice and Exhibits Antimicrobial Activity.

The potential of KDP, a lactic acid bacterial strain of Lactobacillus sakei, to enhance the production of mucosal specific immunoglobulin A (IgA) in mice and thereby enhance gut mucosal immunity was examined. KDP is composed of dead cells isolated from the Korean traditional food kimchi. Female BALB/c mice orally received 0.25 mg KDP once daily for 5 weeks and were co-administrated ovalbumin (OVA) for negative control and cholera toxin for positive control. Mice administered KDP exhibited increased secretory IgA (sIgA) contents in the small intestine, Peyer's patches, serum, colon, and lungs as examined by ELISA. KDP also significantly increased the gene expression of Bcl-6, IL-10, IL-12p40, IL-21, and STAT4. In addition, KDP acted as a potent antioxidant, as indicated by its significant inhibitory effects in the range of 16.5-59.4% for DPPH, nitric oxide, maximum total antioxidant capacity, and maximum reducing power. Finally, KDP exhibited potent antimicrobial activity as evidenced by a significant decrease in the growth of 7 samples of gram-negative and gram-positive bacteria and Candida albicans. KDP's adjuvant effect is shown to be comparable to that of cholera toxin. We conclude that KDP can significantly enhance the intestine's secretory immunity to OVA, as well as act as a potent antioxidant and antimicrobial agent. These results suggest that orally administered KDP should be studied in clinical trials for antigen-specific IgA production.

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

Ceramide structure dictates glycosphingolipid nanodomain assembly and function.

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.

Intestinal lysozyme releases Nod2 ligand(s) to promote the intestinal mucosal adjuvant activity of cholera toxin.

Commensal bacteria boost serum IgG production in response to oral immunization with antigen and cholera toxin (CT) in a manner that depends on Nod2 (nucleotide-binding oligomerization domain-containing protein 2). In this study, we examined the role of intestinal lysozyme (Lyz1) in adjuvant activity of CT. We found that Lyz1 released Nod2 ligand(s) from bacteria. Lyz1 deficiency reduced the level of circulating Nod2 ligand in mice. Lyz1 deficiency also reduced the production of IgG and T-cellspecific cytokines after oral immunization in mice. Supplementing Lyz1-deficient mice with MDP restored IgG production. Furthermore, overexpression of Lyz1 in intestinal epithelium boosted the antigen-specific IgG response induced by CT. Collectively, our results indicate that Lyz1 plays an important role in mediating the immune regulatory effect of commensal bacteria through the release of Nod2 ligand(s).

The quantitative analysis of the mechanism involved in pertussis toxin-mediated cell clustering and its implications in the in vitro quality control of diphtheria tetanus and whole cell pertussis vaccines.

Some of the adverse side-effects such as leukocytosis, hyperinsulinemia, hypoglycemia and sensitization to histamine, caused by diphtheria, tetanus and whole cell pertussis (DTwP) vaccines are related to the presence of non-inactivated pertussis toxin (PTx) residues (NiPTxR). The CHO cell clustering assay is an in vitro assay to measure NiPTxR in DTwP vaccines based on the ability of active PTx to cause cellular clustering. To study the biochemical mechanism involved in the clustering effect in CHO cells induced by PTx and by two DTwP vaccines, the levels of total cyclic cAMP were measured and compared to those obtained after treatment with cholera toxin (CTx) able to induce CHO cells elongation instead of cell clustering. Our results showed an increment of cAMP levels by CTx and total cell elongation in CHO cells. However, changes in cAMP levels were not associated with the total clustering induced by PTx or by DTwP vaccines. The high correlation seen between the levels of NiPTxR in the DTwP vaccines determined by the in vivo lethal histamine sensitization (HIST) assay and the in vitro CHO cell clustering assay indicated that the latter could be a suitable alternative test to HIST assay for the toxicological approval and release of batches of DTwP vaccines in their final formulation for human use in accordance with the application of the 3R's principle.

Tongue and hypoglossal morphology after intralingual cholera toxin B-saporin injection.

INTRODUCTION: We recently developed an inducible model of dysphagia using intralingual injection of cholera toxin B conjugated to saporin (CTB-SAP) to cause death of hypoglossal neurons. In this study we aimed to evaluate tongue morphology and ultrastructural changes in hypoglossal neurons and nerve fibers in this model. METHODS: Tissues were collected from 20 rats (10 control and 10 CTB-SAP animals) on day 9 post-injection. Tongues were weighed, measured, and analyzed for microscopic changes using laminin immunohistochemistry. Hypoglossal neurons and axons were examined using transmission electron microscopy. RESULTS: The cross-sectional area of myofibers in the posterior genioglossus was decreased in CTB-SAP-injected rats. Degenerative changes were observed in both the cell bodies and distal axons of hypoglossal neurons. DISCUSSION: Preliminary results indicate this model may have translational application to a variety of neurodegenerative diseases resulting in tongue dysfunction and associated dysphagia.

Lactate induces synapse-specific potentiation on CA3 pyramidal cells of rat hippocampus.

Neuronal activity within the physiologic range stimulates lactate production that, via metabolic pathways or operating through an array of G-protein-coupled receptors, regulates intrinsic excitability and synaptic transmission. The recent discovery that lactate exerts a tight control of ion channels, neurotransmitter release, and synaptic plasticity-related intracellular signaling cascades opens up the possibility that lactate regulates synaptic potentiation at central synapses. Here, we demonstrate that extracellular lactate (1-2 mM) induces glutamatergic potentiation on the recurrent collateral synapses of hippocampal CA3 pyramidal cells. This potentiation is independent of lactate transport and further metabolism, but requires activation of NMDA receptors, postsynaptic calcium accumulation, and activation of a G-protein-coupled receptor sensitive to cholera toxin. Furthermore, perfusion of 3,5- dihydroxybenzoic acid, a lactate receptor agonist, mimics this form of synaptic potentiation. The transduction mechanism underlying this novel form of synaptic plasticity requires G-protein ßγ subunits, inositol-1,4,5-trisphosphate 3-kinase, PKC, and CaMKII. Activation of these signaling cascades is compartmentalized in a synapse-specific manner since lactate does not induce potentiation at the mossy fiber synapses of CA3 pyramidal cells. Consistent with this synapse-specific potentiation, lactate increases the output discharge of CA3 neurons when recurrent collaterals are repeatedly activated during lactate perfusion. This study provides new insights into the cellular mechanisms by which lactate, acting via a membrane receptor, contributes to the memory formation process.

Inhibition of CFTR-mediated intestinal chloride secretion by a fungus-derived arthropsolide A: Mechanism of action and anti-diarrheal efficacy.

Secretory diarrhea is one of the most common types of diarrhea with high morbidity and mortality. Previous studies showed that inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels alleviated fluid loss in secretory diarrheas. This study aimed to identify novel CFTR inhibitors from fungal metabolites and explore its underlying mechanisms and potential utility in secretory diarrheas. Electrophysiological analyses in human intestinal epithelial (T84) cells were performed to investigate the effect and mechanism of fungal metabolites on CFTR-mediated Cl- secretion. Anti-diarrheal efficacy and the effect of compound on fluid absorption were investigated in mouse closed-loop models. We found that the screening identified arthropsolide A, a fungal metabolite from an endophytic fungus Roussoella sp. PSU-H51, as an inhibitor of CFTR-mediated Cl- secretion in T84 cells (IC50 ~0.8 µM). Arthropsolide A inhibited both CFTR and cAMP-activated basolateral K+ channels. Arthropsolide A had no effect on Na+-K+ ATPase activity. Interestingly, the inhibitory effect of arthropsolide A on CFTR was attenuated by cell depolarization and AMPK inhibition independent of multi-drug resistance protein 4, phosphodiesterases, and protein phosphatases. Importantly, arthropsolide A suppressed cholera toxin (CT)-induced Cl- secretion in T84 cells and CT-induced intestinal fluid secretion in mice by ~75% without affecting intestinal fluid absorption. Taken together, arthropsolide A represents a novel class of fungal metabolites that acts as a potent CFTR inhibitor. Further development of this class of compounds may provide a therapy for secretory diarrheas.

The novel combinations of CTB, CpG, and aluminum hydroxide significantly enhanced the immunogenicity of clumping factor A 221-550 of Staphylococcus aureus.

Here, we prepared the novel combined adjuvants, CTB as intra-molecular adjuvant, CpG and aluminum hydroxide (Alum) to strengthen the immunogenicity of clumping factor A221-550 of Staphylococcus aureus (S. aureus). The protein-immunoactive results showed CTB-ClfA221-550 elicited the strong immune responses to serum from mice immunized with CTB and ClfA221-550, respectively. The mice immunized with CTB-ClfA221-550 plus CpG and Alum adjuvant exhibited significantly stronger CD4+ T cell responses for IFN-γ, IL-2, IL-4, and IL-17 and displayed the higher proliferation response of splenic lymphocytes than the control groups, in addition, these mice generated the strongest humoral immune response against ClfA221-550 among all groups. Our results also showed CTB-ClfA221-550 plus CpG and Alum adjuvant obviously increased the survival percentage of the mice challenged by S. aureus. These data suggested that the novel combined adjuvants, CTB, CpG, and Alum, significantly enhance the immune responses triggered with ClfA221-550, and could provide a new approach against infection of S. aureus. ABBREVIATIONS: CTB: Cholera Toxin B; CpG: Cytosine preceding Guanosine; ODN: Oligodeoxynucleotides; Alum: Aluminum hydroxide; TRAP: Target of RNAIII-activating Protein; TLR9: Toll-like Receptor 9; TMB: 3, 3', 5, 5'-tetramethylbenzidine; mAbs: Monoclonal Antibodies; OD: Optical Densities; S. aureus: Staphylococcus aureus; ClfA: Clumping factor A; FnBPA: Fibronection-binding protein A; IsdB: Iron-regulated surface determinant B; SasA: Staphylococcus aureus Surface Protein A; GapC: Glycer-aldehyde-3-phosphate dehydrogenase-C.

Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin.

AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells, and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells.

Combined Prebiotic and Microbial Intervention Improves Oral Cholera Vaccination Responses in a Mouse Model of Childhood Undernutrition.

Undernourished children in low-income countries often exhibit poor responses to oral vaccination. Perturbed microbiota development is linked to undernutrition, but whether and how microbiota changes affect vaccine responsiveness remains unclear. Here, we show that gnotobiotic mice colonized with microbiota from undernourished Bangladeshi children and fed a Bangladeshi diet exhibited microbiota-dependent differences in mucosal IgA responses to oral vaccination with cholera toxin (CT). Supplementation with a nutraceutical consisting of spirulina, amaranth, flaxseed, and micronutrients augmented CT-IgA production. Mice initially colonized with a microbiota associated with poor CT responses exhibited improved immunogenicity upon invasion of bacterial taxa from cagemates colonized with a more "responsive" microbiota. Additionally, a consortium of five cultured bacterial invaders conferred augmented CT-IgA responses in mice fed the supplemented diet and colonized with the "hypo-responsive" community. These results provide preclinical proof-of-concept that diet and microbiota influence mucosal immune responses to CT vaccination and identify a candidate synbiotic formulation.