Connectivity of the Claustrum-Endopiriform Complex with the Presubiculum and Hippocampal Regions in the Common Marmoset (Callithrix jacchus).

We have investigated the hippocampal connectivity of the marmoset presubiculum (PreS) and reported that major connections of PreS in the rat were conserved in the marmoset. Moreover, our results indicated the presence of several additional projections that were almost absent in the rat brain, but abundant in the marmoset, such as direct projections from CA1 to PreS. However, little is known about the connectivity between the frontal brain regions and PreS or hippocampal formation. Therefore, we investigated the distribution of cells of the origins and terminals of the presubicular and hippocampal projections in the marmoset frontal brain regions using the retrograde and anterograde tracer cholera toxin B subunit. In cases of tracer injections into all layers of PreS, many neurons and terminals were labeled in the claustrum-endopiriform (Cl-En) complex almost entirely along the rostrocaudal axis. Even in cases where the injection site involved the superficial (not deep) layers of PreS, labeled neurons and terminals were distributed over a wide rostrocaudal range of the Cl-En complex, but their number and density were significantly lower than the whole-layer injection cases. In cases where the injection site was confined to the hippocampal formation, labeled cells and terminals were localized at a restricted portion of the Cl-En complex. Here, we demonstrate for what we believe to be the first time the strong, reciprocal connections of the Cl-En complex with PreS and projections from the Cl-En complex to the hippocampal regions (CA1 and the subiculum) in the marmoset. Our findings indicate that the Cl-En complex may exert a strong influence on the cortical and subcortical outputs from PreS and, in turn, the entire memory circuitry in the marmoset brain.

Label-free, real-time monitoring of membrane binding events at zeptomolar concentrations using frequency-locked optical microresonators.

G-protein coupled receptors help regulate cellular function and communication, and are targets of small molecule drug discovery efforts. Conventional techniques to probe these interactions require labels and large amounts of receptor to achieve satisfactory sensitivity. Here, we use frequency-locked optical microtoroids for label-free characterization of membrane interactions in vitro at zeptomolar concentrations for the kappa opioid receptor and its native agonist dynorphin A 1-13, as well as big dynorphin (dynorphin A and dynorphin B) using a supported biomimetic membrane. The measured affinity of the agonist dynorphin A 1-13 to the κ-opioid receptor was also measured and found to be 3.1 nM. Radioligand assays revealed a dissociation constant in agreement with this value (1.1 nM). The limit of detection for the κOR/DynA 1-13 was calculated as 180 zM. The binding of Cholera Toxin B-monosialotetrahexosyl ganglioside was also monitored in real-time and an equilibrium dissociation constant of 1.53 nM was found. Our biosensing platform provides a method for highly sensitive real-time characterization of membrane embedded protein binding kinetics that is rapid and label-free, for drug discovery and toxin screening among other applications.

Mitigating the Functional Deficit after Neurotoxic Motoneuronal Loss by an Inhibitor of Mitochondrial Fission.

Amyotrophic lateral sclerosis (ALS) is an extremely complex neurodegenerative disease involving different cell types, but motoneuronal loss represents its main pathological feature. Moreover, compensatory plastic changes taking place in parallel to neurodegeneration are likely to affect the timing of ALS onset and progression and, interestingly, they might represent a promising target for disease-modifying treatments. Therefore, a simplified animal model mimicking motoneuronal loss without the other pathological aspects of ALS has been established by means of intramuscular injection of cholera toxin-B saporin (CTB-Sap), which is a targeted neurotoxin able to kill motoneurons by retrograde suicide transport. Previous studies employing the mouse CTB-Sap model have proven that spontaneous motor recovery is possible after a subtotal removal of a spinal motoneuronal pool. Although these kinds of plastic changes are not enough to counteract the functional effects of the progressive motoneuron degeneration, it would nevertheless represent a promising target for treatments aiming to postpone ALS onset and/or delay disease progression. Herein, the mouse CTB-Sap model has been used to test the efficacy of mitochondrial division inhibitor 1 (Mdivi-1) as a tool to counteract the CTB-Sap toxicity and/or to promote neuroplasticity. The homeostasis of mitochondrial fission/fusion dynamics is indeed important for cell integrity, and it could be affected during neurodegeneration. Lesioned mice were treated with Mdivi-1 and then examined by a series of behavioral test and histological analyses. The results have shown that the drug may be capable of reducing functional deficits after the lesion and promoting synaptic plasticity and neuroprotection, thus representing a putative translational approach for motoneuron disorders.

Bayesian optimization of glycopolymer structures for the interaction with cholera toxin B subunit.

The optimal structure of synthetic glycopolymers for GM1 mimetics was determined through Bayesian optimization. The interactions of glycopolymers carrying galactose and neuraminic acid units in different compositions with cholera toxin B subunit (CTB) were assessed by an enzyme-linked immunosorbent assay (ELISA). Gaussian process regression, using the ELISA results, predicted the composition of glycopolymers that would exhibit stronger interactions with CTB. Following five cycles of optimization, the glycopolymers carrying 60 mol% galactose and 25 mol% neuraminic acid demonstrated an IC50 value of 75 µM for CTB, representing the lowest value among the synthesized glycopolymers.

Tailored multivalent peptide targeting the B-subunit pentamer of cholera toxin inhibits its intestinal toxicity by inducing aberrant transport of the toxin in cells.

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin.

IRE1α recognizes a structural motif in cholera toxin to activate an unfolded protein response.

IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge ß-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.

Sortase-Modified Cholera Toxoids Show Specific Golgi Localization.

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpectedly specific localization in the medial/trans-Golgi. This study suggests a future role for specifically labeled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labeling of lipid rafts in fixed cells.

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.

Association of intestinal inflammation and permeability markers with clinical manifestations of Parkinson's disease.

INTRODUCTION: Intestinal inflammation and gut microbiota dysbiosis can stimulate degeneration of dopaminergic neurons and development of Parkinson's disease (PD) via the gut-brain axis in certain patients. METHODS: In a case-control study, fecal markers of intestinal inflammation and permeability were measured using the ELISA method in PD patients and healthy controls. Motor and nonmotor symptoms were assessed using the Movement Disorder Society (MDS) Unified PD Rating Scale, Hoehn & Yahr scale, MDS Non-Motor Symptom Scale, Scales for Outcomes in PD - Autonomic Dysfunction, PD Sleep Scale - 2, Montreal Cognitive Assessment, Beck Anxiety Inventory, and Beck Depression Inventory-II. A correlation was established between the intestinal inflammation and permeability markers and PD symptoms. RESULTS: Higher levels of beta-defensin 2, zonulin and lactoferrin were recorded in PD patients compared to controls. Calprotectin and secretory immunoglobulin A showed no significant differences. Regression analysis indicated the roles of beta-defensin 2 and lactoferrin in predicting PD likelihood. Calprotectin yielded positive correlations with disease duration, depression, motor fluctuations, and gastrointestinal symptoms; beta defensin 2 with thermoregulation; and secretory immunoglobulin A with depression. Secretory immunoglobulin A showed negative correlation with age and age at disease onset, while zonulin showed negative correlation with the MDS Unified PD Rating Scale total score. CONCLUSIONS: Fecal markers differed in PD patients compared to controls and correlated with age, disease duration, and some nonmotor symptoms. Future studies should identify the subgroups of PD patients that are likely to develop intestinal inflammation.

Sporadic regional re-emergent cholera: a 19th century problem in the 21st century.

Cholera, caused by Vibrio cholerae, is a severe diarrheal disease that necessitates prompt diagnosis and effective treatment. This review comprehensively examines various diagnostic methods, from traditional microscopy and culture to advanced nucleic acid testing like polymerase spiral reaction and rapid diagnostic tests, highlighting their advantages and limitations. Additionally, we explore evolving treatment strategies, with a focus on the challenges posed by antibiotic resistance due to the activation of the SOS response pathway in V. cholerae. We discuss promising alternative treatments, including low-pressure plasma sterilization, bacteriophages, and selenium nanoparticles. The paper emphasizes the importance of multidisciplinary approaches combining novel diagnostics and treatments in managing and preventing cholera, a persistent global health challenge. The current re-emergent 7th pandemic of cholera commenced in 1961 and shows no signs of abeyance. This is probably due to the changing genetic profile of V. cholerae concerning bacterial pathogenic toxins. Given this factor, we argue that the disease is effectively re-emergent, particularly in Eastern Mediterranean countries such as Lebanon, Syria, etc. This review considers the history of the current pandemic, the genetics of the causal agent, and current treatment regimes. In conclusion, cholera remains a significant global health challenge that requires prompt diagnosis and effective treatment. Understanding the history, genetics, and current treatments is crucial in effectively addressing this persistent and re-emergent disease.

Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration.

Production of soluble proteins is essential for structure/function studies; however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens emerged as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine-binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA, and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein perdeuteration using deuterated minimal media with deuterium oxide as solvent and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news, since isotopic labeling is expensive and often ineffective but represents a necessary prerequisite for some structural biology techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein perdeuteration in amounts suitable for structural biology studies.

Mechanism study of cross presentation of exogenous antigen induced by cholera toxin-like chimeric protein.

Tumor subunit vaccines have great potential in personalized cancer immunotherapy. They are usually administered with adjuvant owing to their low immunogenicity. Cholera toxin (CT) is a biological adjuvant with diverse biological functions and a long history of use. Our earlier study revealed that a CT-like chimeric protein co-delivered with murine granulocyte-macrophage colony stimulating factor (mGM-CSF) and prostate cancer antigen epitope could co-stimulate dendritic cells (DCs) and enhance cross presentation of tumor epitope. To further study the molecular mechanism of CT-like chimeric protein in cross presentation, major histocompatibility complex class I (MHC I)-restricted epitope 257-264 of ovalbumin (OVAT) was used as a model antigen peptide in this study. Recombinant A subunit and pentameric B subunit of CT protein were respectively genetically constructed and purified. Then both assembled into AB5 chimeric protein in vitro. Three different chimeric biomacromolecules containing mGM-CSF and OVAT were constructed according to the different fusion sites and whether the endoplasmic reticulum (ER) retention sequence was included. It was found that A2 domain and B subunit of CT were both available for loading epitopes and retaining GM1 affinity. The binding activity of GM1 was positively correlated with antigen endocytosis. Once internalized, DCs became mature and cross-presented antigen. KDEL helped the whole molecule to be retained in the ER, and this improved the cross presentation of antigen on MHC I molecules. In conclusion, hexameric CT-like chimeric protein with dual effects of GM1 affinity and ER retention sequence were potential in improvement of cross presentation. The results laid a foundation for designing personalized tumor vaccine based on CT-like chimeric protein molecular structure.

The intersection between host-pathogen interactions and metabolism during Vibrio cholerae infection.

Vibrio cholerae (V. cholerae), the etiological agent of cholera, uses cholera toxin (CT) to cause severe diarrheal disease. Cholera is still a significant cause of mortality worldwide with about half of all cholera cases and deaths occurring in children under five. Owing to the lack of cost-effective vaccination and poor vaccine efficacy in children, there is a need for alternative preventative and therapeutic strategies. Recent advances in our knowledge of the interplay between CT-induced disease and host-pathogen metabolism have opened the door for investigating how modulation of intestinal metabolism by V. cholerae during disease impacts host intestinal immunity, the gut microbiota, and pathogen-phage interactions. In this review article, we examine recent progress in our understanding of host-pathogen interactions during V. cholerae infection and discuss future work deciphering how modulation of gut metabolism during cholera intersects these processes to enable successful fecal-oral transmission of the pathogen.

Marked enhancement of the immunogenicity of plant-expressed IgG-Fc fusion proteins by inclusion of cholera toxin non-toxic B subunit within the single polypeptide.

Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.

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.

Efficient Expression in the Prokaryotic Host System, Purification and Structural Analyses of the Recombinant Human ACE2 Catalytic Subunit as a Hybrid Protein with the B Subunit of Cholera Toxin (CTB-ACE2).

Angiotensin-converting enzyme 2 (ACE2) has a specific interaction with the coronavirus spike protein, enabling its entry into human cells. This membrane enzyme converts angiotensin II into angiotensin 1-7, which has an essential role in protecting the heart and improving lung function. Many therapeutic properties have been attributed to the human recombinant ACE2 (hrACE2), especially in combating complications related to diabetes mellitus and hypertension, as well as, preventing the coronavirus from entering the target tissues. In the current study, we designed an appropriate gene construct for the hybrid protein containing the ACE2 catalytic subunit and the B subunit of cholera toxin (CTB-ACE2). This structural feature will probably help the recombinant hybrid protein enter the mucosal tissues, including the lung tissue. Optimization of this hybrid protein expression was investigated in BL21 bacterial host cells. Also, the hybrid protein was identified with an appropriate antibody using the ELISA method. A large amount of the hybrid protein (molecular weight of ~ 100 kDa) was expressed as the inclusion body when the induction was performed in the presence of 0.25 mM IPTG and 1% sucrose for 10 h. Finally, the protein structural features were assessed using several biophysical methods. The fluorescence emission intensity and oligomeric size distribution of the CTB-ACE2 suggested a temperature-dependent alteration. The ß-sheet and α-helix were also dominant in the hybrid protein structure, and this protein also displays acceptable chemical stability. In overall, according to our results, the efficient expression and successful purification of the CTB-ACE2 protein may pave the path for its therapeutic applications against diseases such as covid-19, diabetes mellitus and hypertension.

Unveiling the Anti-Cholera and Active Diabetic Renoprotective Compounds of Maqian Essential Oil: A Computational and Molecular Dynamics Study.

Cholera is an exceptionally aggressive infectious disease characterized by the potential to induce acute, copious, watery diarrhea of considerable severity and renal inflammation. Diabetic nephropathy is a serious complication of diabetes mellitus that can lead to kidney failure through inflammation; thus, anti-inflammatory agents are promising therapies for diabetic nephropathy. Previous studies have shown that the essential oil of Zanthoxylum myriacanthum var. pubescens Huang, Maqian essential oil (MQEO), exhibits potent antibacterial, anti-inflammatory, and renoprotective activities in diabetic mice and has emerged as a potential therapeutic drug for the treatment of diabetic nephropathy complications. Therefore, the present study was carried out to screen the potential inhibition of cholera toxin and the diabetic renoprotective activity of MQEO through computational approaches. Twelve chemical constituents derived from MQEO were docked with cholera toxin and the target proteins involved in diabetic nephropathy, namely, TXNIP, Nrf2, and DPP IV, and, subsequently, the predictions of molecular dynamic simulations, the drug-likeness properties, and the ADMET properties were performed. α-terpineol showed high binding affinities toward the cholera toxin protein. For TXNIP, among all the chemical constituents, α-phellandrene and p-cymene showed strong binding affinities with the TXNIP protein and displayed relatively stable flexibility at the hinge regions of the protein, favorable physicochemical properties in the absence of hepatotoxicity, and low cytotoxicity. For Nrf2, α-terpineol exhibited the highest binding affinity and formed a very stable complex with Nrf2, which displayed high pharmacokinetic properties. All compounds had low free-binding energies when docked with the DPP IV protein, which suggests potent biological activity. In conclusion, based on a computational approach, our findings reveal that MQEO constituents have inhibitory activity against cholera toxin and are promising therapeutic agents for suppressing diabetic inflammation and for the treatment of diabetic nephropathy complications.

Cholera intoxication of human enteroids reveals interplay between decoy and functional glycoconjugate ligands.

Prior research on cholera toxin (CT) binding and intoxication has relied on human colonic cancer derived epithelial cells. While these transformed cell lines have been beneficial, they neither derive from small intestine where intoxication occurs, nor represent the diversity of small intestinal epithelial cells (SI-ECs) and variation in glycoconjugate expression among individuals. Here, we used human enteroids, derived from jejunal biopsies of multipledonors to study CT binding and intoxication of human non-transformed SI-ECs. We modulated surface expression of glycosphingolipids, glycoproteins and specific glycans to distinguish the role of each glycan/glycoconjugate. Cholera-toxin-subunit-B (CTB) mutants were generated to decipher the preference of each glycoconjugate to different binding sites and the correlation between CT binding and intoxication. Human enteroids contain trace amounts of GM1, but other glycosphingolipids may be contributing to CT intoxication. We discovered that inhibition of either fucosylation or O-glycosylation sensitize enteroids to CT-intoxication. This can either be a consequence of the removal of fucosylated "decoy-like-ligands" binding to CTB's non-canonical site and/or increase in the availability of Gal/GalNAc-terminating glycoconjugates binding to the canonical site. Furthermore, simultaneous inhibition of fucosylation and O-glycosylation increased the availability of additional Gal/GalNAc-terminating glycoconjugates but counteracted the sensitization in CT intoxication caused by inhibiting O-glycosylation because of reduction in fucose. This implies a dual role of fucose as a functional glycan and a decoy, the interplay of which influences CT binding and intoxication. Finally, while the results were similar for enteroids from different donors, they were not identical, pointing to a role for human genetic variation in determining sensitivity to CT.

Vibrio cholerae RND efflux systems: mediators of stress responses, colonization and pathogenesis.

Resistance Nodulation Division (RND) efflux systems are ubiquitous transporters in gram-negative bacteria that provide protection against antimicrobial agents and thereby enhance survival in virtually all environments these prokaryotes inhabit. Vibrio cholerae is a dual lifestyle enteric pathogen that spends much of its existence in aquatic environments. An unwitting encounter with a human host can lead to V. cholerae intestinal colonization by strains that encode cholera toxin and toxin co-regulated pilus virulence factors leading to potentially fatal cholera diarrhea and dissemination in the environment. Adaptive response mechanisms to host factors encountered by these pathogens are therefore critical both to engage survival mechanisms such as RND-mediated transporters and to induce timely expression of virulence factors. Sensing of cues encountered in the host may therefore activate more than protective responses such as efflux systems, but also be coordinated to initiate expression of virulence factors. This review summarizes recent advances that contribute towards the understanding of RND efflux physiological functions and how the transport systems interface with the regulation of virulence factor production in V. cholerae.

Sporadic type VI secretion in seventh pandemic Vibrio cholerae.

Vibrio cholerae is a pathogen that causes disease in millions of people every year by colonizing the small intestine and then secreting the potent cholera toxin. How the pathogen overcomes the colonization barrier created by the host's natural microbiota is, however, still not well understood. In this context, the type VI secretion system (T6SS) has gained considerable attention given its ability to mediate interbacterial killing. Interestingly, and in contrast to non-pandemic or environmental V. cholerae isolates, strains that are causing the ongoing cholera pandemic (7PET clade) are considered T6SS-silent under laboratory conditions. Since this idea was recently challenged, we performed a comparative in vitro study on T6SS activity using diverse strains or regulatory mutants. We show that modest T6SS activity is detectable in most of the tested strains under interbacterial competition conditions. The system's activity was also observed through immunodetection of the T6SS tube protein Hcp in culture supernatants, a phenotype that can be masked by the strains' haemagglutinin/protease. We further investigated the low T6SS activity within the bacterial populations by imaging 7PET V. cholerae at the single-cell level. The micrographs showed the production of the machinery in only a small fraction of cells within the population. This sporadic T6SS production was higher at 30 °C than at 37 °C and occurred independently of the known regulators TfoX and TfoY but was dependent on the VxrAB two-component system. Overall, our work provides new insight into the heterogeneity of T6SS production in populations of 7PET V. cholerae strains in vitro and provides a possible explanation of the system's low activity in bulk measurements.