Morphology-Dependent Interaction of Silica Nanoparticles with Intestinal Cells: Connecting Shape to Barrier Function.

The intestinal compartment ensures nutrient absorption and barrier function against pathogens. Despite decades of research on the complexity of the gut, the adaptive potential to physical cues, such as those derived from interaction with particles of different shapes, remains less understood. Taking advantage of the technological versatility of silica nanoparticles, spherical, rod-shaped, and virus-like materials were synthesized. Morphology-dependent interactions were studied on differentiated Caco-2/HT29-MTX-E12 cells. Contributions of shape, aspect ratio, surface roughness, and size were evaluated considering the influence of the mucus layer and intracellular uptake pathways. Small particle size and surface roughness favored the highest penetration through the mucus but limited interaction with the cell monolayer and efficient internalization. Particles of a larger aspect ratio (rod-shaped) seemed to privilege paracellular permeation and increased cell-cell distances, albeit without hampering barrier integrity. Inhibition of clathrin-mediated endocytosis and chemical modulation of cell junctions effectively tuned these responses, confirming morphology-specific interactions elicited by bioinspired silica nanomaterials.

Enhancing Vitamin D3 Efficacy: Insights from Complexation with Cyclodextrin Nanosponges and Its Impact on Gut-Brain Axes in Physiology and IBS Syndrome.

Vitamin D3 (VitD3) plays a crucial role in various cellular functions through its receptor interaction. The biological activity of Vitamin D3 can vary based on its solubility and stability. Thus, the challenge lies in maximizing its biological effects through its complexation within cyclodextrin (ßNS-CDI 1:4) nanosponges (NS) (defined as VitD3NS). Therefore, its activity has been evaluated on two different gut-brain axes (healthy gut/degenerative brain and inflammatory bowel syndrome gut/degenerative brain axis). At the gut level, VitD3-NS mitigated liposaccharide-induced damage (100 ng/mL; for 48 h), restoring viability, integrity, and activity of tight junctions and reducing ROS production, lipid peroxidation, and cytokines levels. Following intestinal transit, VitD3-NS improved the neurodegenerative condition in the healthy axis and the IBS model, suggesting the ability of VitD3-NS to preserve efficacy and beneficial effects even in IBS conditions. In conclusion, this study demonstrates the ability of this novel form of VitD3, named VitD3-NS, to act on the gut-brain axis in healthy and damaged conditions, emphasizing enhanced biological activity through VitD3 complexation, as such complexation increases the beneficial effect of vitamin D3 in both the gut and brain by about 50%.

Identification and Molecular Mechanism of Anti-inflammatory Peptides Isolated from Jack Bean Protein Hydrolysates: in vitro Studies with Human Intestinal Caco-2BBe Cells.

Jack bean (JB), Canavalia ensiformis (L.) DC, is a commonly cultivated legume in Indonesia. It is rich in protein, which can be hydrolyzed, making it potentially a good source of bioactive peptides. Intestinal inflammation is associated with several diseases, and the production of interleukin-8 (IL-8) in intestinal epithelial cells induced by tumor necrosis factor (TNF)-α has an important role in inflammatory reaction. The present study investigated the anti-inflammatory effects of peptides generated from enzymatic hydrolysis of JB protein on human intestinal Caco-2BBe cells. Additionally, in silico approaches were used to identify potential bioactive peptides. JB protein hydrolysate (JBPH) prepared using pepsin and pancreatin reduced the IL-8 expression at protein and mRNA levels in Caco-2BBe cells stimulated with TNF-α. Immunoblot analysis showed that the JBPH reduced the TNF-α-induced phosphorylation of c-Jun-NH(2)-terminal kinase, nuclear factor kappa B (NF-κB), and p38 proteins. Anti-inflammatory activity was observed in the 30% acetonitrile fraction of JBPH separated on a Sep-Pak C18 column. An ultrafiltration method revealed that relatively small peptides (< 3 kDa) had a potent inhibitory effect on the IL-8 production. Purification of the peptides by reversed-phase and anion-exchange high performance chromatography produced three peptide fractions with anti-inflammatory activities. A combination of mass spectrometry analysis and in silico approaches identified the potential anti-inflammatory peptides. Peptides derived from JB protein reduces the TNF-α-induced inflammatory response in Caco-2BBe cells via NF-κB and mitogen-activated protein kinase signaling pathways. Our results may lead to a novel therapeutic approach to promote intestinal health.

Effects of propofol on IGF-1 activity and cell behaviour in the GES 1 mucosal cell model.

Propofol is an important and widely used anaesthetic drug in the clinic. Many works have shown that propofol has important biological functions except as an anaesthetic. In the current study, we mainly explored the effect of propofol on the biological activity of IGF-1, which is an important growth factor involved in regulating the growth and development of the stomach. Here, we explored the effect of propofol on the biological activity of IGF-1 in a GES-1-cell model. We found that propofol affected the biological activity of IGF-1. It not only reduces IGF-1/IGF-1R signalling but also changes IGF-1R cell characteristics. We further explored the mechanism by which propofol affected IGF-1 activity. Through a series of experiments, we found that propofol affected the stability of membrane-localised IGF-1R. It also affects the recycling of the IGF-1R receptor Propofol can affect the degradation of IGF-1R by changing the endocytosis of IGF-1R. In short, the current study found that propofol affected the biological activity of IGF-1, which laid the foundation for related research.

Systematic Investigation of Metabolic Oligosaccharide Engineering Efficiency in Intestinal Cells Using a Dibenzocyclooctyne-Monosaccharide Conjugate.

Metabolic oligosaccharide engineering (MOE) of cells with synthetic monosaccharides can introduce functionality to the glycans of cell membranes. Unnatural sugars (e. g., peracetylated mannose-azide) can be expressed on the cell surface with the azide group in place. After MOE, the azide group can participate in a copper-free click reaction with an alkyne (e. g., dibenzocyclooctyne, DBCO) probe. This allows the metabolic fate of monosaccharides in cells to be understood. However, in a drug delivery context it is desirable to have azide groups on the probe (e. g. a drug delivery particle) and the alkyne (e. g. DBCO) on the cell surface. Consequently, the labelling efficiency of intestinal cell lines (Caco-2 and HT29-MTX-E12) treated with N-dibenzocyclooctyne-tetra-acetylmannosamine, and the concentration- and time-dependent labelling were determined. Furthermore, the labelling of mucus in HT29-MTX-E12 cells with DBCO was shown. This study highlights the potential for using MOE to target azide-functionalised probes to intestinal tissues for drug delivery applications.

Cadmium affects autophagy in the human intestinal cells Caco-2 through ROS-mediated ERK activation.

Cadmium is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium has the capacity to accumulate high levels of this metal. We have previously shown that Cd induces ERK1/2 activation in differentiated but not proliferative human enterocytic-like Caco-2 cells. As autophagy is a dynamic process that plays a critical role in intestinal mucosa, we aimed the present study 1) to investigate the role of p-ERK1/2 in constitutive autophagy in proliferative Caco-2 cells and 2) to investigate whether Cd-induced activation of ERK1/2 modifies autophagic activity in postconfluent Caco-2 cell monolayers. Western blot analyses of ERK1/2 and autophagic markers (LC3, SQSTM1), and cellular staining with acridine orange showed that ERK1/2 and autophagic activities both decreased with time in culture. GFP-LC3 fluorescence was also associated with proliferative cells and the presence of a constitutive ERK1/2-dependent autophagic flux was demonstrated in proliferative but not in postconfluent cells. In the latter condition, serum and glucose deprivation triggered autophagy via a transient phosphorylation of ERK1/2, whereas Cd-modified autophagy via a ROS-dependent sustained activation of ERK1/2. Basal autophagy flux in proliferative cells and Cd-induced increases in autophagic markers in postconfluent cells both involved p-ERK1/2. Whether Cd blocks autophagic flux in older cell cultures remains to be clarified but our data suggest dual effects. Our results prompt further studies investigating the consequences that Cd-induced ERK1/2 activation and the related effect on autophagy may have on the intestinal cells, which may accumulate and trap high levels of Cd under some nutritional conditions.

Developing New Cyclodextrin-Based Nanosponges Complexes to Improve Vitamin D Absorption in an In Vitro Study.

Vitamin D plays an important role in numerous cellular functions due to the ability to bind the Vitamin D receptor (VDR), which is present in different tissues. Several human diseases depend on low vitamin D3 (human isoform) serum level, and supplementation is necessary. However, vitamin D3 has poor bioavailability, and several strategies are tested to increase its absorption. In this work, the complexation of vitamin D3 in Cyclodextrin-based nanosponge (CD-NS, in particular, ßNS-CDI 1:4) was carried out to study the possible enhancement of bioactivity. The ßNS-CDI 1:4 was synthesized by mechanochemistry, and the complex was confirmed using FTIR-ATR and TGA. TGA demonstrated higher thermostability of the complexed form. Subsequently, in vitro experiments were performed to evaluate the biological activity of Vitamin D3 complexed in the nanosponges on intestinal cells and assess its bioavailability without cytotoxic effect. The Vitamin D3 complexes enhance cellular activity at the intestinal level and improve its bioavailability. In conclusion, this study demonstrates for the first time the ability of CD-NS complexes to improve the chemical and biological function of Vitamin D3.

Cellular and Molecular Mechanisms of Toxicity of Ingested Titanium Dioxide Nanomaterials.

An exponential increase in products containing titanium dioxide nanomaterials (TiO2), in agriculture, food and feed industry, lead to increased oral exposure to these nanomaterials (NMs). Thus, the gastrointestinal tract (GIT) emerges as a possible route of exposure that may drive systemic exposure, if the intestinal barrier is surpassed. NMs have been suggested to produce adverse outcomes, such as genotoxic effects, that are associated with increased risk of cancer, leading to a concern for public health. However, to date, the differences in the physicochemical characteristics of the NMs studied and other variables in the test systems have generated contradictory results in the literature. Processes like human digestion may change the NMs characteristics, inducing unexpected toxic effects in the intestine. Using TiO2 as case-study, this chapter provides a review of the works addressing the interactions of NMs with biological systems in the context of intestinal tract and digestion processes, at cellular and molecular level. The knowledge gaps identified suggest that the incorporation of a simulated digestion process for in vitro studies has the potential to improve the model for elucidating key events elicited by these NMs, advancing the nanosafety studies towards the development of an adverse outcome pathway for intestinal effects.

Molecular Effects of Chronic Exposure to Palmitate in Intestinal Organoids: A New Model to Study Obesity and Diabetes.

Intestinal cell dysfunctions involved in obesity and associated diabetes could be correlated with impaired intestinal cell development. To date, the molecular mechanisms underlying these dysfunctions have been poorly investigated because of the lack of a good model for studying obesity. The main aim of this study was to investigate the effects of lipotoxicity on intestinal cell differentiation in small intestinal organoid platforms, which are used to analyze the regulation of cell differentiation. Mouse intestinal organoids were grown in the presence/absence of high palmitate concentrations (0.5 mM) for 48 h to simulate lipotoxicity. Palmitate treatment altered the expression of markers involved in the differentiation of enterocytes and goblet cells in the early (Hes1) and late (Muc2) phases of their development, respectively, and it modified enterocytes and goblet cell numbers. Furthermore, the expression of enteroendocrine cell progenitors (Ngn3) and I cells (CCK) markers was also impaired, as well as CCK-positive cell numbers and CCK secretion. Our data indicate, for the first time, that lipotoxicity simultaneously influences the differentiation of specific intestinal cell types in the gut: enterocytes, goblet cells and CCK cells. Through this study, we identified novel targets associated with molecular mechanisms affected by lipotoxicity that could be important for obesity and diabetes therapy.

Dietary Polyphenols and In Vitro Intestinal Fructose Uptake and Transport: A Systematic Literature Review.

Recent evidence links chronic consumption of large amounts of fructose (FRU) with several non-communicable disease. After ingestion, dietary FRU is absorbed into the intestinal tract by glucose transporter (GLUT) 5 and transported to the portal vein via GLUT2. GLUT2 is primarily localized on the basolateral membrane, but GLUT2 may be dislocated post-prandially from the basolateral membrane of intestinal cells to the apical one. Polyphenols (PP) are plant secondary metabolites that exert hypoglycemic properties by modulating intracellular insulin signaling pathways and by inhibiting intestinal enzymes and transporters. Post-prandially, PP may reach high concentrations in the gut lumen, making the inhibition of FRU absorption a prime target for exploring the effects of PP on FRU metabolism. Herein, we have systematically reviewed studies on the effect of PP and PP-rich products on FRU uptake and transport in intestinal cells. In spite of expectations, the very different experimental conditions in the various individual studies do not allow definitive conclusions to be drawn. Future investigations should rely on standardized conditions in order to obtain comparable results that allow a credible rating of polyphenols and polyphenol-rich products as inhibitors of fructose uptake.

Hydrogen sulfide suppresses the proliferation of intestinal epithelial cells through cell cycle arrest.

The pathogenesis of chronic kidney disease (CKD) is closely related to the changes in the intestinal microbiota and integrity. Our previous studies have shown the accumulation of hydrogen sulfide (H2S)-producing bacterial family, Desulfovibrionacea, in the colon of a murine model of CKD, suggesting that the increased H2S contributes to the impaired intestinal integrity in CKD. Here, we investigated the anti-proliferative effect of H2S in the intestinal epithelial cells. A slow- H2S releasing molecule GYY4137 ((p-methoxyphenyl)morpholino-phosphinodithioic acid) reduced the proliferation of Caco-2 and IEC-6 cells. Flow cytometric analysis demonstrated that GYY4137 accumulated Caco-2 cells in the S phase fraction, suggesting that H2S arrested the cell cycle at G2 and/or M phases. The RNA sequencing analysis demonstrated that GYY4137 modulated the mRNA expression of the genes involved in the G2/M and the spindle assembly checkpoints; increased mRNA levels of Cdkn1a, Gadd45a, and Sfn and decreased mRNA levels of Cdc20, Pttg1, and Ccnb1 were observed. These alterations were confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot analyses. Besides, studies exploring the MEK inhibitor indicated that MEK activation is involved in the GYY4137-mediated increase in the Sfn expression. Altogether, our data showed that H2S reduced the proliferation of intestinal epithelial cells through transcriptional regulation in G2/M and the spindle assembly checkpoints. This may be one of the underlying mechanisms for the observed impaired intestinal integrity in CKD.

Effect of fumonisin B1 on oxidative stress and gene expression alteration of nutrient transporters in porcine intestinal cells.

Fumonisin B1 (FB1 ) is a common environmental mycotoxin produced by molds such as Fusarium verticillioides. The toxin poses health risks to domestic animals, including pigs, through FB1 -contaminanted feed. However, the cytotoxicity of FB1 to porcine intestines has not been fully analyzed. In the present study, the effects of FB1 on oxidative stress and nutrient transporter-associated genes of the porcine intestinal IPEC-J2 cells were explored. FB1 decreased IPEC-J2 proliferation but did not trigger reactive oxygen species (ROS) overproduction. Meanwhile, FB1 reduced the expression levels of the transporters l-type amino acid transporter-1 (y+ LAT1), solute carrier family 7 member 1 (SLC7A1), solute carrier family 1 member 5 (ASCT2), and excitatory amino acid carrier 1 (EAAC1); in addition, FB1 reduced the levels of the fatty acid transporters long-chain fatty acid transport protein 1 (FATP1) and long-chain fatty acid transport protein 4 (FATP4) as well as glucose transporters Na+ /glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2). FB1 stimulation increased the expression levels of peptide transporter peptide transporter 1 (PepT1) and metal ion transport-related gene zinc transporter 1 (ZNT1). Moreover, metal ion transporter divalent metal transporter 1 (DMT1) expression was depressed by a higher dosage of FB1 . The data indicate that FB1 results in aberrant expression of nutrient transporters in IPEC-J2 cells, thereby exerting its toxicity even though it fails to exert ROS-dependent oxidative stress.

Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor.

Disabled 1 (Dab1) is an adapter protein for very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) and an integral component of the Reelin pathway which orchestrates neuronal layering during embryonic brain development. Activation of Dab1 is induced by binding of Reelin to ApoER2 and VLDLR and phosphorylation of Dab1 mediated by Src family kinases. Here we show that Dab1 also acts as an adaptor for epidermal growth factor receptor (EGFR) and can be phosphorylated by epidermal growth factor (EGF) binding to EGFR. Phosphorylation of Dab1 depends on the kinase activity of EGFR constituting a signal pathway independent of Reelin and its receptors.

Determination of Two Differently Manufactured Silicon Dioxide Nanoparticles by Cloud Point Extraction Approach in Intestinal Cells, Intestinal Barriers and Tissues.

Food additive amorphous silicon dioxide (SiO2) particles are manufactured by two different methods-precipitated and fumed procedures-which can induce different physicochemical properties and biological fates. In this study, precipitated and fumed SiO2 particles were characterized in terms of constituent particle size, hydrodynamic diameter, zeta potential, surface area, and solubility. Their fates in intestinal cells, intestinal barriers, and tissues after oral administration in rats were determined by optimizing Triton X-114-based cloud point extraction (CPE). The results demonstrate that the constituent particle sizes of precipitated and fumed SiO2 particles were similar, but their aggregate states differed from biofluid types, which also affect dissolution properties. Significantly higher cellular uptake, intestinal transport amount, and tissue accumulation of precipitated SiO2 than of fumed SiO2 was found. The intracellular fates of both types of particles in intestinal cells were primarily particle forms, but slowly decomposed into ions during intestinal transport and after distribution in the liver, and completely dissolved in the bloodstream and kidneys. These findings will provide crucial information for understanding and predicting the potential toxicity of food additive SiO2 after oral intake.

Protective Role of Flavonoids against Intestinal Pro-Inflammatory Effects of Silver Nanoparticles.

Silver nanoparticles (AgNP) have been increasingly incorporated into food-related and hygiene products for their unique antimicrobial and preservative properties. The consequent oral exposure may then result in unpredicted harmful effects in the gastrointestinal tract (GIT), which should be considered in the risk assessment and risk management of these materials. In the present study, the toxic effects of polyethyleneimine (PEI)-coated AgNP (4 and 19 nm) were evaluated in GIT-relevant cells (Caco-2 cell line as a model of human intestinal cells, and neutrophils as a model of the intestinal inflammatory response). This study also evaluated the putative protective action of dietary flavonoids against such harmful effects. The obtained results showed that AgNP of 4 and 19 nm effectively induced Caco-2 cell death by apoptosis with concomitant production of nitric oxide, irrespective of the size. It was also observed that AgNP induced human neutrophil oxidative burst. Interestingly, some flavonoids, namely quercetin and quercetagetin, prevented the deleterious effects of AgNP in both cell types. Overall, the data of the present study provide a first insight into the promising protective role of flavonoids against the potentially toxic effects of AgNP at the intestinal level.

Melatonin reduces inflammation in intestinal cells, organoids and intestinal explants.

Inflammatory bowel diseases (IBDs) are chronic and recurrent diseases that often occur in young people and place a heavy burden on public health in both developed and developing countries. Melatonin has been confirmed to be useful in various diseases, including Alzheimer's disease, liver injuries and diseases, and cancers, while its role in IBDs remains unclear. To uncover the function of melatonin in IBDs, three intestinal models, including Caco-2 cells, 3D intestinal organoids and intestinal explants, were used. It was found that different concentrations of melatonin could significantly inhibit the expression levels of NFκB and its downstream cytokines, including IL6 and IL8 in Caco-2 cells (*P < 0.05, **P < 0.01), 3D intestinal organoids (*P < 0.05, **P < 0.01) and intestinal explants (*P < 0.05, **P < 0.01). Melatonin abolished the activation of LPS on the expression levels of NFκB, IL6, and IL8 in three intestinal models (*P < 0.05, **P < 0.01, ***P < 0.001). Importantly, the roles of melatonin in the regulation of inflammation was dependent on its receptor (i.e., MTNR1), since it was found that silencing of the melatonin receptor (MTNR1A) abolished the reduction in inflammation induced by melatonin in Caco-2 cells (***P < 0.001) and 3D intestinal organoids (***P < 0.01, ****P < 0.0001). Herein, the findings in this study might provide useful information for understanding the pathogenesis of IBDs and developing novel drugs to treat the diseases.

Studies on host-foodborne bacteria in intestinal three-dimensional cell culture model indicate possible mechanisms of interaction.

Spheroids of intestinal cells (Caco-2) were used to evaluate the adhesion/invasion ability of Listeria monocytogenes (pathogen) and Lactobacillus sakei 1 (potential probiotic). Besides, transcriptomic analyses of Caco-2 cells in three dimensional cultures were done, with the aim of revealing possible host-foodborne bacteria interactions. Result of adhesion assay for L. monocytogenes in Caco-2 spheroids was 22.86 ± 0.33%, but it was stimulated in acidic pH (4.5) and by the presence of 2% sucrose (respectively, 32.56 ± 1.35% and 33.25 ± 1.26%). Conversely, the invasion rate of L. monocytogenes was lower at pH 4.5, in comparison with non-stressed controls (18.89 ± 1.05% and 58.65 ± 0.30%, respectively). L. sakei 1 adhered to Caco-2 tridimensional cell culture (27.30 ± 2.64%), with no invasiveness. There were 19 and 21 genes down and upregulated, respectively, in tridimensional Caco-2 cells, upon infection with L. monocytogenes, which involved immunity, apoptosis; cytoprotective responses, cell signalling-regulatory pathways. It was evidenced despite activation or deactivation of several pathways in intestinal cells to counteract infection, the pathogen was able to hijack many host defense mechanisms. On the other hand, the probiotic candidate L. sakei 1 was correlated with decreased transcription of two genes in Caco-2 cells, though it stimulated the expression of 14 others, with diverse roles in immunity, apoptosis, cytoprotective response and cell signalling-regulatory pathways. Our data suggest the use of tridimensional cell culture to mimic the intestinal epithelium is a good model for gathering broad information on the putative mechanisms of interaction between host and bacteria of importance for food safety, which can serve as a basis for further in-depth investigation.

Zinc interference with Cd-induced hormetic effect in differentiated Caco-2 cells: Evidence for inhibition downstream ERK activation.

Cadmium (Cd) is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium represents an effective protective barrier against Cd toxicity, but it is also a target tissue that may accumulate and trap high levels of the ingested metal. Using human enterocytic-like Caco-2 cells, we have previously shown that Cd may induce a concentration and time-dependent increase in 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT)-reducing activity in differentiated cultures with correlation to ERK1/2 activation. The present study shows that (a) Zn prevents the Cd-induced hormesis effect on MTT reduction in a concentration-dependent manner, without inhibiting Cd-induced ERK1/2 activation; (b) Zn also induces similar hormetic stimulation of MTT-reducing activity but without ERK1/2 activation. The effect of both metals was sensitive to inhibitors of translation during protein synthesis. There is evidence for the involvement of reactive oxygen species (ROS) in Cd-induced ERK1/2 activation. In contrast, the Zn effect on the MTT-reducing activity would not be triggered by ROS but it would be sensitive to the redox state of the cell. Steps downstream ERK1/2 activation by Cd does not involve eIF4E which is rather downregulated by Cd. In conclusion, Cd and Zn both can modify translation processes during protein synthesis via different signaling cascades with crosstalk, and cross-inhibition may occur. This phenomenon is observed over a small range of metal concentrations and is characterized by a hormesis-like response. Considering that the hormetic effect on dehydrogenase activity could reflect an adaptive response to the metals whether cross-inhibition is beneficial is an open question.

HtrA-dependent adherence and invasion of Campylobacter jejuni in human vs avian cells.

The aim of this study was to investigate whether HtrA is responsible for differences in adherence and invasion of Campylobacter jejuni towards human and chicken cell lines. Gentamicin protection assays were performed with either human Caco-2 or chicken 2G4 cells using C. jejuni strain NCTC11168 to compare the adhesion and invasion rates towards these two cell types. The results revealed significant differences in the adhesion and invasion rates between the human and avian cells. Deletion of the Campylobacter htrA gene, coding for the dual function of serine protease and chaperonin with a role in pathogenesis, led to a reduction of the rates in both cell lines. Using a single-amino acid substitution mutant (ΔhtrA/htrAS197A ) that lacked protease activity, but retained chaperonin activity, we show that the first is involved in the invasion of human Caco-2 and chicken 2G4 cells, whereas the latter mutant invaded at lower levels. Adherence towards the chicken cells is higher than towards Caco-2 cells and this is also dependent on HtrA. Together, these data suggest that the proteolytic activity of HtrA is involved in the difference in host response of C. jejuni towards human and chicken-derived cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter jejuni is the main cause for bacterial foodborne enterocolitis worldwide. While colonization of the human intestine can lead to severe problems, avian hosts - as the major source of infection - remain unaffected by the bacteria. We showed that the bacterial serine protease and chaperonin HtrA are involved in adhesion and invasion in both species and not responsible for the discrepancy of virulence between the different hosts. In future, HtrA might act as a target for inhibitors to avoid or eradicate colonization in chickens as a less problematic alternative to antibiotics in commercial livestock breeding.

Botulinum neurotoxin type B uses a distinct entry pathway mediated by CDC42 into intestinal cells versus neuronal cells.

Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis by inhibiting the release of acetylcholine at the neuromuscular junctions. BoNT type B (BoNT/B) most often induces mild forms of botulism with predominant dysautonomic symptoms. In food borne botulism and botulism by intestinal colonisation such as infant botulism, which are the most frequent naturally acquired forms of botulism, the digestive tract is the main entry route of BoNTs into the organism. We previously showed that BoNT/B translocates through mouse intestinal barrier by an endocytosis-dependent mechanism and subsequently targets neuronal cells, mainly cholinergic neurons, in the intestinal mucosa and musculosa. Here, we investigated the entry pathway of BoNT/B using fluorescent C-terminal domain of the heavy chain (HcB), which is involved in the binding to specific receptor(s) and entry process into target cells. While the combination of gangliosides GD1a /GD1b /GT1b and synaptotagmin I and to a greater extent synaptotagmin II constitutes the functional HcB receptor on NG108-15 neuronal cells, HcB only uses the gangliosides GD1a /GD1b /GT1b to efficiently bind to m-ICcl2 intestinal cells. HcB enters both cell types by a dynamin-dependent endocytosis, which is efficiently prevented by Dynasore, a dynamin inhibitor, and reaches a common early endosomal compartment labeled by early endosome antigen (EEA1). In contrast to neuronal cells, HcB uses a Cdc42-dependent pathway to enter intestinal cells. Then, HcB is transported to late endosomes in neuronal cells, whereas it exploits a nonacidified pathway from apical to basal lateral side of m-ICcl2 cells supporting a transcytotic route in epithelial intestinal cells.