Enteropathogenic Escherichia coli inhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level

Enteropathogenic Escherichia coli (EPEC), a food-borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na+/H+ and Cl-/HCO3- exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short-chain fatty acid absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild-type EPEC, various mutant strains, or nonpathogenic E. coli HS4, and [14C]butyrate uptake was determined. EPEC, but not nonpathogenic E. coli, significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB, or espD genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH, and map inhibited butyrate uptake, similar to the wild-type EPEC. Surface expression of MCT1 decreased considerably after EPEC but not after nonpathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, -B, and -D of the translocation apparatus.     

B-cell CLL/Lymphoma 10 (BCL10) Is Required for NF-��B Production by Both Canonical and Noncanonical Pathways and for NF-��B-inducing Kinase (NIK) Phosphorylation

B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-κB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IκB-kinase (IKK)α^−/− mouse embryonic fibroblasts, increases in phospho-IκBα, nuclear NF-κB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKβ^−/− cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-κB-inducing kinase (NIK), cytoplasmic NF-κB p100, and nuclear NF-κB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-κB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKβ^−/− and IKKα^−/− cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKγ, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-κB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.     

Molecular diversity,effectiveness and competitiveness of indigenous rhizobial population infecting mungbean <Emphasis Type="Italic">Vigna radiata</Emphasis> (L. Wilczek) under semi-arid conditions

Nodules from mungbean crop raised for the first time at Ram Dhan Singh (RDS) farm of Chaudhary Charan Singh (CCS) Haryana Agricultural University, Hisar were collected from 17 different locations. Twenty-five mungbean rhizobia were isolated and authenticated by plant infection test. DNA of all these rhizobia was extracted purified and amplified using enterobacterial repetitive intergenic consensus (ERIC) primers. All the mungbean rhizobial isolates were clustered into 4 groups at 65% of similarity and were further divided into 17 subclusters at 80% of similarity. All the 4 types of rhizobia were not present at any of the location and group 2 or 4 rhizobia were invariably present. Efficacy of these rhizobia in terms of nodulation, nitrogen uptake and chlorophyll a fluorescence was determined under pot culture conditions. Strain MB 307 showed maximum nitrogen uptake of 31.9 mg N plant⁻¹ followed by strain MB 1205, MB 1206(2), MB 308, MB 1524 and strain MB 1521 was found to be the least efficient in terms of N 2 fixation. Nodule occupancy by different rhizobia ranged from 5.5 to 40.3%. Most of the strains belonging to the 2nd group which clustered maximum number of strains were comparatively better competitors and formed 19.5–40.3% of the nodules and were also effective. Isolate MB 307, the most efficient strain, was found to have nodule occupancy of 31.5%. Such type of predominant, efficient and better competitor strains should be selected for enhancing nodule competitiveness.     

Serotonin modifies cytoskeleton and brush-border membrane architecture in human intestinal epithelial cells

Serotonin or 5-hydroxytryptamine (5-HT) influences numerous functions in the gastrointestinal tract. We previously demonstrated that 5-HT treatment of Caco-2 cells inhibited Na(+)/H(+) exchangers (NHE) and Cl(-)/OH(-) exchange activities via distinct signaling mechanisms. Since regulation of several ion transporters such as NHE3 is influenced by intact cytoskeleton, we hypothesized that 5-HT modifies actin cytoskeleton and/or brush-border membrane architecture via involvement of signaling pathways. Ultrastructural analysis showed that 5-HT (0.1 muM, 1 h) treatment of Caco-2 cells caused the apical membrane to assume a convex dome shape that was associated with shortening of microvilli. To examine whether these cellular architecture changes are cytoskeleton driven, we analyzed actin cytoskeleton by fluorescence microscopy. 5-HT induced basal stress fibers with prominent cortical actin filaments via 5-HT3 and 5-HT4 receptor subtypes. This induction was partially attenuated by chelation of intracellular Ca(2+) and PKCalpha inhibition (Go6976). In vitro assays revealed that PKCalpha interacted with actin and this association was increased by 5-HT. Our data provide novel evidence that 5-HT-induced signaling via 5-HT3/4 receptor subtypes to cause Ca(2+) and PKCalpha-dependent regulation of actin cytoskeleton may play an important role in modulation of ion transporters that contribute to pathophysiology of diarrheal conditions associated with elevated levels of 5-HT.     

Toll-like receptor 4 mediates induction of the Bcl10-NFkappaB-interleukin-8 inflammatory pathway by carrageenan in human intestinal epithelial cells

The sulfated polysaccharide carrageenan (CGN) induces activation of NFkappaB and interleukin 8 (IL-8) in human colonic epithelial cells through a pathway of innate immunity mediated by Bcl10 (B-cell CLL/lymphoma 10). In this report, we identify Toll-like receptor 4 (TLR4), a member of the family of innate immune receptors, as the surface membrane receptor for CGN in human colonic epithelial cells. Experiments with fluorescence-tagged CGN demonstrated a marked reduction in binding of CGN to human intestinal epithelial cells and to RAW 264.7 mouse macrophages, following exposure to TLR4 blocking antibody (HTA-125). Binding of CGN to 10ScNCr/23 mouse macrophages, which are deficient in the genetic locus for TLR4, was absent. Additional experiments with TLR4 blocking antibody and TLR4 small interfering RNAs showed 80% reductions in CGN-induced increases in Bcl10 and IL-8. Transfection with dominant-negative MyD88 plasmid demonstrated MyD88 dependence of the CGN-TLR4-triggered increases in Bcl10 and IL-8. Therefore, these results indicate that CGN-induced inflammation in human colonocytes proceeds through a pathway of innate immunity, perhaps related to the unusual alpha-1,3-galactosidic linkage characteristic of CGN, and suggest how dietary CGN intake may contribute to human intestinal inflammation. Because CGN is a commonly used food additive in the Western diet, clarification of its effects and mechanisms of action are vital to issues of food safety.     

Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles

Thiamine, a water-soluble vitamin, is essential fornormal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety ofconditions. To date, however, little is known about the mechanism ofthiamine absorption in the native human small intestine. The objectiveof this study was, therefore, to characterize the mechanism of thiaminetransport across the brush-border membrane (BBM) of human smallintestine. With the use of purified BBM vesicles (BBMV) isolated fromthe jejunum of organ donors, thiamine uptake was found to be1) independent of Na⁺ but markedly stimulated byan outwardly directed H⁺ gradient (pH 5.5_in/pH7.5_out); 2) competitively inhibited by thecation transport inhibitor amiloride (inhibitor constant of 0.12 mM);3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structuralanalogs (amprolium, oxythiamine, and pyrithiamine), but not byunrelated organic cations (tetraethylammonium,N-methylnicotinamide, or choline); 5) notaffected by the addition of ATP to the inside and outside of the BBMV;6) potential insensitive; and 7) saturable as afunction of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 ± 0.08 µM and a maximal velocity of 1.00 ± 0.47 pmol · mg protein¹ · 10 s¹. Carrier-mediated thiamine uptake was also found inBBMV of human ileum. These data demonstrate the existence of aNa⁺-independent, pH-dependent, amiloride-sensitive,electroneutral carrier-mediated mechanism for thiamine absorption innative human small intestinal BBMV.

     

Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1

The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was Na(+) independent. Butyrate uptake was unaffected by DIDS; however, alpha-cyano-4-hydroxycinnamate and the butyrate analogs propionate and L-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells.