Up-Regulation of MUC2 and IL-1β Expression in Human Colonic Epithelial Cells by Shigella and Its Interaction with Mucins

BACKGROUND: The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis. METHODS: The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence. RESULTS: The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucin<guinea pig colonic mucin< Human colonic mucin. Invasion of Shigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells. CONCLUSIONS: Our study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells

Apoptosis Inducing Effect of Plumbagin on Colonic Cancer Cells Depends on Expression of COX-2

Plumbagin, a quinonoid found in the plants of the Plumbaginaceae, possesses medicinal properties. In this study we investigated the anti-proliferative and apoptotic activity of plumbagin by using two human colonic cancer cell lines, HT29 and HCT15. IC50 of Plumbagin for HCT15 and HT29 cells (22.5 µM and 62.5 µM, respectively) were significantly different. To study the response of cancer cells during treatment strategies, cells were treated with two different concentrations, 15 µM, 30 µM for HCT15 and 50 µM, 75 µM for HT29 cells. Though activation of NFκB, Caspases-3, elevated levels of TNF-α, cytosolic Cytochrome C were seen in both HCT15 cells HT29 treated with plumbagin, aberrant apoptosis with decreased level of pEGFR, pAkt, pGsk-3β, PCNA and Cyclin D1was observed only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells. This suggests that plumbagin induces apoptosis in both HCT15 cells and HT29 treated, whereas, proliferation was inhibited only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells, but not in 50 µM plumbagin treated HT29 cells. Expression of COX-2 was decreased in 75 µM plumbagin treated HT29 cells when compared to 50 µM plumbagin treated HT29 cells, whereas HCT15 cells lack COX. Hence the observed resistance to induction of apoptosis in 50 µM plumbagin treated HT29 cells are attributed to the expression of COX-2. In conclusion, plumbagin induces apoptosis in colonic cancer cells through TNF-α mediated pathway depending on expression of COX-2 expression

Isolation and Partial Characterisation of a Novel Lectin from Aegle marmelos Fruit and Its Effect on Adherence and Invasion of Shigellae to HT29 Cells

Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 µg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host

The binding parameters of <i>S. dysenteriae</i> and <i>S. flexneri</i> to mucins from different species.

<p>Microtiter plates were coated with a fixed concentration of mucin (12.5 µg for human; 50 µg for guinea pig). 100 µl of bacteria (∼1×10¹⁰ cfu/well) was added. The values are the mean ± S.D triplicate determinations of one representative experiment. (PGM, Pig gastric mucin).</p

RT-PCR analysis of MUC2 and IL-1β expression in <i>Shigella dysenteriae</i> infected HT 29 cells.

<p>HT 29 cells were infected with <i>Shigella dysenteriae</i> for different time intervals. After infection, mRNA was isolated for MUC2 and IL-1β gene expression analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027046#s2" target="_blank">methods</a>. HT-29 control cells showed basal level expression of both MUC2 and IL-1β. Whereas, higher level (intensity) expression of both MUC2 (∼4–5 fold higher for 9 h) and IL-1β (25 fold higher for 9 h) were seen in a time dependent manner upon HT-29 cells infected with <i>S. dysenteriae</i>. HT 29 cells pre-incubated with Actinomycin D (1 µg/ml) for 30 min, prior to <i>S. dysenteriae</i> infection, showed lower level of expression of IL-1β. The band intensity was measured by using image J program. Amplification of β-actin was used as an internal control.</p

Binding of <i>Shigella dysenteriae</i> to mucins.

<p>a. Binding of <i>Shigella dysenteriae</i> to varying concentrations of different mucins. b. Binding of different amount of <i>Shigella dysenteriae</i> to human and guinea pig mucins. Each value is the mean of triplicate determinations. The assay was carried out as described in materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027046#s2" target="_blank">methods</a>.</p

Adherence of <i>S. dysenteriae</i> and <i>S. flexneri</i> to sodium metaperiodate treated mucins.

<p><i>S. dysenteriae</i> (a) and <i>S. flexneri</i> (b) showed significantly reduced adherence to sodium metaperiodate treated mucins in a concentration dependent manner when compared with untreated mucins. Each value is the mean of triplicate determinations. A p value of less than .05 is considered to be significant. Each comparison was made on bacterial adherence to untreated versus treated mucins.</p

Congo-red binding assay.

<p><i>Shigella</i> species were grown in Congo-red agar plate. Orange-pink colour colonies of <i>Shigella dysenteriae</i> (a) and <i>Shigella flexneri</i> (b) indicate the highly virulent nature and plasmid DNA cured non-virulent <i>Shigella flexneri</i> (c) shows white colour colonies.</p

<i>Shigella dysenteriae</i> adherence and invasion of human epithelial cells by Transmission Electron Microscopy.

<p>After 1 h and 2 h of infection, cells were fixed and processed for Transmission Electron Microscopy analysis as described in materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027046#s2" target="_blank">methods</a>. Control cells showed intact nuclei and other cell organelles (a). 1 h of infection of HT 29 cells with <i>Shigellae</i> showed the adherence and pseudopod projection formation (b). Endocytic processes and phagocytosis of bacteria was seen in the vacuoles of HT 29 cells at the end of 2 h infection (c). The scale bars indicate 1 µm. [M – Mucin; A – Adherence; P – Pseudopod formation; I - Invasion].</p