• Journal of Microbiology and Biotechnology
• /
• v.27 no.4
• /
• pp.694-700
• /
• 2017

Clostridium difficile, which causes pseudomembranous colitis, releases toxin A and toxin B. These toxins are considered to be the main causative agents for the disease pathogenesis, and their expression is associated with a marked increase of apoptosis in mucosal epithelial cells. Colonic epithelial cells are believed to form a physical barrier between the lumen and the submucosa, and abnormally increased mucosal epithelial cell apoptosis is considered to be an initial step in gut inflammation responses. Therefore, one approach to treating pseudomembranous colitis would be to develop agents that block the mucosal epithelial cell apoptosis caused by toxin A, thus restoring barrier function and curing inflammatory responses in the gut. We recently isolated an antimicrobial peptide, Periplanetasin-2 (Peri-2, YPCKLNLKLGKVPFH) from the American cockroach, whose extracts have shown great potential for clinical use. Here, we assessed whether Peri-2 could inhibit the cell toxicity and inflammation caused by C. difficile toxin A. Indeed, in human colonocyte HT29 cells, Peri-2 inhibited the toxin A-induced decrease in cell proliferation and ameliorated the cell apoptosis induced by this toxin. Moreover, in the toxin A-induced mouse enteritis model, Peri-2 blocked the mucosal disruption and inflammatory response caused by toxin A. These results suggest that the American cockroach peptide Peri-2 could be a possible drug candidate for addressing the pseudomembranous colitis caused by C. difficile toxin A.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.1
• /
• pp.50-57
• /
• 2012

Phospholipase C-${\gamma}l$ (PLC-${\gamma}l$) expression is associated with cellular transformation. Notably, PLC-${\gamma}$ is up-regulated in colorectal cancer tissue and breast carcinoma. Because exotoxins released by Clostridium botulinum have been shown to induce apoptosis and promote growth arrest in various cancer cell lines, we examined here the potential of Clostridium difficile toxin A to selectively induce apoptosis in cells transformed by PLC-${\gamma}l$ overexpression. We found that PLC-${\gamma}l$-transformed cells, but not vector-transformed (control) cells, were highly sensitive to C. difficile toxin A-induced apoptosis and mitotic inhibition. Moreover, expression of the proapoptotic Bcl2 family member, Bim, and activation of caspase-3 were significantly up-regulated by toxin A in PLC-${\gamma}l$-transformed cells. Toxin A-induced cell rounding and paxillin dephosphorylation were also significantly higher in PLC-${\gamma}l$-transformed cells than in control cells. These findings suggest that C. difficile toxin A may have potential as an anticancer agent against colorectal cancers and breast carcinomas in which PLC-${\gamma}l$ is highly up-regulated.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.39 no.11
• /
• pp.1587-1594
• /
• 2010

This study was designed to investigate the effect of Artemisia capillaris extracts on the intestinal microflora. In agar diffusion method, the solvent fractions of Artemisia capillaris showed growth inhibition against the intestinal microflora. In particular, the chloroform fraction of Artemisia capillaris had strong antibacterial activity against Clostridium perfringens, Clostridium difficile, Eubacterium limosum, and Bacteroides fragilis, but did not show antibacterial activity against Bifidobacterium bifidum and Lactobacillus acidophilus. Most chloroform fraction of Artemisia capillaris inhibitory activities were not reduced by heat treatment or pH variation against C. perfringens, C. difficile, E. limosum, and B. fragilis. MICs of the chloroform fraction were 1.25 mg/mL against C. perfringens, E. limosum and B. fragilis and 2.5 mg/mL against C. difficile. MBCs of chloroform fraction were 5 mg/mL against C. perfringens, E. limosum and 2.5 mg/mL against C. difficile, B. fragilis. The ethyl acetate fraction of Artemisia capillaris showed $3.08{\pm}0.03$ mg/10 mg total polyphenol and $1.91{\pm}0.03$ mg/10 mg total flavonoid contents. In vivo tests were performed to investigate the influence of Artemisia capillaris extract on the intestinal microflora in rats. The results showed the possibilities of utilizing Artemisia capillaris extracts as a functional food component to control intestinal microflora.

• Journal of Life Science
• /
• v.28 no.8
• /
• pp.885-891
• /
• 2018

Clostridium difficile (C. difficile) toxin A is known to cause acute gut inflammation in humans and animals by triggering cytoskeletal disorganization in gut epithelial cells. In human colonocytes, toxin A blocks microtubule assembly by directly increasing the enzymatic activity of histone deacetylase-6 (HDAC-6), a tubulin-specific deacetylase, thereby markedly decreasing tubulin acetylation, which is essential for microtubule assembly. Microtubule assembly dysfunction-associated alterations (i.e., toxin A-exposed gut epithelial cells) are believed to trigger barrier dysfunction and gut inflammation downstream. We recently showed that potassium acetate blocked toxin A-induced microtubule disassembly by inhibiting HDAC-6. Herein, we tested whether acetic acid (AA), another small acetyl residue-containing agent, could block toxin A-induced tubulin deacetylation and subsequent microtubule assembly. Our results revealed that AA treatment increased tubulin acetylation and enhanced microtubule assembly in an HT29 human colonocyte cell line. AA also clearly increased tubulin acetylation in murine colonic explants. Interestingly, the AA treatment also alleviated toxin A-induced tubulin deacetylation and microtubule disassembly, and MTT assays revealed that AA reduced toxin A-induced cell toxicity. Collectively, these results suggest that AA can block the ability of toxin A to cause microtubule disassembly-triggered cytoskeletal disorganization by blocking toxin A-mediated deacetylation of tubulin.

• Pediatric Gastroenterology, Hepatology & Nutrition
• /
• v.15 no.1
• /
• pp.29-37
• /
• 2012

Purpose: To examine the prevalence of $Clostridium$ $difficile$ ($C.$ $difficile$) colonization (CDC) and potential neonatal determinants of CDC in hospitalized preterm infants. Methods: Fecal samples were serially collected within 72 h after birth and at 1, 2, and 4-6 weeks of age from preterm infants in the neonatal intensive care units (NICUs) of two different university hospitals. Total bacterial DNA was extracted from each fecal sample from 49 infants, and polymerase chain reaction (PCR) was performed with primers for the 16S gene of $C.$ $difficile$ and the toxin A and toxin B genes. The correlation between the results of $C.$ $difficile$ PCR assays and the clinical characteristics of the infants was analyzed. Results: The prevalence rates of CDC were 34.7, 37.2, 41.3, and 53.1% within 72 h after birth and at 1, 2, and 4.6 weeks of age, respectively. The toxin positivity rate was significantly higher in the infants with persistent CDC than in those with transient CDC (8/12 [66.7%] vs. 6/25 [24.5%] ($p$=0.001). Among the various neonatal factors, only the feeding method during the first week after birth was significantly associated with persistent CDC. Exclusive breast-milk feeding (EBMF) significantly decreased the risk of persistent CDC compared to formula or mixed feeding (adjusted odds ratio: 0.133, 95% confidence interval: 0.02-0.898, $p$=0.038). Conclusion: The prevalence of CDC increased with the duration of hospitalization in preterm infants in the NICU. EBMF during the first week after birth in hospitalized preterm infants may protect against persistent CDC.

• YAKHAK HOEJI
• /
• v.40 no.3
• /
• pp.343-346
• /
• 1996

The influence of LB20304a, a new fluoroquinolone antibiotic agent, on microflora of caecum in mice was compared with those of ciprofloxacin and piperacillin after administration of drugs for 5 days. Selective medium (CCFMA) was used for the isolation of Clostridium difficile from the specimens of mouse caecum. The emergence of C. difficile in mouse caecum induced by LB20304a was lower than that by ciprofloxacin or piperacillin at day 1 and day 7 after completing administration of drugs.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.4
• /
• pp.693-699
• /
• 2016

Clostridium difficile toxin A is known to cause deacetylation of tubulin proteins, which blocks microtubule formation and triggers barrier dysfunction in the gut. Based on our previous finding that the Clostridium difficile toxin A-dependent activation of histone deacetylase 6 (HDAC-6) is responsible for tubulin deacetylation and subsequent microtubule disassembly, we herein examined the possible effect of potassium acetate (PA; whose acetyl group prevents the binding of tubulin to HDAC-6) as a competitive/false substrate. Our results revealed that PA inhibited toxin A-induced deacetylation of tubulin and recovered toxin A-induced microtubule disassembly. In addition, PA treatment significantly decreased the production of IL-6 (a marker of inflamed tissue) in the toxin A-induced mouse enteritis model. An in vitro HDAC assay revealed that PA directly inhibited HDAC-6-mediated tubulin deacetylation, indicating that PA acted as a false substrate for HDAC-6. These results collectively indicate that PA treatment inhibits HDAC-6, thereby reducing the cytotoxicity and inflammatory responses caused by C. difficile toxin A.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.45 no.7
• /
• pp.1026-1034
• /
• 2016

To develop a natural antimicrobial agent, we investigated the antimicrobial activities of 13 species of edible herbal plant extracts against major Gram-positive foodborne bacteria. Among the 13 screened edible herbal plants, Caesalpinia sappan L. showed the highest antimicrobial activity. In the paper disc agar diffusion assay, Caesalpinia sappan L. extracts had strong antibacterial activities against most Gram-positive bacteria but did not have antibacterial activities against most Gram-negative bacteria. Minimum inhibitory concentrations of the ethanol extract were 0.06 mg/mL against Clostridium difficile and Listeria monocytogenes and 0.03 mg/mL against Staphylococcus aureus. Their inhibitory activities were not reduced by heat treatment or pH adjustment against C. difficile, L. monocytogenes, and S. aureus. Antimicrobial activities were higher in ethanol extract than in distilled water extract. These results support the potential use of Caesalpinia sappan L. ethanol extract as an antimicrobial agent or functional food components against Gram-positive bacteria.

• Journal of Life Science
• /
• v.28 no.9
• /
• pp.1016-1021
• /
• 2018

Clostridium difficile toxin A causes pseudomembranous colitis. The pathogenesis of toxin A-induced colonic inflammation includes toxin A-dependent epithelial cell apoptosis, resulting in the loss of barrier function provided by epithelial cells against luminal pathogens. Toxin A-dependent epithelial cell apoptosis has been linked to toxin A-induced production of reaction oxygen species and subsequent p38MAPK activation; $p21^{CIP1/WAF1}$ upregulation-dependent cell cycle arrest; cytoskeletal disaggregation; and/or the induction of Fas ligand on epithelial cells. However, the molecular mechanisms underlying toxin A-induced apoptosis remain poorly understood. This study tested whether toxin A could block the Wnt signaling pathway, which is involved in gut epithelial cell proliferation, differentiation and antiapoptotic progression. Toxin A treatment of nontransformed human colonocytes (NCM460) rapidly reduced ${\beta}$-catenin protein, an essential component of the Wnt signaling pathway. Exposure of mouse ileum to toxin A also significantly reduced ${\beta}$-catenin protein levels. MG132 inhibition of proteasome-dependent protein degradation resulted in the recovery of toxin A-mediated reduction of ${\beta}$-catenin, indicating that toxin A may activate intracellular processes, such as $GSK3{\beta}$, to promote degradation of ${\beta}$-catenin. Immunoblot analysis showed that toxin A increased active phosphorylation of $GSK3{\beta}$. Because the Wnt signaling pathway is essential for gut epithelial cell proliferation and anti-apoptotic processes, our results suggest that toxin A-mediated inhibition of the Wnt signaling pathway may be required for maximal toxin A-induced apoptosis of gut epithelial cells.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.8
• /
• pp.1446-1451
• /
• 2016

Clostridium difficile toxin A causes acute gut inflammation in animals and humans. It is known to downregulate the tight junctions between colonic epithelial cells, allowing luminal contents to access body tissues and trigger acute immune responses. However, it is not yet known whether this loss of the barrier function is a critical factor in the progression of toxin A-induced pseudomembranous colitis. We previously showed that NADH:quinone oxidoreductase 1 (NQO1) KO (knockout) mice spontaneously display weak gut inflammation and a marked loss of colonic epithelial tight junctions. Moreover, NQO1 KO mice exhibited highly increased inflammatory responses compared with NQO1 WT (wild-type) control mice when subjected to DSS-induced experimental colitis. Here, we tested whether toxin A could also trigger more severe inflammatory responses in NQO1 KO mice compared with NQO1 WT mice. Indeed, our results show that C. difficile toxin A-mediated enteritis is significantly enhanced in NQO1 KO mice compared with NQO1 WT mice. The levels of fluid secretion, villus disruption, and epithelial cell apoptosis were also higher in toxin A-treated NQO1 KO mice compared with WT mice. The previous and present results collectively show that NQO1 is involved in the formation of tight junctions in the small intestine, and that defects in NQO1 enhance C. difficile toxin A-induced acute inflammatory responses, presumably via the loss of epithelial cell tight junctions.