Study On The Mechanism Of Bacillus Subtilis Against Pathogens To Enter Host-Cell

Pathogens invade host cells through three routes:adhesion, activation of signal transduction and destruction of cytoskeletal structure. Bacillus subtilis has been used for a long time as probiotics for antagonistic effects on animals pathogens, knowledge concerning mechanism of its antagonistic is limited. The aim of our study was to study the mechanism of Bacillus subtilis against pathogens to invade host cells. The study included four parts as follows:1. The protection of Bacillus subtilis against the adherence and invasion of pathogens to Caco-2cellsThe test used viable bacteria counting method to evaluate inhibition of enterotoxic Escherichia coli and Salmonella typhimurium adhesion and invasion to Caco-2cells by incubation with Bacillus subtilis and supernatant. The results showed that a decrease in the number of adhering enterotoxic Escherichia coli and Salmonella typhimurium was observed, using either preincubation, postincubation or coincubation of the pathogens with Bacillus subtilis. In competition test, the supernatant could inhibit the adherence and invasion of pathogens. The test reveals that Bacillus subtilis inhibit the adhesion and invasion of pathogens by competing intestinal epithelial cell receptor.2. Effects of Bacillus subtilis against pathogens on actin filamentsThe actin filaments in the cells was dyed by FTTC-phalloidin and the changes of actin filaments was examined by inverted fluorescence microscope, The regulatory protein Racl of actin filaments was measured by Western Blotting. The results showed that the actin filaments of Caco-2cells in normal scattered as bundles in parallel; but the actin filaments aggregated in a small area when Salmonella typhimurium added; the actin filaments rearranged and assemlbed with radial or disorder distribution in the center of cell when enterotoxic Escherichia coli added; Little change occured in actin filaments when Bacillus subtilis added; The actin filaments of most cells scattered as bundles in parallel arrangement, and rearranged only in small amount of cells when two kinds of the pathogens and Bacillus subtilis or supernatant added together. The expression of regulatory protein Racl significantly increased when enterotoxic Escherichia coli or Salmonella typhimurium added to Caco-2cells. Little change occured in the expression of Racl when two kinds of the pathogens and Bacillus subtilis or supernatant added together to Caco-2cells. Our results revealed that enterotoxic Escherichia coli, Salmonella typhimurium could disturb the expression and distribution of actin filaments; while Bacillus subtilis and its supernatant could inhibit the destruction induced by pathogenic bacteria.3. The protection of the host MAPKs Signaling Pathways of Bacillus subtilis against pathogensThe transcription level of MAPK (ERK、p38and JNK) was measured by RT-PCR, the phosphorylation of MAPK was measured by Western Blotting. When enterotoxic Escherichia coli or Salmonella typhimurium added to Caco-2cells, no changes in the transcription level of ERK2、p38、JNK1. ERK、p38and JNK pathway were activated by Salmonella typhimurium, ERK and p38was activated by enterotoxic Escherichia coli. However, Bacillus subtilis inhibited the two kinds of pathogens induced activation of ERK、p38and JNK. Our results revealed that Bacillus subtilis might have effects on controlling infection caused by enterotoxic Escherichia coli and Salmonella typhimurium.4. Effects of Bacillus subtilis against Escherichia coli on pig intestinal epitheliulBacillus subtilis was given to newborn piglets at0,7,14,26days, enterotoxic Escherichia coli was given after weaning, take the duodenum, jejunum, ileum and colon. The results of HE staining showed that the intestinal epithelial cells of duodenum and jejunum lined up in order, the intestinal epithelial cells of ileum became thinness, the intestinal mucosa of colon was almost completely destroyed, extensive inflammation and necrosis when given enterotoxic Escherichia coli without Bacillus subtilis. When given Bacillus subtilis frist then given enterotoxic Escherichia coli, both the intestinal epithelial cells lined up in order and no inflammation. This test reveals that Bacillus subtilis could inhibit the destruction of intestinal epitheliul induced by enterotoxic Escherichia coli in piglets.