|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2003 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 2002 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Our previous studies showed that enterohemorrhagic E. coil (EHEC) has a potential to invade to normal human colon epithelial cells in vitro, which is dependent on the action of cytoskeleton of host cells. The present investigation was conducted to elucidate the bacterial proteins involved in the invasive potential. The trial with mutants, which were defective in various adhesion-related molecules such as intimin, tir or type III secretion systems, resulted in no significant reduction of invasion. An unintended mutant, however, of flagella-related gene showed a remarkable inability of invasion. The defect of flagella in this mutant was suggested by being not mobile and not reactive to 11-7 antibody, and was confirmed by the absence of flagellin protein on 2-D electrophoresis and TOF-MS analysis. A novel mutant with flagellin protein has now been constructed and under investigation.
Next, the effect of probiotic bacteria in normal flora on the invasive potential of EHEC was explored employing Lactobacillus species. Among tested, only L. rhamnosus showed a significant inhibition of EHEC invasion to epithelial cells. On the other hand, the adhesion and colonization of EHEC was not affected by any of the lactobacillus strains tested. Concerning the possible mechanisms, the viabilities of EHEC and host cell were not affected by the presence of L.rhamnosus. Simple competitions at certain receptors were unlikely because the suppressive effect on EHEC invasion was strictly dependent on viable L. rhamnosus and could not be observed with the conditioned medium or killed L.rhamnosus. The fact that L. rhamnosus showed outstanding adhering potential to the colon epithelial cell line, compared with other strains, suggested that an avid interaction of L.rhamnosus to the host cell might be modulating intracellular events responsible for the invasion of EHEC.