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Establishment of Ex Vivo Model Used to Evaluate the Lactobacillus Probiotic Effect and Study of the Lactobacillus Adhesion Mechanism

Author: RenPengFei
Tutor: ZhouZhiGang
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: Intestinal sac Adhesion Lactobacillus Pili protein Toll-like receptor
CLC: Q939.91
Type: Master's thesis
Year: 2013
Downloads: 119
Quote: 0
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Abstract


The adhesion is the first step that microorganism colonizes in the gut of host, which is required forthe probiotics exert their effects maintained in the gut for longe time. Therefore, the adhesive capacity isone of important factors to choose the probiotics from lactic acid bacteria. In aquaculture, the adhesivecapacity of lactic acid bacteria is more important.In order to screen the probiotics for aquaculture in a quick and cheap way, an ex vivo intestinal sacmodel is a good choice before fish animal experiments. Tilapia, a species of warm-water fish, is one ofmain products in Chinese aquacultural industry. Here we established the ex vivo intestinal sac model ofhybrid tilapia (Oreochromis niloticus♀×Oreochromis aureus♂). L. plantarum subsp. JCM1149Tand A.hydrophila NJ-1both could adhere intestine sac through the PCR-DGGE and Scanning electronmicroscopy (SEM) analysis. Besides, the SEM analysis showed the intact intestinal sac. Considering thestable expression of the β-actin gene in the intestinal sac, it demonstrated that the activity of theintestinal sac was maintained. Therefore, the establishment of the intestinal sac model was confirmedsuccessfully, which was the first trial in warm-fish and important for the feasibility as ex vivo intestinalsac model.Our previous work had demonstrated that L. plantarum subsp. JCM1149Tcould colonize in the gutof hybrid tilapia. Based the intestinal sac model, it was studied whether L. plantarum subsp. JCM1149Tadhere the intestinal sac and what would happen to the local immune of the intestinal sac. Hybrid tilapiawere sacrificed to isolate anterior and posterior intestine for incubation with PBS solution only as thecontrol, L. plantarum subsp. JCM1149Tat log9CFU/mL in PBS, A. hydrophila NJ-1at log8CFU/mLin PBS, or the both combination. PCR-DGGE fingerprint and consequent sequence analysis confirmedanterior intestine sac were shown prone to the colonization of L. plantarum subsp. JCM1149Tand A.hydrophila NJ-1than the posterior part. L. plantarum subsp. JCM1149Tand A. hydrophila NJ-1inhibited the population each other in anterior or posterior sac, indicating their competition for thecolonization. The induced expression of Heat shocking protein70(HSP70), Interlukin1β (IL-1β) andTumor nacrosis α (TNF-α) in the anterior sac by the addition L. plantarum subsp. JCM1149Tor A.hydrophila NJ-1demonstrated the activity and a local immune response of ex vivo anterior sac.Compared with posterior intestine, higher population colonization and more sensitive immune responseof anterior sac indicated differential patterns for the probiotic-pathogen-host interactions. SEM analysisshowed that pathogen A. hydrophila NJ-1damaged the anterior intestine, which was alleviated by thepretreatment of L. plantarum subsp. JCM1149T, showing its beneficial effects through competitativeconolization against pathogens and activating the local intestinal immune response.For further investigate the adhesion effects of lactic acid bacteria, we used the biofilm formationand the spa pili gene to evaluate L. rhamnosus CICC6141, L. acidophilus JCM1132T, L. caseiCGMCC1.2435, L. casei BL23, L. plantarum subsp. JCM1149T, L. rhamnosus20300preserved by ourlaboratory. Compared with the other lactic acid bacteria in our laboratory, L. rhamnosus CICC6141 could form the reticular biofilm indicating this strain had the stronger ability of biofilm formation. Inaddition, the pili protein genes, spaABC were expressed in L. rhamnosus CICC6141..Considering thepresent results and our previous work, the stronge adhesion of L. rhamnosus CICC6141in zebra fishintestine and its beneficial effects possibly correlated with pili proteins.To confirm this, the pili protein genes, including spaA, B and C, which was closely related with theadhesion of L. rhamnosus CICC6141, was cloned form the genome of L. rhamnosus CICC6141. Afterexpression and purification of the pili proteins, we investigated the interaction between the spaA, B Cand ZF4cell line. Compared with BSA, spaC, other than spa and spaB, alleviated the apoptosis inducedby A. hydrophila NJ-1, indicating the specific role of spaC. the addition of spaC protein induced theexpression of Toll-like receptor8.2(TLR8.2), NFκB and TNFα, but not myd88in ZF4cell line,indicating the enhanced immune response to spaC through TLR8.2/NFκB pathway, independent ofmyd88. These data demonstrated that the interaction of probiotic and host was mediated by pili proteinspaC, which is important in the field. But the further study to explore the molecular mechanisms forhost and pili protein spaC interaction is still under investigation.

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