| Quorum Sensing(QS)is a signal system mediated by autoinducer(AI)in bacteria.This is the specific way of communication between bacteria,which produce the signal molecules and release them into the environment.When the concentration of signals reaches a threshold,the bacteria initiate the coordinated expression of some specific genes for new behavioral characteristics on the group scale,such as bioluminescence,regulation of virulence factors,spore or biofilm formation,and so on.In quorum sensing,previous sdudies focused on the influence of the signal molecules on bacteria itself or other bacteria.We further focus on the influence of bacterial signal molecules on host cells.This study aims at the direct effects of bacterial quorum sensing signal molecules 30C12-HSL on host cells and its role in the interaction between bacteria and host cells.We used IPEC-J2 cells as model cells for the following studies.1.Application of RNA-Seq technology for screening differentially expressed genes in IPEC-J2 stimulated by bacteria quorum-sensing signal 3OC12-HSLBacteria communicate in a population scale by the synthesis and absorption of quorum-sensing signals,thus regulating a variety of biological functions.For further exploring whether the bacteria quorum-sensing signals have a direct stimulating function on host eukaryotic cells,we selected Porcine Intestinal Epithelial Cell line IPEC-J2,used three important quorum-sensing signals from different bacteria,AI-2,C6-HSL and 30C12-HSL(final concentration 100?M),to stimulate the cells,and screened differentially expressed genes of IPEC-J2 by high-throughput RNA-Seq technology.There are 1 up-regulated and 7 down-regulated genes in group AI-2;2 up-regulated and 8 down-regulated genes in group C6-HSL;20 up-regulated and 15 down-regulated genes in group 3OC12-HSL.Obviously 30C12-HSL signals has most important effects,therefore the following analysis focused on 3OC12-HSL.The GO function classification and KEGG signal pathway analysis were used to analyze the differentially expressed genes.GO showed that these genes mainly related to immune system,structural molecule activity and cell junction;KEGG analysis showed that these differentially expressed genes involved in the pathways of cytoskeleton,pathogenic E.coli infection,cell adhesion molecule(CAM)and extracellular matrix(ECM)-receptor interaction.We validated 8 differentially expressed genes by qRT-PCR,7 of which were related to cytoskeleton/cell junction.It confirmed that the expression of each gene was consistent with transcriptional sequencing results.This study laid a foundation for further study on the role of bacterial quorum-sensing signals in the interaction between pathogen and host,direct stimulation to the host intestinal cells and its associated mechanisms.2.Effects of bacteria quorum sensing signal 30C12-HSL on IPEC-J2Intestinal epithelial cells regulate intracellular homeostasis and maintain intestinal epithelial cell defense ability through the communication by cell junction.Quorum sensing signals regulate the virulence of bacteria,as well as participate in the regulation of host cells to help pathogen pathogenesis,which the whole process is called interkingdom signaling.30C12-HSL had a significant effect on the morphology of IPEC-J2;MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide)results showed that 30C12-HSL had no effect on the activity of IPEC-J2 cells,indicating that the morphological changes were not from the toxicity;using different concentrations of 30C12-HSL to stimulate IPEC-J2 with different times,the results showed that cell morphological changes were positively correlated with signal concentration and interaction time;as low as 25?M could cause significant changes in cell morphology,verified the sensitivity of cells to the signal;the results of bacterial adhesion test showed that 30C12-HSL could enhance the adhesion of enteric pathogenic E.coli to host cell IPEC-J2;the bacterial invasion test results showed that F18ab E.coli invasion to host cells decreased;the results of q-RT-PCR and Western blot revealed that tight junction,rather than gap junction,associated with the mechanism of quorum sensing signal stimulation on intestinal epithelial cells,the expression of tight junction ZO-1 protein significantly increased. |
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