Action Of Bacillus Thuringiensis On The Intestinal Epithelial Junction Of Caenorhabditis Elegans

Bacillus thuringiensis(Bt)is a gram-positive soil bacterium,which can produce parasporal crystal proteins(Cry proteins)that are toxic to agricultural pests and nematodes.Bt is the most successful biological insecticide due to the existence of Cry proteins.So far,the mechanisms of Bt against nematodes are mainly focused on Cry proteins,which are the main virulence factors.Recently,more and more studies gradually acknowledge that Bt is a pathogenic bacterium for both insects and nematodes.Therefore,it is necessary for us to uncover virulence factors besides Cry proteins and unveil their pathogenic mechanisms.As a model organism,Caenorhabditis elegans has a clear genetic background and transparent body which are convenient for us to study the mechanism of pathogenic factors from Bt.The major results are described as follows:1)Excavation and functional verification of a novel pathogenic factor ColB from BtOur group recently found that the culture mixtures of Cry proteins and spores can invade the nematodes and complete their life cycle,leaving the undigested cuticle which serves as a bag to form the "Bob" or bag-of-bacteria phenotype.Here,we proposed spores could germinate in the intestinal lumen of the worms and then secreat some pathogenic factors to help invade the hosts.The transcriptional regulator PlcR is commonly known as a regulator of pathogenic factors in Bt.According to the PlcR box located upstream of regulated genes,we aquired 84 potential pathogenic factors form YBT-1518 through bioinformatics prediction,from which we chosed a metalloproteinase ColB to test its nematicidal activity.We found that ColB could express normly in Bt.Besides,ColB could be highly induced in vivo and purified ColB is toxic to nematodes.In addition,the presence of ColB is conducive to the colonization and germination of Bt in vivo.The conclusions above fully proved that ColB is a novel pathogenic factor which is regulated by PlcR regulon and plays an important role in Bt infecting C.elegans.2)Pathogenic factors disrupt intestinal epithelial junction to help Bt invade Caenorhabditis elegansIn order to better understand the mechanism of pathogenic factors in Bt,we speculated that the pathogenic factors could disrupt the intestinal epithelial junction of C.elegans according to the analysis of transcriptomics as well as the observation of the"Bobs" phenotype.We found that spores can accelerate the disruption of epithelial junction.Besides,deletion of plcR could slower the disruption of epithelial junctions significantly.Moreover,both PlcR regulated pathogenic factors,collagenase ColB and metalloproteinase Bmp1,were found capable of disrupting epithelial junctions directly and eventually leading to the disruption of the whole intestine.In a word,the results above indicates that pathogenic factors are able to disrupt epithelial junctions of intestinal cells in C.elegans to accelerate the invasion of B.thuringiensis,leading eventually to the death of the worms.Our findings uncover the mechanism that how pathogenic factors help Bt efficiently invade and kill nematode hosts.These mechanisms may provide theoretical basis for pathogenic bacteria of nematodes and present a novel model that Bt could taget the host epithelial junction of intestine cells,thus provide an important guidance for the biological control of plant-parasitic nematodes.