Metalloproteinase-like MNP-1 Participates In Caenorhabditis Elegans Defensing Against Cry5Ba Toxin

Bacillus thuringiensis can produce parasporal crystal proteins(Cry)which have high toxicity to insects and nematodes.So far,the mechanism of Cry proteins killing insects is clear.However,the mechanism of Cry proteins killing nematodes is unclear.It has been reported that the glycolipids are receptor of Cry5Ba in C.elegans.Recently,we proved that cadherins are also the receptors of Cry5Ba in C.elegans,which like the pore-forming model in insects.It is very interesting that,when we test whether aminopeptidase N(APN,the secondly receptor of Cry in insets model)is receptor of Cry5Ba in C.elegans,we accidentally found a mutant RB1884 are significant susceptible to Cry5Ba.Hence we speculate MNP-1 maybe involve in C.elegans defence Cry5Ba toxin Considering that MNP-1 has not been reported associate with the host defense,there may be a novel defense mechanism mediate by MNP-1.Therefore,we reseach the pathway and regulation of MNP-1 mediated host defense and got the following conclusion:1.MNP-1 regulates insulin-like signaling pathway to defend PFTs in C.elegansWe found the mutant RB1884(mnp-1)also has allergic phenotypes to other nematicides Cry proteins,such as Cry6Aa,Cry14Aa and Cry21Aa,but no allergic phenotype for hydrogen peroxide and copper sulfate.What's more,mnp-1 gene could be induced by Cry protein,hence we speculate MNP-1 may specificity response to defense Pore Forming Toxins(PFTs)in C.elegans.We then used microarray to anlysis how MNP-1 participates in C.elegans to defense PFTs,we found many defense related effectors such as lysozymes,antimicrobial peptides upregulated significantly.Among these genes,more than 122 defense related effctors has positive correlation with MNP-1,indicating these effectors are regulated by MNP-1.It is known that defense related effectors are regulated by innate immune signaling pathway.We further confirmed that MNP-1 paticipates in regulated insulin-like pathway by qRT-PCR,bioassay and report nematodes,the detailed regulation way is:MNP-1 locates in the upstream of DAF-2 and negative regulates the DAF-2;MNP-1 positive regulates DAF-2 antagonist neuropeptide INS-1;Meanwhile,MNP-1 promotes the localization of DAF-16 in the nuclear,which could active the exression of downstream defense related effctors to defense Cry5Ba.2.MNP-1 regulates proteinase defense effectors to help host degrade Cry5Ba toxinAlthough several innate immune signaling pathways have been reported involved in host defense PFTs,but there have no reports about how the host degrades and removes PFTs.The enrichment GO analysis by microarray data found that a large number of differentially expressed genes related to protein hydrolysis and ubiquitination.These biological processes are associated with protein degradation;Meanwhile,there are 11 defense related effectors that regulated by MNP-1 are proteinase.Therefore,we suppose that MNP-1 may control the expression of proteinase to help host degrades Cry5Ba toxins.We then used bioassay of mutants and RNAi proved that the proteinase participates in C.elegans defensing Cry5Ba;we also proved that 7 of 11 proteinase effectors regulated by DAF-2/DAF-16 signaling pathways;Further,the protein degradation experiments nematodes showed that the crude protease extracts from mnp-1 and insulin-like signaling pathway mutants have less degradation capability against Cry5Ba than that of wild type N2;what's more,the crude protease extracts from protease gene mutants F19C6.4 and R03G8.4 also showed less degradation capability against Cry5Ba than that of wild type N2,Cry5Ba degradation relative to the wild type N2,which confirmed that these protease involved in C.elegans degrading the Cry5Ba toxin.3.MNP-1 regulates multiple innate immune signaling pathways and involved in C.elegans defense pathogens.We have proved that MNP-1 could regulate DAF-2/DAF-16 signaling pathway to defense Cry5Ba,here we verified that the MNP-1 could also regulated other innate immune signaling pathways,such as p38 MAPK and JNK MAPK signaling pathway,in C.elegans to defense Cry5Ba.What's more,we demonstrated that the MNP-1 was also involved in C.elegans defense the invasion of pathogenic bacteria,such as Serratia marcescens and Enterococcus faecalis etc.Because mnp-1 mutant animals are more sensitivity to these pathogenic bacteria compread with that of wild type N2.Finally,we found MNP-1 homologous are conserved exist in nematode and insect species,and also widely found in from Fungus to humanbeing,indicating MNP-1 maybe a conservative defense related protein.Our works revealed MNP-1 is a novel defense related protein in C.elegans,which could regulate multiple innate immune signaling pathways,such as DAF-2/DAF-16,p38 MAPK and JNK MAPK,to participate in host defensing PFTs and pathogen invasion.Meanwhile,we proposeed a novel defense mechanism that MNP-1 regulated defense proteinase expression in C.elegans to help the host degrade and eliminate Cry5Ba toxin.This mechanism may also common in nematodes and even higher animals,which provides a new perspective of toxins-or pathogen-host interaction.