The Role And Mechanism Of Host Derived Novel Pore-forming Toxin-like Protein LIN-24 In Stress Resistance Of Caenorhabditis Elegans

More than 70% of pathogenic bacteria in nature contain pore-forming toxins(PFTs),which account for about 30% of the total virulence proteins of bacteria.Pore-forming toxin like proteins(PFPs),as non-classical membrane proteins,can oligomerize to form pores on the cell membrane,and influence the cell membrane function and internal and external environment.Pore-forming toxin-like proteins are widely distributed in nature,not only in toxic hosts,but also in some non-toxic hosts.At present,the research on pore toxin-like proteins mainly focuses on enhancing immune response.For example,previous studies have shown that βγ-CAT from the skin secretions of the amphibian Bombina Maxima,when it enters the lysosome,leads to activation of NLRP3,maturation and release of IL-1β,which stimulates an immune response and helps the host to clear pathogens.However,it is not clear whether the pore-forming toxin-like proteins have a broader physiological function other than involvement in immune responses.Compared with other model organisms,Caenorhabditis elegans is characterized by simple culture,short development time,clear genetic background,and easy genetic manipulation.LIN-24 is a pore-forming toxin-like protein derived from Caenorhabditis elegans,and its specific physiological function remains unclear.Using Caenorhabditis elegans as a model,we studied the effects of LIN-24 on nematodes,including lifespan,infection resistance and metabolic phenotypes,through omics analysis,genetic manipulation and functional analysis.The main results are as follows:1.The lin-24 intestinal specific overexpression plasmid constructed in vitro was injected into the gonads of N2 nematodes,and the lin-24 intestinal specific overexpression nematode strain was obtained,which was named Lin-24-OE.Nematodes were fed with lin-24 RNA interfering bacteria to construct nematodes model with lin-24 knockdown.The results showed that knockdown of lin-24 and overexpression of lin-24 had no significant effect on the normal lifespan of N2 nematodes.2.Transcriptome sequencing was performed on lin-24 knockdown nematodes,and GO annotation analysis and functional enrichment analysis were performed on these differential genes,which were found to be mainly enriched in immune defense,lipid metabolism and stress response.3.In the experiment of Pseudomonas aeruginosa infection,knocking down lin-24 shortened the infection lifespan of N2 nematodes.However,overexpression of lin-24 can prolong the infection lifespan of nematodes and significantly reduce the number of bacteria in nematodes after infection.By knocking down lin-24 on Lin-24-OE nematodes,the original resistance of the nematode to bacterial infection was abolished.4.DAF-16/FOXO signaling pathway,SKN-1 signaling pathway,DOD-22 signaling pathway and PMK-1 signaling pathway were involved in the process of LIN-24 enhancing nematodes resistance to bacterial infection,and this process does not depend on ZIP-2 signaling pathway.5.LIN-24 can affect nematode growth and development,such as delaying the oviposition cycle of nematode.Lin-24 can also ameliorate the age-related decline of locomotion and physiological function of nematodes and greatly prolong the starvation life of nematodes.Our study found that LIN-24 helps nematodes resist bacterial infection by regulating the Daf-16/FOXO signaling pathway,SKN-1 signaling pathway,DOD-22 signaling pathway and PMK-1 signaling pathway,thus prolongating the infection lifespan of nematodes.LIN-24 can delay growth and development and regulate metabolism by ameliorating age-related motor physiological decline and prolonging lifespan of nematodes under stress conditions such as starvation.These results provide a theoretical basis for other host-derived PFPs to resist stress and regulate metabolism.