Mechanism Of Caenorhabditis Elegans Scavenger Receptor SCAV-5 Involved In Innate Immune Responses

The innate immune system is the first line of defense against pathogens such as gram-positive bacteria,gram-negative bacteria,fungi,and viruses.The components of innate immunity include external physical barrier skin(dermis)and mucous membranes,internal humoral and cellular effector mechanisms.C.elegans is maintained with Escherichia coli,which has advantages of easy cultivation,short life cycle,transparent body structure,and simple genetic manipulation,etc.Its innate immune response signaling pathway is highly conserved in evolution.C.elegans can defend against pathogenic bacteria through molecular and behavioral mechanisms,such as changing the expression of anti-pathogenic response genes and proteins,escaping from pathogenic bacteria,reducing the consumption of pathogenic bacteria,etc.These responses can effectively defend against pathogenic bacteria,increase the survival rate and extend lifespan,which is of great significance for its survival.Scavenger receptors,as a significant kind of intracellular receptor proteins,play an important role in lipid metabolism and innate immune response.In mammals,the scavenger type B receptor(SR-B)as a pattern recognition receptor recognizes the conserved microbial structures lipopolysaccharide and lipoteichoic acid,which promotes the phagocytosis of bacteria and activates p38 MAPK immune signaling pathway.Scavenger receptor SCAV-1-6 in C.elegans are homologous to SR-B in mammals,which mechanisms involved in the innate immune response have not been reported.Thus,this study investigated the effects of SCAV-1-6 on innate immunity response,results as follows:1.All members of the SCAV family except SCAV-3 are mainly expressed in the intestinal tissue in C.elegans.scav-1-6 transgenic C.elegans were generated by microinjection.Transgenic C.elegans displayed that SCAV-3 is expressed in the entire body tissue and SCAV-1,SCAV-2,SCAV-4,SCAV-5,SCAV-6 are mainly expressed in the intestinal tissue.2.scav-5 mutants can effectively defend against pathogenic bacteria.Wild type of scav-1,scav-4,scav-6 RNAi and scav-2,scav-3,scav-5 mutants were fed with E.coli OP50 or S.typhimurium SL1344,separately.The results showed that scav-5 mutants can effectively defend against SL1344,which displayed a significant extension of mean(30.06%)and maximum(15.54%)lifespan compared to wild type.scav-5 mutants also can effectively defend against P.aeruginosa PA14,which displayed extension of mean(21.47%)and maximum(11.88%)lifespan compared to wild type.Transgenic expression of SCAV-5 driven by its own promoter reduced the lifespan extension phenotype in scav-5 mutants,confirming that loss of SCAV-5 was responsible for defense against pathogenic bacteria.3.scav-5 mutants can effectively resist SL1344 by dietary restriction.To quantify bacterial avoidance behavior,scav-5 mutants were scored the number of animals inside and outside the bacterial lawn from L1 to young adult stages on SL1344.Similar to wild type,scav-5 mutants tested on SL1344 did not display avoidance behavior.The rate of pharyngeal pumping be analyzed for scav-5 mutants on SL1344.scav-5 mutants cultured with SL1344 displayed lower rates of pharyngeal pumping on days 6-8 after L4 compared to wild type,indicating that scav-5 mutants reduced bacterial ingestion and had dietary restrictions on SL1344.The levels of PMK-1 phosphorylation in scav-5 mutants cultured on OP50 or SL1344 were significantly downregulated compared to wild type,which were detected by Western blot,respectively.Analyzing expression of pathogenic response genes clec-7,clec-60 clec-82,lys-5,and F53A9.8 by qRT-PCR.The expression of pathogen response genes clec-7,clec-60 clec-82,lys-5,and F53A9.8 were lower in scav-5 mutants compared to wild type when cultured on OP50.The expression of pathogen response genes clec-7,clec-82,F53A9.8 were lower in scav-5 mutants compared to wild type when cultured on SL 1344.These results implied that PMK-1 signaling pathways did not activate in scav-5 mutants under SL1344 infection.4.scav-5 mutants can effectively resist PA14 through activation of its innate immune response pathway.scav-5 mutants were scored the number of animals inside and outside the bacterial lawn from L1 to young adult stages on PA14.Similar to wild type,scav-5 mutants tested on PA 14 displayed avoidance behavior.The rate of pharyngeal pumping be analyzed for scav-5 mutants on PA 14.scav-5 mutants cultured with PA14 displayed higher rates of pharyngeal pumping on hours 48 h-72 h after L4 compared to wild type,indicating that scav-5 mutants increased bacterial ingestion and had no dietary restrictions on PA14.The levels of PMK-1 phosphorylation in scav-5 mutants cultured on PA14 were significantly upregulated compared to wild type,which were detected by Western blot.Analyzing expression of pathogenic response genes clec-7,clec-60 clec-82,lys-5,and F53A9.8 by qRT-PCR.The expression of pathogen response genes clec-60,clec-82,F53A9.8 were higher in scav-5 mutants compared to wild type when cultured on PA14.These suggest that PMK-1 signaling pathways are activated in scav-5 mutants under PA 14 infection.5.The genetic epistatic analysis revealed that scav-5 is upstream of or parallel to tir-1in the innate immune response pathway defense against PA14.Knockdown of tir-1,nsy-1,sek-1,pmk-1 respectively,which is an integral component of the p38 MAPK PMK-1 signaling cassette,caused scav-5 mutants to be more susceptible to killing by PA14.These suggest that scav-5 may be the upstream or parallel of tir-1.SCAV-5 was not able to interact with TIR-1,which were detected by yeast two-hybrid.