Study On The Immune Functions Of The Mythimna Separata Peptidoglycan Recognition Protein PGRP-S1

Peptidoglycan recognition proteins(PGRPs)are widely distributed in insects and higher animals as a family of pattern recognition receptors(PRRs).They can recognize Peptidoglycan(PG)on the surface of bacteria,thus activating immune signaling or performing bacteriostatic functions.At present,studies on PGRPs mainly focus on fruit fly and silkworm,and fewer studies have been performed in other lepidoptera insects.Therefore,in this study,we cloned a PGRP gene from armyworm,Mythimna separata and named it MsePGRP-S1,and its immune-related functions were studied.The main experimental results are as follows:1.Sequence analysis:According to DNAMAN analysis,MsePGRP-S1 cDNA contained an open reading frame(ORF)of 591 bp,encoding 197 amino acids.The protein encoded by this gene was predicted to have a signal peptide sequence(aa:1-18)and a conserved PGRP domain(aa:32-181).The predicted molecular weight of the protein was 19719.7 Da and the isoelectric point was 5.5.Multiple sequence alignment analysis showed that MsePGRP-S1 had 5 Zn2+-dependent amidase activity sites(H-Y-H-T-C),including G-W-R sites.Phylogenetic analysis showed that MsePGRP-S1 was closely related to silkworm PGRP-S3,S4,S5,S6 and Anopheles gambiae PGRP-S2 and S3.2.Tissue distribution and induced expression of the gene:The expression of MsePGRP-S1 in different tissues of armyworm larvae was detected by RT-RCR,and the results showed that it was expressed in hemocytes,midgut,fat body and epidermis.Realtime PCR was used to detect the changes in the expression levels of MsePGRP-S1 in the hemocytes,midgut and fat body of armyworm larvae after the injection of bacteria.The results showed that the expression levels of MsePGRP-S1 could be regulated by the stimulation of Escherichia coli and Staphylococcus aureus.After bacterial stimulation,the expression of MsePGRP-S1 in different tissues was significantly up-regulated at some time points.3.Binding of recombinant protein to bacteria and peptidoglycan:The binding of recombinant protein MsePGRP-S1 to bacteria was detected by Western Blot,and the results showed that it was mainly bound to gram-positive bacteria Staphylococcus aureus.Peptidoglycan was isolated and purified from Staphylococcus aureus and Bacillus subtilis.Binding of recombinant MsePGRP-S1 to peptidoglycans from Staphylococcus aureus and Bacillus subtilis was detected by ELISA assay.The results showed that there was an significant binding,and the binding was enhanced with the increase of MsePGRP-S1 concentration.4.Inhibition of bacterial growth:The inhibition of on bacterial growth by MsePGRP-S1 was detected by CCK-8 method,and the results showed that it could significantly inhibit the growth of Escherichia coli,Staphylococcus aureus,Bacillus subtilis and Serratia marcescens.The effect of MsePGRP-S1 recombinant protein on the bacterial phenotype was detected by scanning electron microscopy.The results showed that the cell membranes of Escherichia coli and Staphylococcus aureus were severely ruptured after treatment.5.Analysis of amidase activity:Realtime PCR was performed to detect the changes in the expression of antimicrobial peptide gene in fruit fly S2 cells stimulated by peptidoglycans treated with MSePGRP-S].The results showed that Escherichia coli and Staphylococcus aureus insoluble peptidoglycans treated with MsePGRP-S1 recombinant protein lost the ability to stimulate antimicrobial peptide gene expression in S2 cells.Amidase activity was further analyzed,the results showed that MsePGRP-S1 had amidase activity against insoluble peptidoglycan of Escherichia coli and Staphylococcus aureus.In the presence of lysozyme,the activity of MsePGRP-S1 against Staphylococcus aureus further increased.In conclusion,MsePGRP-S1 is a typical type PGRP.Its transcription product can be significantly induced by bacteria,the recombinant protein showed immune-related functions:It can recognize bacteria and peptidoglycan,and it can inhibit growth of bacteria due to its amidase activity.These results showed that MsePGRP-S1 played an important role in the immune system of armyworm Mythimna separata.