Antibacterial Activity And Function On Cell Phagocytosis Identification Of BmPGRP-Ss From Bombyx Mori

Peptidoglycan recognition proteins(PGRPs)are important pattern recognition molecules in insects.PGRPscan specifically recognize bacterial peptidoglycan(PGN),and function in antibacterial innate immunity.However,the function of PGRPs in regulating the innate immune response in Drosophila melanogaster has been studied,little is known about PGRPs in Lepidoptera species.In our previous study,we found that there were six long PGRPs(BmPGRP-Ls)and six short PGRPs(BmPGRP-Ss)in the silkworm,Bombyx mori,and their expression varied in different tissues.When infected by bacteria,the expression of BmPGRP-Ss was induced rapidly in fat body,but BmPGRP-Ls was induced late in midgut.It indicated that BmPGRP-Ss may be involved in quick immune responses including phagocytosis and humoral melanization.Moreover,the prediction and analysis of structural domains of BmPGRP-Ss showed that they have amidase catalytic domain and may have amidase activity.So the question is: can BmPGRP-Ss inhibit bacteria through their catalytic activities on amide bonds of PGN?In this study,five BmPGRP-Ss were cloned and expressed,and their functions in innate immunity were investigated.The results of the following studies were obtained:(1)The full-length of five BmPGRP-Ss genes were obtained by RT-PCR from RNA of fat body.The five BmPGRP-Ss were cloned into pIEx-4 and stable expressed in S2 cell lines.Then the recombinant BmPGRP-S proteins were purified byusing N i-NTA affinity chromatography.(2)The results of antibacterial activity assay showed that only BmPGRP-S1 and-S2 had antibacterial activity against gram-negative bacteria(Escherichia coli),and all BmPGRP-Ss were inactive against gram-positive bacteria(Staphylococcus aureus).The results of amidase activity assay showed that BmPGRP-S1 only had amidase activity on DAP-PGN(derived from Gram-negative bacteria),but BmPGRP-S2,-S3,-S4 and-S5 displayed amidase activity on both DAP-PGN and Lys-PGN(derived from Gram-positive bacteria)and were more sensitive to DAP-PGN.(3)Agglutination assay results suggested that BmPGRP-S1,-S2,-S4 and-S5 could agglutinate E.coli and Bacillus megaterium,but BmPGRP-S3 only could agglutinate B.megaterium.(4)Confocal Laser Scanning Microscope results revealed that the phagocytosis of FITC-labeled E.coli by silkworm blood cells was promoted after BmPGRP-S2 or-S5 was added,the phagocytosis of FITC-labeled S.aureus was promoted after BmPGRP-S5 was added,and the phagocytosis of FITC-labeled B.megaterium was promoted after BmPGRP-S4 or S5 was added.All the results from this paper suggested that BmPGRP-Ss may be involved in cell immunity of B.mori.It provides a new target for the development of bactericidal drugs.