Elastase B, A Metalloproteinase Secreted By Pseudomonas Aeruginosa (PAO1) Modulates Immune System Of The Silkworm, Bombyx Mori

As an important economic insect, silkworm, Bombyx mori, has been domesticated for more than 5,000 years and sericiculture, is an important industry in China. However,silkworm’s weak disease resistance owing to long-term artifical breeding limits the development of this industry. This situation impels us to study the pathogenesis and immune system of silkworm for a better control of silkworm diseases. Besides, as a model insect of lepidoptera, the study of silkworm immune system can not only provide important references for lepidopteran insect immunology, but also provide some clues for the control of pests and for the development of new pestcides.Pseudomonas aeruginosa(PAO1), an opportunistic gram-negative bacteria, is ubiquitous in air, water, soil, as well as in skin, respiratory and intestinal tracts. It is usallyused as a test strain to stimulate hosts’ immune responsesin the study of insect immunology.Insect relies on innate immune system to defence against pathogens and parasites. The production of antibacterial peptides(AMP) and melaninization are the two main immune responses. AMP is produced through IMD(immune deficiency) or Toll signaling pathways,while melaninization is depend on the activation of PPO(prophenoloxidase). There are some reports that bacterial secreted virulent factors regulates the immune system of insects, but these results are not consistant. In this study, silkwormwas used as a model to investigate the manipualtion of host immune system by Elastase B secreted by PAO1. Our results show the following:(1) LasB, the gene that encodes Elastase B, is expressed in one hour after injection of PAO1 into the hemocoel of silkworm larvae, resulting in an increase of Elastolytic activity in the hemolymph. This suggests that PAO1 secretes Elastase B after it enters into the body of silkworm and the quantity of Elastase B increases gradually with the propagation of PAO1 inside silkworm.(2) Elastase B modulates AMP and PPO pathways: after injection of purified Elastase B into the silkworm hemocoel, the PPO activition is strongly inhibited leading to reduced PO(phenoloxidase) activity; on the contrary, AMP gloverins are induced.(3) Elastase B degrades some proteins in the silkworm hemolymph, including two serineprotease inhibitors(Serpins), antitrypin isoform1(Serpin 1A) and chymotrypsin CI-8A(Serpin 9), implying that these two serpins might participate in the regulation of AMP pathway rather than PPO pathway.(4) High doses of Elastase B inhibits wound healing and destructs silkworm tissues.(5) Larval silkworms were infected by the mutant of LasB(P.a-ΔLasB) and wild type strains(P.a-WT) respectively, and the results show that the PO activity of the silkworms infected by P.a-ΔLasB is significantly higher than those infected by P.a-WT; while the quantity of gloverins and antimicrobial activity induced by P.a-ΔLasB arelower than thoses induced by P.a-WT. Compared to P.a-WT, P.a-ΔLasB shows a lower multiplication rate in the silkworm hemocoel.Taken together, our results shows that Elastase B secreted by PAO1 functions as a virulent factor to regulate the immune system of silkworm during infection, which can help PAO1 hinder or escape the immune responses of silkworm to make PAO1 grow and reproduce more favorably.