Innate Immune Proteins in a Crustacean Pacifastacus leniusculus

University dissertation from Uppsala : Acta Universitatis Upsaliensis

Abstract: Hemocytes (blood cells) are important in the immune defense against pathogens in invertebrates. In crusteacean, the hemocytes and plasma components mount a strong innate immune response against different pathogens including bacteria and virus. This thesis is aimed to identify marker proteins associated with development of different hemocyte types, and to find a protein involved in the phenoloxidase-induced melanization and other innate immune reactions in freshwater crayfish Pacifastacus leniusculus. In crustaceans, the hemocytes are produced and partly differentiated in the hematopoietic tissue (Hpt) before they are released into the hemolymph circulation. To investigate the connection between semigranular cells, granular cells and precursor cells in Hpt of P. leniusculus and possibly also in other crustaceans, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis was used to identify specific proteins expressed in different hemocytes. The specific expression was analyzed by RT-PCR and western blot. Moreover, RNA interference was used to study the hemocyte differentiation in vivo and in vitro.Melanin formation is essential for host defence in arthopods, and it needs to be tightly regulated since unwanted production of quinone intermediates or melanization is also dangerous to the animal. By using western blot, 2-DE and MS, a melanization inhibiting protein (MIP) was found to have similar function as mealworm Tenebrio molitor MIP. Both of them interfere with the melanization reaction, but do not affect phenoloxidase activity.In order to reveal the mechanism by which peptidoglycan (PGN) induces activation of the prophenoloxidase activating system in P. leniusculus, different forms of Lys-type PGN were used to pull down PGN recognition proteins (PGRPs) from plasma or hemocyte lysate supernatant of crayfish. The binding proteins were separated and then analyzed with MS. Results showed that two serine protease homologues are involved in this activation possibly by forming a complex with lipopolysaccharide and ?-1,3-glucan binding protein (LGBP) and without a PGRP.Besides, two ficolin-like proteins (FLPs) have been found from crayfish plasma by using different bacteria including Staphylocuccus aureus as an affinity matrix to pull down bacterial binding proteins, followed by the analysis with 2-DE and MS. Two FLPs can bind to bacteria, and may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria injected into the crayfish hemolymph, which suggests that FLPs may function as pattern recognition receptors in the immune response of crayfish.