Type I and type II crustins from Penaeus monodon, genetic variation and antimicrobial activity of the most abundant crustinPm4

Abstract

An antimicrobial protein, crustin, is involved in the innate immunity of crustacean by defending the host directly against the microbial pathogens. By data mining the Penaeus monodon EST database, two type I crustins, carcinin. Pm1 and 2, and ten type II crustins, crustin. Pm1-10, were identified. The abundant crustins were crustin. Pm1, 4 and 7, each with variation in the length of Gly-rich repeat among its members. A few crustin. Pm1, 4 and 7 with deletion in the Cys-rich region were also observed. Furthermore, the crustin. Pm4 with the longest N-terminal Gly-rich region was characterized. The crustinPm4 allelic genes were expressed mainly from the hemocytes. Its expression was up-regulated readily by WSSV infection and gradually decreased to normal level afterwards. The recombinant crustin. Pm4-1 (rcrustin. Pm4-1) isoform was produced using the Escherichia coli expression system and tested for its antimicrobial activity. The rcrustin. Pm4-1 was able to inhibit the growth of a Gram-positive bacterium, Bacillus megaterium but not Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus. It also inhibited the growth of two Gram-negative bacteria, E. coli 363 and Vibrio harveyi 639 at lower potency. The rcrustin. Pm4-1 affected the WSSV infection because the expression of an intermediate early gene ie1 in WSSV-infected hemocyte cell culture was reduced. It was shown further that the rcrustin. Pm4-1 could delay by about one and a half days the manifestation of disease by WSSV. © 2014 Elsevier Ltd.