Identification of outer membrane protein ompR from rickettsia-like organism and induction of immune response in Crassostrea ariakensis

Abstract

Rickettsia-like organism (RLO) caused mass mortality of oysters, but little is known about the protective immune response to this microorganism. The present study was undertaken to identify a gene, ompR, encoding an outer membrane protein of rickettsia-like organism from oyster Crassostrea ariakensis. The role of this protein in promoting immune responses was characterized through analyzing the interaction between RLO and oyster. The results indicated: (i) full-length DNA of ompR is 531 bp and encodes 176 amino acid residues. Theoretical isoelectric point and molecular weight for the ompR protein are 9.76 and 19.76 kDa, respectively; (ii) the recombinant ompR was successfully expressed in Escherichia coli BL21 (DE3) cells, and the titre of anti- ompR antibody raised against rabbits was about 1:4100. A specific immunoreactive band was detected when anti- ompR antibody was opposed to the total outer membrane proteins of RLO; (iii) the expression level of TNF-α (tumor necrosis factor-alpha) and Myd88 (myeloid differentiation factor 88) in hemocytes was induced by ompR, whereas TGF-β (transforming growth factor-beta) was not; (iv) in hemocytes monolayers, a rapid and persistent increase in the level of phosphorylated P38 and a large decrease in the level of phosphorylated JNK were induced by ompR, whereas the level of phosphorylated ERK did not change with ompR incubation; (v) the DNA binding activity of NF-κB (nuclear factor κB) in hemocytes increased after ompR stimulation.