Studies On The Molecular Biology Of Pathogen Rickettsia-like Organism Interaction With Its Host, Oyster Crassostrea Ariakensis

Currently, interaction between pathogen and host is a key topic in the frontier of microbiology and immunology in the world. Rickettsia-like organisms (RLOs) are obligate intracellular Gram-negative bacteria and caused mass mortality of oysters. However, little is known about the molecular biology and pathogenesis of rickettsia-like organism The studies on the molecular biology of pathogen rickettsia-like organism interaction with its host, oyster Crassostrea ariakensis were carried out in this scientific dissertation including the immune function and mechanism of transcription factors CREB and LITAF of oyster responsed to RLO stimulation, and signal pathways involved in the host defense under the induction of RLO outer membrane protein ompR.RNA was extracted from hemocytes of oyster stimulated by RLO using TRIzol method and was reverse transcribed into cDNA. Degenerate oligonucleotide primers were designed for RT-PCR and RACE-PCR. Here, we first report that a homologue of CREB, Ca-CREB, was identified and functionally characterized in bivalves. The full-length cDNA of Ca-CREB contains an ORF of 1068 bp encoding a 39.3 kDa protein. Amino acid sequence analysis revealed that Ca-CREB shares conserved signature motifs with other CREB proteins and is highly homologous to Lymnaea stagnalis (56%) and Aplysia californica (58%). The expression level of Ca-CREB in various tissues was investigated by RT-PCR. The results showed that Ca-CREB was ubiquitously expressed in all examined tissues, and expression levels in gills and hemocytes were higher than the others, suggesting that may be involved in immune responses. To investigate the biological activity and functions of Ca-CREB, the recombinant plasmids (pET-CREB) were constructed and identified by sequencing, then transformed into competent cells for expression. The recombinant fusion proteins were purified by affinity chromatography, then the antiserum against New Zealand White rabbits was prepared and the activity of recombinant protein was determined by EMSA. The results of SDS-PAGE. real time RT-PCR.Western blotting and EMSA showed that:①recombinant protein Ca-CREB was successfully expressed in E. coli and purified fusion protein had the specific DNA binding activity.②no obvious changes in the expression level of Ca-CREB in the hemocytes were found after RLO challenge while DNA binding activity and the phosphorylation level of CREB were remarkably modified. The results demonstrated that Ca-CREB was regulated at the protein level, instead of the mRNA level, after RLO stimulation, suggesting that Ca-CREB is involved in immune responses against RLO. To find out the cause of the different migration in the molecular weight of recombinant protein Ca-CREB analyzed by SDS-PAGE method, another experiment was performed from different aspects such as vectors, the concentration of gels and the conditions of electrophoresis. According to our study, different expression vectors and concentrations of gels had no effects on the different migration while different electrophoresis system did.Condering that the host immune responses has been elicited by RLO which was confirmed as above, we decided to carry out the further experiments to investigate whether or not some proteins from RLO could be involved in the immune responses. So it was considered to start a project on studies of the protein of RLO, especially the outer membrane protein of RLO. A DNA fragment of 531 bp encoding ompR was obtained by sequencing the genomic DNA of rickettsia-like organism. It encodes 176 amino acid residues containing a signal peptide and a transmembrane region. Theoretical isoelectric point and molecular weight for this protein are 9.76 and 19.76 kDa, respectively. Comparison of ompR in overall sequence with ompH proteins of Rickettsia grylli, Coxiella burnetii and Thiomicrospira crunogena revealed a similarity ranging from 20%to 27%. Construction of the recombinant plasmid pET-ompR, purification of the recombinant protein and preparation of the antibody against recombinant ompR protein was conducted as described above. The results showed that:①the recombinant ompR was successfully expressed in E. coli cells, a specific immunoreactive band was detected when anti-ompR antibody was opposed to the total outer membrane proteins of RLO.②The expression level of TNF-a and Myd88 in hemocytes was induced by ompR, whereas TGF-(3 was not; 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. while the level of phosphorylated ERK did not change with ompR incubation. Meanwhile, the DNA binding activity of NF-κB in hemocytes increased after ompR stimulation, but it was inhibited by addition of inhibitor for P38 MAPK.Based on our previous studies, a novel gene, Ca-LITAF, and its function in the immune response against RLO was also investigated. The full-length cDNA consists of 750 bp with an ORF of 348 bp encoding a 12.5 kDa protein. Amino acid sequence analysis revealed that Ca-LITAF shares conserved Zn2+ binding domain and LITAF domain. A similarity ranging from 33% to 96% in the entire sequence was revealed by comparison with LITAF proteins from various animals and Cα-LITAF was highly homologous to Crassostrea gigas and Chlamys farreri. As RT-PCR indicated, Ca-LITAF was ubiquitously expressed in all examined tissues, and expression levels in gills and hemocytes were higher than that in the others. Meanwhile, obvious changes in the expression level of Ca-LITAF were found after RLO challenge, suggesting that it may be involved in immune responses.All together, we could draw the conclusion that pathogen/MAPKs/NF-κB (CREB) pathway may exist in the immune system of C. ariakensis and play an immportant role in the immune response against RLO infection.