Recognition Of Vibrio Parahaemolyticus RNA Via TLR13 In Orange-spotted Grouper(Epinephelus Coioid)

Vibrio Parahaemolyticus has caused a huge economic loss in the world-wide grouper aquaculture because of its rapid transmission and high mortality. Toll-like receptors (TLRs) , as an important gene family for host innate immune system,recognize a wide range of pathogen-associated molecular patterns (PAMPs), mediate the MyD88-dependent pathway or TRIF-dependent pathway leading to the activation of cytokine expressions, and finally protect the host against the pathogens.Mouse TLR13 was reported that it sensed a conserved bacterial 23 S ribosomal RNA (rRNA) sequence,and activate the MyD88-dependent pathway leading to the activation of NF-?B and secretion of ILs and TNFs. The immune function of fish TLR13 has not been clarified so far. In present study, we challenged the Orange-spotted grouper spleen cell line (GS cell line) with different Vibrio Parahaemolyticus components and analyze the expression profiles of TLR13 as well as several selected down-stream cytokines. At the same time, we tried to construct a TLR13- recognition-site mutant strain of Vibrio parahaemolyticus to further study the role of Orange-spotted grouper TLR13 in the recognition of Vibrio Parahaemolyticus.The main results are as follows:1. Real-time quantitative PCR was used to identify the expression levels of Orange-spotted grouper TLR13 gene in GS cell line stimulated with different Vibrio parahaemolyticus components, such as RNA, DNA, flagellin, other baterical components including LPS and PGN. The results show that an increased expression of TLR13 was observed in GS cells stimulated with Vibrio parahaemolyticus RNA and flagellin.But the expression of TLR13 has no change stimulated with DNA.In addition, the most dramatic increase of Orange-spotted grouper TLR13 expression was detected in GS cells stimulated with Vibrio parahaemolyticus RNA at 6h. In addition, RNaseA digestion of Vibrio parahaemolyticus RNA significantly reduced the expression of Orange-spotted grouper TLR13, suggesting that the Orange-spotted grouper TLR13 should be involved in the recognition of Vibrio parahaemolyticus RNA.2. To determine whether TLR13 was required for the recognition of Vibrio parahaemolyticus RNA in GS cells, TLR13 expression was silenced by siRNA. The TLR13 mRNA expression in GS cells transfected with TLR13-siRNA was reduced to less than half of that in control group. TLR13-silenced GS cells expressed significantly less IL-1?, IL-12, IL-6 and TNF-a mRNA in response to Vibrio parahaemolyticus RNA than control group. The results revealed that Orange-spotted grouper TLR13 was required for the recognition of Vibrio parahaemolyticus RNA,and induced the production of inflammatory cytokines to resist bacterial damage.3. We syntheticed a sequence ( 5'-ACGGAAAGACCCC-3') named as VP13,which is a Vibrio parahaemolyticus homologue of murine TLR13 ligand. VP 13 was used to stimulate GS cells resulting in the induction of grouper TLR13 mRNA.Orange-spotted grouper TLR13 mRNA had the highest mRNA level at 6h. It showed that Orange-spotted grouper TLR13 was responsive to the stimulation of a Vibrio parahaemolyticus homologue of murine TLR13 ligand.4. Real-time quantitative PCR was introduced to detect the expression patterns of IL-1?,IL-12, IL-6 and TNF-a mRNA when GS cells were stimulated with VP13.After stimulations,the mRNA expressions of four selected cytokines was augmented significantly in GS cells. On the contrary, TLR13-siRNA transfected GS cells expressed significantly less cytokine mRNAs in response to VP13 than those of control group.5. pDS132 suicide vector was used to construct TLR13-recognition-site mutant strain of Vibrio parahaemolyticus. We cloned the upstream and downstream flanking DNA fragments of the target gene sequence, and then successfully subcloned into the suicide vector pDS132. But the target mutant strain has not been obtained yet.