Mechanism Of MAPK And MHC Ⅱ Signaling Pathway In Orange-spotted Grouper(Epinephelus Coioides) After Infected With Cryptocaryon Irritans

Cryptosporidium Irritans is a parasite that seriously harms the marine fish in Southeast Asia.The mechanism of the regulation of MAPK pathway and MHCⅡ on the stimulation of Cryptosporidium Irritans has not been understood.This paper mainly studies MAPK classical pathway and MHC Ⅱ signaling pathway that stimulate Cryptosporidium Irritans infection in grouper.MEK dual-specificity protein kinases are a group of mitogen-activated protein kinase kinases,which act as an integration point by transferringextracellular signals to the nucleus.To investigate the function of MEK in teleost fish,we cloned MEK1 and MEK2 c DNA sequences from the orange-spotted grouper（Epinephelus coioides）.Ec MEK1 and Ec MEK2shared 80%amino acid identity with each other.Ec MEK1 had 89–99%amino acid identity with teleosts or mammals,whereas Ec MEK2 shared 85–97%amino acid identity.The exon structures of the grouper MEK1/2 geneswere conserved with zebrafish and human MEK1/2.Tissue distribution analysis showed that Ec MEK1 and Ec MEK2 had a similar expression pattern in grouper tissues and was mainly transcribe in systemic immune organs.Both Ec MEK1 and Ec MEK2 were distributed throughout the cytoplasm of transfected GS or HEK293T cells.Overexpression of Ec MEK1 or Ec MEK2 activated Activator protein 1dependent luciferase.The phosphorylation levels of Ec MEK1/2 and Ec ERK1/2 were significantly increased in head kidney leukocytes by stimulation with PMA treatment.The grouper MEK1/2-ERK1/2 axis was activated in Cryptocaryon irritans infection and showed an enhanced phosphorylation after immunization.C-Raf proto-oncogene serine/threonine kinase is a mitogen-activated protein kinase（MAP）kinase kinase,which can initiate a mitogen-activated protein kinase（MAPK）cascade by phosphorylating the dual-specific MAP kinase kinases（MEK1/2）,and in turn activate the extracellular signal-regulated kinases（ERK1/2）.To study the function of C-Raf in teleost fish,a C-Raf c DNA sequence from orange-spotted grouper（Epinephelus coioides）was cloned.Ecc-Raf shared 81%–99%amino acid identity with other vertebrate C-Raf molecules,and shared the highest amino acid identity（99%）with Lates calcarifer C-Raf.Genomic structure analysis revealed that grouper C-Raf shared a conserved exon structure with other vertebrates.Tissue distribution showed that Ecc-Raf was mainly transcribed in systemic immune organs.Ecc-Raf was distributed throughout the cytoplasm of transfected GS cells and the overexpression of Ecc-Raf only slightly enhanced the activation of Activator protein 1.The phosphorylation levels of Ecc-Raf can be induced by PMA and H₂O₂treatment,in contrast to DMSO or untreated head kidney leukocytes.The total level and phosphorylation level of C-Raf significantly increased post C.irritans infection and showed an enhanced level post immunization.The results of this study suggested that the Raf-MEK-ERK cascade was involved in the response to viral or parasitic infections.The six genes related to MHC Ⅱ（CTSB,CTSL,CTSS,gilt,MHC ⅡA and MHC ⅡB）were obtained.At almost all time points,all six genes detected by skin tissue were up regulated.At all time,except CTSS gene,the five genes detected by spleen tissue were up regulated.In gill and head kidney tissues,CTSB,CTSL and MHC Ⅱ genes were up regulated at some time points.These results indicate that MHC Ⅱ antigen presentation site may be all on skin tissue and gills.In conclusion,stimulation of Cryptosporidium Irritans to infect grouper can activate MEK1/2-ERK1/2 axis,RAF-MEK-ERK cascade reaction can cause host’s response to virus or parasite infection,and activate host’s specific antibody immune response through MHCⅡ antigen presentation pathway.