Identification And Analysis Of Proteins Involved In Lamprey Adaptive Immunity System Regulated By Intestinal Aeromonas

Bacterial communities colonizing in the intestinal of animals play important regulatory roles in the digestion,defense,and immune function of the host.Lampreys have a high research value in origin and evolution of vertebrate,the mechanism of immune response,and many other fields because of their special evolutionary status and adaptive immune system.So far,there has been no study on the mechanism of the interaction between the lamprey intestinal bacteria and their adaptive immune system.We analyzed the lamprey intestinal bacterial composition by sequencing V3-V4 region of lamprey intestinal 16 S rDNA and constructing intestinal bacterial 16 S rDNA library.Results showed that the bacterial community structure in lamprey intestine was relatively simple,and only eight generas were observed.They are Aeromonas,Shewanella,Enterobacteria,Chitinophagaceae_unclassified,Acinetobacter,Yersinia,Vibrio,and Candidatus Bacilloplasma.The Aeromonas and Chitinophagaceae_unclassified were the dominant members in the lamprey intestine.Then we used the traditional method to isolate a symbiotic bacteria strain from lamprey intestine,and the molecular identification of this strain was carried out.Combined with morphological analysis and phylogenetic analysis,this strain was named as Aeromonas sp.lam-2.The quantitative analysis of Aeromonas by qPCR was established.Compared with PBS control group,the number of Aeromonas in lamprey intestine was two times higher after stimulation by LPS,and the expression of the variable lymphocyte receptors was increased after Aeromonas immunostimulation,indicating that Aeromonas was one of the key intestinal symbiotic bacteria involved in lamprey adaptive immune response.In order to further analyze the molecular mechanism of Aeromonas regulating the lamprey adaptive immunity,we investigated the effect of the Aeromonas immunostimulation on the change of the total proteome of lamprey peripheral blood leukocytes using the label-free quantitative proteomics analysis.There are 34 differentially expressed proteins were identified,and the function annotation,gene ontology(GO)and the KEGG signaling pathway of differentially expressed proteins were analyzed.The expression of differentially expressed proteins in the mRNA level was validated by qPCR.We hypothesized that STAT3 was the key protein molecule participated in lamprey adaptive immune regulation.In conclusion,our study analyzed the effect of Aeromonas on the expression of the total proteome of lamprey peripheral blood leukocytes after determining the composition of the symbiotic bacteria of lamprey intestine.The key protein molecules involved in lamprey adaptive immune response were identified.This study could not only broaden the research approach of lamprey adaptive immunity using the interaction analysis between the intestinal symbiotic bacteria and host immune system as the starting point,but also provide the theoretical basis for studying the origin and evolution of lamprey adaptive immune system.