Functional Study On CTL33 In Intestinal Microbiota Homeostasis And TWD In Antibacterial Response

Shrimp aquaculture is an important industry,however,has long suffered from serious diseases.Studying the shrimp-pathogen interaction mechanism is important to develop new green sustainable strategy for disease prevention and control.Intestinal microbiota exist in the intestinal tract of metazoan host,and is closely related to host immunity,nutrition,metabolism,and so on.The intestinal microbiota homeostasis is critical for maintain host healthy status and inhibiting pathogen invasion.However,the homeostasis mechanism of shrimp intestinal microbiota is largely unknown.In this study,we identified a C-type lectin which is specifically expressed in shrimp digestive tract and obviously respond to oral bacteria infection,and characterized it as a critical factor in shrimp intestinal homeostasis in kuruma shrimp Marsupenaeus japnicus.Silencing CTL33 expression by using RNAi led to intestinal dysbiosis,tissue damage and shrimp death.CTL33 was found able to mediate the biofilm formation by commensal bacteria and facilitate their colonization in shrimp intestine.CTL33 bound to bacteria by interacting with the surface carbohydrates.Its N-terminal region mediated the dimerization of CTL33 and cross-linked the bacteria into a biofilm like complex.The biofilm formation limited the bacteria from direct contact with the intestine epithelium,and increased the resistance of these commensal bacteria against host immune stress.Thus this study demonstrated a new mechanism of CTL-mediated intestinal microbiota homeostasis,and provided new alternative to develop the strategies for disease prevention by maintaining the intestine homeostasis.We also identified a new protein containing three tandem whey acidic protein domains from red swamp crayfish Procambarus clarkii and designated it as PcTWD.This is the first report of a protein with architecture in crustacean.PcTWD played a role in antibacterial immunity through the antibacterial activity and protease inhibitory activity.Thus this study provided new candidate for developing new fishery drug.In particular,we introduced PcTWD into the phylogenetic analysis of the WAPD-containing proteins in crustacean,and successfully divided this into two clusters,namely,Crustins and proteins solely containing WAPDs.This revises the previous understanding that such molecules were collectively known as crustin,and provides new insight into the classification and evolution of WAPDs containing molecules in crustaceans.