Gene Structural And Functional Characterization Of RIOK-1 Protein Kinase Encoding Gene In Strongyloides Stercoralis

Parasitic nematodes cause numerous economic losses in animal husbandry and more serious is that they also cause severe parasitosis in human. There are at least 100 million people with parasitic nematode infection. In ruminant animal breeding, Haemonchus contortus is reported to infect hundreds and millions of sheep, which caused more than 10 billion dollars losses. Because of the abundant species of parasitic nematodes infecting various hosts, the general losses caused by parasitic nematode are far more serious than that of H. contortus in livestock. So far the controlling of parasitic nematodes relies on anthelmintic drugs, and the abuse of drugs has already caused drug resistance in parasitic nematodes. As these parasites have a very complex life cycle and the mechanism of escaping from host immune system are severely hampered parasitic nematode vaccine and new drug development. Drug discovering as well as vaccine candidate screening requires detailed functional knowledge of essential molecules in parasitic nematode development.Therefore, the present research choose a newly distinguished atypical protein kainse RIOK-1, and using the zoonotic parasitic nematode Strongyloides stercoralis to study the functions of RIOK-1 in the development of this parasite. Molecular methods and bioinformatic technology were employed to isolate the gene and assessed its kinase activity as well as its transcription profiles in different developmental stages. Transgenic technology was also used to analyze anatomic localization and function in vivo.(1) The gene characterization of Ss-riok-1The full length of Ss-riok-1 c DNA is 2056 bp, including a 1524 bp coding sequence which encodes 507 amino acids, 377 bp 5’ UTR, 155 bp 3’ UTR. The full length of g DNA of Ss-riok-1 is 6117 bp. There are two introns contained in the coding sequence, which is 134 bp and 64 bp in length, respectively, and two introns in 5’ UTR, which are 711 bp and 3148 bp in size respectively. The length of Ss-riok-1 promoter is 4280 bp long comprising several common regulatory motifs. Ss-RIOK-1 contains the conserved RIO domain which comprises several functional motifs including ATP-binding motif, flexible loop, hinge, catalytic loop, mental-binding loop. Compared to the conserved RIO domain, its N-terminal and C-terminal were more variable. Ss-RIOK-1 has kinase activity in vitro including the ability of auto-phosphorylation and phosphorylation MBP which is a common substrate of Ser/Thr protein kinase. The kinase activity acquires the Asp located in the catalytic loop.(2) Temporal and spatial expression of Ss-riok-1Ss-riok-1 was transcribed in every developmental stage of S. stercoralis, and transcript abundance peaked at the infective third stage larvae. Ss-riok-1 was expressed in cytoplasm of some hypodermal cells and neurons in post free-living larvae including head neurons, tail neurons and some body motor neurons. The anatomic expression pattern shifted during the larvae development. Ss-riok-1 was expressed in whole body when the larvae hatched from eggs, and the expression was shifted to neuron system in post free-living second stage larvae, at last Ss-riok-1 was expressed in the body wall muscle in infective third stage larvae.(3) Mutant Ss-RIOK-1 disturbed larvae developmentMutant Ss-RIOK-1(D282A) that lost its catalytic activity was expressed in the cytoplasm. The expression of Ss-RIOK-1(D282A) in eggs decreased the hatching rate. It also caused mobility defect in post free-living second stage larvae and terminated the development from post free-living second stage to infective third stage with the phenotype of molting defect and failed to complete pharynx development.(4) Identification of potential interacting protein of Ss-RIOK-1Based on the information from gene interaction prediction database of C. elegans and the information of RIOK-1 functional research in yeast, we chose Ss-lin-35, Ss-riok-2, Ss-kin-3, Ss-nob1 and Ss-pno1 to detect the interaction between proteins encoded by these genes and Ss-RIOK-1. However, Ss-RIOK-1 interacted with none of these proteins in yeast cell.This project investigated the functional role of RIOK-1 in S. stercoralis, which helps us to understand how RIOK-1 is involved in regulating developmental processes of multicellular organisms as well as laying a foundation for further assessment on discovering potential drug.