Effect Of Clag9 On The Cytoadherence Function Of Microvascular Endothelial Cell

Malaria still remains a serious health problem. There are an estimated 300-500 million cases and over 1 million deaths from malaria each year. Despite the early success of malaria eradication campaigns in 1960s, the disease has undergone a resurgence as a result of resistance to the commonly used insecticides and drugs respectively. Furthermore, the vaccine development was frustrated because of the complexity of the parasite's life cycle and the antigenic diversity. These brought about the realization that a greater understanding of the pathogenic process associated with the disease is necessary if effective modes of treatment or prevention are to be found. Cerebral Malaria (CM) is the most severe complication and the major cause of death of Malaria. The pathogenic basis of CM is the adherence of the mature-stage-infected erythrocytes (IE) to host endothelium. It has involved multi-factors and different pathways which are still unclear. Our research strategy was to search bibliography and to analyze the malaria genomic database by bioinformatics software to look for the new adherence-associated genes. Moreover, detected by the newly established in vitro model of cytoadherence, these potential adherence-associated genes functions in pathogenesis of CM were investigated, and athogenesis of CM adhesion was discussed.Over the past 10 years, a few of proteins expressed by P.falciparum on the parasite-infected red blood cell (IRBC) surface which involved in cytoadherence in CM has been identified, including the highly diversified PfEMP1 and the highly conserved Clag9. Both of the two proteins are essential for cell-cell interaction between IRBC and HBMVEC.Clag9 was explored in this experiment. Many research work have been done in our laboratory before: Analyzed by bioinformatics softwares, there fragments of Clag9 had no overlap sequence with other malaria sequences were selected (Cg1,Cg2,Cg3). Cg1 was closed to N-terminal of the protein, Cg2 and Cg3 were near to C-terminal of the protein. Then, by RT-PCR, the cDNA sequences of Cg1, Cg2, and Cg3 were obtained from 3D7 strain and cloned to pGEX-4T-1 vector and were expressed in BL21-DE3-RIL. All the polypeptides of Clag9 were used to co-culture with the HBMVEC in vitro with accordingly Endotoxin control. 18 hours later, ICAM-1, the essential receptor on host cell, was measured by flow cytometry. It was evidenced that Cg3 could up-regulate expression of the host ICAM-1, but no such function could be found in Cgl and Cg2.For the endotoxin existing in the recombinent polypeptides solution could infect ICAM-1 expression of the host cell, endotoxin should be removed from the solution. Two methods were used to remove the endotoxin: expressing the recombinent proteins by eukaryotic system or using C8 colomn to remove endotoxin from proteins expressed by E.coli. All the polypeptides of Clag9 were used to co-culture with the HUVEC in vitro with accordingly GST control. 18 hours later, ICAM-1, the essential receptor on host cell, was measured by flow cytometry. The result indicated Cg3 could not up-regulate expression of the host ICAM-1, which was different with the result of polypeptides without removing endotoxin. Literatures published recently suggested that Clag9 may be involved in cytoadherence not binding directly to host receptors but transporting other cytoadherence molecules into or out of parasite.