Gene Expression Profile Analysis Of Aduit Echinococcus Granulosus And Anti-hydatid Drug Screening

Background:Echinococcosis is a serious disease caused by the larval stage of the genus Echinococcus in man and in some animal species. China has a high prevalence of hydatid disease which has two types, cystic echinococcosis and hepatic alveolar. At present, albendazole is the most widely used anti-hydatid agent in clinic, however, albendazole treatment has been found to be ineffective in some instances, is parasitostatic rather than parasiticidal, and uaually involves the long-term uptake of large doses of drugs. Clinical studies has found that only30%of the patients could be cured and abendazole can cause serious adverse drug reactions (SADR), such as encephalitis syndrome (acute demyelinating encephalitis), gastrointestinal disturbances, lession of liver tissue, epilepsy, hemolytic anemia, renal failure, anaphylactic shock, severe drug eruption. In addition, as the application of albendazole in decades, the problems of the drug resistance become serious. Therefore, the search for novel drug candidates with more effective treatment and mild side effects are urgently needed. The development of new therapeutic drugs depends on the further study to the genome and the function of genes of parasites. Currently, the project of genome on Echinococcus granulosus(E.granulosus) and Echinococcus multilocularis(E.multilocularis) has been completed. In addition, in Genbank (NCBI) there are thousands of Expressed Sequence Tag (EST) from protoscolex and germinal layer of E.granulosus and E.multilocularis. The genome and the function of genes of E.granulosus and E.multilocularis can establish the foundation for the comprehensive analyze on the biological characteristics of parasites, as well as accelerate the process of searching for new anti-echinococcosis drugs. In the life cycle of hydatid, artificially destroy any development stages of E.granulosus or E.multilocularis may have the effect of controlling or preventing the transmission of echinococcosis. Every development stages of E.granulosus or E.multilocularis such as eggs, larvae and adult stage can be used as anti-echinococcosis drug targets. Lv Gang et al.have constructed the cDNA plasmid library of adult E.granulosus which has established the foundation for screening, isolating and identify the key genes and potential drug target in growth and development of parasites.Research methods:Based on the cDNA plasmid library from adult stage of E.granulosus, the large-scale5’random cut EST sequencing was carried out. Through the annotation of high-throughput data analysis results, and on the basis of existing hydatid functional genomics information, we analyzed the key genes and potential drug targets. According to the present research of the genes in glycolytic pathway and pentose phosphate pathway, we identified two key genes in these two metabolic pathways. Homological comparison with the Hexokinase of E.granulosus (EgHK) and Transaldolase of E.granulosus(EgTAL) from other species, topology and hydrophilicity were analized online or on the information softwares. Prokaryotic expression plasmid BL21-pET30a-EgTAL and BL21-pET30a-EgHK were constructed and expressed in E.coli. The recombinant protein was purified by His-Bind purification kit. Then the purified protein was immunized mice to obtain the murine antibodies againstTAL andHK. The expression of the recombinant protein was measured by Western blot. Immunohistological detection ofTAL and EgHK in protoscolex and germinal layer of E.granulosus were performed using EgTAL and EgHK antiserum. Based on the research work above, multiple glycolysis inhibitors were selected, such as3-bromopyruvate, lonidamine,2-Deoxy-D-glucose, clorsulon,2,6-dihydroxynaphthalene, sodium oxamate,6-aminonicotinamide, ornidazole and the pentose phosphate metabolism inhibitor oxythiamine chloride hydrochloride, in addition, we also selected a traditional Chinese medicine ampelopsin. The in vitro efficacy of these drugs against E.granulosus and E.multilocularis was measured and the in vivo efficacy of3-bromopyruvate was investigated in E.multilocularis infection mice. The cytotoxicity of3-bromopyruvate, ampelopsin and albendazole was performed on normal human hepatocytes by MTT assay. In additon, the BALB/c mice were administered with effective anti-alveococcus dose of3-bromopyruvate and albendazole and six weeks later, biochemical indicators of liver and kidney and histopathologic analysis were performed to evaluate the adverse effects of3-bromopyruvate.The results:In the cDNA plasmid library of adult stage of E.granulosus, the clones were randomly selected, and after sequencing,1560unigenes were obtained. Among them,155unigenes having no significant matches to any protein sequences in the public database NCBI were identified as novel genes. Based on the Gene Ontology (GO) classification system, matched genes were classed into different GO groups. Among these groups, the groups with the highest percentages of unigenes were binding (23.33%) and catalytic activity (3.45%). Based on the KEGG annotations, the unigenes involved in227metabolic pathways, and the glycolytic pathway genes were highly expressed, this suggested that the genes in this way may be considered as the potential drug targets. Bioinformatic prediction indicated that EgHK and EgTAL have a series of characteristics of potential drug targets. In E.coli expression system, EgGK and EgTAL were expressed as inclusion body and soluble protein, respectively. With his tag the recombinant protein was purified by affinity column chromatography using his-bind purification kit. The results of Western-blot indicated that EgHK and EgTAL were immunogenic. In immunoflurescence assays, EgHK andTAL were detectable in protoscolex and germinal layer of E.granuloaus. Among the glycolysis inhibitors, HK inhibitor3-bromopyruvate exhibited the most effective effect against E.grabulosus and E.multilocularis. At the drug concentration of40μM, the effect against hydatid had significant difference compared with albendazole (P<0.05). The in vivo efficacy of3-bromopyruvate was investigated in mice. The3-bromopyruvate was applied through the oral routeresulted in a significant reduction in parasite burden compared with untreated group (P<0.05). Different concentration of3-bromopyruvate could inhibit HK catalytic activity to different degrees. This indicated that the effect of3-bromopyruvate against hydatid depend on its inhibitation of HK catalytic activity. Traditional Chinese medicine ampelopsin also exhibited the anti-hydatid effect. The survival of protoscolex after7days treatment of160μM ampelopsin was1.5%. When the concentration of ampelopsin was400μM,800μM and1000μM, the effect against E.multilocularis had significant difference compared with40μM albendazole (P<0.05). The results of MTT assay revealed that both3-bromopyruvate and ampelopsin exhibited considerable toxicity on normal human hepatocytes. At the effective anti-hydatid concentration, albendzole seemed to display an even more toxicity than3-bromopyruvate and ampelopsin. The in vivo toxicity test showed that3-bromopyruvate and abendazole administered with effective anti-hydatid concentration had no side effect on renel function. Abendazole could cause serious damage to liver tissue, alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were increased in serum and aspartate aminotransferase (AST) levels was148.000±27.475U/L which was significantly higher than the control group AST values (115.500±18.355U/L)(p<0.05). Histopathological examination showed chronic hepatitis morphological changes.3-bromopyruvate did not induce significant liver damages.Conclusions:In this work, we investigated for the first time the gene expression profile of adult E.granulosus. The set of sequences described here has established the foundation for searching and developping novel potential anti-hydatid drugs and will contribute to the study of E.granulosus genome. Bioinformatics analysis revealed that the key genes EgHK and EgTAL can be considered as potential targets. On the basis of results above, several glycolysis and pentose phosphate metabolism inhibitors were selected. In addition, we also selected a traditional Chinese medicine ampelopsin. The efficacy of these drugs against hydatid was investigated in vitro and in vivo, and glycolysis inhibitor3-bromopyruvate and ampelopsin exhibit effective anti-hydatid effect. Preliminary studies indicate that3-bromopyruvate has less side-effect than albendzole and suggest that3-bromopyruvate and ampelopsin can be used as new drug candidates against hydatid.