Charaterization Of Essential Genes In Blood-stage Plasmodium Parasite By Conditional Knockout System

Malaria caused by Plasmodium spp is one of the major infectious diseases in humans.It is most prevelent in Sub-saharan Africa and Southeast Asia regions and children under the age of five are the most vulnerable groups.The major clinical pathology and symtoms of malaria include severe anemia,metabolic acidosis,multiorgan failure and neurological disorder.So far,anti-malarial drugs are the most effective means to combat malaria.However,emergency of drug resistance strains,especially those to artemisinin,significantly hampers drug efficiency.Malaria vaccines with high efficacy are expceted the most efficient approach to control malaria.Detennination and characterization of the function of genes of Plasmodium parasite would greatly improve our understanding of complex interaction between the parasite and its host and help indentify potential antimalarial drug and vaccine targets.Although a number of methods have been developed for study of gene functions in Plasmodium parasites,their use is often limited due to the constrains of the methods.In my study,the murine malaria parasites,P.berghei and P.chabaudi,were used to establish a new genetic manipulation strategy for study of gene function.The research project consists of following three related parts:1.Establishment of an ATc-inducible gene expression system and generation of whole-pathogen malaria vaccineIn this part of study,I exloited the tetracycline(Tet)-inducible transactivator elements originally identified in Tet-resistant Escherichia coli to establish an anhydrotetracycline(ATc)-inducible transcription(ATc-on)system in blood-stage P.berghei and generated a transgenic parasite line in which the hexose transporter 1(HT1)gene was conditionally knocked out(HT-cKO).The results show that in the presence of ATc,HT-cKO parasites grew normally in mice facilitating the maintenance and production of the transgenic parasites for vaccine use.In the absence of ATc,the transgenic parasites were defective in growth and the infection was self-resolved quickly with no need of drug treatment.Importantly,the mice that cleared the primary HT-cKO infection became fully immune to challenge with wild-type P.berghei.No genetic revertant occurred in HT-cKO parasites upon continuous passage in mice.These conditionally defective blood-stage parasites were highly immunogenic,readily scalable and genetically uniform and stable.These results demonstreated that the transgenic parasites in which an essential gene is conditionally knockedout can be a usefull approach for generation of whole-pathogen malarial vaccine.Analysis of lipoic acid metabolism pathway(s)in P.berghei parasite with ATc-on systemLipoic acid is a cofactor for a-keto acid dehydrogenase system that is involved in the energy metabolism.In apicomplexan parasite Plasmodium,lipoic acid protein ligase 1(LplA1)and LplA2 are known to catalyse the ligation of acquired lipoic acid to the dehydrogenase complexes in mitochondrion.The enzymes LipB and LipA mediate lipoic acid synthesis and ligation to the enzymes in apicoplast.These enzymes in the lipoic acid metabolism machinery have been shown to play important roles in the biology of Plamodium parasites,but the relationship between the enzymes is not fully elucidated.We generated a line of transgenic P.berghei parasite in which the lplA1 gene was conditionally knocked out(LplA1-cKO).It was observed that LplA1-cKO parasites showed severely impaired growth in vivo in the first 8 days of infection,and retarded blood-stage development in vitro,in the absence of ATc.However,these parasites resumed the viability in the late stage of infection and mounted high levels of parasitemia leading to the death of the hosts.Further analysis revealed that,the LplAl-cKO parasites showed significantly increased expression of LplA2 gene in the late stage of infection that might be responsible for the restored growth of the transgenic parasite during the stage of infection.It was concluded that the lplA2 gene can be activated as an alternative pathway to compensate for the loss of LplA1 activity and to maintain lipoic acid metabolism.2.Attempt to establish the ATc-on system in non-lethal P.chabaudi parasiteUnlike P.berghei parasite that causes severe infection with lethal outcome in mice,P.chabaudi parasites develop a self-resolving infection.In addition,P.chabaudi shares many parasitological and immunological features with the human parasite P.falciparum.In the third part of study,I made attempt to generate the ATc-on transgenic P.chabaudi parasite that can be studied in parallel with transgenic P.berghei to analyze the genetic basis of phenotypic difference.Although the ATc-on vector can be succsessfully transfected into P.chabaudi genome and the transgenic parasites showed regulation of GFP expression by ATc,it was difficult to obtain the cloned transgenic parasites,because of the poor growth viability of the parasites in BALB/c mice.