Drug-resistant Analysis Of Plasmodium Falciparum On Compounds Of Malaria Box And Dihydroartemisinin

lasmodium falciparum is the causative agent of malaria.At present,malaria still occurs and is endemic in 91 countries and areas.According to the World Malaria Report 2017,216 million cases and 445,000 deaths occurred in 2016,of which 70%were children under five.Although scientists have been working on the development of malaria vaccine,no effective malaria vaccine is available yet.At the same time,due to the emergence and spread of malaria parasites resistance,the current therapeutic drugs became less effctive.Based on the above problems,this work focused on two aspects.Part one:Resistance of P.falciparum to compounds in the drug development pipeline and and potential mechanism.The previous study in our laboratory found that P.falciparum clinical strains from the borders of China and Myanmar have developed resistance to commonly used antimalarial drugs,increasing in IC₅₀0 values or the survival rate.In this study,eight clinical resistant strain was applied to screen a new compound-set called Malaria Box.Based on the average inhibitory effect of our tested strains to Malaria Box compounds,the top 15%of compounds with good inhibition rates,ranging from 7.7 to 269.1 nM,might be next-genaration antimalarial drugs.At the same time,clinical strais reduced the sensitivity to 17 compounds,compared with IC₅₀ values of3D7.Moving further,comparing with that of well-known multidrug-resistant strains Dd2,clinical strains increased sensitivity to 11 compounds.We found Malaria box compounds with good inhibitory effect,while otential drug resistance were detected in clinical strains.The results of the study are instructive for the development and use of new antimalarial drugs.A total of 11690 single-nucleotide polymorphisms（SNPs）were identified in our tested P.falciparum strains by genome sequencing.The polymorphisms of the56 genes were different from the well-known resistant strain Dd2.In addition,113copy-number variations（CNVs）of 48 genes were defined.The resultes of statistical analysis showed the 21 genes with SNPs were significant association with 8compounds and 11 genes with CNV were related to 4 compounds.The above results provide new clues for the resistance mechanism in P.falciparum.Part two:Phenotypic and Genotypic Analysis of Dihydroartemisinin-resistant P.falciparumWorld Health Organization（WHO）recommended artemisinin combination therapy as the treatment of malaria preferred program.However,in some parts of Southeast Asia ART resistance has been detected,Clinical ART resistance is defined as a reduced parasite clearance rate.Further ring-stage surivival assay（RSA）experiments confirmed the presence of ART resistance.The mechanism of ART resistance has been a research priority.Some potential resistance mechanisms have been identified through laboratory screening and GWAS analysis,but they have not been fully understood yet.After nearly three years of selection with dihydroartemisinin（DHA）,we obtained DHA-resistant P.falciparum parasties.The DHA resistant lines displayed a²1 decrease in sensitivity to DHA.Results of survival experiments of drugs,RSA and trophozoite-stage survival assay（TSA）were consistent with the previous conclusion.In addition,TSA was found to be associated with IC50.However,the DHA-resistant lines showed distinctive responses to other ART family drugs（artesunate and artemether）,revealing structural specificity to the selection of resistance phenotype.The increased resistance to quinine and mefloquine indicated the presence of cross-resistance.Both of clinical and laboratory ART resistant strains exhibit altered patterns of development.Our DHA-resistant lines showed prolonged the ring-stage development,resulting in an erythrocyte development cycle.A total of 190 SNPs distributed in 35 genes were obtained by comparing the differences between the resistant strains and the parental strain Dd2 in the whole genome range.Some of the results validated previously identified resistance loci such as pfATG18（PF3D7₁012900）,protein kinase 7（PF3D7₀213400）,and conserved Plasmodium membrane protein（PF3D7₁464500）,demonstrating the importance of these loci.Novel gene locus,such as DNA repair and recombinant protein 54（PF3D7₀803400）,formate-nitrite transporter（PF3D7₀316600）and double-strand break repair protein（PF3D7₀107800）,provides new ideas for the mechanism of ART resistance.In addition,the study found that 26 gene CNVs were changed,all with an increase in the number of copies.Of interesting note,nine genes ralateding to antioxidant pathways;six mitochondrial proteins;two heat shock proteins;three ion transporters;ubiquitin proteins Ligase E3.They play an anti-drug role in the aspects of redox,oxidative energy supply and ubiquitination,providing new clues for the study of anti-DHA mechanism of P.falciparum.The DHA-resistant mechanism of P.falciparum is a multi-molecular participation process.It is impossible to completely clarified by only a few gene loci（SNP or CNV）.The results in our study provides new clues as much as possible to provide the evidence for future research.