Roles And Molecular Mechanism Of Fatty Acyl Coenzyme A Synthetase-1a (PfACS1a) Of Plasmodium Falciparum In Artemisinin Resistance

Malaria is parasitic disease caused by Plasmodium.It is estimated that there are 104malaria-endemic countries worldwide,with a population of about 3.3 billion people living in endemic areas as yet.Despite great efforts to eradicate malaria had been exerted,the disease has caused enormous damage to human health,particularly in Africa.Since there is no effective commercial vaccine for malaria control,the main treatment for malaria is antimalarials.But with the widely application of these drugs,many endemic areas inevitably emerged drug resistance problems,especially in southeast Asia.Many kinds of antimalarials drug resistance strains emerged and quickly spread around the world,which result in increased malaria prevalence and death rates and cause a great threat to global malaria control and elimination.The world health organization?WHO?recommended artemisinin-based combination therapy?ACTs?as the first-line treatment for malaria in 2005.The drug was characterized by quick work and low toxicity,which effectively reduced the global malaria burden.However,in 2006-2007,artemisinin resistant strains,which were characterized by elongated cleaning time of infected patients in vivo emerged in the Great Mekong subregion?GMSR?and spreaded rapidly to the whole Southeast Asia.The emergence of resistant strains raised concerns among scientists that the malaria eradication program is once again facing serious challenges.The immediate challenge is to find out the molecular mechanism that result in artemisinin resistance.Prospective study by Ariey et al.showed that there was a strong link between the mutation of certain amino acid sites and artemisinin resistance in the Pfkelch13?K13?genes on chromosome 13.Mbengue and his co-worker found that dihydroartemisinin?DHA?can inhibit the activity of P.falciparum phosphatidyl inositol kinase-3?PfPI3K?and decreased the production of phosphatidyl inositol?PI3P?.The uncompleted activation of signaling pathway cause fatal damage to P.falciparum.At the same time,the mutation of K13 reduced the binding of PfPI3K,resulting in the ubiquitination and degradation of PfPI3K decreased so as to increased its catalysate PI3P increased.The increased of PI3P improved the resistance of the P.falciparum strain.These studies suggest that K13-PfPI3K interaction plays a role in the resistance mechanism of artemisinin,but the signaling pathways and molecular mechanisms unclear.The purpose of this project is to further explore the molecular mechanism of P.falciparum artemisinin resistance and find out the specific function of PfPI3K in artemisinin resistance pathway.Six artemisinin resistant strains were developed through three years of drug pressure screen in our lab and two pairs of them were selected to analyze the transcriptional spectrum and found a gene named fatty acyl-CoA synthetase?PF3D7₁479000,PfACS1a?down-regulated in artemisinin resistant strains.We investigated the effect of this gene to artemisinin sensitivity and the related molecular mechanism by means of function inhibition and overexpression.The purpose of this paper is to elucidate the function of PfACS1a in the process of artemisinin resistance and results are as follows:1?The expression of PfACS1a down-regulated in artemisinin resistant P.falciparum.The six artemisinin resistant strains used in this study were obtained by continuous improving drug concentration by early researchers and resistant strains were F0827/F0827ART,F0867/F0867ART,F09P-1/F09P-1ART,F0871/F0871ART,F0870/F0870ART and F0758/F0758ART.PfACS1a was found to be down-regulated all artemisinin resistant strains through qPCR.We know that the mutation of K13 increased the resistance of P.falciparum to artemisinin,but whether the resistance caused the expression of PfACS1a changed in parasites or not?The pARL plasmid was used to construct the recombinant plasmid with F446I,I543T and C580Y mutation respectively,and named them pARL-PfK13^F446Imut,pARL-PfK13^I543Tmut and pARL-PfK13^C580Ymut.These recombinant plasmids were transfected into strains with unmutated K13 gene,like3D7^wt,FCC1/HN^wt,F0829^wt to construct K13 mutant strains.Through single cross exchange,we obtained 3D7^{F446I mut},3D7^{I543T mut},3D7^{C580Y mut},FCC1/HN^{F446I mut},FCC1/HN^C580Y mut580Y mut and F0829^{C580Y mut} strains with F446I,I543T and C580Y mutation successfully.Subsequently we collected the ring stage parasites and detected the expression of PfACS1a of mutant and their parent strains by q-PCR.The result indicated that the expression of PfACS1a in the strains with K13mutation significantly down-regulated compared with their parent strains.Furthermore,29 strains were collected from laza in Myanmar region,of which 12 were wild type K13 strains and 17 were K13mutation strains,were processed to detect the expression of PfACS1a.As revealed by statistical result,PfACS1a in all K13 mutant strains were significantly down-regulated compared with wild type K13 strains?***,p<0.001?.Above experiments proved that the mutation of K13 gene down-regulated the expression level of PfACS1a.2?The expression of PfACS1a regulated by PI3K and its pathway.Previous studies have shown that PI3K-AKT-FOXO was a classic signaling pathway in mammalian cells,which FOXO protein acted as a transcription factor to regulate fatty acid metabolism.Therefore,we hypothesized that whether there was a similar signaling pathway in P.falciparum that the expression of PfACS1a were influenced by PfPI3K.LY294002?50?M?and DHA?10nM?,specific inhibitor of PfPI3K,were selected to work on the ring stage parasites for 3h respectively,and then detected the expression level of PfACS1a by q-PCR.We found that expression levels of PfACS1a in 12 strains under10nM DHA were significantly higher than those without DHA.Then we chose3D7^wt,3D7^C580Ymut,3D7^I543Tmut and FCC1/HN to detect the expression of PfACS1a co-cultured with LY294002 for 3h.The result indicated that LY294002 could significantly up-regulated the expression of PfACS1a compared with control group?*,P?0.05;**,P?0.01;***,P?0.001?.Therefore,we draw a conclusion that PfACS1a may be regulated by PfPI3K by some pathway in P.falciparum.3?The expression level and activity of PfACS1a affected the sensitivity of P.falciparum to DHA.Down-regulated expression level of PfACS1a decreased the sensitivity of P.falciparum to DHA:Through above experiments,we preliminarily confirmed the correlation between the expression level of PfACS1a and the resistance of P.falciparum to DHA.Further,we proposed to verify whether the expression level of PfACS1a affected the survival rate of 3D7^wt and 3D7^{C580Y mut} under DHA.The data showed that the expression level of PfACS1a in 3D7^wt was 3 times that of 3D7^{C580Y mut},which with a significantly difference?***,P?0.001?.The survival rate of 3D7^{C580Y mut} was 27.01%±0.30%,which were significantly higher than 3D7^wt?43.58%±1.76%??*,P?0.05?.Meanwhile,the expression level of PfACS1a in F0871 was 2 times higher than that in F0871ART?***,P?0.001?,and The survival rate of F0871ART was 31.95%±2.55%,which were significantly higher than F0871?23.78%±1.72%??*,P?0.05?.We confirmed that down-regulated expression level of PfACS1a could decreased the sensitivity of P.falciparum to DHA.Inhibited the activity of PfACS1a decreased the sensitivity of P.falciparum to DHA:Because of ACS had many family members with similar gene sequences and protein structure domains in P.falciparum,the method of gene knockout to verify the function of PfACS1a may not suitable for our study.Therefore,we chose TriacsinC,which was the specific inhibitor to ACS,to knockdown the activity of PfACS1a.Preliminary studies showed that TriacsinC worked with DHA could significantly improve the survival rate of parasites when cultured for 3h?***P?0.001?,and TriacsinC had no influenced on the normal growth of P.falciparum.Three P.falciparum with wild type K13 genes,named3D7^wt?F08B-4 and F08B-9 were selected to verify our conclusion.The survival rate of3D7^wt?F08B-4 and F08B-9 that cultured with 10nM DHA were 17.76%±1.62%?33.58%±0.42%and 29.12%±1.58%,but elevated to 27.15%±0.15%?39.13%±1.05%and37.9%±0.47%when added 4?M TriacsinC?**,P?0.01?.Therefore,we concluded that inhibiting the activity of ACS could significantly reduce the sensitivity of P.falciparum to DHA.PfACS1a overexpression significantly increased the sensitivity of P.falciparum to DHA:The above inference could be reverse verified by overexpress of PfACS1a.We firstly optimized the gene sequence of PfACS1a and connected it to pLN plasmid to construct the overexpression plasmid and named pLN-PfACS1a^mut.The pLN-PfACS1a^mutut and pLN empty plasmid were transferred into 3D7^{C580Y mut} and F0871ART to construct overexpression transgenic strains and named them 3D7^{C580Y mut}-PfACS1a and F0871ART-PfACS1a.The plasmid pLN-PfACS1a^mut was successfully expressed in parasites verified by IFA assay.Detected the expression level of PfACS1a of the ring stage parasites after transfected for 48h,we discovered nearly 6000 times of PfACS1a up-regulated in transfected parasites which significantly higher than that of the parent strains?***P?0.001?.The survival rate of 3D7^{C580Y mut-}PfACS1a was 25.04%±2.08%which was significantly lower than that of 3D7^{C580Y mut}?72.90%±2.72%?and 3D7^{C580Y mut}-pLN?61.63%±5.50%??***,P?0.001?,and lower than original 3D7^wt?38.58%±1.59%?as well?*,P?0.05?.Similar results were found in the F0871ART.The survival rate of F0871ART-PfACS1 was 64.37%±1.46%which was significantly lower than that of F0871ART?83.33%±0.67%?and F0871ART-pLN?68.18%±5.75%??**,P?0.01?,but it had no significant difference with F0871?62.99%±2.11%?.The overexpression of PfACS1a in F0871ART-PfACS1reversed the sensitivity to artemisinin the same as the F0871.Accordingly,we inferred that the up-regulated the expression of PfACS1a significantly improved the sensitivity of P.falciparum to DHA.4?PfACS1a affected the survival of parasites by generating ROS to increase mitochondrial membrane potential depolarization and DNA fragmentation.The expression level and activity of PfACS1a affected the production of ROS under the pressure of DHA:The fluorescence intensity of DCFH-DA represents the production of ROS,and the higher the fluorescence intensity meant the higher production of ROS.3D7^wt/3D7^C580Ymut580Ymut and F0870/F0870ART were selected to detect the production of ROS by same parasitemia and HCT when parasites were co-cultured with 10nM DHA.ROS produced by 3D7^C580Ymut was 19050.64±24.96,which were lower than 19555.5±66.5produced by 3D7^wtt significantly?*,P?0.05?.Similarly,ROS produced by F0870ART was27284.75±270.27,which was significant lower than F0870.The results of this study confirmed that down-regulated the expression of PfACS1a decreased production of ROS under DHA.The survival rate of 3D7^C580Ymut was 1.61 times higher than that of 3D7^wt?*,P?0.05?,and F0870ART was 1.67 times higher than that of F0870?***,P?0.001?.We detected ROS produced by 3D7^C580Ymut-PfACS1a and F0871ART-PfACS1a compared with strains transfected with blank plasmid and parent strains and discovered the ROS produced by PfACS1a overexpression strains were significantly higher than other strains?***,P?0.001?.The above studies confirmed that the expression level of PfACS1a could affect the sensitivity of parasites to DHA by the production of ROS.Then,whether the activity of PfACS1a effected the production of the ROS?By preliminary experiment we found the production of ROS were significantly reduced by addition of TriacsinC,but this effect decreased as the concentration of DHA increased.Subsequently,We selected3D7^wt?F0829 and F08B-4,which had no K13 gene mutation to prove our inference.Through the result,we found that TriacsinC and DHA combined to work on P.falciparum could significantly reduce the production of ROS than DHA work alone?*,P?0.05,***,P?0.001?.Above experiments help us to confirm that inhibited the activity of PfACS1a could reduce the generation of ROS,leading to reduced susceptibility of P.falciparum to artemisinin.To sum up,we concluded that the expression level and activity of PfACS1a affected the ROS production by DHA.The expression level and activity of PfACS1a affected the depolarization of mitochondrial membrane potential:According to above study,we speculated that ROS caused oxidative stress in parasites,and activated its apoptosis process so as to leading parasites died.To verify our hypothesis,we took fluorescent probe TMRE to detect the mitochondrial membrane potential of P.falciparum after co-culture parasites with DHA,the low fluoresce intensity meant the high depolarization of mitochondrial membrane potential.Our experiment found that mitochondrial membrane potential of parasites could significantly polarize under 10nM DHA?***,P?0.001?.Through detected mitochondrial membrane potential of 3D7^C580Ymut580Ymut and F0871ART,we concluded that down-regulated the expression of PfACS1a could lead to a decrease in the depolarization of mitochondrial membrane potential significantly under pressure of DHA?*,P?0.05,**,P?0.01?.Then we chose 3D7^wt,3D7^C580Ymut,F0871 and F0871ART to detect fluorescent of TMRE when parasites were co-cultured with 4?M TriacsinC and 10nM DHA and found that 4?M TriacsinC decreased the depolarization degree of mitochondrial membrane potential significantly than parasites cultured with DHA alone?*,P?0.05,***,P?0.001?.Above experiments showed that when the activity of PfACS1a was inhibited,the polarization of mitochondrial membrane potential caused by DHA was significantly reduced.To sum up,the expression level and activity of PfACS1a affected the degree of polarization of mitochondrial membrane potential caused by DHA.DHA promoted the expression level of apoptosis related protein:As we all knows,the depolarization of mitochondrial membrane potential is a sign of early apoptosis.In order to verify whether the parasites processed to apoptosis after mitochondrial membrane potential depolarization or not,we designed specific primers to detect the expression levels of caspase-like protein MCA1 and MCA2 after co-cultured with DHA by qPCR.The results showed that the expression levels of MCA1 in 3D7^wt?3D7^I543Tmut and 3D7^C580Ymut580Ymut elevated by 2.1,1.4 and 2.1 times respectively than control group and MCA2 were elevated by 2.25,1.4 and 2.2 times respectively than control group and MCA2 were elevated by 1.2 and 2.0 times after worked by 10nM DHA?*,P?0.05,**,P?0.01?,but the expression MCA2 in 3D7^I543Tmut543Tmut worked by DHA had no difference compared with control group.In order to further explore whether the up-regulated proteins came into play,we selected an inhibitor named Z-VAD-FMK that could inhibit activity of many kinds of caspases to co-cultured with parasites combined with DHA for 3h.The survival rate of P.falciparum could be significantly improved by Z-VAD-FMK?**,P?0.01,***,P?0.001?.Therefore,we confirmed that DHA could up-regulat expression of apoptosis related proteins and influenced the sensitivity of P.falciparum to DHA.Inhibited the activity of PfACS1a significantly reduced the fragmentation of the DNA caused by DHA.In order to confirm whether PfACS1a influenced the apoptosis process of parasites,we detected the target by TUNEL detection kit.We determined 20nM of DHA fragmented DNA of parasites significantly higher than control group?***,P?0.001?.Furthermore,we statistical compared the fragmentation rate of parasites which were co-cultured with 4?M TriacsinC and 20 nM DHA or with 20 nM DHA alone and found that TriacsinC decreased the proportion of the DNA fracture from 11.13%±0.88%to 6.53%±0.19%,which had significant statistical difference?*,P?0.05?.From experiments above,DHA could cause the fragment of DNA and led parasites to death,while inhibited the activity of PfACS1a could reduce the fragmentation caused by DHA.Summary:Our project obtained artemisinin resistance strains through drug pressure screening,and found a gene named PfACS1a down-regulated in artemisinin resistant strains by analysis of transcription spectrum.On this basis,transgenic strains contain mutation sites of F446I,I543T and C580Y in K13 gene were constructed and confirmed that the expression level of PfACS1a all down-regulated in K13 mutated strains by q-PCR.In addition,the expression level of PfACS1a of clinical strains with K13 mutation was significantly down-regulated than strains without.These results showed that expression level of PfACS1a was associated with artemisinin resistance.Our study preliminarily elucidated the mechanism of artemisinin resistance of P.falciparum:The mutation of PfK13 resulted in the combination of PfK13 and PfPI3K reduced and the affected the degradation of PfPI3K by ubiquitination.PfPI3K was accumulated in parasites and affected the expression of PfACS1a by some regulatory pathways.The down-regulated expression level and inhibited activity of PfACS1a reduced the ROS production by DHA,the degree of polarization of mitochondrial membrane potential in parasites decreased as well.The expression of caspase like proteins were increased by DHA pressure,which resulted in the death of parasites.Inhibition activity of PfACS1a could reduce the proportion of DNA fracture induced by DHA significantly.This article thoroughly clarified the molecular mechanism of PfACS1a in artemisinin resistance,which provided new research ideas and drug targets for malaria control.