Abstract
Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for uncomplicated malaria caused by Plasmodium falciparum parasites. Other metabolites in A. annua or related species, particularly flavonoids, have been proposed to either act as antimalarials on their own or act synergistically with artemisinin to enhance antimalarial activity. We identified a mutation that disrupts the CHALCONE ISOMERASE 1 (CHI1) enzyme that is responsible for the second committed step of flavonoid biosynthesis. Detailed metabolite profiling revealed that chi1-1 lacks all major flavonoids but produces wild-type artemisinin levels, making this mutant a useful tool to test the antiplasmodial effects of flavonoids. We used whole-leaf extracts from chi1-1 and mutant lines impaired in artemisinin production in bioactivity in vitro assays against intraerythrocytic P. falciparum Dd2. We found that chi1-1 extracts did not differ from wild-type extracts in antiplasmodial efficacy nor initial rate of cytocidal action. Furthermore, extracts from the A. annua cyp71av1-1 mutant and RNAi lines impaired in amorpha-4,11-diene synthase gene expression, which are both severely compromised in artemisinin biosynthesis but unaffected in flavonoid metabolism, showed very low or no antiplasmodial activity. These results demonstrate that in vitro bioactivity against P. falciparum of flavonoids is negligible when compared to that of artemisinin.
Highlights
Malaria is one of the most prevalent infectious diseases with 219 million cases and 435,000 deaths reported in 2017 (World Health Organization [WHO], 2018)
We report the identification and characterization of an A. annua mutant in CHALCONE ISOMERASE 1 (CHI1), which encodes the enzyme that catalyzes the second committed step of the flavonoid biosynthesis pathway
The chi1-1 mutation is predicted to result in a truncation that would preclude a sizable portion of the CHI1 functional monomer, including sections that may interact with the product naringenin (Figure 1C and Supplementary Figure S1A)
Summary
Malaria is one of the most prevalent infectious diseases with 219 million cases and 435,000 deaths reported in 2017 (World Health Organization [WHO], 2018). Recent publications have reported that A. annua wholeplant preparations are more effective than artemisinin alone (not ACTs) in treating rodent malaria (Elfawal et al, 2012) and reducing the development of resistance (Elfawal et al, 2015), and that whole-plant preparations may be effective in treating artesunate-resistant malaria patients (Daddy et al, 2017) These results suggest that A. annua produces metabolites that might act together with artemisinin and whole-plant preparations have been proposed as replacement treatments for ACTs (Weathers et al, 2014). Cyp71av completely abolishes artemisinin production and redirects the artemisinin pathway to the synthesis of arteannuin X, a novel sesquiterpene epoxide (Czechowski et al, 2016) Both the AMS silenced and cyp71av mutant lines produce wild-type levels of major flavonoids. We have performed a comparative analysis of wholeleaf extracts from chi, the AMS silenced line, cyp71av, and wild-type A. annua in in vitro P. falciparum Dd2 kill assays (Ullah et al, 2017) to determine the antiplasmodial efficacy and initial cytocidal activity of these extracts
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