Roland A. Cooper

Mutations in the P. falciparum Digestive Vacuole Transmembrane Protein PfCRT and Evidence for Their Role in Chloroquine Resistance

The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K761 mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.

Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum

Widespread use of antimalarial agents can profoundly influence the evolution of the human malaria parasite Plasmodium falciparum. Recent selective sweeps for drug-resistant genotypes may have restricted the genetic diversity of this parasite, resembling effects attributed in current debates to a historic population bottleneck. Chloroquine-resistant (CQR) parasites were initially reported about 45 years ago from two foci in southeast Asia and South America, but the number of CQR founder mutations and the impact of chlorquine on parasite genomes worldwide have been difficult to evaluate. Using 342 highly polymorphic microsatellite markers from a genetic map, here we show that the level of genetic diversity varies substantially among different regions of the parasite genome, revealing extensive linkage disequilibrium surrounding the key CQR gene pfcrt and at least four CQR founder events. This disequilibrium and its decay rate in the pfcrt-flanking region are consistent with strong directional selective sweeps occurring over only ∼20–80 sexual generations, especially a single resistant pfcrt haplotype spreading to very high frequencies throughout most of Asia and Africa. The presence of linkage disequilibrium provides a basis for mapping genes under drug selection in P. falciparum.

Allelic modifications of the cg2 and cg1 genes do not alter the chloroquine response of drug-resistant Plasmodium falciparum

The determinant of chloroquine resistance (CQR) in a Plasmodium falciparum cross was previously mapped by linkage analysis to a 36 kb segment of chromosome 7. Candidate genes within this segment have been previously shown to include two genes, cg2 and cg1, that have complex polymorphisms linked to the CQR phenotype. Using DNA transfection and allelic exchange, we have replaced these polymorphisms in CQR parasites with cg2 and cg1 sequences from chloroquine sensitive parasites. Drug assays of the allelically-modified lines show no change in the degree of CQR, providing evidence against the hypothesis that these polymorphisms are important to the CQR phenotype. Similarly, no change was found in the degree to which verapamil or other chloroquine sensitizers reverse CQR in the transformants. These results and the high though not complete degree of association of CQR with cg2 and cg1 polymorphisms in field isolates suggest involvement of another nearby gene in the P. falciparum CQR mechanism.

Plasmepsin 4, the food vacuole aspartic proteinase found in all Plasmodium spp. infecting man.

Plasmepsins are aspartic proteinases of the malaria parasite, and seven groups of plasmepsins have been identified by comparing genomic sequence data available for the genes encoding these enzymes from Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium berghei, and Plasmodium yoelii. The food vacuole plasmepsins typified by plasmepsin 4 from P. falciparum (PfPM4) constitute one of these groups. Genes encoding the ortholog of PfPM4 have been cloned from Plasmodium ovale, Plasmodium malariae, and P. vivax. In addition, P. falciparum contains three paralagous food vacuole plasmepsins or plasmepsin-like enzymes that appear to have arisen by gene duplication, plasmepsins 1 (PfPM1), 2 (PfPM2) and HAP, and all four were localized to purified food vacuole preparations by two-dimensional gel electrophoresis and mass spectroscopic analysis. The three paralogs of PfPM4 do not have counterparts in the six other Plasmodium spp. examined by genomic DNA blot analysis and by review of available genomic sequence data. The presence of these paralogs among the food vacuole plasmepsins in P. falciparum as compared with the other three species causing malaria in man will impact efforts to rationally design antimalarials targeting the food vacuole plasmepsins.

P. falciparum CG2, linked to chloroquine resistance, does not resemble Na+/H+ exchangers

Matters Arising occurs in known exchanger proteins, is highly im-P. falciparum CG2, Linked probable. homology proposed by Sanchez et al. (1998), and the results of database searches, have been reanalyzed by Understanding the molecular basis for chloroquine re-established statistical methods (Altschul et al., 1994; Altschul and Gish, 1996). No pairwise alignment scores sistance in Plasmodium falciparum will provide important support for the development of new therapies and between the proposed CG2 domain and known NHE transport domains approach even borderline statistical prophylactic measures against malaria. Complementary genetic and biochemical investigations should discrimi-significance. All of these scores have a 0.8 probability or greater of occurring in random pairwise alignments nate among current theories and pinpoint the functional determinants of resistance. With this in mind, Sanchez of protein sequences with the same sizes and compositions. Moreover, the proposed CG2 domain shows con-et al. (1998) have proposed that the P. falciparum cg2 gene, linked by Su et al. (1997) to chloroquine resistance, siderably stronger (but still nonsignificant) scores in alignments with many proteins in current databases that may encode a sodium/hydrogen exchanger (NHE) responsible for drug transport. Here, we present evidence are not transporters. Third, Sanchez et al. (1998) claim that the motif against this proposal. Detailed reanalysis of the CG2 sequence fails to support the claims for significant simi-VFFLFSI in CG2 resembles the characteristic amiloride binding signature VFFLFLL in NHE sequences. They give larity to functional features of well-characterized eukary-otic NHE transport domains. a probability of 8 ϫ 10 Ϫ4 for the chance occurrence of VFFLF in a random sequence of CG2s size and compo-First, although the CG2 sequence has some clusters of hydrophobic amino acids, these are not typical of sition. However, the actual chance of finding a similar or stronger match to VFFLFLL in P. falciparum proteins known types of integral membrane proteins, including those from P. falciparum. Of the seven potential trans-is much greater than this because of the strong, nonran-dom, compositional bias and amino acid clustering membrane segments annotated on the CG2 sequence in Figure 3 of Su et al. (1997), six gave only marginal characteristic of this parasite. The residues encoded by AU-rich codon sets (such as F, L, I, N, K, and Y) in P. predictions in the three algorithms used. Figure 1 shows results from two additional well-validated algorithms, falciparum are unusually abundant, and these amino acids also have a strong tendency to occur clustered …

A potent antimalarial benzoxaborole targets a Plasmodium falciparum cleavage and polyadenylation specificity factor homologue

Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against Plasmodium falciparum laboratory-adapted strains (mean IC50 32 nM), Ugandan field isolates (mean ex vivo IC50 64 nM), and murine P. berghei and P. falciparum infections (day 4 ED90 0.34 and 0.57 mg kg−1, respectively). Multiple P. falciparum lines selected in vitro for resistance to AN3661 harboured point mutations in pfcpsf3, which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of pfcpsf3 mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target.

Impact of Antimalarial Treatment and Chemoprevention on the Drug Sensitivity of Malaria Parasites Isolated from Ugandan Children

ABSTRACT Changing treatment practices may be selecting for changes in the drug sensitivity of malaria parasites. We characterized ex vivo drug sensitivity and parasite polymorphisms associated with sensitivity in 459 Plasmodium falciparum samples obtained from subjects enrolled in two clinical trials in Tororo, Uganda, from 2010 to 2013. Sensitivities to chloroquine and monodesethylamodiaquine varied widely; sensitivities to quinine, dihydroartemisinin, lumefantrine, and piperaquine were generally good. Associations between ex vivo drug sensitivity and parasite polymorphisms included decreased chloroquine and monodesethylamodiaquine sensitivity and increased lumefantrine and piperaquine sensitivity with pfcrt 76T, as well as increased lumefantrine sensitivity with pfmdr1 86Y, Y184, and 1246Y. Over time, ex vivo sensitivity decreased for lumefantrine and piperaquine and increased for chloroquine, the prevalences of pfcrt K76 and pfmdr1 N86 and D1246 increased, and the prevalences of pfdhfr and pfdhps polymorphisms associated with antifolate resistance were unchanged. In recurrent infections, recent prior treatment with artemether-lumefantrine was associated with decreased ex vivo lumefantrine sensitivity and increased prevalence of pfcrt K76 and pfmdr1 N86, 184F, and D1246. In children assigned chemoprevention with monthly dihydroartemisinin-piperaquine with documented circulating piperaquine, breakthrough infections had increased the prevalence of pfmdr1 86Y and 1246Y compared to untreated controls. The noted impacts of therapy and chemoprevention on parasite polymorphisms remained significant in multivariate analysis correcting for calendar time. Overall, changes in parasite sensitivity were consistent with altered selective pressures due to changing treatment practices in Uganda. These changes may threaten the antimalarial treatment and preventive efficacies of artemether-lumefantrine and dihydroartemisinin-piperaquine, respectively.

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite’s food vacuole and alter drug sensitivities

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.

ABSTRACT There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum. Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [14C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.

Changing antimalarial drug sensitivities in Uganda

ABSTRACT Dihydroartemisinin-piperaquine (DP) has demonstrated excellent efficacy for the treatment and prevention of malaria in Uganda. However, resistance to both components of this regimen has emerged in Southeast Asia. The efficacy of artemether-lumefantrine, the first-line regimen to treat malaria in Uganda, has also been excellent, but continued pressure may select for parasites with decreased sensitivity to lumefantrine. To gain insight into current drug sensitivity patterns, ex vivo sensitivities were assessed and genotypes previously associated with altered drug sensitivity were characterized for 58 isolates collected in Tororo, Uganda, from subjects presenting in 2016 with malaria from the community or as part of a clinical trial comparing DP chemoprevention regimens. Compared to community isolates, those from trial subjects had lower sensitivities to the aminoquinolines chloroquine, monodesethyl amodiaquine, and piperaquine and greater sensitivities to lumefantrine and mefloquine, an observation consistent with DP selection pressure. Compared to results for isolates from 2010 to 2013, the sensitivities of 2016 community isolates to chloroquine, amodiaquine, and piperaquine improved (geometric mean 50% inhibitory concentrations [IC50] = 248, 76.9, and 19.1 nM in 2010 to 2013 and 33.4, 14.9, and 7.5 nM in 2016, respectively [P < 0.001 for all comparisons]), the sensitivity to lumefantrine decreased (IC50 = 3.0 nM in 2010 to 2013 and 5.4 nM in 2016 [P < 0.001]), and the sensitivity to dihydroartemisinin was unchanged (IC50 = 1.4 nM). These changes were accompanied by decreased prevalence of transporter mutations associated with aminoquinoline resistance and low prevalence of polymorphisms recently associated with resistance to artemisinins or piperaquine. Antimalarial drug sensitivities are changing in Uganda, but novel genotypes associated with DP treatment failure in Asia are not prevalent.