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Atypical Molecular Basis for Drug Resistance to Mitochondrial Function Inhibitors in Plasmodium falciparum.

ABSTRACT: The continued emergence of drug-resistant Plasmodium falciparum parasites hinders global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Attractive targets for chemotherapeutic intervention are the cytochrome (cyt) bc ₁ complex, which is an essential component of the mitochondrial electron transport chain (mtETC) required for ubiquinone recycling and mitochondrially localized dihydroorotate dehydrogenase (DHODH) critical for de novo pyrimidine synthesis. Despite the essentiality of this complex, resistance to a novel acridone class of compounds targeting cyt bc ₁ was readily attained, resulting in a parasite strain (SB1-A6) that was panresistant to both mtETC and DHODH inhibitors. Here, we describe the molecular mechanism behind the resistance of the SB1-A6 parasite line, which lacks the common cyt bc ₁ point mutations characteristic of resistance to mtETC inhibitors. Using Illumina whole-genome sequencing, we have identified both a copy number variation (∼2×) and a single-nucleotide polymorphism (C276F) associated with pfdhodh in SB1-A6. We have characterized the role of both genetic lesions by mimicking the copy number variation via episomal expression of pfdhodh and introducing the identified single nucleotide polymorphism (SNP) using CRISPR-Cas9 and assessed their contributions to drug resistance. Although both of these genetic polymorphisms have been previously identified as contributing to both DSM-1 and atovaquone resistance, SB1-A6 represents a unique genotype in which both alterations are present in a single line, suggesting that the combination contributes to the panresistant phenotype. This novel mechanism of resistance to mtETC inhibition has critical implications for the development of future drugs targeting the bc ₁ complex or de novo pyrimidine synthesis that could help guide future antimalarial combination therapies and reduce the rapid development of drug resistance in the field.

SUBMITTER: Painter HJ

PROVIDER: S-EPMC8092505 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:INTRODUCTION:Malaria is a human disease of which causes high morbidity and mortality. In Plasmodium falciparum malaria, the resistance to antimalarial drugs, especially chloroquine (CQ) is one of the paramount factors contributing to the global increase in morbidity and mortality, due to malaria. Hence, there is a need for detection of chloroquine drug resistance genes i.e., pfcrt-o (Plasmodium falciparum chloroquine resistance transporter-o) and pfmdr-1 (Plasmodium falciparum multidrug resistance-1) of P. falciparum and pvcrt-o (Plasmodium vivax chloroquine resistance transporter-o) and pvmdr-1 (Plasmodium vivax multidrug resistance-1) of P. vivax by using molecular methods to prevent mortality in malarial cases. AIM:To standardize chloroquine drug sensitivity testing by molecular method so as to provide reports of chloroquine within 6-8 hours to physicians for better treatment. MATERIALS AND METHODS:This study was conducted over a period of one year from January to December 2014. A Total of 300 blood samples were collected from malaria suspected patient attending MGM Hospital, Kamothe, Navi Mumbai, India. Out of 300 blood samples, 44 were malaria positive as assessed by Thick and Thin blood smear stained, by Leishman's method and examination with light microscope. Chloroquine drug sensitivity testing was performed using WHO III plate method (micro test). Nested PCR was done for detection of pfcrt-o and pfmdr-1 for P. falciparum and pvcrt-o, pvmdr-1 genes for P. vivax. RESULTS:Total 44 samples were included in this study, out of which 22 samples confirmed for Plasmodium falciparum and 22 samples confirmed for Plasmodium vivax. Out of 22 P. falciparum 15 (68.18%) samples were chloroquine resistant. P. vivax showed chloroquine resistance to 5 samples (22.73%) by method similar to WHO III plate method (micro test) and nested PCR. CONCLUSION:Drug resistance testing by molecular methods is useful for early detection of antimalarial drug resistance. pfmdr-1 along with pfcrt-o can be used as biomarker for chloroquine drug resistance in P. falciparum and pvmdr-1 along with pvcrt-o for P. vivax.

Project description:This review article aims to investigate the genotypic profiles of Plasmodium falciparum and Plasmodium vivax isolates collected across a wide geographic region and their association with resistance to anti-malarial drugs used in Indonesia. A systematic review was conducted between 1991 and date. Search engines, such as PubMed, Science Direct, and Google Scholar, were used for articles published in English and Indonesian to search the literature. Of the 471 initially identified studies, 61 were selected for 4316 P. falciparum and 1950 P. vivax individual infections. The studies included 23 molecular studies and 38 therapeutic efficacy studies. K76T was the most common pfcrt mutation. K76N (2.1%) was associated with the haplotype CVMNN. By following dihydroartemisinin-piperaquine (DHA-PPQ) therapy, the mutant pfmdr1 alleles 86Y and 1034C were selected. Low prevalence of haplotype N86Y/Y184/D1246Y pfmdr1 reduces susceptibility to AS-AQ. SNP mutation pvmdr1 Y976F reached 96.1% in Papua and East Nusa Tenggara. Polymorphism analysis in the pfdhfr gene revealed 94/111 (84.7%) double mutants S108N/C59R or S108T/A16V in Central Java. The predominant pfdhfr haplotypes (based on alleles 16, 51, 59,108, 164) found in Indonesia were ANCNI, ANCSI, ANRNI, and ANRNL. Some isolates carried A437G (35.3%) or A437G/K540E SNPs (26.5%) in pfdhps. Two novel pfdhps mutant alleles, I588F/G and K540T, were associated with six pfdhps haplotypes. The highest prevalence of pvdhfr quadruple mutation (F57L/S58R/T61M/S117T) (61.8%) was detected in Papua. In pvdhps, the only polymorphism before and after 2008 was 383G mutation with 19% prevalence. There were no mutations in the pfk13 gene reported with validated and candidate or associated k13 mutation. An increased copy number of pfpm2, associated with piperaquine resistance, was found only in cases of reinfection. Meanwhile, mutation of pvk12 and pvpm4 I165V is unlikely associated with ART and PPQ drug resistance. DHA-PPQ is still effective in treating uncomplicated falciparum and vivax malaria. Serious consideration should be given to interrupt local malaria transmission and dynamic patterns of resistance to anti-malarial drugs to modify chemotherapeutic policy treatment strategies. The presence of several changes in pfk13 in the parasite population is of concern and highlights the importance of further evaluation of parasitic ART susceptibility in Indonesia.

Project description:BackgroundPrior to this project, only a handful of online visualizations existed for exploring the published literature on molecular markers of antimalarial drug resistance, and none specifically for the markers associated with Plasmodium falciparum resistance to the partner drugs in artemisinin-based combination therapy (ACT). Molecular information is collected in studies with different designs, using a variety of molecular methodologies and data analysis strategies, making it difficult to compare across studies. The purpose of this project was to develop a free online tool, which visualizes the widely published data on molecular markers of antimalarial drug resistance, starting with the two genes pfcrt and pfmdr-1, associated with resistance to the three most common partner drugs; amodiaquine, lumefantrine and mefloquine.MethodsA literature review was conducted, and a standardized method was used to extract data from publications, and critical decisions on visualization were made. A global geospatial database was developed of specific pfmdr1 and pfcrt single nucleotide polymorphisms and pfmdr1 copy number variation. An informatics framework was developed that allowed flexibility in development of the tool over time and efficient adaptation to different source data.ResultsThe database discussed in this paper has pfmdr1 and pfcrt marker prevalence information, from 579 geographic sites in 76 different countries, including results from over 86,000 samples from 456 articles published January 2001-May 2017. The ACT Partner Drugs Molecular Surveyor was launched by the WorldWide Antimalarial Resistance Network (WWARN) in March 2015 and it has attracted over 3000 unique visitors since then. Presented here is a demonstration of how the Surveyor database can be explored to monitor local, temporal changes in the prevalence of molecular markers. Here publications up to May 2017 were included, however the online ACT partner drug Molecular Surveyor is continuously updated with new data and relevant markers.ConclusionsThe WWARN ACT Partner Drugs Molecular Surveyor summarizes data on resistance markers in the pfmdr1 and pfcrt genes. The database is fully accessible, providing users with a rich resource to explore and analyze, and thus utilize a centralized, standardized database for different purposes. This open-source software framework can be adapted to other data, as demonstrated by the subsequent launch of the Artemisinin Molecular Surveyor and the Vivax Surveyor.

Project description:BackgroundCurrent malaria control and elimination strategies rely mainly on efficacious antimalarial drugs. However, drug resistance is a major threat facing malaria control programs. Determination of drug resistance molecular markers is useful in the monitoring and surveillance of malaria drug efficacy. This study aimed to determine the mutations and haplotypes frequencies of different genes linked with antimalarial drug resistance in certain areas in Sudan.MethodsA total of 226 dried blood spots (DBS) of microscopically diagnosed P. falciparum isolates were collected from Khartoum and three other areas in Sudan during 2015-2017. Plasmodium falciparum confirmation and multiplicity of infection was assessed using the Sanger's 101 SNPs-barcode and speciation was confirmed using regions of the parasite mitochondria. Molecular genotyping of drug resistance genes (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, exonuclease, Pfk13, parasite genetic background (PGB) (Pfarps10, ferredoxin, Pfcrt, Pfmdr2)) was also performed. All genotypes were generated by selective regions amplicon sequencing of the parasite genome using the Illumina MiSeq platform at the Wellcome Sanger Institute, UK then genotypes were translated into drug resistance haplotypes and species determination.FindingsIn total 225 samples were confirmed to be P. falciparum. A higher proportion of multiplicity of infection was observed in Gezira (P<0.001) based on the Sanger 101 SNPs -barcode. The overall frequency of mutant haplotype Pfcrt 72-76 CVIET was 71.8%. For Pfmdr1, N86Y was detected in 53.6%, Y184F was observed in 88.1% and D1246Y was detected in 1.5% of the samples. The most frequently observed haplotype was YFD 47.4%. For Pfdhfr (codons 51, 59,108,164), the ICNI haplotype was the most frequent (80.7%) while for Pfdhps (codons 436, 437, 540, 581, 613) the (SGEAA) was most frequent haplotype (41%). The Quadruple mutation (dhfr N51I, S108N + dhps A437G, K540E) was the highest frequent combined mutation (33.9%). In Pfkelch13 gene, 18 non-synonymous mutations were detected, 7 of them were detected in other African countries. The most frequent Pfk13 mutation was E433D detected in four samples. All of the Pfk13 mutant alleles have not been reported to belong to mutations associated with delayed parasite clearance in Southeast Asia. PGB mutations were detected only in Pfcrt N326S\I (46.3%) and Pfcrt I356T (8.2%). The exonuclease mutation was not detected. There was no significant variation in mutant haplotypes between study areas.ConclusionsThere was high frequency of mutations in Pfcrt, Pfdhfr and Pfdhps in this study. These mutations are associated with chloroquine and sulfadoxine-pyrimethamine (SP) resistance. Many SNPs in Pfk13 not linked with delayed parasite clearance were observed. The exonuclease E415G mutation which is linked with piperaquine resistance was not reported.

Project description:BACKGROUND:Plasmodium falciparum malaria is a public health problem worldwide. Malaria treatment policy has faced periodic changes due to emergence of drug resistant parasites. In Sudan chloroquine has been replaced by artesunate and sulfadoxine/pyrimethamine (AS/SP) in 2005 and to artemether-lumefantrine (AL) in 2017, due to the development of drug resistance. Different molecular markers have been used to monitor the status of drug resistant P. falciparum. This study aimed to determine the frequency of malaria drug resistance molecular markers in Southeast Sudan. METHODS:The samples of this study were day zero dried blood spot samples collected from efficacy studies in the Blue Nile State from November 2015 to January 2016. A total of 130 samples were amplified and sequenced using illumina Miseq platform. The molecular markers included were Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfk13, exonuclease and artemisinin resistant (ART-R) genetic background (Pfmdr2, ferroredoxine, Pfcrt and Pfarps10). RESULTS:Resistance markers for chloroquine were detected in 25.8% of the samples as mutant haplotype Pfcrt 72-76 CVIET and 21.7% Pfmdr1 86Y. Pfdhfr mutations were detected in codons 51, 59 and 108. The ICNI double-mutant haplotype was the most prevalent (69%). Pfdhps mutations were detected in codons 436, 437, 540, 581 and 613. The SGEGA triple-mutant haplotype was the most prevalent (43%). In Pfdhfr/Pfdhps combined mutation, quintuple mutation ICNI/SGEGA is the most frequent one (29%). Six of the seven treatment failure samples had quintuple mutation and the seventh was quadruple. This was significantly higher from the adequately responsive group (P?<?0.01). Pfk13 novel mutations were found in 7 (8.8%) samples, which were not linked to artemisinin resistance. Mutations in ART-R genetic background genes ranged from zero to 7%. Exonuclease mutation was not detected. CONCLUSION:In this study, moderate resistance to chloroquine and high resistance to SP was observed. Novel mutations of Pfk13 gene not linked to treatment failure were described. There was no resistance to piperaquine the partner drug of dihydroartemisinin/piperaquine (DHA-PPQ).

Project description:BackgroundDespite significant progress in eliminating malaria from the state of Odisha, India, the disease is still considered endemic. Artesunate plus sulfadoxine-pyrimethamine (AS + SP) has been introduced since 2010 as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study aimed to investigate the prevalence of mutations associated with resistance to chloroquine (CQ), sulfadoxine-pyrimethamine (SP), and artesunate (ART) in P. falciparum parasites circulating in the state.MethodsA total of 239 isolates of P. falciparum mono infection were collected during July 2018-November 2020 from the four different geographical regions of the state. Genomic DNA was extracted from 200 µL of venous blood and amplified using nested polymerase chain reaction. Mutations on gene associated with CQ (Pfcrt and Pfmdr1) were assessed by PCR amplification and restriction fragment length polymorphism, artemisinin (Pfk13) gene by DNA sequencing and SP (Pfdhfr and Pfdhps) genes by allele-specific polymerase chain reaction (AsPCR).ResultsThe point mutation in Pfcrt (K76T) was detected 2.1%, in Pfmdr1 (N86Y) 3.4%, and no mutations were found in Pfkelch13 propeller domain. Prevalence of Pfdhfr, Pfdhps and Pfhdfr-Pfdhps (two locus) gene mutations were 50.43%, 47.05% and 49.79% respectively. The single, double, triple and quadruple point mutations in Pfdhfr gene was 11.2%, 8.2%, 17.2% and 3.4% while, in Pfdhps gene was 10.9%,19.5%, 9.5% and 2.7% respectively. Of the total 13 haplotypes found in Pfdhfr, 8 were detected for the first time in the state and of the total 26 haplotypes found in Pfdhps, 7 were detected for the fisrt time in the state. The linked quintuple mutation Pfdhfr (N51I-C59R-S108N)-Pfdhps (A437G-K540E) responsible for clinical failure (RIII level of resistance) of SP resistance and A16V-S108T mutation in Pfdhfr responsible for cycloguanil was absent.ConclusionThe study has demonstrated a low prevalence of CQ resistance alleles in the study area. Despite the absence of the Pfkelch13 mutations, high prevalence of Pfdhfr and Pfdhps point mutations undermine the efficacy of SP partner drug, thereby threatening the P. falciparum malaria treatment policy. Therefore, continuous molecular and in vivo monitoring of ACT efficacy is warranted in Odisha.

Project description:The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.

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Atypical Molecular Basis for Drug Resistance to Mitochondrial Function Inhibitors in Plasmodium falciparum.

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