Combined Transcriptome and Proteome Profiling for Role of pfEMP1 in Antimalarial Mechanism of Action of Dihydroartemisinin

Malaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation, with artemisinin combination therapies as the first-line treatments. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach.

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Little Polymorphism at the K13 Propeller Locus in Worldwide Plasmodium falciparum Populations Prior to the Introduction of Artemisinin Combination Therapies

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02370-15 ◽

2016 ◽

Vol 60 (6) ◽

pp. 3340-3347 ◽

Cited By ~ 17

Author(s):

Toshihiro Mita ◽

Richard Culleton ◽

Nobuyuki Takahashi ◽

Masatoshi Nakamura ◽

Takahiro Tsukahara ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Haplotype Diversity ◽

Artemisinin Resistance ◽

Pairwise Comparison ◽

Purifying Selection ◽

Parasite Clearance ◽

Combination Therapies ◽

Content Type ◽

Exposed Population ◽

Artemisinin Combination Therapies

The emergence and spread of artemisinin-resistantPlasmodium falciparumis of huge concern for the global effort toward malaria control and elimination. Artemisinin resistance, defined as a delayed time to parasite clearance following administration of artemisinin, is associated with mutations in thePfkelch13gene of resistant parasites. To date, as many as 60 nonsynonymous mutations have been identified in this gene, but whether these mutations have been selected by artemisinin usage or merely reflect natural polymorphism independent of selection is currently unknown. To clarify this, we sequenced thePfkelch13propeller domain in 581 isolates collected before (420 isolates) and after (161 isolates) the implementation of artemisinin combination therapies (ACTs), from various regions of endemicity worldwide. Nonsynonymous mutations were observed in 1% of parasites isolated prior to the introduction of ACTs. Frequencies of mutant isolates, nucleotide diversity, and haplotype diversity were significantly higher in the parasites isolated from populations exposed to artemisinin than in those from populations that had not been exposed to the drug. In the artemisinin-exposed population, a significant excess of dN compared to dS was observed, suggesting the presence of positive selection. In contrast, pairwise comparison of dN and dS and the McDonald and Kreitman test indicate that purifying selection acts on thePfkelch13propeller domain in populations not exposed to ACTs. These population genetic analyses reveal a low baseline ofPfkelch13polymorphism, probably due to purifying selection in the absence of artemisinin selection. In contrast, variousPfkelch13mutations have been selected under artemisinin pressure.

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In VitroActivities of Primaquine-Schizonticide Combinations on Asexual Blood Stages and Gametocytes of Plasmodium falciparum

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7650-7656 ◽

Cited By ~ 11

Author(s):

Mynthia Cabrera ◽

Liwang Cui

Keyword(s):

Plasmodium Falciparum ◽

World Health ◽

Combination Therapies ◽

Content Type ◽

Synergistic Interactions ◽

Health Organization ◽

Potential Interactions ◽

Artemisinin Combination Therapies ◽

Antagonistic Interactions

ABSTRACTCurrently, the World Health Organization recommends addition of a 0.25-mg base/kg single dose of primaquine (PQ) to artemisinin combination therapies (ACTs) forPlasmodium falciparummalaria as a gametocytocidal agent for reducing transmission. Here, we investigated the potential interactions of PQ with the long-lasting components of the ACT drugs for eliminating the asexual blood stages and gametocytes ofin vitro-culturedP. falciparumstrains. Using the SYBR green I assay for asexual parasites and a flow cytometry-based assay for gametocytes, we determined the interactions of PQ with the schizonticides chloroquine, mefloquine, piperaquine, lumefantrine, and naphthoquine. With the sums of fractional inhibitory concentrations and isobolograms, we were able to determine mostly synergistic interactions for the various PQ and schizonticide combinations on the blood stages ofP. falciparumlaboratory strains. The synergism in inhibiting asexual stages and gametocytes was highly evident with PQ-naphthoquine, whereas synergism was moderate for the PQ-piperaquine, PQ-chloroquine, and PQ-mefloquine combinations. We have detected potentially antagonistic interactions between PQ and lumefantrine under certain drug combination ratios, suggesting that precautions might be needed when PQ is added as the gametocytocide to the artemether-lumefantrine ACT (Coartem).

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Identification of Mutations in Antimalarial Resistance Gene Kelch13 from Plasmodium falciparum Isolates in Kano, Nigeria

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed5020085 ◽

2020 ◽

Vol 5 (2) ◽

pp. 85

Author(s):

Umar F. Abubakar ◽

Ruqayya Adam ◽

Muhammad M. Mukhtar ◽

Abdullahi Muhammad ◽

Adamu A. Yahuza ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Potential Role ◽

Parasite Clearance ◽

Falciparum Infection ◽

Combination Therapies ◽

First Line ◽

High Prevalence ◽

Antimalarial Resistance ◽

Artemisinin Combination Therapies

Malaria control relies on first-line treatments that use artemisinin-combination therapies (ACT). Unfortunately, mutations in the plasmodium falciparum kelch13 gene result in delayed parasite clearance. Research on what is causing ACT failure is non-existent in northwestern Nigeria. Thus, the presence of mutations in kelch13 in P. falciparum isolates from Kano, Nigeria was investigated in this study. Microscopic examination of 154 blood samples obtained from patients revealed a high prevalence of P. falciparum infection (114 positive individuals, slide positivity rate = 74.03%). The 114 patients were administered Cartef® (ACT) and out of the 50 patients that returned for the 14-day follow up, 11 were positive for P. falciparum (slide positivity rate = 22%). On day 0, 80 samples out of 114 and 11 samples on day 14 (91 out of 125 microscopy-positive samples) were positive with Plasmodium according to the PCR of cytochrome oxidase I, which corresponds to 72.8%. A fragment of the kelch13 gene encompassing the propeller domains was sequenced in 49 samples, alongside samples of the susceptible strain pf_3D7. Low polymorphism was observed, suggesting a lack of selection on this gene, and only six mutations (Glu433Gly, Phe434Ile, Phe434Ser, Ile684Asn, Ile684Thr and Glu688Lys) were found. The epidemiologic impact of these mutations and their potential role in ACT resistance needs to be investigated further.

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Comparative Efficacies of Artemisinin Combination Therapies in Plasmodium falciparum Malaria and Polymorphism ofpfATPase6,pfcrt,pfdhfr, andpfdhpsGenes in Tea Gardens of Jalpaiguri District, India

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05388-11 ◽

2012 ◽

Vol 56 (5) ◽

pp. 2511-2517 ◽

Cited By ~ 25

Author(s):

Pabitra Saha ◽

Subhasish K. Guha ◽

Sonali Das ◽

Shrabanee Mullick ◽

Swagata Ganguly ◽

...

Keyword(s):

Plasmodium Falciparum Malaria ◽

Study Groups ◽

Combination Therapies ◽

Antimalarial Drug Resistance ◽

Content Type ◽

Private Practitioners ◽

Mutational Pattern ◽

Sequencing Method ◽

Periodic Monitoring ◽

Artemisinin Combination Therapies

ABSTRACTIn India, chloroquine has been replaced by a combination of artesunate and sulfadoxine-pyrimethamine (AS-SP) for uncomplicatedP. falciparummalaria. Other available combinations, artemether-lumefantrine (AM-LF) and artesunate-mefloquine (AS-MQ), not included in the national program, are widely used by private practitioners. Little is known about the therapeutic efficacy of these artemisinin combinations and the prevalence of molecular markers associated with antimalarial drug resistance. A total of 157 patients withP. falciparummonoinfection were recruited and randomized into three study groups (AS-SP, AM-LF, and AS-MQ). All patients were followed up for 42 days to study the clinical and parasitological responses according to the WHO protocol (2009). We assessed the polymorphism of thepfATPase6,pfcrt,pfdhfr, andpfdhpsgenes by the DNA-sequencing method. The PCR-corrected therapeutic efficacies of AS-SP, AM-LF, and AS-MQ were 90.6% (95% confidence interval [CI], 0.793 to 0.969), 95.9% (95% CI, 0.860 to 0.995), and 100% (95% CI, 0.927 to 1.00), respectively. No specific mutational pattern was observed in thepfATPase6gene. All isolates had a K76T mutation in thepfcrtgene. In thepfdhfr-pfdhpsgenotype, quadruple mutation was frequent, and quintuple mutation was documented in 6.3% ofP. falciparumisolates. The significant failure rate of AS-SP (9.5%), although within the limit (10%) for drug policy change, was due to SP failure because of prevailing mutations inpfdhfr, I51R59N108, withpfdhps, G437and/or E540. The efficacy of this ACT needs periodic monitoring. Artemether-lumefantrine and artesunate-mefloquine are effective alternatives to the artesunate-sulfadoxine-pyrimethamine combination.

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Shedding Light on the Role of the Skin in Vaccine-Induced Protection against the Malaria Sporozoite

mBio ◽

10.1128/mbio.02555-18 ◽

2018 ◽

Vol 9 (6) ◽

Author(s):

Johanna Patricia Daily

Keyword(s):

Plasmodium Falciparum ◽

Blood Vessels ◽

Blood Stream ◽

Mosquito Vectors ◽

Protective Mechanisms ◽

Partial Protection ◽

Content Type ◽

Shed Light ◽

In Infants And Children

ABSTRACT The most advanced vaccine against Plasmodium falciparum malaria, RTS,S/AS01, provides partial protection in infants and children living in areas of malaria endemicity. Further understanding its mechanisms of protection may allow the development of improved second-generation vaccines. The RTS,S/AS01 vaccine targets the sporozoites injected by mosquito vectors into the dermis which then travel into the blood stream to establish infection in the liver. Flores-Garcia et al. (Y. Flores-Garcia, G. Nasir, C. S. Hopp, C. Munoz, et al., mBio 9:e02194-18, 2018, https://doi.org/10.1128/mBio.02194-18) shed light on early protective responses occurring in the dermis in immunized animals. They demonstrated that immunization impairs sporozoite motility and entry into blood vessels. Furthermore, they established that challenge experiments performed using a dermal route conferred greater protection than intravenous challenge in immunized mice. Thus, the dermal challenge approach captures the additional protective mechanisms occurring in the dermis that reflect the natural physiology of infection. Those studies highlighted the fascinating biology of skin-stage sporozoites and provided additional insights into vaccine-induced protection.

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In Silico Investigation of the Decline in Clinical Efficacy of Artemisinin Combination Therapies Due to Increasing Artemisinin and Partner Drug Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01292-18 ◽

2018 ◽

Vol 62 (12) ◽

Cited By ~ 1

Author(s):

Sophie G. Zaloumis ◽

Pengxing Cao ◽

Saber Dini ◽

Miles P. Davenport ◽

Deborah Cromer ◽

...

Keyword(s):

Drug Resistance ◽

Treatment Failure ◽

In Silico ◽

Artemisinin Resistance ◽

Exact Nature ◽

Artemisinin Derivative ◽

Combination Therapies ◽

Content Type ◽

Artemisinin Derivatives ◽

The Impact

ABSTRACT Antimalarial treatment currently relies on an artemisinin derivative and a longer-acting partner drug. With the emergence of resistance to the artemisinin derivatives and the potential pressure this exerts on the partner drugs, the impact of resistance to each drug on efficacy needs to be investigated. An in silico exploration of dihydroartemisinin-piperaquine and mefloquine-artesunate, two artemisinin-based combination therapies that are commonly used in Southeast Asia, was performed. The percentage of treatment failures was simulated from a within-host pharmacokinetic-pharmacodynamic (PKPD) model, assuming that parasites developed increasing levels of (i) artemisinin derivative resistance or (ii) concomitant resistance to both the artemisinin derivative and the partner drug. Because the exact nature of how resistant Plasmodium falciparum parasites respond to treatment is unknown, we examined the impact on treatment failure rates of artemisinin resistance that (i) reduced the maximal killing rate, (ii) increased the concentration of drug required for 50% killing, or (iii) shortened the window of parasite stages that were susceptible to artemisinin derivatives until the drugs had no effect on the ring stages. The loss of the ring-stage activity of the artemisinin derivative caused the greatest increase in the treatment failure rate, and this result held irrespective of whether partner drug resistance was assumed to be present or not. To capture the uncertainty regarding how artemisinin derivative and partner drug resistance affects the assumed concentration-killing effect relationship, a variety of changes to this relationship should be considered when using within-host PKPD models to simulate clinical outcomes to guide treatment strategies for resistant infections.

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Molecular Surveillance of Drug Resistance of Plasmodium falciparum Isolates Imported from Angola in Henan Province, China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00552-19 ◽

2019 ◽

Vol 63 (10) ◽

Cited By ~ 1

Author(s):

Ruimin Zhou ◽

Chengyun Yang ◽

Suhua Li ◽

Yuling Zhao ◽

Ying Liu ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Antimalarial Drug ◽

Henan Province ◽

Nucleotide Polymorphisms ◽

Combination Therapies ◽

Antimalarial Drug Resistance ◽

Single Nucleotide ◽

Molecular Surveillance ◽

Content Type

ABSTRACT Angola was the main origin country for the imported malaria in Henan Province, China. Antimalarial drug resistance has posed a threat to the control and elimination of malaria. Several molecular markers were confirmed to be associated with the antimalarial drug resistance, such as pfcrt, pfmdr1, pfdhfr, pfdhps, and K13. This study evaluated the drug resistance of the 180 imported Plasmodium falciparum isolates from Angola via nested PCR using Sanger sequencing. The prevalences of pfcrt C72V73M74N75K76, pfmdr1 N86Y184S1034N1042D1246, pfdhfr A16N51C59S108D139I164, and pfdhps S436A437A476K540A581 were 69.4%, 59.9%, 1.3% and 6.3%, respectively. Three nonsynonymous (A578S, M579I, and Q613E) and one synonymous (R471R) mutation of K13 were found, the prevalences of which were 2.5% and 1.3%, respectively. The single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, and pfdhps were generally shown as multiple mutations. The mutant prevalence of pfcrt reduced gradually, but pfdhfr and pfdhps still showed high mutant prevalence, while pfmdr1 was relatively low. The mutation of the K13 gene was rare. Molecular surveillance of artemisinin (ART) resistance will be used as a tool to evaluate the real-time efficacy of the artemisinin-based combination therapies (ACTs) and the ART resistance situation.

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Expansion of a Specific Plasmodium falciparum PfMDR1 Haplotype in Southeast Asia with Increased Substrate Transport

mBio ◽

10.1128/mbio.02093-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Carla Calçada ◽

Miguel Silva ◽

Vitória Baptista ◽

Vandana Thathy ◽

Rita Silva-Pedrosa ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Southeast Asia ◽

Gene Copy ◽

Multidrug Resistance Protein ◽

Combination Therapies ◽

Transport Capacity ◽

Multidrug Resistance Protein 1 ◽

Content Type ◽

Resistance Protein

ABSTRACT Artemisinin-based combination therapies (ACTs) have been vital in reducing malaria mortality rates since the 2000s. Their efficacy, however, is threatened by the emergence and spread of artemisinin resistance in Southeast Asia. The Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) transporter plays a central role in parasite resistance to ACT partner drugs through gene copy number variations (CNV) and/or single nucleotide polymorphisms (SNPs). Using genomic epidemiology, we show that multiple pfmdr1 copies encoding the N86 and 184F haplotype are prevalent across Southeast Asia. Applying genome editing tools on the Southeast Asian Dd2 strain and using a surrogate assay to measure transporter activity in infected red blood cells, we demonstrate that parasites harboring multicopy N86/184F PfMDR1 have a higher Fluo-4 transport capacity compared with those expressing the wild-type N86/Y184 haplotype. Multicopy N86/184F PfMDR1 is also associated with decreased parasite susceptibility to lumefantrine. These findings provide evidence of the geographic selection and expansion of specific multicopy PfMDR1 haplotypes associated with multidrug resistance in Southeast Asia. IMPORTANCE Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target Plasmodium asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the P. falciparum multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a pfmdr1 gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of pfmdr1 polymorphisms.

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Protein Kinase A Is Essential for Invasion of Plasmodium falciparum into Human Erythrocytes

mBio ◽

10.1128/mbio.01972-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 8

Author(s):

Mary-Louise Wilde ◽

Tony Triglia ◽

Danushka Marapana ◽

Jennifer K. Thompson ◽

Alexei A. Kouzmitchev ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Protein Kinase ◽

Protein Kinase A ◽

Host Cell ◽

Cell Invasion ◽

Blood Cells ◽

Host Cell Invasion ◽

Parasite Invasion ◽

Content Type ◽

Kinase A

ABSTRACT Understanding the mechanisms behind host cell invasion by Plasmodium falciparum remains a major hurdle to developing antimalarial therapeutics that target the asexual cycle and the symptomatic stage of malaria. Host cell entry is enabled by a multitude of precisely timed and tightly regulated receptor-ligand interactions. Cyclic nucleotide signaling has been implicated in regulating parasite invasion, and an important downstream effector of the cAMP-signaling pathway is protein kinase A (PKA), a cAMP-dependent protein kinase. There is increasing evidence that P. falciparum PKA (PfPKA) is responsible for phosphorylation of the cytoplasmic domain of P. falciparum apical membrane antigen 1 (PfAMA1) at Ser610, a cAMP-dependent event that is crucial for successful parasite invasion. In the present study, CRISPR-Cas9 and conditional gene deletion (dimerizable cre) technologies were implemented to generate a P. falciparum parasite line in which expression of the catalytic subunit of PfPKA (PfPKAc) is under conditional control, demonstrating highly efficient dimerizable Cre recombinase (DiCre)-mediated gene excision and complete knockdown of protein expression. Parasites lacking PfPKAc show severely reduced growth after one intraerythrocytic growth cycle and are deficient in host cell invasion, as highlighted by live-imaging experiments. Furthermore, PfPKAc-deficient parasites are unable to phosphorylate PfAMA1 at Ser610. This work not only identifies an essential role for PfPKAc in the P. falciparum asexual life cycle but also confirms that PfPKAc is the kinase responsible for phosphorylating PfAMA1 Ser610. IMPORTANCE Malaria continues to present a major global health burden, particularly in low-resource countries. Plasmodium falciparum, the parasite responsible for the most severe form of malaria, causes disease through rapid and repeated rounds of invasion and replication within red blood cells. Invasion into red blood cells is essential for P. falciparum survival, and the molecular events mediating this process have gained much attention as potential therapeutic targets. With no effective vaccine available, and with the emergence of resistance to antimalarials, there is an urgent need for the development of new therapeutics. Our research has used genetic techniques to provide evidence of an essential protein kinase involved in P. falciparum invasion. Our work adds to the current understanding of parasite signaling processes required for invasion, highlighting PKA as a potential drug target to inhibit invasion for the treatment of malaria.

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