The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost

AbstractCurrent malaria control efforts rely significantly on artemisinin combinational therapies which have played massive roles in alleviating the global burden of the disease. Emergence of resistance to artemisinins is therefore, not just alarming but requires immediate intervention points such as development of new antimalarial drugs or improvement of the current drugs through adjuvant or combination therapies. Artemisinin resistance is primarily conferred by Kelch13 propeller mutations which are phenotypically characterised by generalised growth quiescence, altered haemoglobin trafficking and downstream enhanced activity of the parasite stress pathways through the ubiquitin proteasome system (UPS). Previous work on artemisinin resistance selection in a rodent model of malaria, which we and others have recently validated using reverse genetics, has also shown that mutations in deubiquitinating enzymes, DUBs (upstream UPS component) modulates susceptibility of malaria parasites to both artemisinin and chloroquine. The UPS or upstream protein trafficking pathways have, therefore, been proposed to be not just potential drug targets, but also possible intervention points to overcome artemisinin resistance. Here we report the activity of small molecule inhibitors targeting mammalian DUBs in malaria parasites. We show that generic DUB inhibitors can block intraerythrocytic development of malaria parasites in vitro and possess antiparasitic activity in vivo and can be used in combination with additive effect. We also show that inhibition of these upstream components of the UPS can potentiate the activity of artemisinin in vitro as well as in vivo to the extent that ART resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream 20s proteasome inhibitors are even more effective at improving the potency of artemisinins than either inhibitors alone providing proof that targeting multiple UPS activities simultaneously could be an attractive approach to overcoming artemisinin resistance. These data further validate the parasite UPS as a target to both enhance artemisinin action and potentially overcome resistance. Lastly, we confirm that DUB inhibitors can be developed into in vivo antimalarial drugs with promise for activity against all of human malaria and could thus further exploit their current pursuit as anticancer agents in rapid drug repurposing programs.Graphical abstract

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Longitudinal genomic surveillance of Plasmodium falciparum malaria parasites reveals complex genomic architecture of emerging artemisinin resistance

Genome Biology ◽

10.1186/s13059-017-1204-4 ◽

2017 ◽

Vol 18 (1) ◽

Cited By ~ 64

Author(s):

Gustavo C. Cerqueira ◽

Ian H. Cheeseman ◽

Steve F. Schaffner ◽

Shalini Nair ◽

Marina McDew-White ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Falciparum Malaria ◽

Artemisinin Resistance ◽

Plasmodium Falciparum Malaria ◽

Malaria Parasites ◽

Genomic Architecture

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Resistance to Artesunate + Mefloquine does not incurin fitness cost in malaria parasites

Malaria Journal ◽

10.1186/1475-2875-9-s2-p43 ◽

2010 ◽

Vol 9 (S2) ◽

Author(s):

Louise A Rodrigues ◽

Gisela Henriques ◽

Pedro Cravo

Keyword(s):

Fitness Cost ◽

Malaria Parasites

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EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

10.1101/752634 ◽

2019 ◽

Author(s):

Timothy Nessel ◽

John M. Beck ◽

Shima Rayatpisheh ◽

Yasaman Jami-Alahmadi ◽

James A. Wohlschlegel ◽

...

Keyword(s):

Genome Editing ◽

Translational Control ◽

Protein Identification ◽

Parasitophorous Vacuole ◽

Critical Role ◽

Transferase Activity ◽

Glutathione S Transferase ◽

Malaria Parasites ◽

Vacuole Membrane

AbstractIntraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane (PPM) and constitutes the barrier between parasite and host compartments. The PVM is the site of several essential transport activities but the basis for organization of this membrane system is unknown. We utilized the second-generation promiscuous biotin ligase BioID2 fused to EXP2 or HSP101 to probe the content of the PVM, identifying known and novel candidate PVM proteins. Among the best represented hits were members of a group of single-pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated the function of EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR-DOZI-aptamers system for conditional translational control. EXP1 knockdown was essential for intraerythrocytic development and accompanied by profound changes in vacuole ultrastructure, including increased separation of the PVM and PPM and formation of abnormal membrane structures in the enlarged vacuole lumen. While previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity, a mutant version of EXP1 lacking a residue important for this activity in vitro still provides substantial rescue of endogenous exp1 knockdown in vivo. Intriguingly, while activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore-forming protein EXP2 was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper PVM organization.ImportanceLike other obligate intracellular apicomplexans, blood-stage malaria parasites reside within a membrane-bound compartment inside the erythrocyte known as the parasitophorous vacuole. Although the vacuole is the site of several transport activities essential to parasite survival, little is known about its organization. To explore vacuole biology, we adopted recently developed proteomic (BioID2) and genetic (CRISPR/Cpf1) tools for use in Plasmodium falciparum, which allowed us to query the function of the prototypical vacuole membrane protein EXP1.Knockdown of EXP1 showed that a previously reported glutathione S-transferase activity cannot fully account for the essential function(s) of EXP1 and revealed a novel role for this protein in maintaining normal vacuole morphology and PVM protein arrangement. Our results provide new insight into vacuole organization and illustrate the power of BioID2 and Cpf1 (which utilizes a T-rich PAM uniquely suited to the P. falciparum genome) for proximity protein identification and genome editing in P. falciparum.

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Seasonal fluctuation of drug-resistant malaria parasites: a sign of fitness cost

Trends in Parasitology ◽

10.1016/j.pt.2009.05.006 ◽

2009 ◽

Vol 25 (8) ◽

pp. 351-352 ◽

Cited By ~ 11

Author(s):

Hamza A. Babiker

Keyword(s):

Seasonal Fluctuation ◽

Fitness Cost ◽

Drug Resistant ◽

Malaria Parasites

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An ER CREC family protein regulates the egress proteolytic cascade in malaria parasites

10.1101/457481 ◽

2018 ◽

Author(s):

Manuel A. Fierro ◽

Beejan Asady ◽

Carrie F. Brooks ◽

David W. Cobb ◽

Alejandra Villegas ◽

...

Keyword(s):

Protein Trafficking ◽

Double Mutant ◽

Essential Role ◽

Parasitophorous Vacuole ◽

Parasitophorous Vacuole Membrane ◽

Malaria Parasites ◽

Vacuole Membrane ◽

Proteolytic Cascade ◽

Family Protein ◽

Parasite Egress

AbstractThe endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be the calcium (Ca2+) signaling hub and required for biogenesis of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparumEndoplasmic Reticulum-resident Calcium-binding protein (PfERC), a member of the CREC family. Knockdown of PfERC shows that this gene is essential for asexual growth of P. falciparum. Analysis of the intraerythrocytic lifecycle revealed that PfERC is essential for parasite egress but not required for protein trafficking or Ca2+ storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease, SUB1. Using double mutant parasites, we show that PfERC is required for the proteolytic maturation of the essential aspartic protease, Plasmepsin X, which cleaves SUB1. Further, we show that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish the ER-resident CREC family protein, PfERC, as a key early regulator of the egress proteolytic cascade of malaria parasites.

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Plasmodium Species and Drug Resistance

10.5772/intechopen.98344 ◽

2021 ◽

Author(s):

Sintayehu Tsegaye Tseha

Keyword(s):

Drug Resistance ◽

Artemisinin Resistance ◽

Plasmodium Species ◽

Public Health Problem ◽

Antimalarial Drugs ◽

Chloroquine Resistance ◽

Resistance Pattern ◽

Malaria Parasites ◽

Human Malaria ◽

The World

Malaria is a leading public health problem in tropical and subtropical countries of the world. In 2019, there were an estimated 229 million malaria cases and 409, 000 deaths due malaria in the world. The objective of this chapter is to discuss about the different Plasmodium parasites that cause human malaria. In addition, the chapter discusses about antimalarial drugs resistance. Human malaria is caused by five Plasmodium species, namely P. falciparum, P. malariae, P. vivax, P. ovale and P. knowlesi. In addition to these parasites, malaria in humans may also arise from zoonotic malaria parasites, which includes P. inui and P. cynomolgi. The plasmodium life cycle involves vertebrate host and a mosquito vector. The malaria parasites differ in their epidemiology, virulence and drug resistance pattern. P. falciparum is the deadliest malaria parasite that causes human malaria. P. falciparum accounted for nearly all malarial deaths in 2018. One of the major challenges to control malaria is the emergence and spread of antimalarial drug-resistant Plasmodium parasites. The P. vivax and P. falciparum have already developed resistance against convectional antimalarial drugs such as chloroquine, sulfadoxine-pyrimethamine, and atovaquone. Chloroquine-resistance is connected with mutations in pfcr. Resistance to Sulfadoxine and pyrimethamine is associated with multiple mutations in pfdhps and pfdhfr genes. In response to the evolution of drug resistance Plasmodium parasites, artemisinin-based combination therapies (ACTs) have been used for the treatment of uncomplicated falciparum malaria since the beginning of 21th century. However, artemisinin resistant P. falciparum strains have been recently observed in different parts of the world, which indicates the possibility of the spread of artemisinin resistance to all over the world. Therefore, novel antimalarial drugs have to be searched so as to replace the ACTs if Plasmodium parasites develop resistance to ACTs in the future.

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Why are there so many independent origins of artemisinin resistance in malaria parasites?

10.1101/056291 ◽

2016 ◽

Cited By ~ 3

Author(s):

Timothy J.C. Anderson ◽

Shalini Nair ◽

Marina McDew-White ◽

Ian H. Cheeseman ◽

Standwell Nkhoma ◽

...

Keyword(s):

Amino Acid ◽

Population Size ◽

Effective Population Size ◽

Clearance Rate ◽

Retrospective Studies ◽

Artemisinin Resistance ◽

Target Size ◽

Effective Population ◽

Malaria Parasites ◽

Multiple Alleles

SummaryMultiple alleles at thekelch13locus conferring artemisinin resistance (ART-R) are currently spreading through malaria parasite populations in Southeast Asia, providing a unique opportunity to directly observe an ongoing soft selective sweep, to investigate why resistance alleles have evolved multiple times and to determine fundamental population genetic parameters for Plasmodium. We sequenced thekelch13gene (n=1,876), genotyped 75 flanking SNPs, and measured clearance rate (n=3,552) in parasite infections from Western Thailand (2001-2014). We describe 32 independent coding mutations: these included common mutations outside thekelch13propeller region associated with significant reductions in clearance rate. Mutations were first observed in 2003 and rose to 90% by 2014, consistent with a selection coefficient of ~0.079. There was no change in diversity in flanking markers, but resistance allele diversity rose until 2012 and then dropped as one allele (C580Y) spread to high frequency. The rapid spread of C580Y suggests that the genomic signature may be considerably harder in the near future, and that retrospective studies may underestimate the complexity of selective sweeps. The frequency with which adaptive alleles arise is determined by the rate of mutation to generate beneficial alleles and the population size. Two factors drive this soft sweep: (1) multiple amino-acid mutations inkelch13can confer resistance providing a large mutational target – we estimate the target size is between 87 and 163bp. (2) The population mutation parameter (Θ=2Neμ) can be estimated from the frequency distribution of resistant alleles and is ~ 5.69, suggesting that short term effective population size is between 88 thousand and 1.2 million. This is 52 to 705-fold greater thanNeestimates based on fluctuation in allele frequencies, suggesting that we have previously underestimated the capacity for adaptive evolution in Plasmodium. Our central conclusions are that retrospective studies may underestimate the complexity of selective events, ART-R evolution is not limited by availability of mutations, and theNerelevant for adaptation for malaria is considerably higher than previously estimated.Significance StatementPrevious work has identified surprisingly few origins of resistance to antimalarial drugs such as chloroquine and pyrimethamine. This has lead to optimism about prospects for minimizing resistance evolution through combination therapy. We studied a longitudinal collection of malaria parasites from the Thai-Myanmar border (2001–14) to examine an ongoing selective event in which ≥32 independent alleles associated with ART-R evolved. Three factors appear to explain the large number of origins observed: the large number of amino acid changes that result in resistance (i.e. large mutational “target size”), the large estimated effective population size (Ne), and the fact that we were able to document this selective event in real time, rather than retrospectively.

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