The first line of defense operates at the parasite membrane. By mutating, deleting, or amplifying transporters, parasites can keep intracellular drug concentrations below toxic thresholds even when patients receive correctly dosed regimens. In Plasmodium falciparum, the digestive-vacuole transporter PfCRT exports protonated chloroquine away from its site of action, undermining the drug’s interference with heme detoxification, while PfMDR1 modulates the partitioning of chloroquine, mefloquine, and artemisinin-combination partner drugs between cytosol and vacuole. Field data from Benin show that more than a decade after artemisinin-based combination therapy deployment, wild-type Pfcrt K76 alleles have re-emerged as predominant, consistent with restored chloroquine sensitivity, while Pfmdr1 N86 and Y184F variants linked to reduced lumefantrine susceptibility remain frequent in artemether-lumefantrine-treated populations. Additional ATP-binding cassette transporters such as PfMRP1, which exports glutathione conjugates and several antimalarials, add functional redundancy that makes transporter-based resistance robust.

Kinetoplastid parasites deploy an equally sophisticated transport strategy. Pentavalent antimonials must be reduced to the active trivalent form, which enters Leishmania mainly through the aquaglyceroporin AQP1; resistant isolates commonly show reduced AQP1 expression, inactivating mutations, or gene deletions. Simultaneously, the ABC transporter MRPA, amplified on episomes or through chromosome 23 polysomy, sequesters antimony-trypanothione conjugates into intracellular vesicles, a mechanism strongly associated with high-grade antimonial resistance on the Indian subcontinent. Miltefosine resistance follows a parallel route centered on the LdMT/LdRos3 flippase complex, where point mutations, truncations, and subtelomeric deletions can reduce drug uptake by more than 95 percent, with accompanying shifts in fatty-acid composition and ergosterol content further limiting drug partitioning into membranes. In Trypanosoma brucei, loss of the aquaglyceroporin AQP2, which imports melarsoprol and pentamidine, is the definitive driver of clinical resistance to these drugs.

The second major mechanism involves changing the molecules drugs bind to. In P. falciparum, antifolate resistance is the textbook case: stepwise accumulation of dhfr mutations N51I, C59R, and S108N together with dhps mutations such as A437G and K540E progressively reduces binding of sulfadoxine-pyrimethamine while preserving folate metabolism. Deep-sequencing surveillance in Benin has documented high frequencies of the dhfr triple mutant and the dhps A437G mutation, though the absence of K540E suggests the highest-level resistance genotypes remain rare, allowing some continued utility for intermittent preventive treatment. Artemisinin resistance adds complexity through mutations in the kelch13 propeller domain, validated by gene editing as causal drivers of reduced susceptibility, alongside pfk13-independent routes involving pfcoronin, chromosome 14 copy-number variants, and the endocytosis regulators pfap2mu and pfubp1.

Where Plasmodium relies on discrete point mutations, Leishmania exploits extreme genomic plasticity. Amphotericin B-resistant isolates remodel sterol biosynthesis, decreasing ergosterol and accumulating alternative intermediates such as cholesta-5,7,24-trien-3β-ol through mutations and copy-number changes in sterol C24-methyltransferase and C5-desaturase, thereby reducing the drug’s preferred membrane binding sites. Whole-genome sequencing of Leishmania donovani demonstrates that aneuploidy is adaptive and environment-dependent: chromosomes bearing resistance or virulence genes can become polysomic under in vitro drug selection and then revert toward disomy in the mammalian host. This reversible gene-dosage modulation provides a rapid mechanism for adjusting effective target abundance, and ribosome profiling of antimony-resistant Leishmania tropica reveals a further post-transcriptional layer, with thousands of transcripts, including those encoding trypanothione metabolism and redox enzymes, differentially loaded onto ribosomes even in the absence of drug pressure.

Metabolic reprogramming forms a third protective tier. Antimony-resistant Leishmania strains consistently elevate intracellular thiols such as trypanothione, glutathione, and cysteine, which facilitate sequestration of antimony-thiol conjugates via MRPA and enhance detoxification of reactive oxygen species. Overexpression of trypanothione pathway enzymes and tryparedoxin peroxidase strengthens antioxidant defenses. For nitroheterocyclic drugs used against trypanosomes, resistance often arises through loss or reduction of the nitroreductase enzymes required to activate the prodrugs, blurring the boundary between metabolic adaptation and target modification. In P. falciparum, resistance to compounds such as fosmidomycin and halofuginone has been linked to adaptive metabolic responses including altered proline metabolism, suggesting that metabolic rewiring acts as a background layer that lowers effective drug potency and facilitates the selection of high-level resistance alleles.

Perhaps the most clinically troubling mechanism is persistence. Subpopulations of Plasmodium, Trypanosoma, and Leishmania can enter dormant, persister-like states characterized by slowed proliferation, reduced metabolic activity, condensed chromatin, and extensive transcriptomic and translational reorganization, evading both immune clearance and chemotherapy. Brief exposure of P. falciparum to dihydroartemisinin enriches for dormant ring-stage parasites that resume proliferation days after treatment ends, a phenotype pronounced in kelch13-mutant parasites. Plasmodium vivax hypnozoites exemplify long-term dormancy, with expression signatures dominated by RNA-binding proteins and translational repressors. Emerging mechanisms extend this theme: kelch13 and pfap2mu mutations reduce hemoglobin endocytosis, lowering the free heme pool needed to activate artemisinin, while mutations in the PfCARL locus and vesicle-associated proteins threaten next-generation antimalarials such as ganaplacide by disrupting intracellular drug trafficking.

Detecting these layered defenses depends on advanced genomics. Next-generation sequencing has replaced phenotypic assays as the gold standard for surveillance, providing the depth needed to detect low-frequency resistant subclones within mixed infections before they become clinically dominant. The computational challenges are substantial: the P. falciparum genome exceeds 80 percent AT content, reaching roughly 90 percent in non-coding regions, causing PCR amplification bias and uneven coverage that demand specialized mapping algorithms. For kinetoplastids, pipelines must resolve aneuploidy, copy-number variation, and environment-dependent polysomy, and integrate ribosome-profiling data to map translational reprogramming. The authors emphasize that resistance must be embedded into the earliest stages of drug discovery rather than treated as a clinical afterthought, incorporating pharmacokinetic and pharmacodynamic modeling with transmission dynamics to predict population-level outcomes.

Looking forward, the review highlights parasite-specific targets that lack human homologues, such as calcium-dependent protein kinases in apicomplexans and parasite-derived secreted kinases in kinetoplastids, as routes to potent efficacy with minimal host toxicity. CRISPR/Cas9 gene editing, now workable even in previously intractable trypanosomatids, enables rigorous validation of candidate targets and resistance loci, while 3D bioengineered tissues, organoids, and humanized mice offer ethically sound, human-relevant alternatives to conventional rodent models. The central message is that durable antiprotozoal therapy will require compounds designed from the outset to withstand transporter redundancy, genomic flexibility, and persistence-related tolerance, combined with genomic surveillance, molecular diagnostics, and mathematical modeling to anticipate rather than merely react to the next wave of resistance.

Subject of Research: Molecular mechanisms of drug resistance in protozoan parasites including Plasmodium, Leishmania, and Trypanosoma

Article Title: Molecular mechanisms underlying drug resistance in protozoan parasites: emerging mechanisms and therapeutic perspectives

Article References: Uduwela, K., & Ranasinghe, K. (2026). Molecular mechanisms underlying drug resistance in protozoan parasites: emerging mechanisms and therapeutic perspectives. Frontiers in Parasitology, 5, Article 1934996. https://doi.org/10.3389/fpara.2026.1934996

Image Credits: AI Generated

DOI: 10.3389/fpara.2026.1934996

Keywords: drug resistance, protozoan parasites, Plasmodium falciparum, Leishmania, Trypanosoma brucei, malaria, artemisinin resistance, PfCRT, genomic plasticity, aneuploidy, parasite persistence, genomic surveillance