Resistance of a Rodent Malaria Parasite to a Thymidylate Synthase Inhibitor Induces an Apoptotic Parasite Death and Imposes a Huge Cost of Fitness

BACKGROUND: The greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy. METHODOLOGY/PRINCIPAL FINDINGS: To generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation. CONCLUSIONS/SIGNIFICANCE: The resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may be exploited as a novel drug target in malaria and other protozoan diseases of medical importance

Resistance induces internucleosomal DNA fragmentation in FOA-resistant parasite grown in mice without drug.

<p>Polyacrylamide gel electrophoretic fraction of DNA was done in two independent experiments, A and B. In A, lanes 1 represent DNA extracted from the wild-type parasite that shows no cleavage, while lanes 2 represent cleaved DNA of FOA-resistant parasite, with the main band at ≈200 bp. Similar results are represented in B, where the wild-type shows no cleaved DNA (lanes 1), while FOA-resistant parasite shows cleaved DNA and a clear band at ≈200 bp (lanes 2). Lane M is the molecular size marker (pSG5/Hinf I and φX174/HincII for A, and pSG5/Hinf I for B).</p

The photomicrographs of FOA-resistant and wild-type parasites during serial passaging in the absence of drug.

<p>A and B show FOA-resistant parasite at passages 2 and 12 respectively, with dark/condensed chromatin dot & cell shrinkage characterized by small and more dense cytosol (red arrowheads), confirming that the death phenomenon was a persistent feature of the FOA-resistant parasite line. The wild-type parasite (C) shows the typical ellipsoidal morphology and full cytoplasm.</p

Parasitaemia patterns of mice infected with FOA-resistant parasite and orally treated with FOA.

<p>The mice were treated twice daily for 3 days with FOA (40 mg/kg cumulative dose) at passages 2, 5, 10 and 12. Note that the patterns mirror each other, confirming that the acquired FOA-resistance is stable. The discontinuous curve indicates that following FOA administration to mice infected with the wild-type parasite, no parasites could be observed under the microscope until day 14 p.i when recrudescent parasites were observed.</p

Electron micrographs of FOA-resistant and wild-type parasites grown in mice in the absence of drug.

<p>In (B), the trophozoite of the wild-type parasite shows normal ultrastructural morphology with various compartments and organelles well defined by membranes that have retained their integrity. In (A), late schizont of FOA-resistant parasite is seen with intact plasmalemma housing non-viable segmenters/merozoites that appear as electron dense bodies probably due to compaction of nuclear chromatin and condensation of cytoplasm. (C) is an immuno-gold electron micrograph adapted from Bhowmick et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021251#pone.0021251-Bhowmick1" target="_blank">[114]</a> showing a similar ‘syncytial’ cell from <i>P. falciparum</i> with viable merozoites. Note the distinct nucleus of segmenters. Abbreviations: CN, condensed segmenter; E, erythrocyte; FV, food vacuole; IPV, intraparasitic vacuole; M, mitochodria; N, nucleus; NM, nuclear membrane; PM, plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuolar membrane; VL, vacuolization.</p