To our knowledge, this work describes for the first time a culture-adapted P. falciparum
strain with a ring stage that can survive high-dose ART pressure. We showed that a P. falciparum
ring-stage subpopulation persists in culture under a very high dose of ART for at least 96 h, suggesting cell cycle arrest under drug pressure. This quiescence phenomenon is temporary, as the parasite can usually continue its cycle development after drug removal and may explain the observation of delayed clearance after treatment (6
). Full elucidation of the signaling pathway trigger and the genetic alterations leading to the cell cycle arrest may permit the development of new antiplasmodial drugs.
Interestingly, ART tolerance did not influence the IC50
s of the parasites, and measurement of the IC50
of ART did not reflect the ability of the parasite to survive ART treatment. This raises the question of the suitability of using the classic chemosensitivity monitoring methods to determine the response to ART. The ability of strain F32-ART to tolerate ART by a quiescence mechanism would be invisible by use of a radioisotopic sensitivity evaluation, as this method determines the rate of DNA synthesis of Plasmodium
continuously exposed to drug. The quiescence of the ring stage could permit parasite survival by slowing its metabolism and limiting the effects of the drug. As no DNA was synthesized by quiescent parasites, drug-tolerant strain F32-ART appeared to be as sensitive as control strain F32-Tanzania in an IC50
assay. These data corroborate the findings of a study by Dondorp et al., in which resistant isolates from western Cambodia did not present higher IC50
). Although delayed clearance after ART treatment is not yet commonly observed in the field, its emergence in southeast Asia represents a glimpse of its global spread (6
), and a protocol better than the traditionally used hypoxanthine microplate method for monitoring of survival during ART treatment is thus urgently needed.
ART acts on F32-ART tolerant parasites via a cytostatic mechanism. In the study of Dondorp et al. (6
), the ring forms were envisaged to be responsible for resistance, whereas they were previously described to be the most sensitive stages (22
). Our data support a new mechanism for P. falciparum
ART tolerance by cell cycle arrest under ART pressure and further parasite development after drug removal.
Our investigations of the molecular markers associated with decreased sensitivity to ART (pfatp6
) showed a lack of association of the known genes with ART tolerance mechanism, as reported in the results of clinical studies (6
). Nevertheless, we highlighted evidence of new potential markers of ART tolerance through microarray studies. Transcriptional analyses suggested a potential role of P. falciparum
exported proteins (KAHRP, PHISTc, RIFIN, STEVOR, and an exported protein of unknown function) in transporting substances from the red blood cell into the parasite (14
). The 70-kDa heat shock protein (HSP70) and the hypoxanthine phosphoribosyltransferase have previously been associated with the P. falciparum
response to lethal doses of artesunate (15
). HSP70 has also been proposed to play an important role during parasite adaptation to different environments (11
), which might be correlated with the physiologic changes that the parasite may undergo when it restarts its cell cycle following its quiescence stage. The PF10_0121 gene product (hypoxanthine phosphoribosyltransferase), an enzyme involved in purine biosynthesis, is essential for DNA synthesis. The modified transcription of PFE1415w supports its role as a cell cycle regulator potentially involved in the quiescence phenomenon in response to ART pressure. PFE1415w could thus be implicated not only in the parasite life cycle but also in ART tolerance; however, because these changes were relatively small and the genes with changed levels of transcription could represent those encoding variant and stress proteins whose levels of expression could change after drug pressure, further investigation is necessary to determine whether these genes play a direct role in the response to ART.
Cell cycle arrest or quiescence is a physiologic feature previously found in other Plasmodium
species, such as P. vivax
and P. ovale
, during hepatic stages, but it has never been demonstrated in P. falciparum
. Quiescence is a phenomenon also found in a wide range of organisms. For example, varicella-zoster virus is able to resist antiviral agents, bacteria in stationary growth phase are resistant to antibiotics, and human cancer cells can survive the assault of chemotherapy by arresting the cell cycle in the G1
). In mammalian cells, P27kip1 can inhibit cyclin-dependent kinase 2 (CDK2), which is indispensable for progression of the cell cycle from G1
phase to S phase (9
), leading to quiescent cells. CDKs exist in P. falciparum
, such as Pfpk6 (whose structure is related to that of human CDK2), and their overexpression was noted in the transition from the ring to the mature trophozoite form (4
). We hypothesize that the quiescence observed here could result in alteration of the cell cycle regulation mediated by CDK and/or CDK inhibitors. Further investigations of P. falciparum
cell cycle regulators (7
) may shed new light on the association between drug tolerance and quiescence.
Finally, our P. falciparum ART-tolerant model offers a major tool for research and screening of molecules active against resistant P. falciparum strains and for a better understanding of the atypical mechanism survival of malaria parasites when they are exposed to ART and its derivatives.