Absence of Association between Polymorphisms in the RING E3 Ubiquitin Protein Ligase Gene and Ex Vivo Susceptibility to Conventional Antimalarial Drugs in Plasmodium falciparum Isolates from Dakar, Senegal

Abstract

The RING E3 ubiquitin protein ligase is crucial for facilitating the transfer of ubiquitin. The only polymorphism identified in the E3 ubiquitin protein ligase gene was the D113N mutation (62.5%) but was not significantly associated with the 50% inhibitory concentration (IC₅₀) of conventional antimalarial drugs. However, some mutated isolates (D113N) present a trend of reduced susceptibility to piperaquine (P = 0.0938). To evaluate the association of D113N polymorphism with susceptibility to antimalarials, more isolates are necessary.

TEXT

Malaria resistance to most antimalarial drugs has developed in southeast Asia and has spread to Africa. The World Health Organization (WHO) has recommended artemisinin-based combination therapy (ACT) as the first-line treatment for malaria since 2005. As recently described in southeast Asia, the emergence of Plasmodium falciparum resistance to artemisinin and its derivatives has manifested in the form of delayed parasite clearance following treatment with artesunate monotherapy or ACT (1, 2). In areas where artemisinin resistance is emerging, the partner drugs within the combination are under increasing pressure for the selection of resistance. In this context, the identification of molecular markers of resistance to these partner drugs is urgently needed for monitoring the emergence and spread of antimalarial drug resistance.

The ubiquitin system is one of the principal pathways used by all eukaryotic cells to regulate protein abundance levels and protein activities. The chemical modification of proteins by ubiquitin, known as ubiquitylation, is an extremely important posttranslational event that is crucial to numerous cellular processes. Ubiquitin is a conserved 76-amino-acid polypeptide that is covalently attached to one or more lysine residues on proteins through a complex enzymatic cascade. This multienzyme complex includes an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin protein ligase (3). Ubiquitylation begins with the activation of ubiquitin via the E1 ubiquitin-activating enzyme. Subsequently, the E2 ubiquitin-conjugating enzyme catalyzes the transfer of ubiquitin from the E1 enzyme to a conserved cysteine on the E2 enzyme by a transesterification reaction. The final step in the ubiquitylation process is the targeted attachment of ubiquitin to a protein via the E3 ubiquitin ligase, creating an isopeptide bond between a lysine on the target protein and the C-terminal glycine on ubiquitin. E3 proteins are categorized into three major classes of enzymes, homologous to E6-associated protein C terminus (HECT) ligases, really interesting new gene (RING) fingers, and U-box E3 ligases, which are the main determinants of substrate specificity. The ubiquitin system could be a target for antimalarial drugs and involved in resistance (4, 5). Using a genome-wide association study (GWAS), mutations in the E2 ubiquitin-conjugating enzyme gene (PF3D7_1243700) and the HECT E3 ubiquitin ligase gene (PF3D7_0826100) were found to be associated with resistance to pyrimethamine in 45 Senegalese isolates (6). The HECT E3 ubiquitin ligase gene may also be involved in reduced susceptibility to quinine and quinidine (7). Another E3 ubiquitin protein ligase (PF3D7_0627300) belonging to the RING-type E3 ligase is crucial for facilitating the transfer of ubiquitin from the E2 enzyme to a primary amino group of the substrate. It was previously shown that P. falciparum parasites carrying the mutant D113N allele for this gene were less susceptible in vitro to chloroquine and amodiaquine (8).

The objective of the current study was to identify polymorphisms in the E3 ubiquitin protein ligase gene (PF3D7_0627300) in P. falciparum Senegalese isolates and to then evaluate the association of polymorphisms with ex vivo susceptibility to chloroquine (CQ), quinine (QN), monodesethylamodiaquine (DQ), mefloquine (MQ), lumefantrine (LMF), piperaquine (PPQ), pyronaridine (PND), dihydroartemisinin (DHA), artesunate (AS), and doxycycline (DOX).

Forty P. falciparum isolates from falciparum malaria patients attending the Hôpital Principal de Dakar from November 2013 to January 2014 and August 2014 to December 2014 were successfully evaluated. Seventy-five percent of the patients were recruited from the emergency department. The other patients were recruited from the intensive care unit (10%), pediatric department (7.5%), and other units (7.5%). There was no information available on antimalarial treatment prior to admission. Despite the WHO's recommendations, the patients were treated with quinine until November 2014 and with artesunate or artemether-lumefantrine at the Hôpital Principal de Dakar. Informed verbal consent was obtained from the patients or their parents/guardians before blood collection. The study was approved by the ethical committee of the Hôpital Principal de Dakar.

Venous blood samples were collected in Vacutainer ACD tubes prior to patient treatment. A malaria diagnosis was confirmed using a thin blood smear and a rapid diagnosis test. Thin blood smears were stained using a RAL kit (Réactifs RAL, Paris, France) based on eosin and methylene blue and were examined to determine P. falciparum density and to confirm species monoinfection. The parasitemia was expressed by the number of parasite-infected red cells as a percentage of the total number of red blood cells. The level of parasitemia ranged from 0.06% to 14.1%.

The susceptibility of the isolates was assessed without culture adaptation. For the ex vivo microtest, 100 μl of parasitized red blood cells (final parasitemia, 0.5%; final hematocrit, 1.5%) was divided into aliquots and placed into 96-well plates predosed with antimalarial drugs (CQ, QN, MQ, DQ, LMF, DHA, AS, PPQ, PND, and DOX). The plates were incubated in a sealed bag for 72 h at 37°C with atmospheric generators for capnophilic bacteria using Genbag CO2 at 5% CO₂ and 15% O₂ (bioMérieux, Marcy l'Etoile, France). The ex vivo HRP2 enzyme-linked immunosorbent assay (ELISA)-based procedure performed using a commercial Malaria Ag CELISA kit (reference KM2159; Cellabs PTY Ltd., Brookvale, Australia) was previously described (9). The batches of plates were tested and validated with the chloroquine-resistant W2 strain (Indochina) (MR4, Virginia, USA) in three to six independent experiments under the same conditions.

E3 ubiquitin protein ligase (PF3D7_0627300) was amplified by PCR using the following primer pair: 5′-AAT-GGT-CCA-GAA-GAA-GAT-TAT-3′ and 5′-AAA-TAT-ATA-AGG-ATA-GGA-AG-3′. The reaction mixture consisted of 200 ng of genomic DNA, 0.32 μM (each) primers, 1× reaction buffer [750 mM Tris-HCl, 200 mM (NH₄)₂SO₄, 0.1% (vol/vol) Tween 20, and stabilizer, pH 8.8], 2.5 mM MgCl₂, 200 μM deoxynucleoside triphosphate (dNTP) mixture, and 0.2 U of Hot Diamond Taq polymerase (Eurogentec) in a final volume of 25 μl. The thermal cycler (T3 Biometra) was programmed as follows: 10 min at 95°C followed by 40 cycles of 30 s at 95°C, 45 s at 45°C, and 90 s at 72°C and a final extension of 10 min at 72°C. The purified amplicons were sequenced using the PCR primers and a sequencing primer (5′-AAT-ACT-TAT-GAT-ATG-ACA-AGT-GA-3′) on an ABI Prism 3100 Analyzer (Applied Biosystems) according to the manufacturer's instructions. The sequences were analyzed using Vector NTI Advance software (version 11; Invitrogen, Cergy Pontoise, France).

The 50% inhibitory concentrations (IC₅₀s) in the ex vivo chemosusceptibility assay ranged from 6.3 to 414.0 nM for CQ, 6.2 to 1,429.8 nM for QN, 1.9 to 227.3 nM for DQ, 0.6 to 45.0 nM for LMF, 3.4 to 74.5 nM for MQ, 3.9 to 241.9 nM for PPQ, 0.4 to 111.6 nM for PND, 0.1 to 17.3 nM for DHA, 0.1 to 18.1 nM for AS, and 0.9 to 79.0 μM for DOX. The only polymorphism identified in the E3 ubiquitin protein ligase gene was the D113N mutation. The sequences of wild-type parasites (Sub 1478173) and mutant parasites (Sub 1478169) were submitted to GenBank. The mutation was present in 25 isolates (62.5% of the 40 samples). However, the D113N mutation was not significantly associated with the IC₅₀ of CQ, QN, DQ, MQ, LMF, PPQ, PND, DHA, AS, and DOX (P value between 0.2872 and 0.9883 [Wilcoxon signed-rank test]) (Table 1). We did not find in field isolates that P. falciparum parasites carrying the mutation D113N allele for this gene were less susceptible in vitro to CQ and DQ as previously shown in genetically modified parasites (8). The isolates were categorized as being susceptible or resistant or showing reduced susceptibility using the following cutoff values: 77 nM (CQ), 61 nM (DQ), 115 nM (LMF), 12 nM (DHA and AS), 611 nM (QN), 30 nM (MQ), 135 nM (PPQ), 60 nM (PND), and 37 μM (DOX) (10, 11). These cutoff values for reduced in vitro susceptibility to antimalarial drugs were previously estimated using the arithmetic mean plus two standard deviations of the IC₅₀s (10, 11). There was no significant difference between the wild-type parasites (wild-type D113N) and the mutated parasites (mutated D113N) in reduced susceptibility to the 10 conventional antimalarial drugs (P value between 0.0938 and 1 [chi-square test]) (Table 2). The most notable difference seen was for PPQ, for which 0% of the wild-type isolates had reduced susceptibility versus 19% of the samples with D113N-mutated parasites (P value = 0.0938). In 2006, the first-line therapy for uncomplicated malaria became artemether-lumefantrine or artesunate-amodiaquine. The dihydroartemisinin-piperaquine combination was then recommended as a second-line treatment for uncomplicated P. falciparum malaria in Senegal. But it seems that the combination dihydroartemisinin-piperaquine is not yet widely used in Dakar. Multidrug resistance to dihydroartemisinin-piperaquine has emerged in Cambodia, where the rate of recrudescent infections in patients increased from 15.4% in 2011 to 2013 to 39% in 2012 to 2014 (12, 13). In 2012 to 2014, 57% of the patients were still parasitemic at 72 h (13). Based on data from 40 P. falciparum isolates, there was no association between the D113N mutation and ex vivo susceptibility to 10 conventional antimalarial drugs, although more data may show a different outcome.

TABLE 1.

Ex vivo susceptibility of 40 Plasmodium falciparum isolates to chloroquine, quinine, monodesethylamodiaquine, mefloquine, lumefantrine, piperaquine, pyronaridine, dihydroartemisinin, artesunate, and doxycycline according to the D113N mutation in the E3 ubiquitin protein ligase gene^a

Drug	Wild-type D113N (no. of isolates)		Mutated D113N (no. of isolates)		P value
	Mean IC50	95% CI	Mean IC50	95% CI
CQ	75.8 nM (15)	43.9–130.8	60.5 nM (25)	40.4–89.3	0.3909
QN	116.2 nM (15)	64.6–209.0	71.4 nM (24)	42.0–121.2	0.4188
DQ	23.5 nM (15)	12.0–46.2	17.1 nM (25)	10.2–28.7	0.4178
MQ	22.4 nM (14)	15.3–32.8	27.5 nM (25)	19.0–39.8	0.2887
LMF	6.6 nM (15)	3.3–13.6	5.7 nM (25)	3.3–9.8	0.6711
PPQ	34.5 nM (13)	27.1–43.9	37.7 nM (25)	23.0–61.9	0.5827
PND	10.3 nM (13)	7.6–13.9	9.7 nM (20)	5.8–16.2	0.6717
DHA	1.6 nM (14)	0.9–2.7	1.4 nM (25)	0.7–2.6	0.9883
AS	3.3 nM (13)	1.9–5.7	2.2 nM (19)	1.2–4.1	0.2872
DOX	12.2 μM (14)	6.7–22.3	13.6 μM (25)	8.4–22.1	0.6539

^aCQ, chloroquine; QN, quinine; DQ, monodesethylamodiaquine; MQ, mefloquine; LMF, lumefantrine; PPQ, piperaquine; PND, pyronaridine; DHA, dihydroartemisinin; AS, artesunate; DOX, doxycycline; mean IC₅₀, geometric mean 50% inhibitory concentration; 95% CI, 95% confidence interval. P values were determined by the Wilcoxon signed-rank test.

TABLE 2.

Prevalences of Plasmodium falciparum isolates with reduced susceptibility to chloroquine, quinine, monodesethylamodiaquine, mefloquine, lumefantrine, piperaquine, pyronaridine, dihydroartemisinin, artesunate, and doxycycline according to the D113N mutation in the E3 ubiquitin protein ligase gene^a

Drug	Reduced-susceptibility cutoff	% of isolates with reduced susceptibility (no. of isolates with reduced susceptibility/total no. of isolates)		P value
		Wild-type group	Mutated group
CQ	77 nM	60.0 (9/15)	52.0 (13/25)	0.6224
QN	611 nM	6.7 (1/15)	4.2 (1/24)	0.7305
DQ	61 nM	33.3 (5/15)	16.0 (4/25)	0.2037
MQ	30 nM	42.9 (6/14)	52.0 (13/25)	0.5807
LMF	115 nM	0 (0/15)	0 (0/25)	1.0000
PPQ	135 nM	0 (0/13)	20.0 (5/21)	0.0938
PND	60 nM	0 (0/13)	10.0 (2/20)	0.2394
DHA	12 nM	0 (0/14)	4.0 (1/25)	0.4423
AS	12 nM	7.7 (1/13)	10.5 (2/19)	0.7870
DOX	37 μM	14.3 (2/14)	28.0 (7/25)	0.3295

^aCQ, chloroquine; QN, quinine; DQ, monodesethylamodiaquine; MQ, mefloquine; LMF, lumefantrine; PPQ, piperaquine; PND, pyronaridine; DHA, dihydroartemisinin; AS, artesunate; DOX, doxycycline. P values were determined using the chi-square test.

The main limitation of the current study was the low number of P. falciparum isolates. It is difficult to extrapolate the results to other parts of the world without a new set of data. Another limitation was the use of the standard in vitro test for artemisinin exploration of resistance. The standard in vitro test was not adapted to follow resistance to artemisinin derivatives. The clinical resistance to artemisinin was manifested by an increase in the ring-stage survival rate after contact with artemisinin (ring survival test) (14). The ubiquitin system remains a target which may be involved in reduced susceptibility to antimalarial drugs. Furthermore, more isolates from different geographical areas, and, more specifically, from Cambodia, where multidrug resistance to dihydroartemisinin-piperaquine has emerged, are needed to ascertain the role of the different enzymes in this system in P. falciparum resistance.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.