Amodiaquine resistance in Plasmodium berghei is associated with PbCRT His95Pro mutation, loss of chloroquine, artemisinin and primaquine sensitivity, and high transcript levels of key transporters

Abstract

The human malaria parasite has evolved complex drug evasion mechanisms to all available antimalarials. To date, the combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short acting, artesunate is partnered with long acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite ANKA as a surrogate of to investigate the mechanisms of amodiaquine resistance.: We used serial technique to select amodiaquine resistance by submitting the parasites to continuous amodiaquine pressure. We then employed the 4-Day Suppressive Test to monitor emergence of resistance and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Submission of ANKA to amodiaquine pressure yielded resistant parasite within thirty-six passages. The effective dosage that reduced 90% of parasitaemia (ED ) of sensitive line and resistant line were 4.29mg/kg and 19.13mg/kg, respectively. After freezing at -80ºC for one month, the resistant parasite remained stable with an ED of 18.22mg/kg. Amodiaquine resistant parasites are also resistant to chloroquine (6fold), artemether (10fold), primaquine (5fold), piperaquine (2fold) and lumefantrine (3fold). Sequence analysis of revealed His95Pro mutation. No variation was identified in or nucleotide sequences. Amodiaquine resistance is also accompanied by high mRNA transcripts of key transporters;, and and Ca /H antiporter. Selection of amodiaquine resistance yielded stable "multidrug-resistant'' parasites and thus may be used to study common resistance mechanisms associated with other antimalarial drugs. Genome wide studies may elucidate other functionally important genes controlling AQ resistance in.

Amodiaquine drug pressure selects nonsynonymous mutations in pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol-4 kinase in Plasmodium berghei ANKA

“…Two parasite reference lines of P. berghei ANKA (the MRA-865 and MRA-868 reference lines obtained from MR4, ATCC ® Manassas, Virginia) were used as the drug-sensitive wild-type (WT) parasites. Also, stable multidrug-resistant parasites: the PQ-resistant (PQ R ) P. berghei ANKA parasites previously selected from the MRA-865 line, the LM-resistant (LM R ), and AQ-resistant (AQ R ) P. berghei ANKA parasites previously selected from MRA-868 line 39 , 41 , were utilized. Male Swiss albino mice weighing 20±2g outbred at KEMRI Animal house Nairobi were employed, housed, and fed as previously described 46 .…”

“…Here, we used in silico computational analysis, PCR, sequencing, and qPCR analysis to interrogate the association of five selected kinases with LM, AQ, and PQ resistance in Plasmodium berghei ANKA. The stable drug-resistant parasites used in this study had previously been selected by submitting P. berghei ANKA parasites separately to LM, AQ, or PQ for at least 40 mechanical passages 39 – 41 . We reveal nonsynonymous mutations in Pantothenate kinase 1 (PANK1) (PBANKA_1022600), Diacylglycerol kinase (DAGK) (PBANKA_1334600), and Phosphatidylinositol 4-kinase beta (PI4Kβ) (PBANKA_1109400), only in the AQr parasites.…”

Genetic alteration of ferredoxin NADP+-reductase or cysteine desulfurase in piperaquine-resistant Plasmodium berghei restores susceptibility to lumefantrine and abolishes the impact of probenecid, verapamil, and cyproheptadine

“…The two parasite lines were confirmed as stable resistant parasites 26,28 . Male Swiss albino mice weighing 20±2g outbred at KEMRI, Animal house Nairobi were utilized, housed, and fed as previously described 50 . The knockout (KO) vector (PbGEM-018972), as well as the overexpression (OE) vector (PbGEM-456502), were kindly provided by the PlasmoGEM project under an agreement of a material transfer (PG-MTA-0093).…”

“…The activity profiles of probenecid, verapamil, or cyproheptadine alone and in combination with PQ, or LM against the wildtype, resistant, or the transgenic parasites were assessed in the standard 4-day suppressive test (4-DT) in which the parasite is exposed to four daily drug doses 51 . Infection, randomization of experimental mice, drug administration, estimation of parasite densities, and calculation of percentage drug killing was performed and determined as previously described 26,50,52 .…”

“…The quantification of mRNA transcripts of Pbcrt, Pbmdr1, Pbmrp2, Pbpank, Pbfnr, PbSufS, Pbpmiv, Pbpmix, Pbpmx , and Pbpi3k was carried out using primer listed on Table S1 https://osf.io/j5w3e. Extraction of total RNA from the wildtype, LM R , PQ R, and transgenic parasites and synthesis of respective first-strand cDNA was performed as previously described 50 . Using the Pbβ-actin I , as the housekeeping gene, the relative expression profile of the ten genes was performed in a final volume of 20 μ l using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific™).…”