Malaria antifolate resistance with contrasting Plasmodium falciparum dihydrofolate reductase (DHFR) polymorphisms in humans and Anopheles mosquitoes

Abstract

Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections-S108N (>90%), with N51I, C59R, and 108N+51I+59R triple mutants (30-80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples-S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2-12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent-S108T (90%), with A16V and the 108T+16V double mutant (49-57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles.

Kinetic and Structural Analysis for Potent Antifolate Inhibition of Pneumocystis jirovecii, Pneumocystis carinii, and Human Dihydrofolate Reductases and Their Active-Site Variants

“…Structural models of the pjDHFR ternary complex with OAAG324 show weaker interactions of the 5=-Cl of OAAG324 with Ser69. Similar observations were made that helped explain the gain of function observed for the S108T mutation in Plasmodium falciparum DHFR (29).…”

“…The K i values for OAAG324 were determined for all native and mutant DHFR forms from pj-, pc-, and hDHFR. Substitution of amino acids from the pjDHFR sequence into the active site of hDHFR lowered the K i , i.e., improved the binding of OAAG324 (and thus, resulted in gain of function [29]) (Table 3 and Fig. 6a); the Q35S/N64S double variant shows a K i value of 2.47 Ϯ 0.5 nM (mean Ϯ standard deviation), which is statistically significantly lower than the value for hDHFR (24.3 Ϯ 4.4 nM) and is similar to that for pjDHFR (2.7 Ϯ 0.4 nM).…”

Epigenetic regulation of Plasmodium falciparum clonally variant gene expression during development in Anopheles gambiae

“…In the context of the natural conditions of transmission in Burkina, the interaction between P. falciparum field isolates with the local mosquito populations could have favored selection of this particular var gene if there is a survival advantage to the mosquito by conferring or increasing resistance to disease. Other types of selection events related to pleiotropy or gene linkage of the selected variant to certain drug-resistance alleles cannot be ruled out63.…”

Genetic diversity and signatures of selection of drug resistance in Plasmodium populations from both human and mosquito hosts in continental Equatorial Guinea

“…Differences have also been found in drug-resistant associated genes. In Gabon, Mharakurwa et al [5] reported that parasites in humans presented high levels of pyrimethamine (PYR)-resistant mutants, whereas parasites in Anopheles mosquitoes showed high levels of cycloguanil-resistant mutants.…”