The interplay between drug resistance and fitness in malaria parasites

Transporter mutations

Informatic studies have identified multiple predicted transporter genes in P. falciparum (Table 1); SNPs in 11 of these genes were associated with decreased sensitivity to chloroquine or quinine (Mu et al., 2003), although a subsequent study could not confirm associations between most of the identified polymorphisms and drug sensitivity in clinical isolates (Anderson et al., 2005). Many predicted P. falciparum transporters are members of the ATP-binding cassette (ABC) transporter superfamily. ABC transporters are responsible for the transfer of a range of substances across concentration gradients in an energy-dependent manner (Koenderink et al., 2010). Polymorphisms in transport proteins can mediate resistance to many agents active against cancer and infectious diseases via enhancing efflux of the drugs from cells (Borges-Walmsley et al., 2003). It appears that a number of plasmodial proteins transport different drugs and that polymorphisms in these proteins may impact upon drug sensitivity (Picot et al., 2009).

Pfmdr1

Polymorphisms in the P. falciparum multidrug resistance-1 (pfmdr1) gene, which encodes the P-glycoprotein homolog, impact on sensitivity to multiple antimalarial drugs (Foote et al., 1990, Sanchez et al., 2010, Valderramos & Fidock, 2006). In humans, P-glycoprotein polymorphisms are associated with resistance to cancer drugs (Sharom, 2011). In P. falciparum, the function of the pfmdr1 product is unknown, but the protein localizes to the membrane of the food vacuole, the site of action of a number of drugs, suggesting that it is a drug transporter (Cowman et al., 1991). Data on associations between pfmdr1 polymorphisms and drug sensitivity are complex, but overall suggest that changes in pfmdr1 sequence or copy number alter transport of multiple drugs in or out of the parasite food vacuole, with individual polymorphisms leading to opposite effects on different drugs (Koenderink et al., 2010). Mutations at pfmdr1 N86Y and D1246Y (for this and other P. falciparum genes wild type sequence is based on the 3D7 reference strain), which are common in Africa, have been linked to decreased sensitivity to chloroquine and amodiaquine, but increased sensitivity to lumefantine, mefloquine, and artemisinins (Duraisingh et al., 2000a, Duraisingh et al., 2000b, Reed et al., 2000, Mwai et al., 2009). Other polymorphisms primarily seen outside Africa (including 1034C, 1042D, and increased gene copy number) are associated with altered sensitivity to lumefantrine, mefloquine, and artemisinins (Reed et al., 2000, Pickard et al., 2003, Sidhu et al., 2005, Sidhu et al., 2006, Veiga et al., 2011). Considering infections that emerge soon after prior therapy, amodiaquine-containing regimens selected for the 86Y and 1246Y mutant alleles (Humphreys et al., 2007, Zongo et al., 2007, Nsobya et al., 2007) and for parasites with decreased in vitro sensitivity to the active metabolite monodesethylamodiaquine (Nawaz et al., 2009) in subsequent infections. In contrast, therapy with artemether-lumefantrine selected for the N86 and D1246 wild type alleles in subsequent infections within 60 days of prior therapy (Sisowath et al., 2005, Humphreys et al., 2007, Zongo et al., 2007, Happi et al., 2009, Some et al., 2010, Baliraine & Rosenthal, 2011). Importantly, impacts of pfmdr1 polymorphisms on drug sensitivity are modest, correlations between particular polymorphisms and treatment efficacy have not been seen, and the ACTs artesunate-amodiaquine and artemether-lumefantrine remain highly efficacious for the treatment of uncomplicated falciparum malaria in Africa (Dorsey et al., 2007, Four Artemisinin-Based Combinations Study Group, 2011). However, as seen for chloroquine and amodiaquine, pfmdr1 polymorphisms may contribute, with additional polymorphisms, to higher level resistance to increasingly used components of ACTs.

Pfcrt

Soon after the identification of pfmdr1 it became clear that polymorphisms in this gene are not the primary mediators of chloroquine resistance. Subsequently, analysis of progeny of a genetic cross between chloroquine sensitive and resistant strains led to the identification of pfcrt (Fidock et al., 2000), which encodes a food vacuole membrane protein that is predicted to be a member of the drug/metabolite transporter superfamily (Martin & Kirk, 2004, Tran & Saier, 2004). The function of pfcrt is unknown, but apparently essential, as disruption of the gene has not been possible (Ecker et al., 2012). Pfcrt is highly polymorphic, but one SNP, K76T, is the primary mediator of chloroquine resistance (Lakshmanan et al., 2005, Ecker et al., 2012). The 76T mutation appears to act principally by increasing the export of chloroquine from the food vacuole, but the mechanism of pfcrt 76T- mediated chloroquine resistance is incompletely understood (Ecker et al., 2012). Other pfcrt SNPs always accompany 76T in field isolates, and these likely encode compensatory mutations that allow parasites containing 76T to maintain adequate fitness; some other SNPs may also contribute directly to the drug resistant phenotype. The 76T mutation also mediates decreased sensitivity to monodesethylamodiaquine, and studies with genetically modified parasites have shown it to mediate increased susceptibility to mefloquine and artemisinins (Sidhu, et al., 2002, Lakshmanan et al., 2005), suggesting the same reciprocal relationship between sensitivities to aminoquinolines and other drugs as described for certain pfmdr1 polymorphisms.

Pfmrp1

P. falciparum multidrug resistance protein-1 (Pfmrp1) is a member of the ABC transporter superfamily (Koenderink et al., 2010). Unlike the products of pfmdr1 and pfcrt, Pfmrp1 localizes principally to the parasite plasma membrane (Kavishe et al., 2009). In studies of culture adapted P. falciparum, SNPs in pfmrp1 were linked to decreased sensitivity to chloroquine and quinine (Mu et al., 2003). Two SNPs that appear to be common in African parasites, I876V and K1466R, were selected by prior treatment with artemether/lumefantrine (Dahlstrom et al., 2009a) and sulfadoxine pyrimethamine (Dahlstrom et al., 2009b), respectively. Disruption of the pfmrp1 gene yielded parasites with diminished growth and increased sensitivity to chloroquine and other drugs, suggesting a role for this protein in the efflux of antimalarial drugs from the parasite and in parasite fitness (Raj et al., 2009).

Resistance to antibiotics

Resistance of P. falciparum to antibiotics has not been well studied clinically, but varied sensitivities to tetracyclines of unknown clinical significance have been seen in vitro (Briolant et al., 2009). Sequence polymorphisms and variation in copy number in P. falciparum homologs of bacterial mediators of tetracycline resistance (pfmdt and pftetQ) were associated with decreased drug sensitivity (Briolant et al., 2010). A survey of parasites from the Amazon identified a SNP in an apicoplast gene encoding a homolog of a ribosomal protein in which mutations mediate clindamycin resistance in bacteria; this SNP was associated with in vitro clindamycin resistance in 3 clinical isolates (Dharia, et al., 2010). Resistance to azithromycin has also been linked to mutations in an apicolast-encoded ribosomal protein (Sidhu, et al., 2007).

Fitness consequences of antimalarial drug resistance: in vitro studies

The first direct comparison of the fitness of drug sensitive and resistant malaria parasites used competition experiments to compare the growth of P. falciparum selected in vitro for resistance to atovaquone with that of the parent strain. Parasites selected for resistance had multiple mutations in pfcytb. Those with a single M133I mutation had 25-fold decreased atovaquone sensitivity and those with the M133I and G280D mutations had 1000-fold decreased sensitivity (Korsinczky et al., 2000). In competition growth experiments, the single mutant line grew as well as the parent parasite, but the more resistant double mutant was out-competed by the parent line, with a 5–9% loss of fitness calculated based on relative growth rates (Peters et al., 2002). Molecular modeling predicted that the G280D mutation altered the orientation of a putative ubiquinone-binding residue, likely altering enzyme function, while the M133I mutation would have minimal effect on enzyme conformation. Another pfcytb mutation, Y268S, was seen in a P. falciparum isolate from Thailand, and led to marked alterations in enzyme function (Fisher et al., 2012). Decreased enzyme activity was associated with increased expression of mitochondrial cytochrome bc₁ complex genes, apparently compensating for detrimental effects of the mutation, and offering an example of parasites reaching a balance to maintain fitness in the setting of drug-resistance.

A similar approach was used to evaluate the relative fitness of P. falciparum engineered to contain different pfmdr1 haplotypes. Competitive growth was compared between the chloroquine sensitive D10 strain, the chloroquine-resistant 7G8 strain, and a strain in which most of the pfmdr1 gene of D10 was replaced by that of 7G8, introducing the S1034C, N1042D and D1246Y mutations (Hayward et al., 2005). Introduction of the mutant pfmdr1 in D10 partially reversed chloroquine resistance and mediated decreased sensitivity to mefloquine, halofantrine, and artemisinin (Reed et al., 2000). These results demonstrate the reciprocal impacts of the pfmdr1 polymorphisms on sensitivity to chloroquine (which is mediated principally by a different mutation, pfcrt K76T) and to mefloquine, halofantrine, and artemisinin. Considering fitness, the chloroquine-resistant 7G8 strain was outcompeted by the chloroquine-sensitive D10 strain and by the intermediate sensitivity 7G8 strain containing D10 pfmdr1 sequence. Improved fitness of the more chloroquine sensitive strains was evidenced by gradual increases in chloroquine sensitivity of mixed cultures until, after 34 days, sensitivities were identical to that of a pure D10 culture (Hayward et al., 2005). Comparison of growth rates led to an estimate of ~25% loss of fitness mediated by introduction of the three pfmdr1 mutations. However, the relative importance of the individual SNPs in mediating resistance is unclear, and the N86Y mutation, which appears to be the principle mediator of altered drug sensitivity in Africa, was not studied.

Increases in the copy number of pfmdr1 have an important impact on sensitivity to numerous antimalarials, with increased copy number increasing sensitivity to chloroquine, but decreasing sensitivity to mefloquine, quinine, and artemisinins (Pickard et al., 2003, Phompradit et al., 2011, Veiga et al., 2011). Increased pfmdr1 copy number is common in Asia, but not Africa. As is the case with resistance-mediating SNPs, copy number variation is likely to impact upon parasite fitness. This factor was studied by selecting in vitro for resistance to mefloquine in a Thai strain of P. falciparum (Preechapornkul et al., 2009). Decreasing susceptibility to mefloquine was associated with increasing copy number, but pfmdr1 SNPs were not selected. Mefloquine-resistant clones with average copy numbers of 2.3 (~ 5-fold less sensitive to mefloquine than the parental strain) and 3.1 (~6-fold less sensitive) were co-cultured with the parent strain (1 copy number), and the selected strains showed a fitness disadvantage, with overgrowth of single copy number strains over 3–4 weeks. Modeling predicted a loss of fitness, compared to the parental strain, of 6.3% and 8.7% for parasites with 2.3 and 3.1 copies of pfmdr1, respectively. Modeling further suggested that under drug pressure pfmdr1 amplification is a common event, with increases from one to two copies occurring once in every 10⁸ parasites and from two to three copies once in every 5000 parasites. Thus, the selective pressure for increased pfmdr1 copy number is great, but gene amplification is apparently kept in check by the fitness disadvantage of increased copy number.

Considering impacts of pfmdr1 polymorphisms on the fitness of clinical isolates, P. falciparum was cultured from children with malaria in a region of Uganda with high multiplicity of infection (and thus high likelihood of mixed infections), and changes in the prevalence of the pfmdr1 N86Y and D1246Y alleles were followed over time (Ochong, et. al., 2013). Most cultures did not undergo changes in culture, but for those that did show selection 8/11 selected toward mutant 86Y, 9/14 selected toward wild type D1246, and 5/7 with selection at both alleles selected toward 86Y and D1246. Surprisingly, the results suggest a mixed picture, with fitness advantages for parasites with pfmdr1 mutant 86Y and wild type D1246 alleles.

The gch1 gene encodes GTP cyclohydrolase, which acts upstream of DHPS and DHFR in the folate synthesis pathway. This gene had increased copy number in Asian P. falciparum with folate resistance mutations, suggesting a compensatory mechanism to increase fitness in these drug-resistant parasites (Nair et al., 2008). Genetic manipulation of P. falciparum to increase gch1 copy number led to modest decreases in pyrimethamine sensitivity in most tested parasite lines (Heinberg et al., 2013). However, results were complex, with varied effects in different strains, and with evidence for detrimental effects of marked overexpression of GTP cyclohydrolase. These results offer another example of how selection of parasite alterations by drug pressure is balanced by effects on parasite fitness.

Fitness consequences of antimalarial drug resistance: animal model studies

Studies using murine malaria models have also attempted to assess the fitness costs of drug resistance. Growth of a pyrimethamine-resistant clone of the rodent parasite P. chabaudi was compared with that of its drug-sensitive parent strain by co-infection of mice. The sensitive strain appeared to outgrow the resistant strain in two of three experiments (Rosario et al., 1978). In the same studies, a chloroquine resistant strain of P. chabaudi outgrew a chloroquine sensitive strain in 4 of 4 experiments, in one case even when the inoculum was 90% sensitive, and only 10% resistant parasites. This experiment suggests a surprising fitness advantage for chloroquine-resistant parasites that is difficult to reconcile with other reports. In another study P. chabaudi selected for resistance to pyrimethamine were outcompeted by sensitive parasites, but after many passages the resistant strain gained a fitness advantage over both the parent strain and the orginial resistant strain (Walliker, et al, 2005). Thus, determinants of fitness are complex, and compensatory mutations gained over time may mediate fitness improved even over that of the parental drug-sensitive strain.

In another strategy to study fitness, P. chabaudi was passaged in mice to increase virulence (Mackinnon et al., 2002). In this case the features described as virulence (increased parasite growth rate and mouse mortality) can also be seen to represent fitness. The parasites selected for virulence were consistently less susceptible than parent strains to treatment with pyrimethamine or artemisinin, and differences were not explained by differences in known resistance-mediating polymorphisms (Schneider et al., 2008, Schneider et al., 2012). Thus, independent of specific resistance mediators, parasites selected for increased fitness were more resistant to drug treatment. In another study P. chabaudi were selected by passages in mice for resistance to artesunate-mefloquine. The parasites selected for resistance grew as well as parent drug-sensitive parasites, in this case showing no apparent fitness cost of drug resistance (Rodrigues et al., 2013).

To study impacts of resistance on fitness in mosquito stages of malaria parasites, mice infected with P. berghei were treated with pyrimethamine to select for resistance. Selected parasites were similar to sensitive parasites in the mouse, but they proceeded more slowly through mosquito development (Shinondo et al., 1994). Thus, drug-resistant strains might be less capable than sensitive parasites to transmit malaria. In contrast, when mosquitoes were fed on mice infected with a mixture of parasites differing in virulence as described above, the more virulent parasites were more readily transmitted (Schneider et al., 2012). In other studies, when mice were infected with P. berghei encoding wild type or resistance-mediating pfcrt haplotypes, blood-stage parasites were equally sensitive to chloroquine, but mutant parasites had enhanced mosquito infectivity in the presence of chloroquine, suggesting that drug resistance enhanced transmission (Ecker, et al., 2011).