Intrachromosomal Amplification, Locus Deletion and Point Mutation in the Aquaglyceroporin AQP1 Gene in Antimony Resistant Leishmania (Viannia) guyanensis

Abstract

BackgroundAntimony resistance complicates the treatment of infections caused by the parasite Leishmania.Methodology/Principal FindingsUsing next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR) mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1). Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion.Conclusions/SignificanceThis is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.

Drug resistance in protozoan parasites

“…Common adaptations include: target enzyme mutations reducing interactions with the drug (antifolates in malaria [10]); reduced drug uptake (diminazene aceturate in T. b. brucei [11] or antimonials in L. donovani [12]); up-regulation of a metabolic bypass (methotrexate in Leishmania [13]); failing to activate a prodrug (nifurtimox in T. cruzi [14]); increased drug efflux (chloroquine in malaria [15]); and even the failure to produce the target (amphotericin B in Leishmania [16]). The genetic mechanisms of resistance include gene deletions [17], point mutations in targets [10] or transporters (enabling [15] or disabling [18]), copy number variations [19,20], base pair insertions/deletions causing frame shifts in the target gene [12] and the formation of chimeric genes through recombination [21].…”

“…phenanthridines, diamidines [24][25][26]), or e.g. to haem (chloroquine [27]), or be more generally cytotoxic, with multiple targets ( polypharmacology), as may be the case for heavy metal drugs (arsenicals, antimonials) and suramin [28], in which case selectivity depends mostly on selective entry into the parasite rather than into the host cells, through unique transporters or other uptake mechanisms [20,28,29].…”

“…In some such cases, resistance still occurs, through mutation or deletion of these transporters [20,29], but in the case of pentamidine, for instance, clinical resistance for sleeping sickness has not been reported despite being virtually the only drug used against early-stage T. b. gambiense sleeping sickness since the mid-1930s, including population-scale mass treatment campaigns [30], although various levels of resistance can be induced in vitro [31]. The lack (of reports) of clinical resistance, in this case, can be traced to the fact that pentamidine is taken up by three different transport mechanisms, at least two of which, the aminopurine transporter AT1 and the High Affinity Pentamidine Transporter, HAPT1 (now identified as an aquaglyceroporin, TbAQP2 [21]), are highly efficient, allowing a fast accumulation of the drug [32]; moreover, the drug action becomes irreversible after only a brief exposure [33], giving little scope for resistance to develop.…”

Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species

“…(37). However, we have recently identified an Sb-resistant L. guyanensis strain from which a locus containing LgAQP1 was deleted during an in vitro stepwise drug selection process (16). Additionally, LmAQP1 was successfully disrupted in L. major null mutants (38).…”

“…This phenotype is a result of posttranscriptionally regulated AQP1 expression (13). Although the molecular basis of the mechanisms of resistance are not fully understood, the downregulation, mutation, and deletion of the AQP1-encoding gene have been clearly associated with Sb resis-tance in both laboratory-selected and field isolates of Leishmania (14)(15)(16). In Leishmania, AQP1 has also been implicated in the transport of the Sb-related metal arsenite (As III ) (17).…”