Cation ATPase (ATP4) Orthologue Replacement in the Malaria Parasite Plasmodium knowlesi Reveals Species-Specific Responses to ATP4-Targeting Drugs

Several unrelated classes of antimalarial compounds developed against Plasmodium falciparum target a parasite-specific P-type ATP-dependent Na(+) pump, PfATP4. We have previously shown that other malaria parasite species infecting humans are less susceptible to these compounds. Here, we generated a series of transgenic Plasmodium knowlesi orthologue replacement (OR) lines in which the endogenous pkatp4 locus was replaced by a recodonized P. knowlesi atp4 (pkatp4) coding region or the orthologous coding region from P. falciparum, Plasmodium malariae, Plasmodium ovale subsp. curtisi, or Plasmodium vivax. Each OR transgenic line displayed a similar growth pattern to the parental P. knowlesi line. We found significant orthologue-specific differences in parasite susceptibility to three chemically unrelated ATP4 inhibitors, but not to comparator drugs, among the P. knowlesi OR lines. The PfATP4(OR) transgenic line of P. knowlesi was significantly more susceptible than our control PkATP4(OR) line to three ATP4 inhibitors: cipargamin, PA21A092, and SJ733. The PvATP4(OR) and PmATP4(OR) lines were similarly susceptible to the control PkATP4(OR) line, but the PocATP4(OR) line was significantly less susceptible to all ATP4 inhibitors than the PkATP4(OR) line. Cipargamin-induced inhibition of Na(+) efflux was also significantly greater with the P. falciparum orthologue of ATP4. This confirms that species-specific susceptibility differences previously observed in ex vivo studies of human isolates are partly or wholly enshrined in the primary amino acid sequences of the respective ATP4 orthologues and highlights the need to monitor efficacy of investigational malaria drugs against multiple species. P. knowlesi is now established as an important in vitro model for studying drug susceptibility in non-falciparum malaria parasites.