Multiple genetic loci define Ca++ utilization by bloodstream malaria parasites.

BACKGROUND: Bloodstream malaria parasites require Ca++ for their development, but the sites and mechanisms of Ca++ utilization are not well understood. We hypothesized that there may be differences in Ca++ uptake or utilization by genetically distinct lines of P. falciparum. These differences, if identified, may provide insights into molecular mechanisms. RESULTS: Dose response studies with the Ca++ chelator EGTA (ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid) revealed stable differences in Ca++ requirement for six geographically divergent parasite lines used in previous genetic crosses, with the largest difference seen between the parents of the HB3 x Dd2 cross. Genetic mapping of Ca++ requirement yielded complex inheritance in 34 progeny clones with a single significant locus on chromosome 7 and possible contributions from other loci. Although encoded by a gene in the significant locus and a proposed Ca++ target, PfCRT (P. falciparum chloroquine resistance transporter), the primary determinant of clinical resistance to the antimalarial drug chloroquine, does not appear to contribute to this quantitative trait. Stage-specific application of extracellular EGTA also excluded determinants associated with merozoite egress and erythrocyte reinvasion. CONCLUSIONS: We have identified differences in Ca++ utilization amongst P. falciparum lines. These differences are under genetic regulation, segregating as a complex trait in genetic cross progeny. Ca++ uptake and utilization throughout the bloodstream asexual cycle of malaria parasites represents an unexplored target for therapeutic intervention.

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors.

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad-selectivity channel known as the plasmodial surface anion channel, increased Ca++ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca++ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N-hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca++ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca++ permeability, suggesting involvement of parasite-encoded proteins trafficked to the host membrane. A high-throughput chemical screen identified the first Ca++ transport inhibitors active against Plasmodium-infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca++ ] is consistent with parasite killing specifically via action on one or more Ca++ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca++ transport and may be starting points for new antimalarial drugs.