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

Abstract

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.

ATPe Dynamics in Protozoan Parasites. Adapt or Perish

“…In addition, P. falciparum can activate Ca 2+ influx of the RBC host. Early 45 Ca 2+ flux studies indicated that net Ca 2+ entry into infected RBCs was approximately 20 times faster than into uninfected cells [79], which agrees well with the reported elevated Ca 2+ conductance of patched membranes from infected RBCs [80]. This increased accumulation rate could not be explained by inhibition of the Ca 2+ pump, a cell membrane ATPase that keeps the Ca 2+ of uninfected RBCs very low.…”

“…I.e., when RBCs were infected with a parasite knocked down for PTEX (a translocon for export of parasite proteins into the host cell), Ca 2+ uptake was highly reduced. Moreover, a cell-based high-throughput screen for specific Ca 2+ transport inhibitors confirmed a protein-mediated uptake mechanism on infected cells distinct from the low capacity Ca 2+ carrier of RBCs [79] (Figure 1).…”

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells

“…The membrane-permeable dye crosses into the cell, where it is cleaved by esterases and retained inside the cell; the fluorescence intensity is proportional to the calcium concentration [ 68 ]. Cal520-AM and other calcium-sensitive AM dyes have previously been applied to uRBCs and iRBCs [ 67 , 69 ], supporting the notion that the RBC cytoplasm contains sufficient esterase activity to ensure this is not a rate-limiting step in fluorescence intensity. We observed significantly higher fluorescence in whole iRBCs compared to uRBCs, equalling approximately a 7.5-fold-change (± 0.9, p < 0.001) ( Fig 3A ).…”

“…We next quantified the intracellular calcium content of uRBCs and late-stage (trophozoite/schizont) iRBCs using the calcium-sensitive fluorescent dye Cal520-AM [ 67 ]. The membrane-permeable dye crosses into the cell, where it is cleaved by esterases and retained inside the cell; the fluorescence intensity is proportional to the calcium concentration [ 68 ].…”

Human plasma plasminogen internalization route in Plasmodium falciparum-infected erythrocytes

“…Most studies of incorporation and utilization of host factors have focused on low-molecular-weight compounds such as sugars, lipids, vitamins, and ions [19][20][21][22][23][24][25]. However, in recent decades, there has been accumulating evidence for the import of functional proteins by malariainfected erythrocytes.…”