An engineered bifunctional high affinity iron uptake protein in the yeast plasma membrane

High affinity iron uptake in fungi is supported by a plasma membrane protein complex that includes a multicopper ferroxidase enzyme and a ferric iron permease. In Saccharomyces cerevisiae, this complex is composed of the ferroxidase Fet3p and the permease Ftr1p. Fe(II) serves as substrate for Fe-uptake by being substrate for Fet3p; the resulting Fet3p-produced Fe(III) is then transported across the membrane via Ftr1p. A model of metabolite channeling of this Fe(III) is tested here by first constructing and kinetically characterizing in Fe-uptake two Fet3p-Ftr1p chimeras in which the multicopper oxidase/ferroxidase domain of Fet3p has been fused to the Ftr1p iron permease. Although the bifunctional chimeras are as kinetically efficient in Fe-uptake as is the wild type two-component system, they lack the adaptability and fidelity in Fe-uptake of the wild type. Specifically, Fe-uptake through the Fet3p, Ftr1p complex is insensitive to a potential Fe(III) trapping agent - citrate - whereas Fe-uptake via the chimeric proteins is competitively inhibited by this Fe(III) chelator. This inhibition does not appear to be due to scavenging Fet3p-produced Fe(III) that is in equilibrium with bulk solvent but could be due to leakiness to citrate found in the bifunctional but not the two-component system. The data are consistent with a channeling model of Fe-trafficking in the Fet3p, Ftr1p complex and suggest that in this system, Fet3p serves as a redox sieve that presents Fe(III) specifically for permeation through Ftr1p.     

Targeted suppression of the ferroxidase and iron trafficking activities of the multicopper oxidase Fet3p from<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The Fet3 protein in Saccharomyces cerevisiae is a multicopper oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe(2+) to Fe(3+); this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe(2+), one of which is essential also to the coupling of the ferroxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K(M) for Fe(2+) exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferroxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in (59)Fe uptake. For the Fet3p(E185D) mutant, K(M) for iron was 300-fold greater than the wild-type K(M), while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe(2+ )binding to Fet3p and to the subsequent trafficking of the Fe(3+) produced to Ftr1p.     

Evidence for iron channeling in the Fet3p-Ftr1p high-affinity iron uptake complex in the yeast plasma membrane

In high-affinity iron uptake in the yeast Saccharomyces cerevisiae, Fe(II) is oxidized to Fe(III) by the multicopper oxidase, Fet3p, and the Fe(III) produced is transported into the cell via the iron permease, Ftr1p. These two proteins are likely part of a heterodimeric or higher order complex in the yeast plasma membrane. We provide kinetic evidence that the Fet3p-produced Fe(III) is trafficked to Ftr1p for permeation by a classic metabolite channeling mechanism. We examine the (59)Fe uptake kinetics for a number of complexes containing mutant forms of both Fet3p and Ftr1p and demonstrate that a residue in one protein interacts with one in the other protein along the iron trafficking pathway as would be expected in a channeling process. We show that, as a result of some of these mutations, iron trafficking becomes sensitive to an added Fe(III) chelator that inhibits uptake in a strictly competitive manner. This inhibition is not strongly dependent on the chelator strength, however, suggesting that Fe(III) dissociation from the iron uptake complex, if it occurs, is kinetically slow relative to iron permeation. Metabolite channeling is a common feature of multifunctional enzymes. We constructed the analogous ferroxidase, permease chimera and demonstrate that it supports iron uptake with a kinetic pattern consistent with a channeling mechanism. By analogy to the Fe(III) trafficking that leads to the mineralization of the ferritin core, we propose that ferric iron channeling is a conserved feature of iron homeostasis in aerobic organisms.     

Fre1p Cu2+ reduction and Fet3p Cu1+ oxidation modulate copper toxicity in Saccharomyces cerevisiae

Fre1p is a metalloreductase in the yeast plasma membrane that is essential to uptake of environmental Cu2+ and Fe3+. Fet3p is a multicopper oxidase in this membrane essential for high affinity iron uptake. In the uptake of Fe3+, Fre1p produces Fe2+ that is a substrate for Fet3p; the Fe3+ produced by Fet3p is a ligand for the iron permease, Ftr1p. Deletion of FET3 leads to iron deficiency; this deletion also causes a copper sensitivity not seen in wild type. Deletion of FTR1 leads to copper sensitivity also. Production in the ftr1delta strain of an iron-uptake negative Ftr1p mutant, Ftr1p(RAGLA), suppressed this copper sensitivity. This Ftr1p mutant supported the plasma membrane targeting of active Fet3p that is blocked in the parental ftr1delta strain. A ferroxidase-negative Fet3p did not suppress the copper sensitivity in a fet3delta strain, although it supported the plasma membrane localization of the Fet3p.Ftr1p complex. Thus, loss of membrane-associated Fet3p oxidase activity correlated with copper sensitivity. Furthermore, in vitro Cu1+ was shown to be an excellent substrate for Fet3p. Last, the copper sensitivity of the fet3delta strain was suppressed by co-deletion of FRE1, suggesting that the cytotoxic species was Cu1+. In contrast, deletion of CTR1 or of FET4 did not suppress the copper sensitivity in the fet3delta strain; these genes encode the two major copper transporters in laboratory yeast strains. This result indicated that the apparent cuprous ion toxicity was not due to excess intracellular copper. These biochemical and physiologic results indicate that at least with respect to cuprous and ferrous ions, Fet3p can be considered a metallo-oxidase and appears to play an essential role in both iron and copper homeostasis in yeast. Its functional homologs, e.g. ceruloplasmin and hephaestin, could play a similar role in mammals.     

Assembly, activation, and trafficking of the Fet3p.Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae

The high affinity iron uptake complex in the yeast plasma membrane (PM) consists of the ferroxidase, Fet3p, and the ferric iron permease, Ftr1p. We used a combination of yeast two-hybrid analysis, confocal fluorescence microscopy, and fluorescence resonance energy transfer (FRET) quantification to delineate the motifs in the two proteins required for assembly and maturation into an uptake-competent complex. The cytoplasmic, carboxyl-terminal domain of each protein contains a four-residue motif adjacent to the cytoplasm-PM interface that supports an interaction between the proteins. This interaction has been quantified by two-hybrid analysis and is required for assembly and trafficking of the complex to the PM and for the approximately 13% maximum FRET efficiency determined. In contrast, the Fet3p transmembrane domain (TM) can be exchanged with the TM domain from the vacuolar ferroxidase, Fet5p, with no loss of assembly and trafficking. A carboxyl-terminal interaction between the vacuolar proteins, Fet5p and Fth1p, also was quantified. As a measure of the specificity of interaction, no interaction between heterologous ferroxidase permease pairs was observed. Also, whereas FRET was quantified between fluorescent fusions of the copper permease (monomers), Ctr1p, none was observed between Fet3p and Ctr1p. The results are consistent with a (minimal) heterodimer model of the Fet3p.Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM.     

Structural basis of the ferrous iron specificity of the yeast ferroxidase, Fet3p

Fet3p is a multicopper oxidase (MCO) that functions together with the iron permease, Ftr1p, to support high-affinity Fe uptake in yeast. Fet3p is a ferroxidase that, like ceruloplasmin and hephaestin, couples the oxidation of 4 equiv of Fe(II) to the reduction of O2 to 2 H2O. The ferrous iron specificity of this subclass of MCO proteins has not been delineated by rigorous structure-function analysis. Here the crystal structure of Fet3p has been used as a template to identify the amino acid residues that confer this substrate specificity and then to quantify the contributions they make to this specific reactivity by thermodynamic and kinetic analyses. In terms of the Marcus theory of outer-sphere electron transfer, we show here that D283, E185, and D409 in Fet3p provide a Fe(II) binding site that actually favors ferric iron; this site thus reduces the reduction potential of the bound Fe(II) in comparison to that of aqueous ferrous iron, providing a thermodynamically more robust driving force for electron transfer. In addition, E185 and D409 constitute parts of the electron-transfer pathway from the bound Fe(II) to the protein's type 1 Cu(II). This electronic matrix coupling relies on H-bonds from the carboxylate OD2 atom of each residue to the NE2 NH group of the two histidine ligands at the type 1 Cu site. These two acidic residues and this H-bond network appear to distinguish a fungal ferroxidase from a fungal laccase since the specificity that Fet3p has for Fe(II) is completely lost in a Fet3pE185A/D409A mutant. Indeed, this double mutant functions kinetically better as a laccase, albeit a relatively inefficient one.     

Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stability

Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N‐linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high‐molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N → A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high‐affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N‐linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N‐linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex.     

The yeast multicopper oxidase Fet3p and the iron permease Ftr1p physically interact

High affinity iron uptake in yeast is carried out by a multicomponent system formed by the ferroxidase Fet3p and the iron permease Ftr1p. The currently accepted model predicts that Fet3p and Ftr1p are functionally associated, however, a structural interaction between these two proteins has not been proven yet. The methylotrophic yeast Pichia pastoris has been used to perform cross-linking studies aimed to demonstrate the existence of a Fet3p-Ftr1p complex. Cross-linking of membrane suspensions with the membrane-impermeable reagents DTSSP and BS(3) has evidenced the presence of a high molecular weight band with Fet3p oxidase activity. This band has been purified and subjected to N-terminal sequence analysis. Two sequences were found in the cross-linked species, one of which could be assigned to Fet3p and the other to Ftr1p. This is the first experimental demonstration that Fet3p and Ftr1p are physically associated.     

Antifungal and Other Biological Activities of Two 2S Albumin-Homologous Proteins Against Pathogenic Fungi

The aim of this study was to determine whether 2S albumins from Passiflora edulis f. flavicarpa and Capsicum annuum seeds inhibit growth, induce plasma membrane permeabilization and induce endogenous production of nitric oxide in different pathogenic and non-pathogenic yeasts. The 2S albumin from P. flavicarpa (Pf-Alb) inhibited the growth of Kluyveromyces marxiannus, Candida albicans and Candida parapsilosis. The membranes of these yeast strains were permeabilized in the presence of Pf-Alb. The Pf-Alb also inhibited the glucose-stimulated acidification of the medium by Saccharomyces cerevisiae and C. albicans cells, which indicates a probable impairment of fungal metabolism because the inhibition of acidification occurred at various Pf-Alb concentrations and pre-incubation times. The 2S albumin from C. annuum (Ca-Alb) inhibited the growth of the yeasts K. marxiannus, C. tropicalis, C. albicans and S. cerevisiae. These yeast strains exhibited NO induction in the presence of Ca-Alb and displayed cellular agglomeration, elongated cells and the induction of pseudohyphae. Pf-Alb and Ca-Alb at various concentrations also inhibited the glucose-stimulated acidification of the medium by S. cerevisiae cells. Our results indicate that the ability of antimicrobial plant proteins such as 2S albumins to induce microbial inhibition could be an important factor in determining pathogen virulence. Therefore, 2S albumins might be targets for the design of new antifungal drugs.     

Bovine pancreatic trypsin inhibitor is a new antifungal peptide that inhibits cellular magnesium uptake

Antimicrobial peptides (AMPs) are promising agents for control of bacterial and fungal infections. Traditionally, AMPs were thought to act through membrane disruption but recent experiments have revealed a diversity of mechanisms. Here we describe a novel antifungal activity for bovine pancreatic trypsin inhibitor (BPTI). BPTI has several features in common with a subset of antimicrobial proteins in that it is small, cationic and stabilized by disulphide bonds. BPTI inhibits growth of Saccharomyces cerevisiae and the human pathogen Candida albicans. Screening of the yeast heterozygous essential deletion collection identified the magnesium transporter Alr1p as a potential BPTI target. BPTI treatment of wild type cells resulted in a lowering of cellular Mg²⁺ levels. Populations treated with BPTI had fewer cells in S‐phase of the cell cycle and a corresponding increase of cells in G₀/G₁ and G₂ phases. The same patterns of cell cycle arrest obtained with BPTI were also obtained with the magnesium channel inhibitor hexamine(III)cobalt chloride. Analysis of the growth inhibition of C. albicans revealed that BPTI is inhibiting growth via the same mechanism in the two yeast species. Inhibition of magnesium uptake by BPTI represents a novel mechanism of action for AMPs.     

EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport

The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component—the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved ‘HxxE’ motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu²⁺ using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe³⁺ and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the ‘EfeO-Cup’ family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology.     

Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a d-octapeptide derivative inhibitor

Overexpression of the Candida albicans ATP‐binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ～ 1.89 × 10⁶ member d ‐octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4‐methoxy‐2,3,6‐trimethylbenzenesulphonyl derivative of the d ‐octapeptide d ‐NH₂‐FFKWQRRR‐CONH₂, as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization‐resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug‐like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin β9 have modes of action similar to RC21v3.     

Grd19/Snx3p functions as a cargo-specific adapter for retromer-dependent endocytic recycling

Amajor function of the endocytic system is the sorting of cargo to various organelles. Endocytic sorting of the yeast reductive iron transporter, which is composed of the Fet3 and Ftr1 proteins, is regulated by available iron. When iron is provided to iron-starved cells, Fet3p–Ftr1p is targeted to the lysosome-like vacuole and degraded. In contrast, when iron is not available, Fet3p–Ftr1p is maintained on the plasma membrane via an endocytic recycling pathway requiring the sorting nexin Grd19/Snx3p, the pentameric retromer complex, and the Ypt6p Golgi Rab GTPase module. A recycling signal in Ftr1p was identified and found to bind directly to Grd19/Snx3p. Retromer and Grd19/Snx3p partially colocalize to tubular endosomes, where they are physically associated. After export from the endosome, Fet3p–Ftr1p transits through the Golgi apparatus for resecretion. Thus, Grd19/Snx3p, functions as a cargo-specific adapter for the retromer complex, establishing a precedent for a mechanism by which sorting nexins expand the repertoire of retromer-dependent cargos.     

Extensive functional redundancy in the regulation of Candida albicans drug resistance and morphogenesis by lysine deacetylases Hos2, Hda1, Rpd3 and Rpd31

Current treatment efforts for fungal infections are hampered by the limited availability of antifungal drugs and by the emergence of drug resistance. A powerful strategy to enhance the efficacy of antifungal drugs is to inhibit the molecular chaperone Hsp90. Hsp90 governs drug resistance, morphogenesis and virulence in a leading fungal pathogen of humans, Candida albicans. Our previous work with Saccharomyces cerevisiae established acetylation as a novel mechanism of posttranslational control of Hsp90 function in fungi. We implicated lysine deacetylases (KDACs) as key regulators of resistance to the most widely deployed class of antifungals, the azoles, in both S. cerevisiae and C. albicans. Here, we demonstrate high levels of functional redundancy among the KDACs that are important for regulating Hsp90 function. We identify Hos2, Hda1, Rpd3 and Rpd31 as the KDACs mediating azole resistance and morphogenesis in C. albicans. Furthermore, we identify lysine 30 and 271 as critical acetylation sites on C. albicans Hsp90, and substitutions at these residues compromise Hsp90 function. Finally, we show that pharmacological inhibition of KDACs phenocopies pharmacological inhibition of Hsp90 and abrogates Hsp90‐dependent azole resistance in numerous Candida species. This work illuminates new facets to the impact of KDACs on fungal drug resistance and morphogenesis, provides important insights into the divergence of the C. albicans Hsp90 regulatory network and reveals new targets for development of antifungal drugs.     

Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole Drugs

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H⁺-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma ⁻ phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca²⁺ and H⁺ surges triggered by the antimicrobial agent amiodarone, and impaired Ca²⁺ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.     

Candida albicans possess a highly versatile and dynamic high‐affinity iron transport system important for its commensal‐pathogenic lifestyle

Iron is an essential nutrient for nearly all organisms, but iron overdose is toxic. The human commensal‐pathogenic fungus Candida albicans traverses host niches with markedly different iron availability. During systemic infection, C. albicans must activate the high‐affinity iron permease Ftr1 to acquire iron sequestered by the host's iron‐withholding defense and suppresses iron uptake while residing in the iron‐rich gut to avoid toxicity. Ftr1 associates with a ferroxidase to form an iron transporter. C. albicans contains four permeases and five ferroxidase homologs, suggesting 20 possible subunit combinations. Here, we investigated the iron‐dependent expression, cellular localization and interacting partners of all permeases and ferroxidases and the significance of each subunit for gastrointestinal colonization and systemic infection in mice. We uncovered three distinct patterns of iron‐dependent expression and highly flexible ferroxidase‐permease partnerships, which underlie a dynamic iron transport system that can be deftly tuned according to iron availability. We found functional differentiation as well as redundancy among the ferroxidases and permeases during both gastrointestinal colonization and bloodstream infection. We propose that C. albicans possesses a sophisticated iron acquisition and utilization system befitting its commensal‐pathogenic lifestyle. Our findings reveal new possibilities for medical intervention of C. albicans infection.     

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.