Iron transport pathways in the human malaria parasitePlasmodium falciparumrevealed by RNA-sequencing

Plasmodium falciparum, malaria, drug target, iron deficiency, transporters, 22 nutrient uptake, gene expression, AlphaFold. 23 24 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 2

25 Host iron deficiency is protective against severe malaria as the human malaria parasite 26 Plasmodium falciparum depends on free iron from its host to proliferate. Due to the absence 27 of transferrin, ferritin, ferroportin, and a functional heme oxygenase, the parasite’s essential 28 pathways of iron acquisition, storage, export, and detoxification differ from those in humans 29 and may thus be excellent targets for therapeutic development. However, the P. falciparum 30 proteins involved in these processes remain largely unknown. 31 Here, we show that parasites cultured in erythrocytes from an iron-deficient donor displayed 32 significantly reduced growth rates compared to those grown in red blood cells from healthy 33 controls. Sequencing of parasite RNA revealed diminished expression of genes involved in 34 overall metabolism, hemoglobin digestion, and metabolite transport under low-iron versus 35 control conditions. Supplementation with hepcidin, a specific ferroportin inhibitor, resulted in 36 increased free iron levels in erythrocytes, enhanced parasite replication, and transcriptional 37 upregulation of genes responsible for merozoite motility and host cell invasion. Based on 38 endogenous GFP tagging of differentially expressed putative transporter genes followed by 39 confocal live-cell imaging, proliferation assays with knockout and knockdown lines, and 40 protein structure predictions, we identified six proteins that are likely required for ferrous iron 41 transport in P. falciparum. PfE140 may be involved in iron uptake into the parasite cytosol 42 across the plasma membrane and PfMRS3 could mediate import of the metal ion into the 43 mitochondrion. PfVIT may transport excess iron from the cytosol into cytoplasmic vesicles, 44 and PfZIPCO could be implicated in Zn2+ and Fe2+ export from these organelles, while 45 PfNRAMP and PfCRT are likely responsible for Fe2+ efflux from the digestive vacuole. Our 46

provide new insights into the mechanisms of iron transport in P. falciparum and the 47 parasite’s response to iron status alterations in the host. PfE140 and PfCRT are particularly 48 promising candidate targets for novel antimalarial drugs, as these are essential to the 49 parasite and lack human orthologs. 50 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 3

51 Iron is an essential micronutrient for all living organisms and has been associated with 52 virulence of many pathogens. Iron abundance increases the replication of human 53 immunodeficiency virus (HIV) (1) and Mycobacterium tuberculosis (2), and promotes biofilm 54 formation in Pseudomonas aeruginosa (3). A “fight for iron” has been described between 55 bacteria and the human host in the gastrointestinal tract (4), where the metal skews the 56 composition of the gut microbiome by facilitating the growth of enteropathogenic Escherichia 57 coli and Salmonella (5). Similarly, cancer cells require more iron compared to healthy cells 58 (6) and higher ferritin levels in individuals diagnosed with COVID-19 were associated with 59 increased disease severity and lethality (7). 60 61 Host iron deficiency is known to be protective against severe malaria (8-11) and iron-62 chelators have cytocidal effects on the human malaria parasite Plasmodium falciparum (12). 63 This obligate intracellular parasite depends on free iron for its proliferation and relies entirely 64 on the host to meet its nutrient requirements (13). Furthermore, P. falciparum senses 65 environmental fluctuations (14-16) and modulates its virulence in response (16). While iron is 66 crucial for DNA replication and repair, mitochondrial electron transport, and redox regulation, 67 it becomes toxic when in excess, as it is a source of damaging reactive oxygen species (17). 68 Importantly for therapeutic development, the mechanisms of iron acquisition, storage, 69 detoxification, and export in the parasite are different from those in humans due to the 70 absence of transferrin, ferritin, ferroportin, and a functional heme oxygenase in Plasmodium 71 (18). 72 73 While human blood plasma contains between 10 and 30 µM total iron and an erythrocyte 74 carries approximately 20 mM Fe (19), only 2.5 – 4.7 µM of iron is unbound and bioavailable 75 in uninfected red blood cells, and 1.5 – 1.8 µM in P. falciparum-infected ones in the form of 76 ferrous iron, Fe2+ (20). An estimated total iron concentration of 500 mM (21) is reached 77 within the parasite’s digestive vacuole (DV), where iron-containing hemoglobin (Hb) is 78 digested and the released heme is detoxified by biocrystallization into hemozoin (22). 79 However, P. falciparum cannot access this iron source and is thought to acquire free Fe2+ 80 from the host cell (23). Over-elevated concentrations of free iron likely compromise the 81 integrity of the DV membrane and cytosolic iron also needs to be regulated to prevent 82 oxidative stress (18). Iron detoxification in the parasite may be achieved by translocating the 83 metal ion into dynamic intracellular Fe2+ stores, which may include acidocalcisomes – 84 cytoplasmic vesicles that contain high concentrations of phosphate, calcium, iron, and zinc 85 (24). In contrast to Trypanosoma brucei (24), no transport proteins have yet been 86 experimentally shown to localize to the acidocalcisome membrane in P. falciparum (25, 26). 87 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 4 Like the DV, these organelles are thought to be acidified by the plant-like V-ATPase and 88 their low internal pH may fuel secondary active transport processes (22, 27). 89 90 P. falciparum encodes approximately 200 transmembrane or membrane-associated 91 transport proteins (channels, pores, carriers, and pumps), many of which are essential for 92 parasite growth and lack orthologs in humans (28). For instance, the vacuolar iron 93 transporter PfVIT (PF3D7_1223700), an ortholog of Arabidopsis thaliana VIT1 (expect value 94 (E) = 5 x 10-29, 30.5% identity, 87% coverage, as determined by position-specific iterated 95 BLAST (29)), is a Fe2+/H+ exchanger with unknown subcellular localization that likely plays a 96 role in iron detoxification (30-32). The chloroquine resistance transporter PfCRT 97 (PF3D7_0709000) and the natural resistance-associated macrophage protein PfNRAMP 98 (PF3D7_0523800) were both localized to the digestive vacuolar (DV) membrane (33, 34), 99 and have been suggested to export Fe2+ into the cytosol in symport with protons. These 100 predicted functions are based on transport assays with Xenopus oocytes (35) and homology 101 to the human endosomal Fe2+ transporter 2/DMT1 (36), respectively. In Saccharomyces 102 cerevisiae, a model organism for eukaryotic iron homeostasis, the mitochondrial carrier 103 protein MRS3 (mitochondrial RNA-splicing protein 3) was shown to ensure Fe2+ supply to the 104 mitochondrion (37-39). In P. falciparum, the mitochondrion is also the focal point for cellular 105 iron metabolism and contains iron-dependent proteins implicated in the biosynthesis of heme 106 and iron-sulfur clusters, redox reactions, and electron transport (18). It was proposed that its 107 ortholog PfMRS3 (also known as mitoferrin (PfMFRN), PF3D7_0905200, E = 3 x 10-14, 108 35.1% identity, 26% coverage) mediates Fe2+ import into the mitochondrion in P. falciparum 109 (40), but its localization had not been experimentally determined prior to this study. 110 111 Despite the importance of iron for P. falciparum virulence, understanding of the molecular 112 mechanisms of iron sensing, acquisition, utilization, and regulation in the parasite remains 113 limited. The goal of this exploratory study was to dissect how the parasite responds to 114 differences in iron availability in its environment and to identify putative iron transporters as 115 potential new antimalarial drug targets. We investigated growth and gene expression 116 patterns of the laboratory P. falciparum strain 3D7 under control (iron-replete), high-iron and 117 low-iron conditions, and in the presence of the iron-regulatory peptide hormone hepcidin. In 118 the human body, hepcidin is produced to reduce the concentration of serum iron when it 119 rises above a certain threshold. The hormone binds specifically to ferroportin on the surface 120 of many cell types including erythrocytes and sterically inhibits the transporter’s iron export 121 activity, thereby increasing the concentration of bioavailable intracellular iron and decreasing 122 serum iron levels (41, 42). Here, whole-transcriptome sequencing was used to identify 123 putative iron transport proteins based on differential gene expression patterns between high 124 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 5 vs. low-iron conditions. We then further studied these proteins by assessing their subcellular 125 localization in live parasites, determining the growth rates of knockout or knockdown parasite 126 lines, and analyzing their predicted 3D structures. 127 128

129 Elevated erythrocyte free iron levels promote P. falciparum proliferation in vitro 130 To investigate whether free iron levels in the erythrocyte correlate with parasite replication 131 rates, we established different iron conditions in vitro. The first approach was to culture P. 132 falciparum 3D7 parasites in erythrocytes from an individual with high iron status (231 µg/L 133 ferritin, 18.2 g/dL Hb, 51.5% hematocrit), an iron-deficient (43) person (3 µg/L ferritin, 11.4 134 g/dL Hb, 36.3% hematocrit) and a healthy donor (21 µg/L ferritin, 15.0 g/dL Hb, 42.3% 135 hematocrit). Secondly, infection of red blood cells from other healthy individuals with or 136 without the addition of 0.7 µM hepcidin to the culture medium was compared. This 137 concentration of the iron-regulatory hormone was reported to reduce binding of ferrous iron 138 to ferroportin by 50% in vitro (44), which is expected to increase intracellular Fe2+ 139 concentrations. 140 141 Relative free iron levels in uninfected erythrocytes were estimated by determining the mean 142 fluorescence intensity (MFI) of the iron-sensitive dye Phen Green SK per cell using flow 143 cytometry. As binding of ferrous iron to the metal-binding moiety causes fluorescence 144 quenching of the fluorophore, a reduction in fluorescence intensity indicates higher free iron 145 levels (45). In erythrocytes from the iron-deficient donor, the Phen Green SK MFI was 43% 146 higher relative to control, confirming the reduced free iron levels (Fig. 1A). The parasite 147 replication rate after one intraerythrocytic developmental cycle (IDC) decreased by 16% (Fig. 148 1B), the DNA content of late schizonts by 19% (Fig. 1C) and the number of merozoites 149 counted per late schizont by 14% (Fig. 1D). In contrast, bioavailable iron levels of 150 erythrocytes from the donor with high iron status were only slightly increased (without 151 statistical support, two-tailed unpaired t tests with Welch’s correction for unequal variances 152 and adjusted with the Holm-Šídák method for multiple comparisons, P = 0.25) relative to 153 blood with normal iron level (healthy control) – as were the parasite proliferation rate, the 154 DNA content and the merozoite number of mature schizonts (Fig. 1). To further increase 155 intracellular free iron levels, we incubated parasites with 0.7 µM hepcidin during one IDC, 156 resulting in 11% reduced Phen Green SK MFI compared to control (Fig. 1A). Under these 157 conditions, the parasite growth rate increased by 57% (Fig. 1B), the DNA content per 158 schizont by 16% (Fig. 1C), and the number of merozoites produced per schizont by 15% 159 (Fig. 1D). 160 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 6 161 Taken together, these data show that parasites grown in erythrocytes from an iron-deficient 162 donor displayed significantly reduced growth rates compared to healthy control. Our in vitro 163

also demonstrate that hepcidin treatment of control erythrocytes elevated intracellular 164 Fe2+ concentrations and promoted parasite proliferation. For each condition, independent 165 experiments were carried out on multiple occasions with consistent results. 166 167 RNA-sequencing reveals differential expression of putative iron transporters 168 To identify iron-regulated mechanisms and putative iron transporters in P. falciparum, we 169 carried out whole-transcriptome profiling using bulk RNA-sequencing (Fig. 2). P. falciparum 170 3D7 parasites were cultured either using erythrocytes from a donor with high, control 171 (healthy) or low iron status (experiment 1); or with red blood cells from another healthy donor 172 in the presence or absence of 0.7 µM hepcidin (experiment 2). Samples from three biological 173 replicates per condition were harvested at the ring and trophozoite stage (6 – 9 and 26 – 29 174 hours post invasion, hpi) during the second IDC under the conditions specified. 175 176 To exclude the possibility that differences in mRNA abundance were due to divergent 177 progression through the IDC under different nutritional conditions, we assessed the average 178 developmental age of the parasites in each sample based on a statistical maximum 179 likelihood estimation (MLE) method of transcriptional patterns according to Lemieux et al. 180 (46). The general transcriptional patterns of parasites were highly similar at individual time 181 points and consistent across different experimental treatments, corresponding to those of a 182 3D7 reference strain (47) at approximately 10 hpi and 35 hpi (Fig. 2A). This indicates that 183 differences in mRNA abundance of parasites were not caused by divergent progression 184 through the IDC but by direct effects of the experimental treatments. As the 3D7 strain we 185 used for the experiments had a reduced total IDC length of 44 h instead of 48 h, possibly 186 due to gene deletions that may have occurred during long-term culturing (48, 49), it 187 progresses through the cycle faster than the 3D7 reference strain (47). This may explain why 188 the calculated MLEs of parasite age were higher than the actual values of 6 – 9 hpi and 26 – 189 29 hpi (Fig. 2A). 190 191 Using a threshold of 1.5 for the fold change (FC) in gene expression (log2 FC of 0.585 or -192 0.585) yielded twelve significantly upregulated and 175 downregulated genes in ring-stage 193 parasites under high vs. low-iron conditions (P < 0.05, exact test for the negative binomial 194 distribution with Benjamini-Hochberg correction (50)). As differences in transporter gene 195 transcription are typically small (51, 52), we examined the 351 upregulated and 770 196 downregulated genes with a significant expression change and a minimum absolute value of 197 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 7 the log2 FC of 0.2 for this comparison (Fig. 2B). The full RNA-seq datasets are available in 198 the BioStudies repository (53) under accession number E-MTAB-13411 199 (https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13411) and differential gene expression 200 test results for individual genes are shown in Supplementary Tables S1 and S2. The highly 201 polymorphic var, stevor, and rifin gene families were excluded from downstream analyses 202 due to their significant sequence diversity between parasites of the same strain during 203 mitotic growth (54, 55). Functional Gene Ontology (GO), Kyoto Encyclopedia of Genes and 204 Genomes (KEGG) and Reactome (REAC) term enrichment analyses of differentially 205 expressed genes (DEGs) were performed using the g:Profiler web server (56). 206 207 Under high vs. low-iron conditions at the ring stage (6 – 9 hpi), the GO term for biological 208 process GO:0055085 “transmembrane transport” was 2.8-fold enriched (P = 0.007, 209 hypergeometric test) among significantly upregulated parasite genes (Fig. 2C). Using the 210 recently updated P. falciparum transporter list (28), all genes with differential expression 211 levels at the ring stage were then screened for transport proteins and all of the five putative 212 iron transporters previously proposed for Plasmodium (VIT, ZIPCO, NRAMP, CRT, 213 MRS3/MFRN (18, 57)) were found differentially expressed (Table 1). Other significantly 214 enriched functional terms at the ring stage under high-iron conditions were GO:0009056 215 “catabolic process”, GO:0020020 “food vacuole”, KEGG:01100 “metabolic pathways”, and 216 GO:0005737 “cytoplasm” (Fig. 2C). Among downregulated genes under high vs. low-iron 217 conditions at the ring stage, KEGG:03440 “homologous recombination”, KEGG:03410 “base 218 excision repair”, GO:0007049 “cell cycle”, and GO:0015630 “microtubule cytoskeleton” were 219 enriched (Fig. 2C). At the more metabolically active trophozoite stage (26 – 29 hpi), 220 processes related to mRNA splicing and protein production were overrepresented in 221 upregulated genes, as indicated by the 2.9-fold enrichment (P = 0.00006) of the 222 KEGG:03040 pathway “spliceosome” and the 2.5-fold enrichment (P < 0.05) of the 223 GO:0015934 term “large ribosomal subunit” (Fig. 2C). 224 225 In contrast, hepcidin treatment resulted in reduced metabolism compared to control 226 conditions, as KEGG:00040 “pentose and glucuronate interconversions”, REAC:R-PFA-227 71291 “metabolism of amino acids and derivatives”, GO:0005737 “cytoplasm”, and 228 GO:0015934 “large ribosomal subunit” were significantly enriched in downregulated genes 229 during the parasite ring stage at 6 – 9 hpi (Fig. 2E). Among significantly upregulated genes 230 in the presence vs. absence of hepcidin, the terms GO:0070258 “inner membrane pellicle 231 complex” (P < 0.05), KEGG:03430 “mismatch repair” (P = 0.04), and GO:0015630 232 “microtubule cytoskeleton” (P = 0.04) were enriched at the ring stage. GO:0044409 “entry 233 into host” (P = 0.00008) and GO:0052126 “movement in host environment” (P = 0.0001) 234 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 8 were overrepresented at the trophozoite stage (Fig. 2E), possibly linked to the observed 235 increase in parasite proliferation (Fig. 1B). 236 237 Our RNA-seq data also revealed the differential expression of genes involved in epigenetic, 238 transcriptional, translational, and post-translational regulation. Under high vs. low-iron 239 conditions, histone deacetylation and chromatin organization processes as well as 240 GO:1990904 “ribonucleoprotein complex” were significantly enriched in upregulated genes 241 at the trophozoite stage, and GO:000370 “DNA binding transcription factor activity” in 242 downregulated genes at the ring stage (Fig. 2C). Furthermore, the known iron-regulatory 243 protein PfIRP or aconitate hydratase (58, 59) was upregulated during the ring stage under 244 high vs. low-iron conditions (log2 FC = +0.49, P = 0.00003) and downregulated in the 245 presence of hepcidin (log2 FC = -0.27, P = 0.01) as compared to control. Many protein 246 kinases involved in post-translational modifications and endocytosis were also upregulated 247 at 26 – 29 hpi at high vs. low iron levels, as indicated by the enriched terms GO:0043170 248 “macromolecule metabolic process” and KEGG:04070 “phosphatidylinositol signaling 249 system” (Fig. 2C). 250 251 Localization of putative iron transporters in P. falciparum 252 Based on transcriptomic profiles and data from the literature, six proteins with a potential role 253 in iron transport were identified (Table 1 and 2). Of these, PfCRT (60) and PfNRAMP (34) 254 had already been shown to localize to the digestive vacuole (DV) in live parasites (61). The 255 subcellular localization of the other four putative iron transporters (PfMRS3, PfVIT, PfZIPCO, 256 and PfE140) was then examined by endogenous tagging with GFP and confocal imaging of 257 live parasites under physiological control conditions. At least two cell lines were generated 258 per candidate with consistent results and representative example images are shown in Fig. 259 3. Diagnostic PCRs confirmed the fusion of gfp to the respective gene of interest and the 260 absence of parental DNA at the original locus (Supplementary Fig. S2). Only the PfMRS3 261 reporter cell line still contained wild-type DNA of the parental parasites even after prolonged 262 WR99210/neomycin selection and limiting dilution cloning (Supplementary Fig. S2), 263 indicating the importance of this mitochondrial transporter for asexual parasite growth during 264 the blood stage. 265 266 The GFP-tagged mitochondrial carrier protein PfMRS3 exclusively localized to the 267 mitochondrion, as determined by colocalization with MitoTracker Red (Fig. 3A, 268 Supplementary Video S1). PfVIT-GFP displayed a punctate fluorescence pattern within the 269 cytoplasm (Fig. 3B, Supplementary Video S2), resembling that of PfZIPCO-GFP (ZIP 270 domain-containing protein, PF3D7_1022300, Fig. 3C, Supplementary Video S3). These 271 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 9 structures did not colocalize with ER Tracker Red in live cells (Fig. 3B, Supplementary Video 272 S2). For both PfVIT-GFP and PfZIPCO-GFP, the number of cytoplasmic foci increased as 273 the parasites matured from the ring to the late schizont stage (Fig. 3B and C). To test 274 whether these could be acidocalcisomes, we employed LysoTracker Deep Red, commonly 275 used to visualize small acidic organelles in T. brucei (24). However, the fluorescent dye only 276 stained the DV in P. falciparum (Fig. 3C, Supplementary Video S3) and no acidocalcisome-277 specific marker is currently available for this parasite. 278 279 GFP-tagged PfE140 (PF3D7_0104100), also known as conserved Plasmodium membrane 280 protein or CPMP (62), localized to the parasite plasma membrane, as evidenced by the ring-281 like fluorescence pattern around newly formed merozoites (Fig. 3D, Supplementary Video 282 S4). The fluorescence intensity was very low at the ring and early trophozoite stage 283 compared to schizonts. Based on amino acid sequence similarity (E = 9 x 10-5, 22.5% 284 identity, 66% coverage) to the essential apicoplast transporter PfDER1-2 (29, 63), we also 285 investigated the potential colocalization with the apicoplast marker PfACP (acyl carrier 286 protein), which could not be detected (Fig. 3D, Supplementary Video S4). 287 288 Functional assessment of PfVIT, PfZIPCO and PfE140 289 To study the function of the putative transport proteins identified, we used targeted gene 290 disruption (TGD) by selection-linked integration (SLI) to generate the corresponding 291 knockout parasite lines for the putative iron transporters that are non-essential during P. 292 falciparum blood stage: PfVIT and PfZIPCO (Fig. 4A, Supplementary Fig. S2). As GFP was 293 cloned in frame with the truncated version of the respective transporter (the N-terminal 143 294 amino acids (aa) of 274-aa PfVIT or 117 of the 325 aa of PfZIPCO), the subcellular 295 localization of the resulting GFP fusion protein was also assessed. PfVIT(1-143)-GFP 296 localized to cytoplasmic structures and PfZIPCO(1-117)-GFP to the DV and cytoplasmic 297 vesicles (Fig. 4A). Proliferation assays were then performed to determine the importance of 298 the respective transporter for parasite growth. While the PfVIT knockout had no effect on 299 parasite growth under standard conditions, addition of hepcidin reduced the growth rate of 300 the Δ VIT line by 30% (Fig. 4B). Of note, hepcidin generally had a smaller effect after two 301 cycles of incubation (Fig. 4B) than after one cycle compared to the first IDC (Fig. 1B). 302 Unexpectedly, knocking out PfZIPCO led to a growth rate increase by 42% after two IDCs 303 relative to wild-type 3D7 parasites (Fig. 4B). 304 305 For an inducible knockdown of PfE140, which is predicted to be essential (64), a glmS 306 ribozyme sequence (65) was introduced upstream of the 3’ untranslated region in the pSLI 307 plasmid, allowing for conditional mRNA degradation by adding 2.5 mM glucosamine (GlcN) 308 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 10 to the culture media (Fig. 4C, Supplementary Fig. S2). This led to a 61% decrease in total 309 parasite fluorescence intensity after 36 hours of GlcN treatment (Fig. 4D) without affecting 310 parasite size compared to untreated control (Fig. 4E). GlcN addition also caused a 38% 311 growth rate reduction of the PfE140-GFP-glmS line in the absence of hepcidin vs. no 312 significant alteration in the presence of hepcidin relative to standard culture conditions (Fig. 313 4F). The generation of a PfMRS3-knockdown line was not successful after four independent 314 attempts, supporting the potential essentiality of the gene for asexual growth (64). 315 316 Characterization and functional implications of predicted protein structures 317 We next took advantage of the recent progress in protein structure prediction and generated 318 models of the putative iron transport proteins identified (Table 2) using AlphaFold2 (66, 67) 319 and AlphaFold2-multimer (68). The transmembrane regions of the proteins typically 320 exhibited the highest confidence score, while some other protein portions appeared 321 unstructured (Fig. 5A). Regions that are likely located within a membrane were validated by 322 inspecting the molecular lipophilicity potential of the protein surfaces (Fig. 5B). A clear 323 hydrophobic belt was observed for all proteins and their orientation in the membrane was 324 determined based on that of orthologous proteins. As transport cavities with negatively 325 charged residues are a hallmark of heavy metal ion transporters, we analyzed the 326 distribution of charge on the surface of the proteins and looked for negatively charged 327 regions to assess the capacity to bind cations like Fe2+ (Fig. 5C). To gain further insights into 328 the functions of the proteins identified, we also compared the predicted structures with those 329 of well-characterized putative orthologs from S. cerevisiae, Eucalyptus grandis, Bordetella 330 bronchiseptica and Staphylococcus capitis (Table 2, Supplementary Fig. S3 and S4). 331 332 The outer surface of PfMRS3 (transport classification (TC): 2.A.29, mitochondrial carrier 333 family) is positively charged (Fig. 5C) and there is a clear negatively charged patch in the 334 putative binding pocket facing the mitochondrial intermembrane space. We compared the 335 predicted PfMRS3 structure with that of S. cerevisiae MRS3, which is known to import 336 ferrous iron into the mitochondrial matrix across the inner membrane (37-39). The predicted 337 structures of PfMRS3 and S. cerevisiae MRS3 were superimposed with an average root 338 mean square deviation of Cα atoms (Cα RMSD) of the 205 matched residues of 2.3 Å 339 (Supplementary Fig. S3A and S4A). The conserved histidine residues His48 and His105 that 340 were required for Fe2+ transport by S. cerevisiae MRS3 in reconstituted liposomes (37) are 341 also present in PfMRS3 and the three functionally relevant histidine residues identified in 342 yeast are in a similar functional context in both structures (Supplementary Fig. S3A and 343 S4A). This suggests that MRS3 may elicit similar molecular functions in S. cerevisiae and P. 344 falciparum. 345 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 11 346 PfVIT is highly similar to VIT1 from E. grandis (E = 7 x 10-27, 30.3% identity, 84% coverage), 347 for which an experimental structure is available (PDB 6IU4). The plant protein crystallized as 348 a homodimer (69), and the same oligomeric state was suggested for the vacuolar iron 349 transporter family (TC: 2.A.89) protein in P. falciparum (31). A PfVIT monomer also has five 350 transmembrane domains and comprises a negatively charged region facing the cytosol that 351 may enable cation transport (Fig. 5C). In agreement with this, one Fe2+ ion and two Zn2+ ions 352 were bound by a strongly charged region on the cytosolic side of the E. grandis VIT1 353 monomer (69) and a highly similar putative binding pocket is present in the parasite protein 354 (Supplementary Fig. S4B). In the structural alignment, 219 of the 227 residues of the 355 experimental EgVIT123-249 structure are within 5 Å of the predicted structure of PfVIT with an 356 average Cα RMSD of 1.9 Å and the key residues in the metal-binding domain (Glu102, Glu105, 357 Glu113, Glu116, using EgVIT123-249 numbering) are placed in a similar molecular context in the 358 predicted structure of PfVIT (Supplementary Fig. S3B and S4B). The residues in the 359 transmembrane domain that are in the vicinity of the Co2+ ion in the EgVIT123-249 structure 360 (Met80 and Asp43) are also conserved (Supplementary Fig. S3B). These results strongly 361 suggest that PfVIT functions similarly to EgVIT1. 362 363 PfZIPCO contains seven transmembrane domains and was modeled as a homodimer (Fig. 364 5A), as it is part of the zinc (Zn2+)-iron (Fe2+) permease (ZIP) family (TC: 2.A.5), whose 365 members usually function as homo- or heterodimers (70). The negatively charged patch in 366 each binding pocket facing the vesicle lumen (Fig. 5C) may be involved in cation transport to 367 the cytosolic side. In an overlay of the PfZIPCO model with the cryo-EM structure (PDB 368 8GHT) of a ZIP transporter from B. bronchiseptica in the presence of either Zn2+ or Cd2+ ions 369 (71), the average Cα RMSD of 140 sequence-aligned residues was 2.0 Å (Supplementary 370 Fig. S3C and S4C). Several key residues of the metal binding site M1 of BbZIP (Met99, 371 His177, Glu181, Glu211) were also found in PfZIPCO, whereas others (Asn178, Gln207, Asp208, 372 Glu240) were different (Supplementary Fig. S3C), possibly resulting in divergent substrate 373 specificity. 374 375 Like PfCRT (TC 2.A.7, drug/metabolite exporter family), for which a recent cryo-EM structure 376 (PDB: 6UKJ) is available (72), the predicted structure of PfNRAMP (TC: 2.A.55, metal ion 377 (Mn2+-iron) transporter family) contains ten transmembrane domains (Fig. 5A) and a 378 negatively charged region within its binding pocket facing the digestive vacuolar lumen (Fig. 379 5C). This is consistent with binding of cations such as Fe2+. The PfNRAMP model was 380 superimposed on the solved crystal structure of S. capitis NRAMP/DMT (PDB code: 5M95, E 381 = 1 x 10-32, 26.3% identity, 60% coverage), which was shown to bind Mn2+, Fe2+, Co2+, Ni2+, 382 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 12 Cd2+ and Pb2+ (73). In the overlay, the average Cα RMSD of the 349 matched residues was 383 1.6 Å and the negatively charged cavity of PfNRAMP was in close proximity to the Mn2+ ion 384 bound to S. capitis NRAMP (Supplementary Fig. S3D and Supplementary Fig. S4D). Two of 385 the four key residues required for ion coordination in the binding pocket of the bacterial 386 protein (Asn52 and Asp49) are present in PfNRAMP, while the two other residues (Met226 and 387 Ala223) are changed to serine (73). PfNRAMP is thus likely to perform cation transport from 388 the DV into the cytosol. 389 390 PfE140 is predicted to be anchored in the parasite plasma membrane by a bundle of five 391 transmembrane domains (74) and forms a coiled coil with a hydrophilic region that displays 392 negatively charged patches exposed to the extracellular side (Fig. 5A and B). No human 393 orthologs could be identified for this highly conserved Plasmodium protein (29). As there is 394 no obvious channel or cavity in the transmembrane region of the PfE140 monomer (Fig. 5B 395 and C), the helical bundles may form a dimer to enable ion transport. However, we were not 396 able to obtain a PfE140 dimer model with AlphaFold2-multimer due to its sequence length. 397 To predict functional residues based on the amino acid sequence and the AlphaFold2 398 structure of PfE140, we used DeepFRI graph convolutional network (75), which has 399 significant denoising capability and can reliably assign GO terms to residues in the protein. 400 In particular, the terms GO:0022857 “transmembrane transporter activity” (DeepFRI 401 gradCAM score 0.94), GO:0015075 “monoatomic ion transmembrane transporter activity“ 402 (score 0.78), and GO:0046873 “metal ion transmembrane transporter activity” (score 0.67) 403 were assigned to a putative transmembrane region of PfE140 with high confidence 404 (Supplementary Fig. S5). We thus speculate that the protein is a transporter of metal ions. 405 406

407 Here, we studied the role of iron in growth and transcription of P. falciparum using blood 408 from individuals of different iron status and by adding hepcidin as an iron-regulatory 409 hormone and ferroportin inhibitor. Overall, our data demonstrate the importance of Fe2+ in 410 parasite replication and development and highlight areas for further study. We showed that 411 in vitro growth rates of P. falciparum 3D7 and the number of merozoites formed per schizont 412 were reduced within erythrocytes that contain lower concentrations of free iron, while 413 culturing in blood from an individual with high iron status did not lead to a significant increase 414 in free iron levels within erythrocytes or in parasite growth relative to control (Fig. 1). 415 Consistent with this, reduced propagation of P. falciparum 3D7, Dd2, and FCR3-FMG was 416 reported when erythrocyte samples from iron-deficient individuals used for parasite culture 417 (10, 76). This effect was eliminated after these donors were iron-supplemented, while 418 supplementation of healthy (iron-replete) donors did not significantly promote parasite 419 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 13 growth (10). The strong increase in parasite replication in the presence of hepcidin relative to 420 control conditions (Fig. 1B) may have been due to enhanced invasion efficiency in addition 421 to the increased number of merozoites formed (Fig. 1D). Previous reports also indicated that 422 higher hepcidin levels in blood samples were associated with elevated P. falciparum growth 423 rates in vitro (76) and severe malaria in vivo (77). 424 425 To identify putative iron transporters and iron-regulated processes, we carried out RNA-426 sequencing analyses of P. falciparum during the ring (6 – 9 hpi) and trophozoite (26 – 29 427 hpi) stages cultured under the different iron conditions described above. A higher number of 428 biological processes and pathways were significantly enriched among DEGs when 429 erythrocytes from donors with different iron status were used for parasite culture (Fig. 2C) 430 compared to red blood cells from the same healthy donor in the presence vs. absence of 431 hepcidin (total of 28 vs. 13 functional terms, Fig. 2E). This may reflect greater differences in 432 the culture conditions; for instance, blood from the donor with high ferritin and Hb levels may 433 have also contained more glucose or copper (78, 79), potentially explaining the more diverse 434 physiological response of the parasite. Including erythrocyte samples from more individuals 435 in the growth experiments and RNA-seq analysis would have provided further insights, 436 however, the provision of sufficient blood from iron-deficient donors is limited by ethical 437 constraints. 438 439 The availability of additional nutrients likely resulted in increased endocytosis and digestion 440 of host cell contents in the DV of the parasite, leading to enhanced metabolism, mRNA 441 splicing, and protein production. Interestingly, the terms KEGG:01100 “metabolic pathways” 442 and GO:0005737 “cytoplasm” were also found to be enriched in upregulated parasite genes 443 in children with high vs. low parasitemia (80, 81). RNA binding and mRNA splicing 444 processes were previously reported to be overrepresented in upregulated genes in severe 445 malaria linked to high parasite density (80-82). Hence, an increase in overall parasite fitness 446 under high vs. low-iron conditions may explain the increase in parasite multiplication (Fig. 447 1B) and could be associated with higher parasitemia and disease severity. Consistent with 448 the observed upregulation of transmembrane transporters at the parasite ring stage (6 – 9 449 hpi) under high vs. low-iron conditions, Mancio-Silva et al. found that the functional term “ion 450 transporter activity” was enriched in Plasmodium berghei genes that were downregulated 451 under caloric restriction at 6 and 10 hpi (16). Thus, transmembrane transporter genes may 452 need to be transcribed at the beginning of the IDC to ensure that the appropriate level of 453 transport proteins is available for nutrient acquisition and metabolite efflux during the 454 subsequent metabolically active trophozoite and schizont stages. 455 456 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 14 Hepcidin plays a central role in mammalian iron homeostasis and reduces serum iron 457 concentrations (83). It is also known that hepcidin levels are elevated in P. falciparum-458 infected individuals, especially those with high parasitemia (77, 84), and that malaria causes 459 iron deficiency (77). The transcription profile of parasites treated with hepcidin showed 460 similarities to those cultured in erythrocytes from the iron-deficient donor compared to 461 standard conditions in terms of downregulated catabolic and translation processes as well as 462 transport protein regulation (Fig. 2, Table 1). The upregulation of genes involved in 463 merozoite motility (PfMTIP, PfGAP45, and various inner membrane complex proteins) and 464 host cell entry (such as PfAMA1, PfMSP3, PfMSP7, and PfEBA181) when hepcidin was 465 present (Fig. 2) may suggest an improved ability of the released merozoites to invade 466 erythrocytes. Thus, the addition of the peptide hormone to the culture media may be a signal 467 for the parasite to reduce metabolic processes and to increase its replication and invasion 468 efficiency. 469 470 In addition to roles in parasite proliferation and development, different levels of bioavailable 471 iron may induce regulatory processes at various levels. Under high-iron conditions, the 472 observed upregulation of histone deacetylation (Fig. 2C) may lead to the condensation and 473 thus deactivation of certain chromatin regions (85). Similarly, iron-mediated regulation of 474 mRNA translation by iron-regulatory proteins has been described in yeast, trypanosomes 475 and mammals (86-88). The binding sites and target genes of the differentially expressed 476 transcription factors and of PfIRP remain to be identified in P. falciparum. Moreover, protein 477 phosphorylation may play a role in iron-dependent regulatory mechanisms. As a 478 serine/threonine kinase (KIN) serves as a nutrient sensor in P. berghei, driving a fast 479 response that leads to increased parasite multiplication and virulence (16), a similar kinase 480 may sense iron and lead to increased replication in P. falciparum. 481 482 Based on our RNA-sequencing results and data from the literature (Fig. 2 and Table 1), we 483 identified six proteins that are likely involved in P. falciparum iron transport (Table 2 and 484 Figure 6) and analyzed their subcellular localization (Fig. 3), their importance for growth (Fig. 485 4), and their predicted structures (Fig. 5). PfMRS3 transcription was upregulated at the ring 486 stage under high vs. low-iron conditions (log2 FC = 0.33, P = 0.002, Fig. 2B), and 487 fluorescence of the GFP-tagged protein was exclusively detected at the mitochondrion (Fig. 488 3A). As a disruption of the gene was reported to fail (64), we were not able to generate a 489 knockdown line after four independent attempts, and parental DNA of the original gene locus 490 was still present in the GFP reporter line (Supplementary Fig. S2), PfMRS3 is likely essential 491 for asexual growth like PBANKA_041620 (E = 1 x 10-69, 71.4% identity, 25% coverage) in P. 492 berghei (89). The orthologous mitochondrial iron transporter (TgMIT, TGME49_277090, E = 493 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 15 7 x 10-19, 26.0% identity, 28% coverage) also localized to the mitochondrion in T. gondii and 494 was upregulated at the protein level upon iron overload due to a TgVIT knock out in the 495 related apicomplexan parasite (90). In line with our structural analyses (Fig. 5, 496 Supplementary Fig. S3A and S4A), PfMRS3 may thus import ferrous iron into the 497 mitochondrion – the main iron user of the cell – and thereby reduce the cytosolic Fe2+ 498 concentration (Fig. 6) as a means of detoxification, as previously described in yeast (91). 499 The protein’s substrate specificity as well as iron binding and transport activity remain to be 500 confirmed experimentally. 501 502 PfVIT was suggested to be involved in iron detoxification in Plasmodium (30) and its 503 expression was upregulated under high vs. low-iron conditions (log2 FC = +0.29, P = 0.02, 504 Fig. 2B). The fluorescence pattern of PfVIT-GFP in live cells (Fig. 3B) was consistent with 505 cytoplasmic vesicles that may be acidocalcisomes, as described for T. brucei VIT1 (24). An 506 increase in the number of fluorescent punctate structures during parasite development (Fig. 507 3B) was also observed for VIT in Toxoplasma gondii (90). PfVIT shares 47.0% identity with 508 TgVIT (E = 8 x 10-84, 95% coverage) and 36.9% identity with TbVIT1 (E = 9 x 10-39, 98% 509 coverage). In contrast, P. berghei VIT (PBANKA_143860, E = 3 x 10-160, 79.3% identity, 98% 510 coverage) was shown to localize to the ER in indirect immunofluorescence assays (30). This 511 may be explained by differences between species or variation in methodology such as 512 fixation, permeabilization, and immunolabeling techniques as opposed to live-cell imaging 513 (92-94). While not essential during asexual blood stages (64), a knockout of VIT resulted in 514 reduced liver stage development in P. berghei (30) and increased sensitivity to high iron 515 levels in both P. berghei (30) and T. gondii (90). Similarly, growth of the Δ VIT P. falciparum 516 line was not affected under standard conditions, whereas the addition of hepcidin – which 517 increases free intracellular iron levels (Fig. 1A) – compromised parasite proliferation in our 518 study (Fig. 4B). The high similarity of the putative Fe2+-binding pocket in the predicted PfVIT 519 structure with that of the experimentally characterized EgVIT1 (69) and the conservation of 520 key residues for metal ion binding indicate that PfVIT is capable of Fe2+ translocation (Fig. 5, 521 Supplementary Fig. S3B and S4B). Thus, the transporter may contribute to iron 522 detoxification by Fe2+ sequestration into cytoplasmic vesicles (Fig. 6) and Δ VIT parasites 523 may be more sensitive to elevated intracellular iron concentrations due to impaired removal 524 of excess iron from the cytosol. 525 526 PbZIPCO (PBANKA_050650) was localized to the parasite plasma membrane (PPM) in P. 527 berghei sporozoites in indirect immunofluorescence assays and suggested to transport both 528 Zn2+ and Fe2+ (95). In P. falciparum, however, the GFP fusion protein caused a punctate 529 fluorescence pattern in the cytoplasm of live cells (Fig. 3C), similar to that of PfVIT-GFP (Fig. 530 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 16 3B). While the PfZIPCO knockout caused a growth increase under standard conditions (Fig. 531 4B), Δ ZIPCO P. berghei parasites displayed normal blood stage development and impaired 532 sporozoite infectivity in mice (95). Interestingly, the ortholog TGME49_225530 in T. gondii is 533 also dispensable with a phenotype score of −2.94 (values below -1.5 are considered non-534 essential (96)). Hence, Fe2+ efflux via PfZIPCO from cytoplasmic vesicles (potentially 535 acidocalcisomes) may be dispensable in P. falciparum under iron-replete conditions during 536 the blood stage, and the production of the transporter may come at a fitness cost, while 537 sporozoites may rely on its activity in low-iron environments. As the transcription of PfZIPCO 538 was upregulated at low vs. control iron levels (log2 FC = 0.55, P = 0.04, Table 1) and in 539 response to hepcidin treatment (log2 FC = 0.63, P = 0.006, Fig. 2D, Table 1), the transport 540 protein may release Zn2+ and Fe2+ ions from intracellular stores, in this case cytoplasmic 541 vesicles (Fig. 6), in case of scarcity, thereby increasing cytosolic ion levels like other ZIP 542 transporters (57). While our analyses of the predicted structure and the structural alignment 543 with BbZIP indicate that PfZIPCO likely has the capacity to bind and transport cations like 544 Zn2+ or Fe2+ (Fig. 5, Supplementary Fig. S3C and S4C), its substrate specificity can only be 545 conclusively established by characterizing the purified protein. Liposomal assays with the 546 putative zinc transporter PfZIP1 (PF3D7_0609100, 24.5% identity with PfZIPCO, E = 1 x 10-547 19, 78% coverage), which localized to the plasma membrane in schizonts, demonstrated that 548 this ZIP transporter preferentially binds Zn2+ over Fe2+ (97). Interestingly, the preference was 549 abolished if the histidine-rich loop at the C-terminus of PfZIP1, which is not present in 550 PfZIPCO, was truncated. As mRNA levels of PfZIP1 were enhanced at low cytosolic Zn2+ 551 levels (97) but not differentially regulated under various iron conditions (Supplementary 552 Tables S1 and S2), it may play a role in zinc rather than iron homeostasis under 553 physiological conditions. 554 555 As the highest intracellular iron concentration in P. falciparum is reached within the DV (21, 556 98), the free form of the metal may need to be exported from this compartment under high-557 iron conditions to prevent damage to the DV membrane (Fig. 6). This function may be 558 performed by PfCRT (35) and / or PfNRAMP (36), which were both upregulated under high 559 vs. low-iron conditions in our RNA-seq analysis (log2 FC = 0.26, P = 0.007 and log2 FC = 560 0.28, P = 0.003, respectively, Fig. 2B, Table 1) and are essential in asexual parasites (33, 561 34, 64). The predicted structure of PfNRAMP (Fig. 5) likely reflects the state that is open 562 towards the cytosol as in the crystal structure of NRAMP from Deinococcus radiodurans 563 (99). While a negatively charged cavity inside the protein is clearly visible in the PfNRAMP 564 model, the proposed outward-facing permeation pathway for metal ions is likely occluded in 565 this conformation (Fig. 5C). It is conceivable that Fe2+ ions permeate through this pathway 566 from the DV lumen and bind to the charged cavity like the Mn2+ ion to S. capitis 567 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 17 NRAMP/DMT (Supplementary Fig. S4D). PfNRAMP might function similarly to its ortholog in 568 D. radiodurans, which was shown to mediate pH-dependent transport of Fe2+ and Mn2+ in 569 symport with H+ using uptake assays in E. coli, HEK293T cells, and proteoliposomes (99, 570 100). 571 572 Expression of the surface protein PfE140 was upregulated when iron levels were low 573 compared to standard conditions (log2 FC = 0.65, P = 0.0006, Table 1) and the GFP fusion 574 protein localized to the PPM only, as evidenced by the fluorescent edges of free merozoites 575 (Fig. 3D). This observation is consistent with the fact that the extracellular portions of this 576 protein are highly polymorphic due to their exposure to the immune system at the sporozoite 577 stage (74). Interestingly, vaccines targeting PyE140 in Plasmodium yoelii were reported to 578 induce up to 100% sterile protection mediated by antibodies in mice (101). The reduced 579 parasite replication rate upon its conditional knockdown (Fig. 4D) demonstrates the 580 importance of PfE140 for parasite growth. Its predicted essential nature (64), in addition to 581 the absence of orthologs in humans, make it an excellent drug target candidate. While our P. 582 falciparum gene expression data (Fig. 2B and D) point towards a role of PfE140 in iron 583 homeostasis, its precise function is still unclear and it remains to be clarified whether the 584 large coiled-coil domain exposed to the extracellular space (Fig. 5) can mediate dimerization 585 upon substrate binding. Given our experimental results and the functional annotations (Fig. 586 5C, Supplementary Fig. S5), we hypothesize that PfE140 is a plasma membrane transporter 587 for inorganic cations such as metal ions. 588 589 In conclusion, this is the first study to investigate P. falciparum transcriptomics under 590 different iron conditions and to determine the subcellular localization of the putative iron 591 transport proteins PfMRS3, PfVIT, PfZIPCO and PfE140. These results improve our 592 understanding of how the human malaria parasite reacts to alterations in the iron status of its 593 host and provide new insights into the mechanisms of iron transport in P. falciparum. We 594 propose a new model for iron homeostasis in the P. falciparum-infected erythrocyte (Fig. 6) 595 with a series of six organelle-specific iron transport proteins that were identified based on 596 differential gene expression patterns, subcellular localization, proliferation assays, protein 597 structure predictions, and the assessment of putative binding sites using structural 598 alignments with well-characterized proteins. Formal demonstration of the substrate 599 specificity and activity of the transporters will require transport assays with purified proteins 600 in future studies. 601 602 Further functional characterization of the parasite transporters identified in this exploratory 603 research is expected to facilitate the development of new therapeutic strategies against 604 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 18 malaria. Due to the absence of human orthologs of the essential proteins PfE140 and 605 PfCRT, which both likely increase iron transport into the parasite cytosol, these provide 606 candidate targets for urgently needed antimalarial drugs. In addition, dissecting how P. 607 falciparum senses changes in micronutrient availability in its environment and how it 608 modulates its virulence accordingly is an area of considerable interest for future 609 investigation, as iron is an essential regulatory signal for virulence factors in many 610 pathogens. 611 612

613 P. falciparum culture and proliferation assays 614 The P. falciparum strain 3D7 was cultured according to modified standard procedures (102) 615 at 5% hematocrit using human 0 Rh+ erythrocytes from the University Medical Center 616 Hamburg-Eppendorf (UKE), Germany, at 1% O2, 5% CO2 and 94% N2. RPMI 1640 medium 617 was supplemented with 0.5% (w/v) AlbuMAX II, 20 µg/mL gentamicin and 100 µM 618 hypoxanthine (Thermo Fisher Scientific). Mature schizonts were obtained by treating 619 schizonts at 40 hpi with 1 mM compound 2 (4-[7-[(dimethylamino)methyl]-2-(4-620 fluorphenyl)imidazo[1,2-α ]pyridine-3-yl]pyrimidin-2-amine, LifeArc) for 8 h. To count the 621 number of merozoites per mature schizont, Giemsa-stained blood smears were analyzed by 622 light microscopy. Only single-infected cells with one digestive vacuole were taken into 623 account. 624 625 To assess parasite proliferation over six days, a previously described assay based on flow 626 cytometry was employed (103). Parasites were synchronized to a 3-h age window by 627 isolating late schizonts from a 60% Percoll (GE Healthcare, Life Sciences) gradient and 628 culturing these for 3 h with fresh erythrocytes (104), followed by controlled elimination of 629 advanced parasite stages using 5% (w/v) D-sorbitol (Carl Roth) for 10 min at 37°C (105). 630 The growth assay was started at 0.1% parasitemia using the resulting ring-stage parasites at 631 0 – 3 hpi. The parasitemia was determined at the trophozoite stage every two days by flow 632 cytometry and culture media with the respective supplements were exchanged daily. 633 634 Flow cytometry 635 To determine parasitemia, 20 µL of resuspended parasite culture was added to 80 μ L culture 636 medium and stained with 5 μ g/mL SYBR Green I (Thermo Fisher Scientific) and 4.5 μ g/mL 637 dihydroethidium (DHE, Sigma) in the dark for 20 min at room temperature. Stained cells 638 were washed with PBS three times and analyzed with an ACEA NovoCyte flow cytometer 639 and NovoExpress Software (version 1.6.1, Agilent). Forward and side scatter gating was 640 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 19 used to identify erythrocytes and SYBR Green I fluorescence intensity to determine the 641 number of parasitized cells per 100,000 events recorded for each sample. For Phen Green 642 SK measurements, uninfected erythrocytes were washed with PBS and incubated with 10 643 µM Phen Green SK in PBS at 37°C for 60 min. DHE at 4.5 μ g/mL was added during the last 644 20 min of incubation. After three washes with PBS, the cells were analyzed as described 645 above. 646 647 Cloning of DNA constructs 648 For generating the GFP reporter lines, a homologous region of approximately 800 bp at the 649 3’ end of the respective gene was amplified without the stop codon from 3D7 gDNA using 650 Phusion high fidelity DNA polymerase (NEB). A homology region of about 400 bp at the 5’ 651 end of the respective gene was used for targeted gene disruption. The fragments were then 652 inserted into pSLI-GFP (106) using NotI and AvrII restriction sites. For glmS constructs, 653 pSLI-GFP-glmS (107) was used as a vector instead. All oligonucleotides and plasmids used 654 in this study are listed in Supplementary Table S3. 655 656 Transfection of P. falciparum 657 As described previously (108), parasites at the late schizont stage were purified using 60% 658 Percoll (104) and electroporated with 50 μ g DNA of the respective plasmid in a 0.2-cm gap 659 cuvette (Bio-Rad Laboratories) using Amaxa Nucleofector 2b (Lonza, Switzerland). Either 4 660 nM WR99210 (Jacobus Pharmaceuticals) or 2 μ g/mL blasticidin S (Life Technologies, USA) 661 was used for selecting transfectants. For the selection of parasites that were genomically 662 modified using the SLI system (106), 400 μ g/mL G418 (ThermoFisher, USA) was added to 663 the culture medium once the parasitemia reached 5%. After the selection of modified 664 parasites, genomic DNA was isolated with the QIAamp DNA Mini Kit (Qiagen) and 665 diagnostic tests for correct integration into the genome were performed as specified earlier 666 (106). 667 668 Confocal live-cell microscopy 669 Erythrocytes infected with parasites at different stages at 3 – 6% parasitemia were incubated 670 in culture medium with 20 nM MitoTracker Red, 200 nM ER Tracker Red or 100 nM 671 LysoTracker Deep Red (if applicable) at 37°C for 20 min. Then, 200 nM Hoechst-33342 672 (Invitrogen) was added for 10 min prior to washing the cells with Ringer’s solution (122.5 mM 673 NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 0.8 mM MgCl2, 11 mM D-glucose, 25 mM HEPES, 1 mM 674 NaH2PO4, pH 7.4) prewarmed to 37°C and seeding on a chambered No. 1.5 polymer cover 675 slip (Ibidi GmbH). After 5 min, unbound erythrocytes were removed by washing with Ringer’s 676 solution and the sample was placed into an incubation chamber that maintained the 677 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 20 microscope work area including the objective at 37°C. Images and videos were acquired 678 using an SP8 confocal microscope system with a 63x oil-corrected lens (C-Apochromat, 679 numerical aperture, NA = 1.4) and Lightning deconvolution software (Leica), and processed 680 using ImageJ version 2.9.0/1.53t (109). If fluorescence intensities were to be quantified, no 681 averaging or deconvolution software was applied. 682 683 glmS-based knockdown 684 For glmS-based knockdown induction (65), highly synchronous parasites at early ring stage 685 were cultured with or without supplementation with 2.5 mM glucosamine (GlcN, Sigma-686 Aldrich). The knockdown was quantified by confocal live-cell microscopy using schizonts 36 687 h post GlcN treatment initiation. Images of parasites of similar size were acquired with the 688 same settings and background-corrected fluorescence intensities (integrated density) as well 689 as the size of the region of interest were determined using ImageJ version 2.9.0/1.53t (109), 690 and the data visualized using Graph Pad Prism version 9.4.1. 691 692 Sample collection for RNA extraction and RNA sequencing 693 Parasites were synchronized within a 3-h window after invasion of erythrocytes from the 694 respective donor as described above. Samples for RNA-sequencing were prepared in 695 triplicate for each condition and time point, i.e. three separate parasite cultures each were 696 grown in parallel for a total of at least two weeks. During the second IDC, two 10-mL dishes 697 each were harvested for parasites at the ring stage (6 – 9 hpi) and one 10-mL dish each for 698 trophozoites (26 – 29 hpi). Samples were collected by centrifuging the culture for 5 min at 699 800 g and 37°C and dissolving the erythrocyte pellet using 5 mL TRIzol prewarmed to 37°C, 700 followed by immediate transfer to -80°C for storage. The parasitemia was 0.3% at the start of 701 the experiments with high, control and low-iron donor blood and 2 – 3% at the time of 702 harvest. Parasite cultures treated with 0.7 µM hepcidin (Bachem) had a starting parasitemia 703 of 0.6% and untreated cultures 1% to reach a parasitemia of 4 – 5% during the second 704 cycle. For each experiment, the parasitemia was kept consistent at the point of harvest as 705 high parasite densities can affect transcription (15). 706 707 For RNA extraction, the samples frozen in TRIzol were thawed, mixed thoroughly with 0.1 708 volume cold chloroform, and incubated at room temperature for 3 min. Following 709 centrifugation at 20,000 g and 4°C for 30 min, the supernatants were transferred to fresh 710 vials and combined with 70% ethanol of equal volume. RNA was purified using the RNeasy 711 MinElute Kit (Qiagen) by on-column DNase I digest for 30 min and elution with 14 µL water. 712 The GLOBINclear Human Kit (ThermoFisher Scientific) was then employed to deplete 713 human globin mRNA in all samples. The Qubit RNA HS Assay Kit and Qubit 3.0 fluorometer 714 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 21 (ThermoFisher Scientific) were used for RNA quantification. Upon arrival at the EMBL 715 Genomics Core Facility (GeneCore Heidelberg, Germany), the RNA quality of each sample 716 was evaluated using the RNA 6000 Nano kit and Bioanalyzer 2100 (Agilent). The median 717 RNA integrity number (RIN) of all samples was 7.30 (IQR: 6.85 – 8.15, Supplementary Fig. 718 S1). Individually barcoded strand-specific libraries for mRNA sequencing were prepared 719 from total RNA samples of high quality (approximately 150 ng per sample) using the 720 NEBNext® RNA Ultra II Directional RNA Library Prep Kit (New England Biolabs) for 12 PCR 721 cycles on the liquid handler Biomek i7 (Beckman Coulter GmbH) at GeneCore. Libraries that 722 passed quality control were pooled in equimolar amounts, and a 2 pM solution of this pool 723 was sequenced unidirectionally on a NextSeq® 500 System (Illumina) at GeneCore, 724 resulting in about 500 million reads of 85 bases each. 725 726 RNA sequencing read mapping and data analysis 727 Following successful initial quality control of the RNA-sequencing reads with FastQC version 728 0.11.8 (110), sequencing adapters were trimmed using Cutadapt version 2.10 (111). A 729 genome index was generated using the FASTA sequence file of the P. falciparum 3D7 730 genome release 46 (PlasmoDB-46_Pfalciparum3D7_Genome.fasta) and the GFF3 731 annotation file (PlasmoDB-46_Pfalciparum3D7.gff), both obtained from PlasmoDB (112), 732 with STAR version 2.7.5c (113). The same R package was used to align reads to 733 the genome with a maximum of three allowed mismatches (--outFilterMismatchNmax 3). To 734 consolidate the results obtained with FastQC and STAR alignments, a single report file was 735 created using MultiQC version 1.9 (114). 736 737 The mapped reads were then summarized in Sequence Alignment/Map (SAM) format using 738 featureCounts (115) from the R package Rsubread version 2.2.1 (116). For counting 739 mapped reads per gene using featureCounts, fragments with a minimum length of 50 bases 740 were considered (minFragLength = 50). Therefore, gene IDs and lengths of transcripts were 741 extracted from PlasmoDB-46_Pfalciparum3D7_AnnotatedTranscripts.fasta with SAMtools 742 faidx version 1.10.2 (117). The R package edgeR 3.30.3 (118) was used to compute RPKM 743 values (reads per kilobase per million mapped reads) and for differential gene expression 744 analysis. Gene annotations were retrieved from PlasmoDB (112) and PhenoPlasm (119). 745 The results of these analyses were visualized with volcano plots using the R package 746 Enhanced Volcano version 1.15.0 (120). The raw and processed data (FASTA files, RPKM 747 values and results of the differential gene expression analysis) can be accessed at 748 https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13411. 749 750 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 22 The highly polymorphic var, stevor, and rifin gene families were excluded from downstream 751 analyses due to their great sequence diversity between parasites of the same strain during 752 mitotic growth (54, 55). Genes that were significantly regulated (defined as P < 0.05 753 according to the exact test for the negative binomial distribution with Benjamini-Hochberg 754 correction (50) and an absolute value of log2 FC ≥ 0.2) were subjected to functional 755 enrichment analysis with g:Profiler (https://biit.cs.ut.ee/gprofiler/gost (56), accessed on 756 August 17, 2022). The resulting GO, KEGG and REAC terms were summarized using 757 REVIGO (http://revigo.irb.hr/) with the similarity value set to 0.5 (121) and visualized as in 758 Thomson-Luque et al. (81) using the scientific color map “roma” (122). To estimate parasite 759 age, an algorithm developed by Avi Feller and Jacob Lemieux (46) was adapted to use 760 expression data from Broadbent et al. (47) with the time points 6, 14, 20, 24, 28, 32, 36, 40, 761 44, and 48 hpi as reference. The code and data used were deposited to Zenodo with the 762 record ID 7996302 (https://zenodo.org/record/7996302). 763 764 Protein structure prediction 765 Structure predictions for monomeric proteins were obtained from AlphaFold Protein 766 Structure Database version 3 (66, 67) and homodimeric proteins were predicted using 767 AlphaFold2-multimer version 2.2.2, database version 2.2.0 (68) deployed at the EMBL 768 Hamburg computer cluster. Molecular visualization was performed with UCSF ChimeraX 769 version 1.3 (123). UCSF Chimera MatchMaker and Match → Align tools with default settings 770 were used for structural comparison of the predicted structures of P. falciparum proteins with 771 putative orthologs and sequence alignments were generated using the Match → Align tool 772 (124). The DeepFRI server (https://beta.deepfri.flatironinstitute.org) was used to identify 773 possible functional residues with the DeepFRI graph convolutional network (75). 774 775

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J Infect 1171 Dis. 2011;203(2):228-36. 1172 1173 Figures: 1174 Figure 1: Effects of the iron status of the blood donor and of hepcidin on (A) 1175 erythrocyte free iron levels, (B) P. falciparum 3D7 growth rates, (C) DNA content per 1176 mature schizont, and (D) the number of merozoites per mature schizont. 1177 The relative free iron level and DNA content per cell were assessed by measuring the mean 1178 fluorescence intensity (MFI) of Phen Green SK or SYBR Green I compared to control (Ctrl, 1179 untreated, normal hemoglobin level) using flow cytometry. 0.7 µM hepcidin (Hep) was 1180 chosen as a concentration reported to reduce binding of ferrous iron to the erythrocyte iron 1181 exporter ferroportin by 50% in vitro (44). Parasite growth rates refer to the fold change in 1182 parasitemia after one intraerythrocytic developmental cycle in vitro relative to control as 1183 determined by flow cytometry with SYBR Green I (103). Mature schizonts were obtained by 1184 treating schizonts at 40 hpi with 1 mM compound 2 (4-[7-[(dimethylamino)methyl]-2-(4-1185 fluorphenyl)imidazo[1,2-α ]pyridine-3-yl]pyrimidin-2-amine) for 8 h. To count the number of 1186 merozoites, Giemsa-stained blood smears were analyzed microscopically and only single-1187 infected cells with one digestive vacuole were considered. Means and 95% confidence 1188 intervals (indicated by error bars) are shown. Statistical significance was calculated with two-1189 tailed unpaired t tests with Welch’s correction for unequal variances and adjusted with the 1190 Holm-Šídák method for multiple comparisons except for merozoite numbers, which were 1191 compared using Mann-Whitney test. N represents the number of parasites and n the number 1192 of independent experiments. 1193 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 30 1194 Figure 2: Differential expression of P. falciparum 3D7 genes under various iron 1195 conditions. 1196 Parasites were cultured with erythrocytes from an individual with high, medium (healthy) or 1197 low iron status (experiment 1) or with red blood cells from another healthy donor in the 1198 presence or absence of 0.7 µM hepcidin (experiment 2). Samples were harvested at the ring 1199 and trophozoite stage (6 – 9 and 26 – 29 hours post invasion, hpi) with three biological 1200 replicates per time point and condition. The maximum likelihood estimation (MLE) of the 1201 average developmental age of the parasites for each condition and time point (A) was 1202 calculated using an algorithm developed by Avi Feller and Jacob Lemieux (46). CI, 1203 confidence interval. The volcano plots (B and D) show transcriptional changes of all parasite 1204 genes. Red dots indicate significantly (P < 0.05, exact test for negative binomial distribution) 1205 upregulated genes (log2 (fold change) ≥ 0.2), blue dots stand for significantly downregulated 1206 genes (log2 (fold change) ≤ -0.2), while grey dots represent genes that did not significantly 1207 differ in transcription under the conditions described (P ≥ 0.05 and / or -0.2 < log2 (fold 1208 change) < 0.2). Differentially expressed genes encoding putative iron transporters (see 1209 Table 1) are labeled. Panels C and E show the enrichment of Gene Ontology (GO), Kyoto 1210 Encyclopedia of Genes and Genomes (KEGG) and Reactome (REAC) terms among 1211 significantly regulated genes excluding var, stevor and rifin gene families at the two time 1212 points. The functional terms were summarized using REVIGO (121) to remove 1213 redundancies, represented by circles and plotted according to the significance of their 1214 enrichment (-log10 (adjusted P), hypergeometric test). The size of the circle is proportional to 1215 the number of differentially regulated genes in the dataset that are associated with the 1216 respective term, while the color stands for the fold enrichment. The gray dashed line 1217 indicates the threshold of the adjusted P value (-log10 0.05 = 1.3). 1218 1219 Figure 3: Subcellular localization of four putative iron transport proteins. 1220 Representative erythrocytes infected with P. falciparum 3D7 parasites endogenously 1221 expressing GFP-tagged PfMRS3 (A), PfVIT (B), PfZIPCO (C) or PfE140 (D) were 1222 additionally stained with the fluorescent dyes Hoechst-33342, MitoTracker Red, ER Tracker 1223 Red and/or LysoTracker Deep Red. Co-transfection with a construct that encodes the 60 N-1224 terminal amino acids of acyl carrier protein (PfACP) tagged with mCherry (125) resulted in 1225 red fluorescence of the apicoplast. Live-cell images were taken under physiological 1226 conditions at 37°C using an SP8 confocal laser-scanning microscope (Leica). DIC, 1227 differential interference contrast. Scale bar, 2 µm. 1228 1229 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 31 Figure 4: PfVIT and PfE140 are important for P. falciparum growth and may be 1230 involved in intracellular iron homeostasis. 1231 A Representative erythrocytes infected with P. falciparum 3D7 parasites that endogenously 1232 express a truncated version of PfVIT or PfZIPCO tagged with GFP (green). B Growth rates 1233 of knockout parasite lines generated. C Reduction of PfE140-GFP fluorescence (green) in 1234 live 3D7 parasites caused by glmS-mediated knockdown that was induced by treatment with 1235 2.5 mM glucosamine (GlcN) for 36 h compared to untreated control (Ctrl). D Total parasite 1236 fluorescence intensities were quantified as background-corrected integrated densities using 1237 ImageJ version 2.9.0/1.53t (109) and compared using Mann-Whitney test. E The size of the 1238 parasites was measured as the area of the region of interest and compared using equal 1239 variance unpaired t test. F Conditional knockdown of PfE140 induced by treatment with 2.5 1240 mM GlcN results in a growth defect during asexual blood stage development. Live parasites 1241 were stained with Hoechst-33342 (blue) and imaged under physiological conditions at 37°C 1242 using an SP8 confocal laser-scanning microscope (Leica). DIC, differential interference 1243 contrast. Scale bar, 2 µm. Error bars represent 95% confidence intervals of the mean, N the 1244 number of parasites analyzed, n the number of independent experiments and Hep treatment 1245 with 0.7 µM hepcidin. Growth rates refer to the fold change in parasitemia after two 1246 intraerythrocytic developmental cycles in vitro relative to untreated wild-type 3D7 parasites 1247 (WT) as determined by flow cytometry with SYBR Green I (103). Statistical significance of 1248 growth differences was calculated with two-tailed unpaired t tests with Welch’s correction for 1249 unequal variances and adjusted with the Holm-Šídák method for multiple comparisons. 1250 1251 Figure 5: Predicted structures of putative iron transporters identified in P. falciparum 1252 as viewed from the membrane plane. 1253 A Predicted protein structures with per-residue pLDDT (predicted local distance difference 1254 test) confidence scores on a scale from 0 to 100, where blue represents high and red low 1255 confidence, respectively. The experimentally determined structure of PfCRT is shown in 1256 gray. B Molecular lipophilicity potential of the protein surfaces as implemented in UCSF 1257 ChimeraX; tan is hydrophobic and cyan hydrophilic. Dashed lines above and below the tan 1258 regions of all proteins indicate the respective membrane and disordered loops were removed 1259 for clarity. C Surface charge of the proteins with positively charged areas colored blue and 1260 negatively charged ones red. Putative cation-binding site are indicated with an asterisk and 1261 transport directions by arrows. PfE140 likely forms a dimer but is shown as a monomer, as 1262 no predicted dimer structure could be obtained using AlphaFold2-multimer. The putative 1263 cation-binding sites for this protein are based on DeepFRI gradCAM scores for the functional 1264 term GO:0015075 “monoatomic ion transmembrane transporter activity” (Supplementary Fig. 1265 S5). 1266 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 32 1267 Figure 6: Iron homeostasis in a P. falciparum-infected erythrocyte. 1268 The human blood plasma contains between 10 and 30 µM total Fe and the erythrocyte 1269 cytosol approximately 20 mM (19). However, only 2.5 – 4.7 µM of Fe2+ is bioavailable in an 1270 uninfected erythrocyte, and 1.5 – 1.8 µM in a P. falciparum-infected one (20). Human 1271 ferroportin (FPN) at the host cell surface (erythrocyte plasma membrane, EPM) exports free 1272 iron from the erythrocyte (126), the nutrient pore formed by PfEXP1 and PfEXP2 allows the 1273 passage of the ions through the parasitophorous vacuole membrane (PVM) (127). PfE140 at 1274 the parasite plasma membrane (PPM) may mediate iron uptake iron into the parasite cytosol 1275 and the mitochondrial carrier protein PfMRS3 likely translocates Fe2+ into the mitochondrion, 1276 a site of de novo heme biosynthesis. The digestive vacuole (DV) contains a high amount of 1277 total Fe as it is the site of hemoglobin degradation. The chloroquine resistance transporter 1278 (PfCRT) and the natural resistance-associated macrophage protein (PfNRAMP) were 1279 suggested to mediate proton-coupled export of Fe2+ from the DV into the parasite cytosol 1280 (35, 36). The vacuolar iron transporter (PfVIT) may be involved in iron detoxification by 1281 transporting excess iron from the cytosol into cytoplasmic vesicles that may be 1282 acidocalcisomes, whereas PfZIPCO may release zinc and ferrous iron from these organelles 1283 under low-iron conditions (this study). Both acidocalcisomes and the DV are likely acidified 1284 by the plant-like H+-pump V-ATPase, which can fuel secondary active transport processes 1285 (22, 27). Parasite-encoded proteins are shown in orange and human-encoded transporters 1286 in blue. 1287 1288 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 33 Table 1: P. falciparum transport proteins with differential gene expression under various iron conditions. 1289 Putative and known transporter genes were filtered from differentially expressed genes in the described RNA-seq experiments usi ng an updated list of P. falciparum transport 1290 proteins (28). The log2 (fold change) of gene expression at the ring stage (6 – 9 hours post invasion) and known or proposed functions are indicated fo r significantly regulated 1291 genes (exact P < 0.05) and putative iron transport proteins are highlighted in red. DV: digestive vacuole, EPM: erythrocyte plasma membrane, PPM: parasite plasma 1292 membrane. 1293 Gene product and ID Log 2 (fold change) high vs. low Fe Log2 (fold change) low Fe vs. control Log2(fold change) hepcidin vs. control Known or putative function PLP5 (PF3D7_0819200) + 0.49 (P = 0.0002) n.s. - 0.29 ( P = 0.003) Host cell permeabilization and rupture (128) ABCG (PF3D7_1426500) + 0.45 (P = 0.02) - 0.57 ( P = 0.006) n.s. Putative metabolite exporter at PPM (61), human ortholog ABCG2 exports heme (129) VP1 (PF3D7_1456800) + 0.39 (P = 0.0006) - 0.34 ( P = 0.004) n.s. Active H + export across PPM (130) TOM7 (PF3D7_0823700) + 0.38 (P = 0.02) n.s. n.s. Protein import across outer mitochondrial membrane (131, 132) HlyIII (PF3D7_1455400) + 0.38 (P = 0.0009) n.s. n.s. Forms pore (~3.2 nm) for solutes and ions in EPM (133) TPT (PF3D7_0508300) + 0.37 (P = 0.001) - 0.40 ( P = 0.002) n.s. Imports phosphoenolpyruvate, dihydroxyacetone, and 3-phosphoglycerate across outer apicoplast membrane (134) Vo c (PF3D7_0519200) + 0.36 (P = 0.0006) n.s. - 0.16 (P = 0.007) V-ATPase subunit: active H+ export from cytosol (135) MDR1 (PF3D7_0523000) + 0.34 (P = 0.0009) - 0.23 ( P = 0.03) n.s. Active drug and solute import into DV (136) NT3 (PF3D7_1469400) + 0.34 (P = 0.03) n.s. n.s. Putative nucleoside transporter (36) SEC61α (PF3D7_1346100) + 0.33 (P = 0.0006) - 0.22 ( P = 0.04) - 0.17 ( P = 0.005) ER import of proteins destined for export (137) MRS3 (PF3D7_0905200) + 0.33 (P = 0.002) - 0.19 ( P = 0.04) n.s. Putative Fe 2+ importer into mitochondrial matrix (38) HT1 (PF3D7_0204700) + 0.33 (P = 0.002) - 0.30 ( P = 0.006) n.s. Imports glucose and fructose across PPM (138) ATP4 (PF3D7_1211900) + 0.33 (P = 0.005) - 0.40 (P = 0.002) n.s. H + import, Na+ export across PPM (139) MCT2 (PF3D7_0926400) + 0.32 (P = 0.005) n.s. n.s. Exports organic solutes from apicoplast, imports H + (140) V1 B (PF3D7_0406100) + 0.30 (P = 0.001) - 0.25 (P = 0.008) - 0.13 (P = 0.04) V-ATPase subunit: active H+ export from cytosol (135) V1 A (PF3D7_1311900) + 0.30 (P = 0.001) - 0.28 (P = 0.003) - 0.20 (P = 0.001) V-ATPase subunit: active H+ export from cytosol (135) TIM16 (PF3D7_0513500) + 0.29 (P = 0.03) n.s. n.s. Protein import across inner mitochondrial membrane (131, 141) VIT (PF3D7_1223700) + 0.29 (P = 0.02) n.s. n.s. Fe 2+ sequestration from cytosol in exchange for H + (30-32) ApiAT2 (PF3D7_0914700) + 0.28 (P = 0.006) n.s. n.s. Putative amino acid transporter at PPM (142) NRAMP (PF3D7_0523800) + 0.28 (P = 0.003) n.s. n.s. Symport of Mn 2+ or Fe2+ with H+ from DV into cytosol (22, 34) AMC1 (PF3D7_0108800) + 0.26 (P = 0.03) n.s. n.s. Putative mitochondrial transporter (143) CRT (PF3D7_0709000) + 0.26 (P = 0.007) n.s. n.s. Symport of positively charged dipeptides or Fe 2+ with H+ from DV into cytosol (35) V1 H (PF3D7_1306600) + 0.26 (P = 0.01) n.s. n.s. V-ATPase subunit: active H + export from cytosol (135) AAC2 (PF3D7_1004800) + 0.25 (P = 0.03) n.s. - 0.21 (P = 0.04) Mitochondrial ADP/ATP antiporter (141) PiT (PF3D7_1340900) + 0.24 (P = 0.02) - 0.21 ( P = 0.04) n.s. Imports phosphate and Na + into cytosol across PPM (36) Vo d (PF3D7_1464700) + 0.24 (P = 0.02) - 0.23 ( P = 0.03) - 0.15 ( P = 0.02) V-ATPase subunit: active H+ export from cytosol (135) Vo c" (PF3D7_1354400) + 0.23 (P = 0.03) n.s. n.s. V-ATPase subunit: active H + export from cytosol (135) ATP10 (PF3D7_0727800) + 0.23 (P = 0.01) n.s. n.s. Active apicoplast Mn2+ transporter (144) SulP (PF3D7_1471200) + 0.23 (P = 0.02) n.s. n.s. Inorganic anion antiporter at PPM (36) ATP2 (PF3D7_1219600) + 0.22 (P = 0.02) - 0.22 (P < 0.05) - 0.14 (P = 0.03) Putative phospholipid flippase at PPM (36) NT1 (PF3D7_1347200) + 0.22 (P = 0.02) n.s. n.s. Purin base import across PPM (145) AAT1 (PF3D7_0629500) - 0.26 (P = 0.007) n.s. n.s. Putative amino acid transporter at PPM and DV (143) MIT1 (PF3D7_1120300) - 0.27 (P = 0.02) n.s. n.s. Putative mitochondrial magnesium/nickel/cobalt ion channel (141) AAT2 (PF3D7_1208400) - 0.29 (P = 0.009) n.s. n.s. Putative amino acid transporter at PPM (36) MDR7 (PF3D7_1209900) - 0.34 (P = 0.04) n.s. n.s. Active efflux of peptides from mitochondrion (36, 40) CuTP (PF3D7_0904900) - 0.36 (P = 0.008) + 0.52 ( P = 0.004) n.s. Active Cu2+ export from cytoplasmic vesicle (36, 146) GCβ (PF3D7_1360500) - 0.36 (P = 0.007) n.s. n.s. Putative phospholipid flippase at PPM (36) TPC (PF3D7_1368700) - 0.37 (P = 0.01) + 0.33 ( P = 0.02) n.s. Thiamine pyrophosphate import, nucleotide export from mitochondrion (144) F1 γ (PF3D7_1311300) - 0.38 (P = 0.03) n.s. + 0.32 (P = 0.03) Subunit of mitochondrial H+-importing ATP synthase (143) .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 34 PF3D7_0614900 - 0.43 (P = 0.005) + 0.72 ( P = 0.0001) + 0.29 ( P = 0.0003) Putative ABC transporter at PPM (28) MFS4 (PF3D7_1203400) - 0.44 (P = 0.0008) + 0.56 ( P = 0.0005) + 0.27 ( P = 0.02) Putative transporter (36) Fo d (PF3D7_0311800) - 0.46 (P = 0.004) + 0.34 ( P = 0.02) n.s. Subunit of mitochondrial H +-importing ATP synthase (144) PF3D7_1004600 - 0.47 (P = 0.01) + 0.58 ( P = 0.003) + 0.28 ( P = 0.03) Putative ABC transporter linked to drug resistance (147) MPC2 (PF3D7_1470400) - 0.47 (P = 0.03) + 0.66 ( P = 0.005) n.s. Subunit of mitochondrial putative pyruvate:H + importer (144) E140 (PF3D7_0104100) - 0.47 (P = 0.03) + 0.65 ( P = 0.006) n.s. Putative transport protein at PPM (28) MDR4 (PF3D7_0302600) - 0.55 (P = 0.001) + 0.63 ( P = 0.001) + 0.33 ( P = 0.02) Active drug export across innermost apicoplast membrane (148, 149) ZIPCO (PF3D7_1022300) - 0.57 (P = 0.03) + 0.55 ( P = 0.04) + 0.63 ( P = 0.006) Zn2+/Fe2+ import into cytosol (95) F1 δ (PF3D7_1147700) - 0.59 (P = 0.004) + 0.63 ( P = 0.005) n.s. Subunit of mitochondrial H +-importing ATP synthase (143) TRP-ML (PF3D7_1313500) - 0.59 (P = 0.01) + 0.64 ( P = 0.005) n.s. Putative Ca 2+ channel (150) F1 ε (PF3D7_0715500) - 0.67 (P = 0.04) + 0.94 (P = 0.007) n.s. Subunit of mitochondrial H+-importing ATP synthase (143) MFS3 (PF3D7_0919500) - 0.85 (P = 0.01) + 0.72 (P = 0.01) n.s. Putative sugar transporter (143) ATP11 (PF3D7_1468600) n.s. + 1.14 (P = 0.005) n.s. Putative phospholipid flippase at PPM (28, 36) NGT (PF3D7_0505300) n.s. + 0.91 (P = 0.00008) + 0.79 (P = 0.00004) UDP-N-acetylglucosamine import, UMP export from Golgi (36) SAMC (PF3D7_1241600) n.s. + 0.77 (P = 0.03) + 0.68 (P = 0.02) S-adenosylmethionine import into mitochondrion (151) PF3D7_0614900 n.s. + 0.72 ( P = 0.0001) + 0.29 ( P = 0.0003) Putative active transporter at PPM (28) GPH (PF3D7_0529200) n.s. + 0.70 (P = 0.03) n.s. Putative sugar:cation symporter (36) ATP9 (PF3D7_1348800) n.s. + 0.62 (P = 0.04) n.s. Active Ca 2+ import into DV? (144) CTR2 (PF3D7_1421900) n.s. + 0.52 (P = 0.004) n.s. Putative apicoplast copper channel (144) MDR5 (PF3D7_1339900) n.s. + 0.51 (P = 0.02) n.s. Active solute export across PPM (144) TIC20 (PF3D7_1144700) n.s. + 0.49 (P = 0.007) n.s. Protein import across innermost apicoplast membrane (152) PF3D7_0924500 n.s. + 0.47 (P = 0.002) n.s. Putative Na +:H+ exchanger (70) PPT (PF3D7_0530200) n.s. + 0.46 (P = 0.02) Imports phosphoenolpyruvate, dihydroxyacetone, and 3-phosphoglycerate across inner apicoplast membrane (134) CLAG3.1 (PF3D7_0302500) n.s. + 0.45 (P < 0.05) n.s. Purine, amino acid, sugar, and vitamin import across EPM (153) CDF (PF3D7_0715900) n.s. + 0.44 (P = 0.02) n.s. Putative Zn 2+ importer (57) into cytoplasmic vesicles (34) ATP7 (PF3D7_0319000) n.s. + 0.44 (P = 0.007) n.s. Putative phospholipid flippase at PPM (28, 36) RhopH3 (PF3D7_0905400) n.s. + 0.42 (P = 0.04) n.s. Purine, amino acid, sugar, and vitamin import across EPM (153) AQP2 (PF3D7_0810400) n.s. + 0.34 (P = 0.02) - 0.19 (P < 0.05) Water channel at PPM (154) MFS2 (PF3D7_0916000) n.s. + 0.33 (P = 0.04) n.s. Putative sugar transporter (36) TFP1 (PF3D7_0206200) n.s. + 0.33 (P = 0.02) n.s. Putative metabolite transporter at PPM (144) ATP6 (PF3D7_0106300) n.s. - 0.22 (P = 0.03) n.s. Active Ca2+ import into ER for storage (144) V1 G (PF3D7_1323200) n.s. - 0.23 (P = 0.04) - 0.12 ( P = 0.04) V-ATPase subunit: active H+ export from cytosol (135) FNT (PF3D7_0316600) n.s. - 0.27 (P = 0.02) - 0.16 ( P = 0.004) Lactate/formate and H+ release from cytosol (144) MFR1 (PF3D7_0614300) n.s. - 0.33 (P = 0.004) - 0.14 ( P = 0.02) Putative organic anion transporter (36) PMRT1 (PF3D7_1135300) n.s. - 0.36 (P = 0.009) n.s. Putative transporter at PPM (34) PLP3 (PF3D7_0923300) n.s. - 0.42 (P = 0.015) n.s. Host cell permeabilization and rupture (128) OSCP (PF3D7_1310000) n.s. n.s. + 0.58 (P = 0.01) Subunit of mitochondrial H+-importing ATP synthase (143) GFT (PF3D7_0212000) n.s. n.s. + 0.54 (P = 0.03) GDP-fucose import, GMP export from Golgi (36) SEC61γ (PF3D7_0210000) n.s. n.s. - 0.23 (P = 0.001) ER import of proteins destined for export (137) SEC61β (PF3D7_0821800) n.s. n.s. - 0.31 (P = 0.0005) ER import of proteins destined for export (137) DTC (PF3D7_0823900) n.s. n.s. - 0.34 (P = 0.004) Imports dicarboxylate, exports tricarboxylate from mitochondrion (143) 1294 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 35 Table 2: Proteins identified by RNA-sequencing that may be involved in iron transport in P. falciparum. 1295 The classification of the proteins identified is indicated according to the Transport Classification Database (70). Data on hum an orthologs was retrieved using the NCBI 1296 position-specific iterated (PSI) BLAST with default settings at https://blast.ncbi.nlm.nih.gov/Blast.cgi (29). DV: digestive vacuole, E: expect value, EM: electron microscopy, hpi: 1297 hours post invasion, MIS: mutagenesis index score, PDB: Protein Data Bank, PPM: parasite plasma membrane. 1298 Gene product, gene ID Blood stage tran- scription peak (47) Classifi- cation (70) Solved structure of a similar protein Potential human ortholog(s) Localization in P. falciparum Transport assays and proposed function Mutability in P. falciparum Essentiality in P. berghei MRS3, PF3D7_0905200 20 hpi 2.A.29.5.9 None Mitoferrin-1 (E = 7 x 10 -10, 27.0% identity, 26% coverage); Mitoferrin-2 (E = 6 x 10 -8, 27.9% identity, 12% coverage) Mitochondrion (this study) Liposomal transport assays with S. cerevisiae MRS3 (37): Fe 2+ import into mitochondrial matrix across inner membrane Non- disruptable, MIS: 0.135 (64) PBANKA_041620: essential at asexual blood stage (89) VIT, PF3D7_1223700 36 hpi 2.A.89.1.13 Crystal structure of VIT1 from Eucalyptus grandis: PDB 6IU9 (69) None Cytoplasmic vesicles (this study) Transport assays using inverted vesicles with PfVIT (32): Fe 2+ export from cytoplasmic vesicles (this study) into cytosol in exchange for H + Disruptable, MIS: 0.903 (64) PBANKA_143860: dispensable at asexual blood stage with growth rate of 1 (95% CI: 0.94 – 1.05) (89), and at mosquito and liver stage (155); reduced growth in blood and liver in mice (30) ZIPCO, PF3D7_1022300 32 hpi 2.A.5.3.12 Cryo-EM structure of ZIP from Bordetella bronchiseptica: PDB 8GHT (71) ZIP1 (E = 2 x 10 -5, 21.7% identity, 48% coverage) Cytoplasmic vesicles (this study) Zn 2+ uptake assays with BbZIP in Escherichia coli (71), Liposomal assays with PfZIP1 (97): Zn2+/Fe2+ import into cytosol from cytoplasmic vesicles (this study) Disruptable, MIS: 1 (64), growth increase at asexual blood stage (this study) PBANKA_050650: growth rate of 0.86 (95% CI: 0.74 – 0.98) at asexual blood stage (89); dispensable at blood and mosquito stage but reduced sporozoite infectivity and inefficient schizogony in hepatocytes (95) NRAMP, PF3D7_0523800 14 hpi 2.A.55.2.25 Crystal structure of NRAMP/DMT from Staphylo- coccus capitis: PDB 5M95 (73) NRAMP1 (E = 2 x 10 -38, 28.4% identity, 56% coverage); NRAMP2 (E = 2 x 10 -38, 27.6% identity, 65% coverage) DV (34) Uptake assays with Deinococcus radiodurans NRAMP in E. coli and proteoliposomes (99, 100): pH-dependent symport of Mn 2+/Fe2+ with H+ into cytosol from DV Non- disruptable, MIS: 0.123 (64) PBANKA_123860: no mutants generated CRT, PF3D7_0709000 14 hpi 2.A.7.20.1 Cryo-EM structure of PfCRT from strain 7G8: PDB 6UKJ (72) None DV (60) Transport assays using Xenopus oocytes with PfCRT (35): symport of Fe 2+ with H+ into cytosol from DV Non- disruptable, MIS: 0.127 (64), essential at asexual blood stage PBANKA_121950: essential at asexual blood stage (89, 156) .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 36 (33) E140, PF3D7_0104100 40 hpi Unknown None None PPM (this study) Putative Fe2+ importer at PPM (this study) Non- disruptable, MIS: 0.119 (64) PBANKA_0209000: growth rate of 0.48 (95% CI: 0.32 – 0.63) at asexual blood stage (89), dispensable in mosquito and liver stages (155) .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 37 Supplementary Material 1299 mentary Figures 1300 Supplementary Figure S1: RNA quality. 1301 Supplementary Figure S2: Cloning strategy and confirmation of correct DNA integration 1302 into the genome of the cell lines generated. 1303 Supplementary Figure S3: Sequence alignments of P. falciparum proteins with their 1304 potential orthologs from other species that have been functionally characterized. 1305 Supplementary Figure S4: Alignments of functional sites in the predicted P. falciparum 1306 protein structures with those of potential orthologs using UCSF Chimera. 1307 1308 Supplementary Videos 1309 Supplementary Video S1: Parasite endogenously expressing PfMRS3-GFP stained with 1310 MitoTracker Red and Hoechst-33342. 1311 Supplementary Video S2: Parasite endogenously expressing PfVIT-GFP stained with ER 1312 Tracker Red and Hoechst-33342. 1313 Supplementary Video S3: Parasite endogenously expressing PfZIPCO-GFP stained with 1314 LysoTracker Deep Red and Hoechst-33342. 1315 Supplementary Video S4: Parasite expressing PfE140-GFP endogenously, PfACP(1-60)-1316 mCherry episomally and stained with Hoechst-33342. 1317 1318 Supplementary Tables 1319 Supplementary Table S1: Differentially expressed genes of P. falciparum 3D7 cultured with 1320 erythrocytes from a donor with high, control or low iron status. 1321 Supplementary Table S2: Differentially expressed genes of P. falciparum 3D7 cultured with 1322 erythrocytes from a healthy donor with or without addition of 0.7 µM hepcidin. 1323 Supplementary Table S3: Oligonucleotides (A) and plasmids (B) used in this study. 1324 1325 Supplementary PDB files 1326 Top-scoring PDB files for the multimers that were computed at EMBL: 1327 PfVIT.pdb 1328 PfZIPCO.pdb 1329 1330 Funding 1331 This work was supported by a Boehringer Ingelheim Foundation Exploration Grant, the 1332 Partnership for Innovation, Education and Research (PIER) of Hamburg University and 1333 DESY (project PIF-2018-87) and the European Molecular Biology Laboratory (EMBL). JW 1334 was additionally funded by the European Research Council under the European Union’s 1335 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 38 Horizon 2020 Research and Innovation Programme (grant agreement 759534) and VK by a 1336 research fellowship from the EMBL Interdisciplinary Postdoc (EIPOD) Programme under 1337 Marie Curie Cofund Actions MSCA-COFUND-FP (grant agreement 847543). The funders 1338 had no role in study design, data collection and analysis, decision to publish, or preparation 1339 of the manuscript. 1340 1341 Authors’ contributions 1342 JS and JW designed the study; MG and SPe recruited the blood donors; JW and CN 1343 performed the experiments; JW, JS, VK and LVN analyzed the data; VK generated the 1344 structural models, and JW wrote the manuscript with contributions from VK, JS and SPo. All 1345 authors read and approved the submitted version. 1346 1347

1348 The authors thank the Genomics Core Facility at EMBL Heidelberg, especially Vladimir 1349 Benes, for the RNA-sequencing service, and EMBL Hamburg for the provision of research 1350 and technical support as well as access to research infrastructures. Grzegorz Chojnowski 1351 and the group of Jan Kosinski at EMBL Hamburg enabled the AlphaFold2 workflow at the 1352 EMBL Hamburg computer cluster. The Advanced Light and Fluorescence Microscopy 1353 Facility at CSSB Hamburg, in particular Roland Thünauer, supported microscopy 1354 experiments, and the Bernhard Nocht Institute for Tropical Medicine (BNITM) provided lab 1355 space. We gratefully acknowledge Tobias Spielmann for pSLI-GFP and pARL-PfACP(1-60)-1356 mCherry, Paul Burda for pSLI-GFP-glmS, Jacobus Pharmaceuticals for WR99210, Anna 1357 Bachmann and Mayka Sánchez for helpful advice, Eileen Devaney, Katharina Jungnickel, 1358 and Samuel Pažický for critical reading of the manuscript, and Heidrun von Thien, Yannick 1359 Höppner, and Gabriela Guédez for technical assistance. 1360 1361 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint A C B D Low Fe Healthy High Fe 0 20 40 60 80 100 120 140 160 180 200Phen Green SK MFI [%] P = 0.0006 P = 0.25 n = 8 n = 9 n = 9 P = 0.0002 Low Fe Healthy High Fe 0 20 40 60 80 100 120 140 160 180 200Growth [%] after one IDC n = 7 n = 7 n = 7 P = 0.035 P = 0.067 P = 0.02 Low Fe Healthy High Fe 0 5 10 15 20 25 30 35 40 Merozoites / schizont N = 37 N = 30 N = 30 P < 0.0001 P = 0.11 P < 0.0001 Ctrl Hep 0 20 40 60 80 100 120 140 160 180 200Growth [%] after one IDC n = 10 n = 10 P = 0.0003 Ctrl Hep 0 20 40 60 80 100 120 140 160 180 200Phen Green SK MFI [%] n = 8 n = 8 P = 0.0005 Ctrl Hep 0 5 10 15 20 25 30 35 40Merozoites / schizont N = 36 N = 44 P < 0.0001 Low Fe Healthy High Fe 0 20 40 60 80 100 120 140 160 180 200SYBR Green I MFI [%] n = 5 n = 5 n = 5 P = 0.02 P = 0.17 P = 0.007 Ctrl Hep 0 20 40 60 80 100 120 140 160 180 200SYBR Green I MFI [%] n = 8 n = 8 P = 0.06 Erythrocyte iron levels P. falciparum replication rate DNA content of late schizonts Merozoite number per late schizont Figure 1: Effects of the iron status of the blood donor and of hepcidin on (A) erythrocyte free iron levels, (B) P. falciparum 3D7 growth rates, (C) DNA content per mature schizont, and (D) the number of merozoites per mature schizont. The relative free iron level and DNA content per cell were assessed by measuring the mean fluorescence intensity (MFI) of Phen Green SK or SYBR Green I compared to control (Ctrl, untreated, normal hemoglobin level) using flow cytometry. 0.7 µM hepcidin (Hep) was chosen as a concentration reported to reduce binding of ferrous iron to the erythrocyte iron exporter ferroportin by 50% in vitro (44). Parasite growth rates refer to the fold change in parasitemia after one intraerythrocytic developmental cycle in vitro relative to control as determined by flow cytometry with SYBR Green I (103). Mature schizonts were obtained by treating schizonts at 40 hpi with 1 mM compound 2 (4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-α]pyridine-3- yl]pyrimidin-2-amine) for 8 h. To count the number of merozoites, Giemsa-stained blood smears were analyzed microscopically and only single-infected cells with one digestive vacuole were considered. Means and 95% confidence intervals (indicated by error bars) are shown. Statistical significance was calculated with two-tailed unpaired t tests with Welch’s correction for unequal variances and adjusted with the Holm-Šídák

for multiple comparisons except for merozoite numbers, which were compared using Mann-Whitney test. N represents the number of parasites and n the number of independent experiments. .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint P. falciparum ring stage P. falciparum trophozoite stage Experiment 1 Experiment 2 Low Fe Healthy control High Fe Hepcidin-treated Untreated control MLE of parasite age [hpi] at ring stage 9.9 (97.5% CI: 9.6 – 10.3) 10.2 (97.5% CI: 9.8 – 10.7) 10.0 (97.5% CI: 9.6 – 10.5) 10.2 (97.5% CI: 9.8 – 10.7) 10.6 (97.5% CI: 10.2 – 11.0) MLE of parasite age [hpi] at trophozoite stage 35.4 (97.5% CI: 34.8 – 35.8) 35.5 (97.5% CI: 35.0 – 36.0) 35.4 (97.5% CI: 34.8 – 36.0) 34.7 (97.5% CI: 34.0 – 35.3) 34.5 (97.5% CI: 33.8 – 35.0) Enriched functional terms under high vs. low Fe conditions Enriched functional terms in the presence vs. absence of hepcidin Fold enrichment of underrepresented terms overrepresented terms Number of genes per term Ring stage Trophozoite stage - log10 P - log10 P Fold enrichment of underrepresented terms overrepresented terms Number of genes per term Ring stage Trophozoite stage A C B D E P. falciparum ring stage P. falciparum trophozoite stage BP-GO:0055085-transmembrane transport REAC:R-PFA-382551-transport of small molecules .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint Figure 2: Differential expression of P. falciparum 3D7 genes under various iron conditions. Parasites were cultured with erythrocytes from an individual with high, medium (healthy) or low iron status (experiment 1) or with red blood cells from another healthy donor in the presence or absence of 0.7 µM hepcidin (experiment 2). Samples were harvested at the ring and trophozoite stage (6 – 9 and 26 – 29 hours post invasion, hpi) with three biological replicates per time point and condition. The maximum likelihood estimation (MLE) of the average developmental age of the parasites for each condition and time point (A) was calculated using an algorithm developed by Avi Feller and Jacob Lemieux (46). CI, confidence interval. The volcano plots (B and D) show transcriptional changes of all parasite genes. Red dots indicate significantly (P < 0.05, exact test for negative binomial distribution) upregulated genes (log2 (fold change) ≥ 0.2), blue dots stand for significantly downregulated genes (log2 (fold change) ≤ -0.2), while grey dots represent genes that did not significantly differ in transcription under the conditions described (P ≥ 0.05 and / or -0.2 < log2 (fold change) < 0.2). Differentially expressed genes encoding putative iron transporters (see Table 1) are labeled. Panels C and E show the enrichment of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome (REAC) terms among significantly regulated genes excluding var, stevor and rifin gene families at the two time points. The functional terms were summarized using REVIGO (121) to remove redundancies, represented by circles and plotted according to the significance of their enrichment (-log10 (adjusted P), hypergeometric test). The size of the circle is proportional to the number of differentially regulated genes in the dataset that are associated with the respective term, while the color stands for the fold enrichment. The gray dashed line indicates the threshold of the adjusted P value (-log10 0.05 = 1.3). .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint A C B D DIC PfVIT-GFP ER Tracker Hoechst Merge DIC PfMRS3-GFP MitoTracker Hoechst Merge DIC PfZIPCO-GFP LysoTracker Hoechst Merge DIC PfE140-GFP PfACP-mCherry Hoechst Merge Figure 3: Subcellular localization of four putative iron transport proteins. Representative erythrocytes infected with P. falciparum 3D7 parasites endogenously expressing GFP-tagged PfMRS3 (A), PfVIT (B), PfZIPCO (C) or PfE140 (D) were additionally stained with the fluorescent dyes Hoechst-33342, MitoTracker Red, ER Tracker Red and/or LysoTracker Deep Red. Co-transfection with a construct that encodes the 60 N-terminal amino acids of acyl carrier protein (PfACP) tagged with mCherry (125) resulted in red fluorescence of the apicoplast. Live-cell images were taken under physiological conditions at 37°C using an SP8 confocal laser-scanning microscope (Leica). DIC, differential interference contrast. Scale bar, 2 µm. .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint 2.5 mM GlcN Control A C B D DIC PfVIT(1-143)-GFP Hoechst Merge DIC PfZIPCO(1-117)-GFP Hoechst Merge DIC PfE140-GFP-glmS Hoechst Merge Ctrl GlcN 0 10 20 30 40 Parasite size [µm2] N = 30 N = 34 P = 0.48 Ctrl GlcN 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150Total fluorescence [AU] N = 30 N = 34 P < 0.0001 E F WT WT+GlcNE140-glmS E140-glmS+GlcN E140-glmS+GlcN+Hep E140-glmS+Hep 0 20 40 60 80 100 120 140 160 180 200 220Growth [%] after two IDCs P = 0.56 P = 0.68 P = 0.56 P = 0.016 P = 0.25 n = 6 n = 6 n = 6 n = 6 n = 6 n = 3 WT WT+Hep ΔVIT ΔVIT+Hep ΔZIPCO 0 20 40 60 80 100 120 140 160 180 200 220Growth [%] after two IDCs P = 0.04 P = 0.92 P = 0.03 n = 15 n = 9 n = 8 n = 5 n = 8 P = 0.03 PfE140-GFP-glmS PfE140-GFP-glmS Figure 4: PfVIT and PfE140 are important for P. falciparum growth and may be involved in intracellular iron homeostasis. A Representative erythrocytes infected with P. falciparum 3D7 parasites that endogenously express a truncated version of PfVIT or PfZIPCO tagged with GFP (green). B Growth rates of knockout parasite lines generated. C Reduction of PfE140-GFP fluorescence (green) in live 3D7 parasites caused by glmS-mediated knockdown that was induced by treatment with 2.5 mM glucosamine (GlcN) for 36 h compared to untreated control (Ctrl). D Total parasite fluorescence intensities were quantified as background-corrected integrated densities using ImageJ version 2.9.0/1.53t (109) and compared using Mann-Whitney test. E The size of the parasites was measured as the area of the region of interest and compared using equal variance unpaired t test. F Conditional knockdown of PfE140 induced by treatment with 2.5 mM GlcN results in a growth defect during asexual blood stage development. Live parasites were stained with Hoechst-33342 (blue) and imaged under physiological conditions at 37°C using an SP8 confocal laser-scanning microscope (Leica). DIC, differential interference contrast. Scale bar, 2 µm. Error bars represent 95% confidence intervals of the mean, N the number of parasites analyzed, n the number of independent experiments and Hep treatment with 0.7 µM hepcidin. Growth rates refer to the fold change in parasitemia after two intraerythrocytic developmental cycles in vitro relative to untreated wild-type 3D7 parasites (WT) as determined by flow cytometry with SYBR Green I (103). Statistical significance of growth differences was calculated with two-tailed unpaired t tests with Welch’s correction for unequal variances and adjusted with the Holm-Šídák method for multiple comparisons. .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint A B C MIT VIT ZIPCO Confidence score Surface charge E140 NRAMP CRT Molecular lipophilicity potential Mitochondrial matrix Intermembrane space Cytoplasmic vesicle lumen Cytosol Cytosol Extracellular space Digestive vacuole lumen Cytosol * Intermembrane space Mitochondrial matrix Cytoplasmic vesicle lumen Cytosol * * * Mitochondrial matrix Inter- membrane space Cytoplasmic vesicle lumen Cytosol * * * Extracellular space Extracellular space Cytosol Cytosol Cytosol Cytosol Digestive vacuole lumen Digestive vacuole lumen * low high -20 0 20 -10 0 10 Inner mito- chondrial membrane Inner mito- chondrial membrane Inner mito- chondrial membrane Vesicle membrane Vesicle membrane Vesicle membrane DV membrane DV membrane DV membrane Plasma membrane Plasma membrane Plasma membrane * Figure 5: Predicted structures of putative iron transporters i dentified in P. falciparum as viewed from the membrane plane. A Predicted protein structures with per-residue pLDDT (predicted local distance difference test) confidence scores on a scale from 0 to 100, where blue represents high and red low confidence, respectively. The experimentally determined structure of PfCRT is shown in gray. B Molecular lipophilicity potential of the protein surfaces as implemented in UCSF ChimeraX; tan is hydrophobic and cyan hydrophilic. Dashed lines above and below the tan regions of all proteins indicate the respective membrane and disordered loops were removed for clarity. C Surface charge of the proteins with positively charged areas colored blue and negatively charged ones red. Putative cation-binding site are indicated with an asterisk and transport directions by arrows. PfE140 likely forms a dimer but is shown as a monomer, as no predicted dimer structure could be obtained using AlphaFold2-multimer. The putative cation-binding sites for this protein are based on DeepFRI gradCAM scores for the functional term GO:0015075 Òmonoatomic ion transmembrane transporter activityÓ (Supplementary Fig. S5). .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint Figure 6: Iron homeostasis in a P. falciparum-infected erythrocyte. The human blood plasma contains between 10 and 30 µM total Fe and the erythrocyte cytosol approximately 20 mM (19). However, only 2.5 – 4.7 µM of Fe2+ is bioavailable in an uninfected erythrocyte, and 1.5 – 1.8 µM in a P. falciparum-infected one (20). Human ferroportin (FPN) at the host cell surface (erythrocyte plasma membrane, EPM) exports free iron from the erythrocyte (126), the nutrient pore formed by PfEXP1 and PfEXP2 allows the passage of the ions through the parasitophorous vacuole membrane (PVM) (127). PfE140 at the parasite plasma membrane (PPM) may mediate iron uptake iron into the parasite cytosol and the mitochondrial carrier protein PfMRS3 likely translocates Fe2+ into the mitochondrion, a site of de novo heme biosynthesis. The digestive vacuole (DV) contains a high amount of total Fe as it is the site of hemoglobin degradation. The chloroquine resistance transporter (PfCRT) and the natural resistance-associated macrophage protein (PfNRAMP) were suggested to mediate proton-coupled export of Fe2+ from the DV into the parasite cytosol (35, 36). The vacuolar iron transporter (PfVIT) may be involved in iron detoxification by transporting excess iron from the cytosol into cytoplasmic vesicles that may be acidocalcisomes, whereas PfZIPCO may release zinc and ferrous iron from these organelles under low-iron conditions (this study). Both acidocalcisomes and the DV are likely acidified by the plant-like H+-pump V-ATPase, which can fuel secondary active transport processes (22, 27). Parasite-encoded proteins are shown in orange and human- encoded transporters in blue. .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 20, 2024. ; https://doi.org/10.1101/2024.04.18.590068doi: bioRxiv preprint