Mutant/mutation
The mutant expresses a mutated version of the gene encoding DMT1. The 5' UTR of the dmt1 gene is truncated.

Protein (function)
Divalent metal transporter (DMT1) is a critical iron transporter with 12 transmembrane structures responsible for iron uptake in many organisms.
Through bioinformatics analysis, we found a homolog of human Dmt1 in the malaria parasite genome, which we named PyDMT1 in P. yoelii. Bioinformatic analysis shows that all Plasmodium genomes encode one gene product with homology to Saccharomyces cerevisiae (the yeast) Smf1-3, Homo sapiens Nramp1 and Dmt1 (Nramp2). PyDMT1 shares 29.53% amino-acid sequence identity with Homo sapiens Dmt1, and 24.94% with S. cerevisiae Smf3.

Phenotype
A significantly reduction of parasitemia in mice infected with the mutant; 3 days after infection the parasitemia for Pydmt1-C2 clone and Pydmt1-A1 clone was each reduced by 50 and 60%, respectively.

To analyse the function of DMT1, a partial loss of function mutant (hypomorph) was generated. Truncating the regulatory region by inserting a selectable cassette into the upstream of a gene could reduce its expression. We decided to insert an hdhfr(coding for the human dihydrofolate reductase) selectable cassette at the Pydmt1 locus upstream of the coding region of Pydmt1-HA to knock down its expression. In the Plasmodium database, we found that Pydmt1 shares the same regulatory region of approximately 4.1 kb with an adjacent gene, PY17X_1241700, whose function remains unknown. To minimize the undesirable effect on this neighbor gene, we designed a single insertion strategy to shorten the regulatory region of Pydmt1 without altering the entire 4.1 kb regulatory region of PY17X_1241700. Multiple attempts to put the cassette in at − 400, − 600, − 800, − 1200, and − 1600 bp upstream of Pydmt1 were unsuccessful. However, a recombinant clone was obtained by inserting the hdhfr cassette at the − 2000 bp position. After limiting dilution and mice infection, we obtained six independent clones, among which two clones (Pydmt1-A1 and Pydmt1-C2; PyDMT hypomorph) PyDMT1 hypomorph) were selected for Pydmt1 expression analysis. Inserting hdhfr at the − 2000 bp led to a reduction of Pydmt1 expression in the clones, demonstrated at both the mRNA and protein levels. This insertion, however, had little influence on the mRNA expression of the flanking gene PY17X_1241700. This roughly halving of PyDMT1 expression led to a significantly reduction of parasitemia; 3 days after infection the parasitemia for Pydmt1-C2 clone and Pydmt1-A1clone was each reduced by 50 and 60%, respectively

Additional information:
The unsuccessful attempts to disrupt DMT1 indicate an essential function during asexual blood stage growth/multiplication (see RMgm-5515)

By analysis of a mutant expressing a C-terminal HA tagged version of DMT1 (RMgm-5516) the following was shown: Confocal analysis after immunostaining with HA antibody showed that PyDMT-HA surrounded the hemozoin in the blood stage, implying that PyDMT1 is likely localized on the membrane of the food vacuole.

To test whether PyDMT1 acts as an iron importer in P. yoelii, a mutant was generated with a partial loss of function allele of PyDMT1 by a single crossover to the regulatory region of Pydmt1. This mutation caused a partial loss of PyDMT1 expression but resulted in severe defects, rescued by iron supplementation.

From the paper: 'Plasmodium CRT (chloroquine-resistant transport) is known to localize on the vacuolar membrane, responsible for chloroquine resistance. To further confirm that PyDMT1 is localized on the food vacuole membrane, we fused a FLAG tag at the C-terminal of CRT by single crossover to label the food vacuole. Confocal imaging revealed that PyDMT1 indeed mainly colocalized with CRT at all blood stages, and slightly with the Golgi marker Rab6, but completely not with the endoplasmic reticulum marker PMV. During the schizont stage, a small amount of PyDMT1 signal is found in the merozoites, mainly in the hemozoin region. These data strongly suggest that PyDMT1 mainly localizes on the digestive vacuole membrane'.

From the paper: 'To test the hypothesis that the slow growth of the hypomorphic PyDMT1 parasite was due to iron deficiency caused by lower iron uptake, we counted the merozoites of the mutant Pydmt1 parasite and the parent strain without or with exogenous iron in vitro by adding Fe2+.  After 18 h incubation, the number of merozoites for Pydmt1 mutant parasites (clone A1 and clone C2) was about 9, in comparison to 14 for the parent strain, consistent with the slower in vivo growth of the mutant. Meanwhile, addition of Fe2+ did not significantly increase the number of merozoites for the parent strain, indicating that iron was not a limiting factor for the normal parasite under our experimental condition. In contrast, iron supplements significantly increased the number of merozoites for the Pydmt1 mutant parasite, to a comparable level with that in the control parasite. The data indicate that it is primarily iron deficiency and not anything else that underlies the growth defects from reduced PyDMT1 expression. Next, we compared the labile iron pool (LIP) in the control parasite and clone A1. To that end, we used a similar strategy adopted by other groups; iRBCs containing both the parent parasite and clone A1 were stained with the iron- sensitive fluorescent probe Calcein-AM, and the signals were analyzed by flow cytometry. The LIP of the Pydmt1 mutant parasite iRBC was significantly lower than that of the control parasite iRBC. Noteworthy is that Fe2+ quenches the fluorescence so that higher Fe2+ levels result in lower signals. In order to make sure that these signal changes were primarily from the parasites instead of the RBC, a confocal image was further taken. The primary signal of the Calcein-AM stain indeed appeared from the parasites. Furthermore, the diffuse and stronger signal across the whole parasite suggests a reduced LIP of Pydmt1-A1 in the cytosol. We then determined whether the number of merozoites in Pydmt1 mutant parasite could be reverted by other metals. Given the same nature of the different Pydmt1 hypomorph mutants, essentially identical results obtained with A1 and C2, and the laborious amount of work involved, we chose A1 for this work. Zinc addition partially restored the number of merozoites of clone A1, meaning that zinc supplement could, to some extent, rescue the growth defect of Pydmt1 mutant parasites. However, adding copper or manganese ions did not affect the merozoite maturation. Taken together, we conclude that the primary physiological function of PyDMT1 is iron uptake, and decreased PyDMT1 expression causes parasite iron deficiency, leading to severe physiological consequences. The perfect rescue of the PyDMT1 hypomorphs with iron, together with the observation of the inability of iron addition to enhance the growth of the wild-type pathogen, confirms our suspicion that the normal pathogen may obtain just enough iron for its optimal growth.

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