Assigning function to orphan membrane transport proteins and prioritizing candidates for detailed biochemical characterization remain fundamental challenges and are particularly important for medically relevant pathogens, such as malaria parasites. Here we present a comprehensive genetic analysis of 35 orphan transport proteins of Plasmodium berghei during its life cycle in mice and Anopheles mosquitoes. Six genes, including four candidate aminophospholipid transporters, are refractory to gene deletion, indicative of essential functions. We generate and phenotypically characterize 29 mutant strains with deletions of individual transporter genes. Whereas seven genes appear to be dispensable under the experimental conditions tested, deletion of any of the 22 other genes leads to specific defects in life cycle progression in vivo and/or host transition. Our study provides growing support for a potential link between heavy metal homeostasis and host switching and reveals potential targets for rational design of new intervention strategies against malaria.

Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein-protein interfaces that are present repetitively along naturally occurring PKS assembly lines.A fundamental challenge to our understanding of multimodular polyketide synthases (PKSs) is the ability to explain the unidirectional translocation of growing polyketide chains through these enzymatic assembly lines. The 6-deoxyerythronolide B synthase (DEBS; Fig. 1) is arguably the most well studied example of assembly line PKSs (1-4). Each module of DEBS has an acyl carrier protein (ACP) that collaborates with the β-ketosynthase (KS) domain of the same module to catalyze a single round of polyketide chain elongation ( Fig. 2). At this point, the ACP-bound intermediate is precluded from back-transfer to the same KS domain and is instead translocated to the KS domain of the downstream module (Fig. 2).In the course of our investigations into the mechanism of intermodular chain translocation (Fig. 2, reaction 1) and intramodular chain elongation (Fig. 2, reaction 4) within DEBS, we discovered that the specificity of these two reactions is controlled by protein-protein interfaces involving distinct regions of the ACP domain (5). In the present study, we have used site-directed mutagenesis to identify ACP residues that contribute to the observed specificity. In turn, these residue-level constraints were exploited to validate the proposed structural model for ACP docking during chain elongation (5), as well as to develop an analogous in silico model for chain translocation. Generalization of these models to three naturally occurring PKSs has revealed a programming pattern that establishes a ratchet mechanism that accurately explains the unique chain translocation pathway of each PKS. As a test of this ratcheting mechanism in PKS assembly lines, we engineered a module of DEBS to iteratively catalyze two successive rounds of chain elongation instead of only one. Results and DiscussionIdentification of ACP Residues That Contribute to Chain Elongation Specificity. All ACPs from fatty acid and polyketide synthases are all-helical bundles comprised of three major α-helices connected by two structured loops (Fig. 3A). In earlier work (5) we showe...

Graphical abstractHighlights► Rapid, robust isolation of recombinant Plasmodium berghei lines by flow cytometry. ► Reduction by >80% of animal use for the generation of mutant parasite lines. ► Plasmodium berghei aquaglyceroporin is dispensable for all life cycle stages. ► Tagged aquaglyceroporin localizes to perinuclear structures inside the parasite.

Homeostasis of the trace element copper is essential to all eukaryotic life. Copper serves as a cofactor in metalloenzymes and catalyses electron transfer reactions as well as the generation of potentially toxic reactive oxygen species. Here, we describe the functional characterization of an evolutionarily highly conserved, predicted copper-transporting P-type ATPase (CuTP) in the murine malaria model parasite Plasmodium berghei. Live imaging of a parasite line expressing a fluorescently tagged CuTP demonstrated that CuTP is predominantly located in vesicular bodies of the parasite. A P. berghei loss-of-function mutant line was readily obtained and showed no apparent defect in in vivo blood stage growth. Parasite transmission through the mosquito vector was severely affected, but not entirely abolished. We show that male and female gametocytes are abundant in cutp− parasites, but activation of male microgametes and exflagellation were strongly impaired. This specific defect could be mimicked by addition of the copper chelator neocuproine to wild-type gametocytes. A cross-fertilization assay demonstrated that female fertility was also severely abrogated. In conclusion, we provide experimental genetic and pharmacological evidence that a healthy copper homeostasis is critical to malaria parasite fertility of both genders of gametocyte and, hence, to transmission to the mosquito vector.

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species.

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