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The sexual phase of the malaria parasite Plasmodium falciparum is accompanied by the coordinated expression of stagespecific adhesive proteins. Among these are six secreted proteins with multiple adhesion domains, termed P. falciparum LCCL domain-containing protein (PfCCp) proteins, which are expressed in the parasitophorous vacuole of the differentiating gametocytes and which are later associated with macrogametes. Although the majority of the PfCCp proteins are implicated in parasite development in the mosquito vector, their functions remain unknown. In the present study we investigated the molecular interactions between the PfCCp proteins during gametocyte development and emergence. Using five different gene-disruptant parasite lines, we show that the lack of one PfCCp protein leads to the loss of other PfCCp family members. Co-immunoprecipitation assays on gametocyte lysates revealed formation of complexes involving all PfCCp proteins, and affinity chromatography co-elution binding assays with recombinant PfCCp domains further indicated direct binding between distinct adhesion domains. PfCCp-coated latex beads bind to newly formed macrogametes but not to gametocytes or older macrogametes 6 or 24 h post-activation. In view of these data, we propose that the PfCCp proteins form multi-protein complexes that are exposed during gametogenesis, thereby mediating cell contacts of macrogametes.

Human complement is a first line defense against infection in which circulating proteins initiate an enzyme cascade on the microbial surface that leads to phagocytosis and lysis. Various pathogens evade complement recognition by binding to regulator proteins that protect host cells from complement activation. We show that emerging gametes of the malaria parasite Plasmodium falciparum bind the host complement regulator factor H (FH) following transmission to the mosquito to protect from complement-mediated lysis by the blood meal. Human complement is active in the mosquito midgut for approximately 1 hr postfeeding. During this period, the gamete surface protein PfGAP50 binds to FH and uses surface-bound FH to inactivate the complement protein C3b. Loss of FH-mediated protection, either through neutralization of FH or blockade of PfGAP50, significantly impairs gametogenesis and inhibits parasite transmission to the mosquito. Thus, Plasmodium co-opts the protective host protein FH to evade complement-mediated lysis within the mosquito midgut.

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70°DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90°angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

During differentiation in the human, the gametocytes of the malaria parasite Plasmodium falciparum display a remarkable number of adhesive proteins on their plasma membrane. These include the PfCCp family of six secreted proteins that assemble to multimeric protein complexes (MPCs) within the gametocyte parasitophorous vacuole. We now show that the PfCCp-based MPCs are linked to the gametocyte plasma membrane via interactions with Pfs230, a binding-partner of the GPI-anchored Pfs48/45. Upon onset of gametogenesis, which takes place after gametocyte uptake by bloodfeeding mosquitoes, GPI-anchored Pfs25 joins the MPC, providing an additional link of its components to the plasma membrane. Gametogenesis also initiates cleavage of Pfs230 at its Nterminal site, resulting in its increased interaction with the MPC. Either lack of Pfs230 or impaired Pfs230 processing causes proteolysis of the PfCCp proteins and release from the MPC. Our data point to MPC reassembly as a crucial step for sexual reproduction.

A substantial challenge worldwide is emergent drug resistance in malaria parasites against approved drugs, such as chloroquine (CQ). To address these unsolved CQ resistance issues, only rare examples of artemisinin (ART)‐based hybrids have been reported. Moreover, protein targets of such hybrids have not been identified yet, and the reason for the superior efficacy of these hybrids is still not known. Herein, we report the synthesis of novel ART–isoquinoline and ART–quinoline hybrids showing highly improved potencies against CQ‐resistant and multidrug‐resistant P. falciparum strains (EC50 (Dd2) down to 1.0 nm; EC50 (K1) down to 0.78 nm) compared to CQ (EC50 (Dd2)=165.3 nm; EC50 (K1)=302.8 nm) and strongly suppressing parasitemia in experimental malaria. These new compounds are easily accessible by step‐economic C−H activation and copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) click reactions. Through chemical proteomics, putatively hybrid‐binding protein targets of the ART‐quinolines were successfully identified in addition to known targets of quinoline and artemisinin alone, suggesting that the hybrids act through multiple modes of action to overcome resistance.

Malaria parasites express a broad repertoire of proteins whose expression is tightly regulated depending on the life-cycle stage of the parasite and the environment of target organs in the respective host. Transmission of malaria parasites from the human to the anopheline mosquito is mediated by intraerythrocytic sexual stages, termed gametocytes, which circulate in the peripheral blood and are essential for the spread of the tropical disease. In Plasmodium falciparum, gametocytes express numerous extracellular proteins with adhesive motifs, which might mediate important interactions during transmission. Among these is a family of six secreted proteins with adhesive modules, termed PfCCp proteins, which are highly conserved throughout the apicomplexan clade. In P. falciparum, the proteins are expressed in the parasitophorous vacuole of gametocytes and are subsequently exposed on the surface of macrogametes during parasite reproduction in the mosquito midgut. One characteristic of the family is a co-dependent expression, such that loss of all six proteins occurs if expression of one member is disrupted via gene knockout. The six PfCCp proteins interact by adhesion domain-mediated binding and thus form complexes on the sexual stage surface having adhesive properties. To date, the PfCCp proteins represent the only protein family of the malaria parasite sexual stages that assembles to multimeric complexes, and only a small number of such protein complexes have so far been identified in other life-cycle stages of the parasite.

BackgroundDuring development in human erythrocytes, Plasmodium falciparum parasites display a remarkable number of adhesive proteins on their plasma membrane. In the invasive merozoites, these include members of the PfMSP1 and PfAMA1/RON complexes, which facilitate contact between merozoites and red blood cells. In gametocytes, sexual precursor cells mediating parasite transmission to the mosquito vector, plasma membrane-associated proteins primarily belong to the PfCCp and 6-cys families with roles in fertilization. This study describes a newly identified WD40-repeat protein unique to Plasmodium species that associates with adhesion protein complexes of both merozoites and gametocytes.MethodsThe WD40-repeat protein-like protein PfWLP1 was identified via co-immunoprecipitation assays followed by mass spectrometry and characterized using biochemical and immunohistochemistry methods. Reverse genetics were employed for functional analysis.ResultsPfWLP1 is expressed both in schizonts and gametocytes. In mature schizonts, the protein localizes underneath the merozoite micronemes and interacts with PfAMA1, while in gametocytes PfWLP1 primarily accumulates underneath the plasma membrane and associates with PfCCp1 and Pfs230. Reverse genetics failed to disrupt the pfwlp1 gene, while haemagglutinin-tagging was feasible, suggesting a crucial function for PfWLP1 during blood stage replication.ConclusionsThis is the first report on a plasmodial WD40-repeat protein associating with cell adhesion proteins. Since WD40 domains are known to mediate protein–protein contact by serving as a rigid scaffold for protein interactions, the presented data suggest that PfWLP1 supports the stability of adhesion protein complexes of the plasmodial blood stages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0967-x) contains supplementary material, which is available to authorized users.