The Multiple Roles of LCCL Domain-Containing Proteins for Malaria Parasite Transmission.

Multi-protein complexes are crucial for various essential biological processes of the malaria parasite Plasmodium, such as protein synthesis, host cell invasion and adhesion. Especially during the sexual phase of the parasite, which takes place in the midgut of the mosquito vector, protein complexes are required for fertilization, sporulation and ultimately for the successful transmission of the parasite. Among the most noticeable protein complexes of the transmission stages are the ones formed by the LCCL domain-containing protein family that play critical roles in the generation of infective sporozoites. The six members of this protein family are characterized by numerous adhesive modules and domains typically found in secreted proteins. This review summarizes the findings of expression and functional studies on the LCCL domain-containing proteins of the human pathogenic P. falciparum and the rodent-infecting P. berghei and discusses the common features and differences of the homologous proteins.

The Plasmodium falciparum CCCH zinc finger protein MD3 regulates male gametocytogenesis through its interaction with RNA-binding proteins.

The transmission of malaria parasites to mosquitoes is dependent on the formation of gametocytes. Once fully matured, gametocytes are able to transform into gametes in the mosquito's midgut, a process accompanied with their egress from the enveloping erythrocyte. Gametocyte maturation and gametogenesis require a well-coordinated gene expression program that involves a wide spectrum of regulatory proteins, ranging from histone modifiers to transcription factors to RNA-binding proteins. Here, we investigated the role of the CCCH zinc finger protein MD3 in Plasmodium falciparum gametocytogenesis. MD3 was originally identified as an epigenetically regulated protein of immature gametocytes and recently shown to be involved in male development in a barcode-based screen in P. berghei. We report that MD3 is mainly present in the cytoplasm of immature male P. falciparum gametocytes. Parasites deficient of MD3 are impaired in gametocyte maturation and male gametocytogenesis. BioID analysis in combination with co-immunoprecipitation assays unveiled an interaction network of MD3 with RNA-binding proteins like PABP1 and ALBA3, with translational initiators, regulators and repressors like elF4G, PUF1, NOT1 and CITH, and with further regulators of gametocytogenesis, including ZNF4, MD1 and GD1. We conclude that MD3 is part of a regulator complex crucial for post-transcriptional fine-tuning of male gametocytogenesis.

The patatin-like phospholipase PfPNPLA2 is involved in the mitochondrial degradation of phosphatidylglycerol during Plasmodium falciparum blood stage development.

Plasmodium falciparum is an Apicomplexa responsible for human malaria, a major disease causing more than ½ million deaths every year, against which there is no fully efficient vaccine. The current rapid emergence of drug resistances emphasizes the need to identify novel drug targets. Increasing evidences show that lipid synthesis and trafficking are essential for parasite survival and pathogenesis, and that these pathways represent potential points of attack. Large amounts of phospholipids are needed for the generation of membrane compartments for newly divided parasites in the host cell. Parasite membrane homeostasis is achieved by an essential combination of parasite de novo lipid synthesis/recycling and massive host lipid scavenging. Latest data suggest that the mobilization and channeling of lipid resources is key for asexual parasite survival within the host red blood cell, but the molecular actors allowing lipid acquisition are poorly characterized. Enzymes remodeling lipids such as phospholipases are likely involved in these mechanisms. P. falciparum possesses an unusually large set of phospholipases, whose functions are largely unknown. Here we focused on the putative patatin-like phospholipase PfPNPLA2, for which we generated an glmS-inducible knockdown line and investigated its role during blood stages malaria. Disruption of the mitochondrial PfPNPLA2 in the asexual blood stages affected mitochondrial morphology and further induced a significant defect in parasite replication and survival, in particular under low host lipid availability. Lipidomic analyses revealed that PfPNPLA2 specifically degrades the parasite membrane lipid phosphatidylglycerol to generate lysobisphosphatidic acid. PfPNPLA2 knockdown further resulted in an increased host lipid scavenging accumulating in the form of storage lipids and free fatty acids. These results suggest that PfPNPLA2 is involved in the recycling of parasite phosphatidylglycerol to sustain optimal intraerythrocytic development when the host resources are scarce. This work strengthens our understanding of the complex lipid homeostasis pathways to acquire lipids and allow asexual parasite survival.

Naturally occurring phenylethanoids and phenylpropanoids: antimalarial potential.

Malaria as an infectious disease is one of the world's most dangerous parasitic diseases. There is an urgent need for the development of new antimalarial drugs. Natural products are a very rich source of new bioactive compounds. Our research aims to shed light on the recent studies which demonstrated the antimalarial potential of phenylpropanoids as a major natural-products class. This study involves an in silico analysis of naturally-occurring phenylpropanoids and phenylethanoids which showed 25 compounds with moderate to strong binding affinity to various amino acid residues lining the active site; P. falciparum kinase (PfPK5), P. falciparum cytochrome bc1 complex (cyt bc1), and P. falciparum lysyl-tRNA synthetase (PfKRS1); of Plasmodium falciparum parasite, a unicellular protozoan which causes the most severe and life-threatening malaria. Furthermore, the study was augmented by the assessment of antiplasmodial activity of glandularin, a naturally occurring dibenzylbutyrolactolic lignan, against chloroquine-sensitive 3D7 strain of P. falciparum using SYBR green I-based fluorescence assay, which showed high antimalarial activity with IC50 value of 11.2 µM after 24 hours of incubation. Our results highlight phenylpropanoids and glandularin in particular as a promising chemical lead for development of antimalarial drugs.

Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells.

Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside-out mode during which the membrane of the parasitophorous vacuole (PV) ruptures prior to the erythrocyte membrane. Membrane rupture requires exocytosis of specialized egress vesicles of the parasites; that is, osmiophilic bodies (OBs) involved in rupturing the PV membrane, and vesicles that harbor the perforin-like protein PPLP2 (here termed P-EVs) required for erythrocyte lysis. While some OB proteins have been identified, like G377 and MDV1/Peg3, the majority of egress vesicle-resident proteins is yet unknown. Here, we used high-resolution imaging and BioID methods to study the two egress vesicle types in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the P-EVs and that exocytosis of the P-EVs, but not of the OBs, is calcium sensitive. Both vesicle types exhibit distinct proteomes with the majority of proteins located in the OBs. In addition to known egress-related proteins, we identified novel components of OBs and P-EVs, including vesicle-trafficking proteins. Our data provide insight into the immense molecular machinery required for the inside-out egress of P. falciparum gametocytes.

Plasmodium falciparum Gametes and Sporozoites Hijack Plasmin and Factor H To Evade Host Complement Killing.

Plasmodium parasites are the etiological agents of malaria, a disease responsible for over half a million deaths annually. Successful completion of the parasite's life cycle in the vertebrate host and transmission to a mosquito vector is contingent upon the ability of the parasite to evade the host's defenses. The extracellular stages of the parasite, including gametes and sporozoites, must evade complement attack in both the mammalian host and in the blood ingested by the mosquito vector. Here, we show that Plasmodium falciparum gametes and sporozoites acquire mammalian plasminogen and activate it into the serine protease plasmin to evade complement attack by degrading C3b. Complement-mediated permeabilization of gametes and sporozoites was higher in plasminogen-depleted plasma, suggesting that plasminogen is important for complement evasion. Plasmin also facilitates gamete exflagellation through complement evasion. Furthermore, supplementing serum with plasmin significantly increased parasite infectivity to mosquitoes and lowered the transmission-blocking activity of antibodies to Pfs230, a potent vaccine candidate currently in clinical trials. Finally, we show that human factor H, previously shown to facilitate complement evasion by gametes, also facilitates complement evasion by sporozoites. Plasmin and factor H simultaneously cooperate to enhance complement evasion by gametes and sporozoites. Taken together, our data show that Plasmodium falciparum gametes and sporozoites hijack the mammalian serine protease plasmin to evade complement attack by degrading C3b. Understanding of the mechanisms of complement evasion by the parasite is key to the development of novel effective therapeutics. IMPORTANCE Current approaches to control malaria are complicated by the development of antimalarial-resistant parasites and insecticide-resistant vectors. Vaccines that block transmission to mosquitoes and humans are a plausible alternative to overcome these setbacks. To inform the development of efficacious vaccines, it is imperative to understand how the parasite interacts with the host immune response. In this report, we show that the parasite can co-opt host plasmin, a mammalian fibrinolytic protein to evade host complement attack. Our results highlight a potential mechanism that may reduce efficacy of potent vaccine candidates. Taken together, our results will inform future studies in developing novel antimalarial therapeutics.

Characterization of Plasmodium falciparum prohibitins as novel targets to block infection in humans by impairing the growth and transmission of the parasite.

Prohibitins (PHBs) are highly conserved pleiotropic proteins as they have been shown to mediate key cellular functions. Here, we characterize PHBs encoding putative genes ofPlasmodium falciparum by exploiting different orthologous models. We demonstrated that PfPHB1 (PF3D7_0829200) and PfPHB2 (PF3D7_1014700) are expressed in asexual and sexual blood stages of the parasite. Immunostaining indicated hese proteins as mitochondrial residents as they were found to be localized as branched structures. We further validated PfPHBs as organellar proteins residing in Plasmodium mitochondria, where they interact with each other. Functional characterization was done in Saccharomyces cerevisiae orthologous model by expressing PfPHB1 and PfPHB2 in cells harboring respective mutants. The PfPHBs functionally complemented the yeast PHB1 and PHB2 mutants, where the proteins were found to be involved in stabilizing the mitochondrial DNA, retaining mitochondrial integrity and rescuing yeast cell growth. Further, Rocaglamide (Roc-A), a known inhibitor of PHBs and anti-cancerous agent, was tested against PfPHBs and as an antimalarial. Roc-A treatment retarded the growth of PHB1, PHB2, and ethidium bromide petite yeast mutants. Moreover, Roc-A inhibited growth of yeast PHBs mutants that were functionally complemented with PfPHBs, validating P. falciparum PHBs as one of the molecular targets for Roc-A. Roc-A treatment led to growth inhibition of artemisinin-sensitive (3D7), artemisinin-resistant (R539T) and chloroquine-resistant (RKL-9) parasites in nanomolar ranges. The compound was able to retard gametocyte and oocyst growth with significant morphological aberrations. Based on our findings, we propose the presence of functional mitochondrial PfPHB1 and PfPHB2 in P. falciparum and their druggability to block parasite growth.

Evaluation of Chiral Organosulfur Compounds on Their Activity against the Malaria Parasite Plasmodium falciparum.

Malaria is one of the deadliest tropical diseases, especially causing havoc in children under the age of five in Africa. Although the disease is treatable, the rapid development of drug resistant parasites against frontline drugs requires the search for novel antimalarials. In this study, we tested a series of organosulfur compounds from our internal library for their antiplasmodial effect against Plasmodium falciparum asexual and sexual blood stages. Some active compounds were also obtained in enantiomerically pure form and tested individually against asexual blood stages of the parasite to compare their activity. Out of the 23 tested compounds, 7 compounds (1, 2, 5, 9, 15, 16, and 17) exhibited high antimalarial activity, with IC50 values in the range from 2.2 ± 0.64 to 5.2 ± 1.95 µM, while the other compounds showed moderate to very low activity. The most active compounds also exhibited high activity against the chloroquine-resistant strain, reduced gametocyte development and were not toxic to non-infected red blood cells and Hela cells, as well as the hematopoietic HEL cell line at concentrations below 50 µM. To determine if the enantiomers of the active compounds display different antimalarial activity, enantiomers of two of the active compounds were separated and their antimalarial activity compared. The results show a higher activity of the (-) enantiomers as compared to their (+) counterparts. Our combined data indicate that organosulfur compounds could be exploited as antimalarial drugs and enantiomers of the active compounds may represent a good starting point for the design of novel drugs to target malaria.

Metabolomic Profiling, In Vitro Antimalarial Investigation and In Silico Modeling of the Marine Actinobacterium Strain Rhodococcus sp. UR111 Associated with the Soft Coral Nephthea sp.

Malaria is a persistent illness with a great public health concern. To combat this fatal disease, developing effective antimalarial medications has become a necessity. In the present study, we described the actinomycetes associated with the Red Sea soft coral Nephthea sp. and isolated a strain that was sub-cultured in three different media (M1, ISP2, and OLIGO). Actinomycete isolate's phylogenetic analysis of the 16S rRNA gene revealed that it belongs to the genus Rhodococcus. In vitro screening of the antimalarial activity for three extracts against Plasmodium falciparum was carried out. Non-targeted metabolomics for the chemical characterization of the isolated actinomycete species UA111 derived extracts were employed using high-resolution liquid chromatography-mass spectrometry (LC-HR-MS) for dereplication purposes. Additionally, statistical analysis of the vast LC-MS data was performed using MetaboAnalyst 5.0. Finally, an in silico analysis was conducted to investigate the potential chemical compounds that could be the source of the antimalarial potential. The results revealed that ISP2 media extract is the most effective against Plasmodium falciparum, according to antimalarial screening (IC50 8.5 µg/mL), in contrast, OLIGO media extract was inactive. LC-HRMS-based metabolomics identified a range of metabolites, mainly alkaloids, from the genus Rhodococcus. On the other hand, multivariate analysis showed chemical diversity between the analyzed samples, with ISP2 extract being optimal. The docking analysis was able to anticipate the various patterns of interaction of the annotated compounds with three malarial protein targets (P. falciparum kinase, P. falciparum cytochrome bc1 complex, and P. falciparum lysyl-tRNA synthetase). Among all of the test compounds, perlolyrine (11) and 3097-B2 (12) displayed the best docking profiles. In conclusion, this work demonstrated the value of the established method for the metabolic profiling of marine actinomycetes using the data from liquid chromatography-mass spectrometry (LC-MS), which helps to streamline the difficult isolation stages required for their chemical characterization. In addition, the antimalarial efficacy of this strain has intriguing implications for future pharmaceutical development.

New Halogenated Compounds from Halimeda macroloba Seaweed with Potential Inhibitory Activity against Malaria.

Malaria is one of the most important infectious diseases worldwide. The causative of the most severe forms of malaria, Plasmodium falciparum, has developed resistances against all the available antimalarial drugs. In the present study, the phytochemical investigation of the green seaweed Halimeda macroloba has afforded two new compounds 1-2, along with 4 known ones 3-6. The structures of the compounds had been confirmed using 1& 2D-NMR and HRESIMS analyses. Extensive machine-learning-supported virtual-screening suggested cytochrome-C enzyme as a potential target for compound 2. Docking, absolute-binding-free-energy (ΔGbinding) and molecular-dynamics-simulation (MDS) of compound 2 revealed the strong binding interaction of this compound with cytochrome-C. In vitro testing for crude extract and isolated compounds revealed the potential in vitro inhibitory activity of both extract and compound 2 against P. falciparum. The crude extract was able to inhibit the parasite growth with an IC50 value of 1.8 ± 0.35 µg/mL. Compound 2 also showed good inhibitory activity with an IC50 value of 3.2 ± 0.23 µg/mL. Meanwhile, compound 6 showed moderate inhibitory activity with an IC50 value of 19.3 ± 0.51 µg/mL. Accordingly, the scaffold of compound 2 can be considered as a good lead compound for the future development of new antimalarial agents.

The impact of human complement on the clinical outcome of malaria infection.

The tropical disease malaria remains a major cause of global morbidity. Once transmitted to the human by a blood-feeding mosquito, the unicellular malaria parasite comes into contact with the complement system and continues to interact with human complement during its intraerythrocytic replication cycles. In the course of infection, both the classical and the alternative pathway of complement are activated, leading to parasite opsonization and lysis as well as the induction of complement-binding antibodies. While complement activity can be linked to the severity of malaria, it remains to date unclear, whether human complement is beneficial for protective immunity or if extensive complement reactions may rather enhance pathogenesis. In addition, the parasite has evolved molecular strategies to circumvent attack by human complement and has even developed means to utilize complement factors as mediators of host cell infection. In this review, we highlight current knowledge on the role of human complement for the progression of malaria infection. We discuss the various types of interactions between malaria parasites and complement factors with regard to immunity and infection outcome and set a special emphasis on the dual role of complement in the context of parasite fitness.

The WD40-Protein PfWLP1 Ensures Stability of the PfCCp-Based Adhesion Protein Complex in Plasmodium falciparum Gametocytes.

Members of the WD40-repeat protein family can be found in all eukaryotic proteomes where they usually serve as interaction platforms for the assembly of large protein complexes and are therefore essential for the integrity of these complexes. In the malaria parasite Plasmodium falciparum, the WD40-repeat protein PfWLP1 has been shown to interact with members of distinct adhesion protein complexes in the asexual blood stages and gametocyte stages. In this study, we demonstrate that the presence of PfWLP1 is crucial for both the stability of these gametocyte-specific adhesion complexes as well as for gametocyte maturation and gametogenesis. Using reverse genetics, we generated a PfWLP1-knockdown parasite line for functional characterization of the protein. Knockdown of PfWLP1 resulted in a slight reduction of gametocyte numbers and significantly the impaired ability of the gametocytes to exflagellate. PfWLP1-knockdown further led to reduced protein levels of the Limulus coagulation factor C-like (LCCL)-domain proteins PfCCp1 and PfCCp2, which are key components of the adhesion complexes. These findings suggest that the interaction of PfWLP1 with members of the PfCCp-based adhesion complex ensures complex stability and thereby contributes to gametocyte viability and exflagellation.

Plasmodium falciparum S-Adenosylmethionine Synthetase Is Essential for Parasite Survival through a Complex Interaction Network with Cytoplasmic and Nuclear Proteins.

S-adenosylmethionine synthetase (SAMS) is a key enzyme for the synthesis of the lone methyl donor S-adenosyl methionine (SAM), which is involved in transmethylation reactions and hence required for cellular processes such as DNA, RNA, and histone methylation, but also polyamine biosynthesis and proteostasis. In the human malaria parasite Plasmodium falciparum, PfSAMS is encoded by a single gene and has been suggested to be crucial for malaria pathogenesis and transmission; however, to date, PfSAMS has not been fully characterized. To gain deeper insight into the function of PfSAMS, we generated a conditional gene knockdown (KD) using the glmS ribozyme system. We show that PfSAMS localizes to the cytoplasm and the nucleus of blood-stage parasites. PfSAMS-KD results in reduced histone methylation and leads to impaired intraerythrocytic growth and gametocyte development. To further determine the interaction network of PfSAMS, we performed a proximity-dependent biotin identification analysis. We identified a complex network of 1114 proteins involved in biological processes such as cell cycle control and DNA replication, or transcription, but also in phosphatidylcholine and polyamine biosynthesis and proteasome regulation. Our findings highlight the diverse roles of PfSAMS during intraerythrocytic growth and sexual stage development and emphasize that PfSAMS is a potential drug target.

The Plasmodium falciparum CCCH Zinc Finger Protein ZNF4 Plays an Important Role in Gametocyte Exflagellation through the Regulation of Male Enriched Transcripts.

CCCH zinc finger proteins (ZFPs) function mainly as RNA-binding proteins (RBPs) and play a central role in the mRNA metabolism. Over twenty seven CCCH-ZFPs are encoded in the genome of the human malaria parasite Plasmodium falciparum, the causative agent of malaria tropica. However, little is known about their functions. In this study, we characterize one member of the PfCCCH-ZFP named ZNF4. We show that ZNF4 is highly expressed in mature gametocytes, where it predominantly localizes to the cytoplasm. Targeted gene disruption of ZNF4 showed no significant effect in asexual blood stage replication and gametocyte development while male gametocyte exflagellation was significantly impaired, leading to reduced malaria transmission in the mosquito. Comparative transcriptomics between wildtype (WT) and the ZNF4-deficient line (ZNF4-KO) demonstrated the deregulation of about 473 genes (274 upregulated and 199 downregulated) in mature gametocytes. Most of the downregulated genes show peak expression in mature gametocyte with male enriched genes associated to the axonemal dynein complex formation, and cell projection organization is highly affected, pointing to the phenotype in male gametocyte exflagellation. Upregulated genes are associated to ATP synthesis. Our combined data therefore indicate that ZNF4 is a CCCH zinc finger protein which plays an important role in male gametocyte exflagellation through the regulation of male gametocyte-enriched genes.

Cytotoxic potential of Nephthea sp.-derived actinomycetes supported by metabolomics analysis.

Soft corals and associated microorganisms are known to produce leads for anticancer drugs. Keeping this in mind, Nephthea sp.; a Red Sea soft coral was investigated for the first time using the OSMAC approach. Two isolates, Streptomyces sp. UR63 and Micrococcus sp. UR67 were identified. Their extracts revealed the presence of alkaloids, macrolides, quinones, fatty acids and terpenoids. Further comparison through a set of multivariate data analyses revealed their unique chemical profiles. The extracts displayed inhibitory potencies against HepG-2, Caco-2 and MCF-7 tumor cell lines with IC50 values ranging from 11.4 to 38.7 µg/mL when compared with the positive control, doxorubicin. The study not only highlights the cytotoxic potential of soft coral-associated actinomycetes but also shows the advantage of using the OSMAC approach in this regard.

Potent antiplasmodial alkaloids from the rhizobacterium Pantoea agglomerans as hemozoin modulators.

Global health concern regarding malaria has increased since the first report of artemisinin-resistant Plasmodium falciparum (Pf) two decades ago. The current therapies suffer various drawbacks such as low efficacy and significant side effects, alarming for an urgent need of more effective and less toxic drugs with higher patient compliance. Chemical entities with natural origins become progressively attractive as new drug leads due to their structural diversity and bio-compatibility. This study initially aimed at the targeted isolation of hydroxyquinoline derivatives following our published genomics and metabolomics study of Pantoea agglomerans (Pa). Fermentation of Pa on a pre-selected medium followed by chromatographic isolation, NMR and HRMS analyses led to the characterisation of one new hydroxyquinoline alkaloid together with another six known congeners and two known hydroxyquinolone derivatives. When screened for their antimalarial activity by high throughput screening against asexual blood-stage parasites, almost all compounds showed potent and selective sub-micromolar activities. Computational investigation was performed to identify the antiplasmodial potential targets. Ligand-based similarity search predicted the tested compounds to act as hemozoin inhibitors. Computational target identification results were further validated by competitive hemozoin inhibitory properties of hydroxyquinoline and hydroxyquinolone derivatives in vitro. The overall results suggest this natural scaffold is of potential to be developed as antimalarial drug lead.

Zinc finger proteins of Plasmodium falciparum.

Zinc finger proteins (ZFPs) are a large diverse family of proteins with one or more zinc finger domains in which zinc is important in stabilising the domain. ZFPs can interact with DNA, RNA, lipids or even other proteins and therefore contribute to diverse cellular processes including transcriptional regulation, ubiquitin-mediated protein degradation, mRNA decay and stability. In this review, we provide the first comprehensive classification of ZFPs of the malaria parasite Plasmodium falciparum and provide a state of knowledge on the main ZFPs in the parasite, which include the C2H2, CCCH, RING finger and the PHD finger proteins. TAKE AWAYS: The Plasmodium falciparum genome encodes 170 putative Zinc finger proteins (ZFPs). The C2H2, CCCH, RING finger and PHD finger subfamilies of ZFPs are most represented. Known ZFP functions include the regulation of mRNA metabolism and proteostasis.

Vesicle dynamics during the egress of malaria gametocytes from the red blood cell.

Malaria parasites are obligate intracellular pathogens that live in human red blood cells harbored by a parasitophorous vacuole. The parasites need to exit from the red blood cell to continue life-cycle progression and ensure human-to-mosquito transmission. Two types of blood stages are able to lyse the enveloping red blood cell to mediate egress, the merozoites and the gametocytes. The intraerythrocytic parasites exit the red blood cell via an inside-out mode during which the membrane of the parasitophorous vacuole ruptures prior to the red blood cell membrane. Membrane rupture is initiated by the exocytosis of specialized secretory vesicles following the perception of egress triggers. The molecular mechanisms of red blood cell egress have particularly been studied in malaria gametocytes. Upon activation by external factors, gametocytes successively discharge at least two types of vesicles, the osmiophilic bodies needed to rupture the parasitophorous vacuole membrane and recently identified egress vesicles that are important for the perforation of the erythrocyte membrane. In recent years, important components of the signaling cascades leading to red blood cell egress have been investigated and several proteins of the osmiophilic bodies have been identified. We here report on the newest findings on the egress of gametocytes from the red blood cell. We further focus on the content and function of the egress-related vesicles and discuss the molecular machinery that might drive vesicle discharge.