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.

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.

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.

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.

Bio-Guided Isolation of Antimalarial Metabolites from the Coculture of Two Red Sea Sponge-Derived Actinokineospora and Rhodococcus spp.

Coculture is a productive technique to trigger microbes' biosynthetic capacity by mimicking the natural habitats' features principally by competition for food and space and interspecies cross-talks. Mixed cultivation of two Red Sea-derived actinobacteria, Actinokineospora spheciospongiae strain EG49 and Rhodococcus sp. UR59, resulted in the induction of several non-traced metabolites in their axenic cultures, which were detected using LC-HRMS metabolomics analysis. Antimalarial guided isolation of the cocultured fermentation led to the isolation of the angucyclines actinosporins E (1), H (2), G (3), tetragulol (5) and the anthraquinone capillasterquinone B (6), which were not reported under axenic conditions. Interestingly, actinosporins were previously induced when the axenic culture of the Actinokineospora spheciospongiae strain EG49 was treated with signalling molecule N-acetyl-d-glucosamine (GluNAc); this finding confirmed the effectiveness of coculture in the discovery of microbial metabolites yet to be discovered in the axenic fermentation with the potential that could be comparable to adding chemical signalling molecules in the fermentation flask. The isolated angucycline and anthraquinone compounds exhibited in vitro antimalarial activity and good biding affinity against lysyl-tRNA synthetase (PfKRS1), highlighting their potential developability as new antimalarial structural motif.

The G9a Histone Methyltransferase Inhibitor BIX-01294 Modulates Gene Expression during Plasmodium falciparum Gametocyte Development and Transmission.

Transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is initiated by specialized sexual cells, the gametocytes. In the human, gametocytes are formed in response to stress signals and following uptake by a blood-feeding Anopheles mosquito initiate sexual reproduction. Gametocytes need to fine-tune their gene expression in order to develop inside the mosquito to continue life-cycle progression. Previously, we showed that post-translational histone acetylation controls gene expression during gametocyte development and transmission. However, the role of histone methylation remains poorly understood. We here use the histone G9a methyltransferase inhibitor BIX-01294 to investigate the role of histone methylation in regulating gene expression in gametocytes. In vitro assays demonstrated that BIX-01294 inhibits intraerythrocytic replication with a half maximal inhibitory concentration (IC50) of 13.0 nM. Furthermore, BIX-01294 significantly impairs gametocyte maturation and reduces the formation of gametes and zygotes. Comparative transcriptomics between BIX-01294-treated and untreated immature, mature and activated gametocytes demonstrated greater than 1.5-fold deregulation of approximately 359 genes. The majority of these genes are transcriptionally downregulated in the activated gametocytes and could be assigned to transcription, translation, and signaling, indicating a contribution of histone methylations in mediating gametogenesis. Our combined data show that inhibitors of histone methylation may serve as a multi-stage antimalarial.

Pyrethroids and DDT tolerance of Anopheles gambiae s.l. from Sengerema District, an area of intensive pesticide usage in north-western Tanzania.

OBJECTIVE: To assess the susceptibility status of malaria vectors to pyrethroids and dichlorodiphenyltrichloroethane (DDT), characterise the mechanisms underlying resistance and evaluate the role of agro-chemical use in resistance selection among malaria vectors in Sengerema agro-ecosystem zone, Tanzania. METHODS: Mosquito larvae were collected from farms and reared to obtain adults. The susceptibility status of An. gambiae s.l. was assessed using WHO bioassay tests to permethrin, deltamethrin, lambdacyhalothrin, etofenprox, cyfluthrin and DDT. Resistant specimens were screened for knock-down resistance gene (kdr), followed by sequencing both Western and Eastern African variants. A gas chromatography-mass spectrophotometer (GC-MS) was used to determine pesticide residues in soil and sediments from mosquitoes' breeding habitats. RESULTS: Anopheles gambiae s.l. was resistant to all the insecticides tested. The population of Anopheles gambiae s.l was composed of Anopheles arabiensis by 91%. The East African kdr (L1014S) allele was found in 13 of 305 specimens that survived insecticide exposure, with an allele frequency from 0.9% to 50%. DDTs residues were found in soils at a concentration up to 9.90 ng/g (dry weight). CONCLUSION: The observed high resistance levels of An. gambiae s.l., the detection of kdr mutations and pesticide residues in mosquito breeding habitats demonstrate vector resistance mediated by pesticide usage. An integrated intervention through collaboration of agricultural, livestock and vector control units is vital.

Lysine acetylation in sexual stage malaria parasites is a target for antimalarial small molecules.

Therapies to prevent transmission of malaria parasites to the mosquito vector are a vital part of the global malaria elimination agenda. Primaquine is currently the only drug with such activity; however, its use is limited by side effects. The development of transmission-blocking strategies requires an understanding of sexual stage malaria parasite (gametocyte) biology and the identification of new drug leads. Lysine acetylation is an important posttranslational modification involved in regulating eukaryotic gene expression and other essential processes. Interfering with this process with histone deacetylase (HDAC) inhibitors is a validated strategy for cancer and other diseases, including asexual stage malaria parasites. Here we confirm the expression of at least one HDAC protein in Plasmodium falciparum gametocytes and show that histone and nonhistone protein acetylation occurs in this life cycle stage. The activity of the canonical HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; Vorinostat) and a panel of novel HDAC inhibitors on early/late-stage gametocytes and on gamete formation was examined. Several compounds displayed early/late-stage gametocytocidal activity, with TSA being the most potent (50% inhibitory concentration, 70 to 90 nM). In contrast, no inhibitory activity was observed in P. falciparum gametocyte exflagellation experiments. Gametocytocidal HDAC inhibitors caused hyperacetylation of gametocyte histones, consistent with a mode of action targeting HDAC activity. Our data identify HDAC inhibitors as being among a limited number of compounds that target both asexual and sexual stage malaria parasites, making them a potential new starting point for gametocytocidal drug leads and valuable tools for dissecting gametocyte biology.

Changes in the transcriptome of the malaria parasite Plasmodium falciparum during the initial phase of transmission from the human to the mosquito.

BACKGROUND: The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector. RESULTS: To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence.For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using quantitative confocal microscopy. CONCLUSIONS: The obtained transcriptome data demonstrate the regulations of gene expression immediately following malaria parasite transmission to the mosquito. Our findings support the identification of proteins important for sexual reproduction and further development of the mosquito midgut stages and provide insights into the genetic basis of the rapid adaption of Plasmodium to the insect vector.

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.

Harmonine, a defence compound from the harlequin ladybird, inhibits mycobacterial growth and demonstrates multi-stage antimalarial activity.

The harlequin ladybird beetle Harmonia axyridis has been introduced in many countries as a biological control agent, but has become an invasive species threatening the biodiversity of native ladybirds. Its invasive success has been attributed to its vigorous resistance against diverse pathogens. This study demonstrates that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), which is present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited. Harmonine displays gametocytocidal activity, and inhibits the exflagellation of microgametocytes and zygote formation. In an Anopheles stephensi mosquito feeding model, harmonine displays transmission-blocking activity.

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.

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.

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.

Organoarsenic Compounds with In Vitro Activity against the Malaria Parasite Plasmodium falciparum.

The rapid development of parasite drug resistance as well as the lack of medications targeting both the asexual and the sexual blood stages of the malaria parasite necessitate the search for novel antimalarial compounds. Eleven organoarsenic compounds were synthesized and tested for their effect on the asexual blood stages and sexual transmission stages of the malaria parasite Plasmodium falciparum using in vitro assays. The inhibitory potential of the compounds on blood stage viability was tested on the chloroquine (CQ)-sensitive 3D7 and the CQ-resistant Dd2 strain using the Malstat assay. The most effective compounds were subsequently investigated for their effect on impairing gametocyte development and gametogenesis, using the gametocyte-producing NF54 strain in respective cell-based assays. Their potential toxicity was investigated on leukemia cell line Nalm-6 and non-infected erythrocytes. Five out of the 11 compounds showed antiplasmodial activities against 3D7, with half-maximal inhibitory concentration (IC50) values ranging between 1.52 and 8.64 µM. Three of the compounds also acted against Dd2, with the most active compound As-8 exhibiting an IC50 of 0.35 µM. The five compounds also showed significant inhibitory effects on the parasite sexual stages at both IC50 and IC90 concentrations with As-8 displaying the best gametocytocidal activity. No hemolytic and cytotoxic effect was observed for any of the compounds. The organoarsenic compound As-8 may represent a good lead for the design of novel organoarsenic drugs with combined antimalarial and transmission blocking activities.

Coming soon: probiotics-based malaria vaccines.

The beneficial health effect of gut probiotics has been known since its discovery by Élie Metchnikoff. A new study has linked the microbiome of the human gut with immunity against malaria infections. Gut probiotics represent innovative tools for malaria prevention and lead the way to novel types of vaccination strategies.