Combination of computational techniques and RNAi reveal targets in Anopheles gambiae for malaria vector control.

Increasing reports of insecticide resistance continue to hamper the gains of vector control strategies in curbing malaria transmission. This makes identifying new insecticide targets or alternative vector control strategies necessary. CLassifier of Essentiality AcRoss EukaRyote (CLEARER), a leave-one-organism-out cross-validation machine learning classifier for essential genes, was used to predict essential genes in Anopheles gambiae and selected predicted genes experimentally validated. The CLEARER algorithm was trained on six model organisms: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Mus musculus, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and employed to identify essential genes in An. gambiae. Of the 10,426 genes in An. gambiae, 1,946 genes (18.7%) were predicted to be Cellular Essential Genes (CEGs), 1716 (16.5%) to be Organism Essential Genes (OEGs), and 852 genes (8.2%) to be essential as both OEGs and CEGs. RNA interference (RNAi) was used to validate the top three highly expressed non-ribosomal predictions as probable vector control targets, by determining the effect of these genes on the survival of An. gambiae G3 mosquitoes. In addition, the effect of knockdown of arginase (AGAP008783) on Plasmodium berghei infection in mosquitoes was evaluated, an enzyme we computationally inferred earlier to be essential based on chokepoint analysis. Arginase and the top three genes, AGAP007406 (Elongation factor 1-alpha, Elf1), AGAP002076 (Heat shock 70kDa protein 1/8, HSP), AGAP009441 (Elongation factor 2, Elf2), had knockdown efficiencies of 91%, 75%, 63%, and 61%, respectively. While knockdown of HSP or Elf2 significantly reduced longevity of the mosquitoes (p<0.0001) compared to control groups, Elf1 or arginase knockdown had no effect on survival. However, arginase knockdown significantly reduced P. berghei oocytes counts in the midgut of mosquitoes when compared to LacZ-injected controls. The study reveals HSP and Elf2 as important contributors to mosquito survival and arginase as important for parasite development, hence placing them as possible targets for vector control.

The circadian clock modulates Anopheles gambiae infection with Plasmodium falciparum.

Key behaviours, physiologies and gene expressions in Anopheles mosquitoes impact the transmission of Plasmodium. Such mosquito factors are rhythmic to closely follow diel rhythms. Here, we set to explore the impact of the mosquito circadian rhythm on the tripartite interaction between the vector, the parasite and the midgut microbiota, and investigate how this may affect the parasite infection outcomes. We assess Plasmodium falciparum infection prevalence and intensity, as a proxy for gametocyte infectivity, in Anopheles gambiae mosquitoes that received a gametocyte-containing bloodfeed and measure the abundance of the midgut microbiota at different times of the mosquito rearing light-dark cycle. Gametocyte infectivity is also compared in mosquitoes reared and maintained under a reversed light-dark regime. The effect of the circadian clock on the infection outcome is also investigated through silencing of the CLOCK gene that is central in the regulation of animal circadian rhythms. The results reveal that the A. gambiae circadian cycle plays a key role in the intensity of infection of P. falciparum gametocytes. We show that parasite gametocytes are more infectious during the night-time, where standard membrane feeding assays (SMFAs) at different time points in the mosquito natural circadian rhythm demonstrate that gametocytes are more infectious when ingested at midnight than midday. When mosquitoes were cultured under a reversed light/dark regime, disrupting their natural physiological homeostasis, and infected with P. falciparum at evening hours, the infection intensity and prevalence were significantly decreased. Similar results were obtained in mosquitoes reared under the standard light/dark regime upon silencing of CLOCK, a key regulator of the circadian rhythm, highlighting the importance of the circadian rhythm for the mosquito vectorial capacity. At that time, the mosquito midgut microbiota load is significantly reduced, while the expression of lysozyme C-1 (LYSC-1) is elevated, which is involved in both the immune response and microbiota digestion. We conclude that the tripartite interactions between the mosquito vector, the malaria parasite and the mosquito gut microbiota are finely tuned to support and maintain malaria transmission. Our data add to the knowledge framework required for designing appropriate and biologically relevant SMFA protocols.

Identifying essential genes across eukaryotes by machine learning.

Identifying essential genes on a genome scale is resource intensive and has been performed for only a few eukaryotes. For less studied organisms essentiality might be predicted by gene homology. However, this approach cannot be applied to non-conserved genes. Additionally, divergent essentiality information is obtained from studying single cells or whole, multi-cellular organisms, and particularly when derived from human cell line screens and human population studies. We employed machine learning across six model eukaryotes and 60 381 genes, using 41 635 features derived from the sequence, gene function information and network topology. Within a leave-one-organism-out cross-validation, the classifiers showed high generalizability with an average accuracy close to 80% in the left-out species. As a case study, we applied the method to Tribolium castaneum and Bombyx mori and validated predictions experimentally yielding similar performances. Finally, using the classifier based on the studied model organisms enabled linking the essentiality information of human cell line screens and population studies.

Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement.

Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an acceptable testing pathway of gene drives for malaria eradication.

Synthesis and biological evaluation of benzhydryl-based antiplasmodial agents possessing Plasmodium falciparum chloroquine resistance transporter (PfCRT) inhibitory activity.

Due to the surge in resistance to common therapies, malaria remains a significant concern to human health worldwide. In chloroquine (CQ)-resistant (CQ-R) strains of Plasmodium falciparum, CQ and related drugs are effluxed from the parasite's digestive vacuole (DV). This process is mediated by mutant isoforms of a protein called CQ resistance transporter (PfCRT). CQ-R strains can be partially re-sensitized to CQ by verapamil (VP), primaquine (PQ) and other compounds, and this has been shown to be due to the ability of these molecules to inhibit drug transport via PfCRT. We have previously developed a series of clotrimazole (CLT)-based antimalarial agents that possess inhibitory activity against PfCRT (4a,b). In our endeavor to develop novel PfCRT inhibitors, and to perform a structure-activity relationship analysis, we synthesized a new library of analogues. When the benzhydryl system was linked to a 4-aminoquinoline group (5a-f) the resulting compounds exhibited good cytotoxicity against both CQ-R and CQ-S strains of P. falciparum. The most potent inhibitory activity against the PfCRT-mediated transport of CQ was obtained with compound 5k. When compared to the reference compound, benzhydryl analogues of PQ (5i,j) showed a similar activity against blood-stage parasites, and a stronger in vitro potency against liver-stage parasites. Unfortunately, in the in vivo transmission blocking assays, 5i,j were inactive against gametocytes.

PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector.

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.

Male-Specific Protein Disulphide Isomerase Function is Essential for Plasmodium Transmission and a Vulnerable Target for Intervention.

Inhibiting transmission of Plasmodium is an essential strategy in malaria eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. Lack of knowledge of the mechanisms underlying fertilization have been a hindrance in the development of transmission-blocking interventions. Here we describe a protein disulphide isomerase essential for malarial transmission (PDI-Trans/PBANKA_0820300) to the mosquito. We show that PDI-Trans activity is male-specific, surface-expressed, essential for fertilization/transmission, and exhibits disulphide isomerase activity which is up-regulated post-gamete activation. We demonstrate that PDI-Trans is a viable anti-malarial drug and vaccine target blocking malarial transmission with the use of PDI inhibitor bacitracin (98.21%/92.48% reduction in intensity/prevalence), and anti-PDI-Trans antibodies (66.22%/33.16% reduction in intensity/prevalence). To our knowledge, these results provide the first evidence that PDI function is essential for malarial transmission, and emphasize the potential of anti-PDI agents to act as anti-malarials, facilitating the future development of novel transmission-blocking interventions.

Effects of Azadirachta indica seed kernel extracts on early erythrocytic schizogony of Plasmodium berghei and pro-inflammatory response in inbred mice.

BACKGROUND: Medicinal plant research may contribute to develop new pharmacological control tools for vector borne diseases, such as malaria. METHODS: The effects of methanol extracts (ME) obtained from seed kernel of ripe and unripe Azadirachta indica fruits were studied on erythrocytic proliferation of the rodent malaria parasite Plasmodium berghei strain ANKA and on mice pro-inflammatory response, as evaluated by measuring the matrix-metalloproteinase-9 (MMP-9) and tumour necrosis factor (TNF) plasma levels, in two mouse strains (C57BL/6 and BALB/c) which are considered as prototypical of Th1 and Th2 immune response, respectively. RESULTS: ME obtained from seed kernel of unripe Azadirachta indica fruits decreased by about 30% the proportion of erythrocytes infected with the malaria parasite in C57BL/6 mice in the 4 days suppressive test. In this treatment group, MMP-9 and TNF levels were notably higher than those measured in the same mouse strain treated with the anti-malarial drug artesunate, Azadirachta indica kernel extracts from ripe fruits or solvent. In BALB/c mice, treatment with kernel extracts did not influence parasitaemia. MMP-9 and TNF levels measured in this mouse strain were notably lower than those recorded in C57BL/6 mice and did not vary among treatment groups. CONCLUSIONS: The effects of the ME on the parasite-host interactions appeared to be mouse strain-dependent, but also related to the ripening stage of the neem fruits, as only the unripe fruit seed kernel extracts displayed appreciable bioactivity.

Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity.

BACKGROUND: The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive. METHODS: The SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed. RESULTS: A detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector. CONCLUSION: A robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

Angeloylated Germacranolides from Daucus virgatus and Their Plasmodium Transmission Blocking Activity.

Phytochemical investigation of the aerial parts of the Tunisian plant Daucus virgatus led to the isolation of eight new germacranolides named daucovirgolides A-H (1-8). The stereostructures of these sesquiterpene lactones, decorated by either one or two angeloyl groups, have been determined by a combination of MS, NMR spectroscopy, chemical derivatization, and comparison of experimental electronic circular dichroism curves with TDDFT-predicted data. Daucovirgolide G (7) proved to be the single member of this family to possess a marked inhibitory activity (92% at 50 µg/mL) on the development of Plasmodium early sporogonic stages, the nonpathogenic transmissible stages of malaria parasites, devoid of general cytotoxicity. The selective activity of daucovirgolide G points to the existence of strict structural requirements for this transmission-blocking activity and therefore of a well-defined, although yet unidentified, biological target.

Novel metalloantimalarials: Transmission blocking effects of water soluble Cu(I), Ag(I) and Au(I) phosphane complexes on the murine malaria parasite Plasmodium berghei.

The water soluble phosphane complexes [M(L)4]PF6 (M=Cu(I), Ag(I)) and [Au(L)4]Cl (L=thp (tris(hydroxymethyl)phosphane) or PTA (1,3,5-triaza-7-phosphaadamantane)) showed notable in vitro activity against Plasmodium early sporogonic stages, the sexual forms of the malaria parasite that are responsible for infection of the mosquito vector. Effects varied according to both, the type of metal and phosphane ligands. [Ag(thp)4]PF6 was the best performing complex exhibiting a half inhibitory concentration (IC50) value in the low micromolar range (0.3-15.6µM). The silver complex [Ag(thp)4]PF6 was characterized by X-ray crystallography revealing that the structure comprises the cationic complex [Ag(thp)4]+, the PF6- anion, and a water molecule of crystallization. Our results revealed that Cu(I), Ag(I) and Au(I) phosphanes complexes elicited similar activity profiles showing potential for the development of antimalarial, transmission blocking compounds. Molecules targeting the sexual parasite stages in the human and/or mosquito host are urgently needed to complement current artemisinin based treatments and next generation antimalarials in a vision not only to cure the disease but to interrupt its transmission.

Transmission blocking effects of neem (Azadirachta indica) seed kernel limonoids on Plasmodium berghei early sporogonic development.

Azadirachta indica, known as neem tree and traditionally called "nature's drug store" makes part of several African pharmacopeias and is widely used for the preparation of homemade remedies and commercial preparations against various illnesses, including malaria. Employing a bio-guided fractionation approach, molecules obtained from A. indica ripe and green fruit kernels were tested for activity against early sporogonic stages of Plasmodium berghei, the parasite stages that develop in the mosquito mid gut after an infective blood meal. The limonoid deacetylnimbin (3) was identified as one the most active compounds of the extract, with a considerably higher activity compared to that of the close analogue nimbin (2). Pure deacetylnimbin (3) appeared to interfere with transmissible Plasmodium stages at a similar potency as azadirachtin A. Considering its higher thermal and chemical stability, deacetylnimbin could represent a suitable alternative to azadirachtin A for the preparation of transmission blocking antimalarials.