Let it glow: genetically encoded fluorescent reporters in Plasmodium.

The use of fluorescent proteins (FPs) in Plasmodium parasites has been key to understand the biology of this obligate intracellular protozoon. FPs like the green fluorescent protein (GFP) enabled to explore protein localization, promoter activity as well as dynamic processes like protein export and endocytosis. Furthermore, FP biosensors have provided detailed information on physiological parameters at the subcellular level, and fluorescent reporter lines greatly extended the malariology toolbox. Still, in order to achieve optimal results, it is crucial to know exactly the properties of the FP of choice and the genetic scenario in which it will be used. This review highlights advantages and disadvantages of available landing sites and promoters that have been successfully applied for the ectopic expression of FPs in Plasmodium berghei and Plasmodium falciparum. Furthermore, the properties of newly developed FPs beyond DsRed and EGFP, in the visualization of cells and cellular structures as well as in the sensing of small molecules are discussed.

Proteomic Profiling of Antimalarial Plasmodione Using 3-Benz(o)ylmenadione Affinity-Based Probes.

Understanding the mechanisms of drug action in malarial parasites is crucial for the development of new drugs to combat infection and to counteract drug resistance. Proteomics is a widely used approach to study host-pathogen systems and to identify drug protein targets. Plasmodione is an antiplasmodial early-lead drug exerting potent activities against young asexual and sexual blood stages in vitro with low toxicity to host cells. To elucidate its molecular mechanisms, an affinity-based protein profiling (AfBPP) approach was applied to yeast and P. falciparum proteomes. New (pro-) AfBPP probes based on the 3-benz(o)yl-6-fluoro-menadione scaffold were synthesized. With optimized conditions of both photoaffinity labeling and click reaction steps, the AfBPP protocol was then applied to a yeast proteome, yielding 11 putative drug-protein targets. Among these, we found four proteins associated with oxidoreductase activities, the hypothesized type of targets for plasmodione and its metabolites, and other proteins associated with the mitochondria. In Plasmodium parasites, the MS analysis revealed 44 potential plasmodione targets that need to be validated in further studies. Finally, the localization of a 3-benzyl-6-fluoromenadione AfBPP probe was studied in the subcellular structures of the parasite at the trophozoite stage.

The salivary protein Saglin facilitates efficient midgut colonization of Anopheles mosquitoes by malaria parasites.

Malaria is caused by the unicellular parasite Plasmodium which is transmitted to humans through the bite of infected female Anopheles mosquitoes. To initiate sexual reproduction and to infect the midgut of the mosquito, Plasmodium gametocytes are able to recognize the intestinal environment after being ingested during blood feeding. A shift in temperature, pH change and the presence of the insect-specific compound xanthurenic acid have been shown to be important stimuli perceived by gametocytes to become activated and proceed to sexual reproduction. Here we report that the salivary protein Saglin, previously proposed to be a receptor for the recognition of salivary glands by sporozoites, facilitates Plasmodium colonization of the mosquito midgut, but does not contribute to salivary gland invasion. In mosquito mutants lacking Saglin, Plasmodium infection of Anopheles females is reduced, resulting in impaired transmission of sporozoites at low infection densities. Interestingly, Saglin can be detected in high amounts in the midgut of mosquitoes after blood ingestion, possibly indicating a previously unknown host-pathogen interaction between Saglin and midgut stages of Plasmodium. Furthermore, we were able to show that saglin deletion has no fitness cost in laboratory conditions, suggesting this gene would be an interesting target for gene drive approaches.

Activation of complement-like antiparasitic responses in Anopheles mosquitoes.

During their development in mosquitoes, malaria parasites undergo massive losses that are in part due to a potent antiparasitic response mounted by the vector. The most efficient and best-characterized response relies on a complement-like system particularly effective against parasites as they cross the mosquito midgut epithelium. While our vision of the molecular and cellular events that lead to parasite elimination is still partial, our understanding of the steps triggering complement activation at the surface of invading parasites has considerably progressed, not only through the identification of novel contributing genes, but also with the recent in-depth characterization of the different mosquito blood cell types, and the ability to track them in live mosquitoes. Here, we propose a simple model based on the time of invasion to explain how parasites may escape complement-like responses during midgut infection.

A toolbox of engineered mosquito lines to study salivary gland biology and malaria transmission.

Mosquito saliva is a vehicle for the transmission of vector borne pathogens such as Plasmodium parasites and different arboviruses. Despite the key role of the salivary glands in the process of disease transmission, knowledge of host-pathogen interactions taking place within this organ is very limited. To improve the experimental tractability of the salivary glands, we have generated fluorescent reporter lines in the African malaria mosquito Anopheles coluzzii using the salivary gland-specific promoters of the anopheline antiplatelet protein (AAPP), the triple functional domain protein (TRIO) and saglin (SAG) coding genes. Promoter activity was specifically observed in the distal-lateral lobes or in the median lobe of the salivary glands. Besides a comparison of the expression patterns of the selected promoters, the fluorescent probes allowed us to evaluate the inducibility of the selected promoters upon blood feeding and to measure intracellular redox changes. We also combined the aapp-DsRed fluorescent reporter line with a pigmentation-deficient yellow(-) mosquito mutant to assess the feasibility of in vivo microscopy of parasitized salivary glands. This combination allowed locating the salivary gland through the cuticle and imaging of individual sporozoites in vivo, which facilitates live imaging studies of salivary gland colonization by Plasmodium sporozoites.

Synthesis of plasmodione metabolites and 13C-enriched plasmodione as chemical tools for drug metabolism investigation.

Malaria is a tropical parasitic disease threatening populations in tropical and sub-tropical areas. Resistance to antimalarial drugs has spread all over the world in the past 50 years, thus new drugs are urgently needed. Plasmodione (benzylmenadione series) has been identified as a potent antimalarial early lead drug, acting through a redox bioactivation on asexual and young sexual blood stages. To investigate its metabolism, a series of plasmodione-based tools, including a fully 13C-labelled lead drug and putative metabolites, have been designed and synthesized for drug metabolism investigation. Furthermore, with the help of UHPLC-MS/MS, two of the drug metabolites have been identified from urine of drug-treated mice.

Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae.

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites.

Tools for Anopheles gambiae Transgenesis.

Transgenesis is an essential tool to investigate gene function and to introduce desired characters in laboratory organisms. Setting-up transgenesis in non-model organisms is challenging due to the diversity of biological life traits and due to knowledge gaps in genomic information. Some procedures will be broadly applicable to many organisms, and others have to be specifically developed for the target species. Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects. Here, we report a compilation of the transgenesis tools that we have designed for the malaria vector Anopheles gambiae, including new docking strains, convenient transgenesis plasmids, a puromycin resistance selection marker, mosquitoes expressing cre recombinase, and various reporter lines defining the activity of cloned promoters. This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis. Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects.

Mosquito genomics. Highly evolvable malaria vectors: the genomes of 16 Anopheles mosquitoes.

Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution. Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila. Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts.

Silencing of genes and alleles by RNAi in Anopheles gambiae.

Anopheles gambiae mosquitoes are the major vectors of human malaria parasites. However, mosquitoes are not passive hosts for parasites, actively limiting their development in vivo. Our current understanding of the mosquito antiparasitic response is mostly based on the phenotypic analysis of gene knockdowns obtained by RNA interference (RNAi), through the injection or transfection of long dsRNAs in adult mosquitoes or cultured cells, respectively. Recently, RNAi has been extended to silence specifically one allele of a given gene in a heterozygous context, thus allowing to compare the contribution of different alleles to a phenotype in the same genetic background.

Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae.

The ability of Anopheles gambiae mosquitoes to transmit Plasmodium parasites is highly variable between individuals. However, the genetic basis of this variability has remained unknown. We combined genome-wide mapping and reciprocal allele-specific RNA interference (rasRNAi) to identify the genomic locus that confers resistance to malaria parasites and demonstrated that polymorphisms in a single gene encoding the antiparasitic thioester-containing protein 1 (TEP1) explain a substantial part of the variability in parasite killing. The link between TEP1 alleles and resistance to malaria may offer new tools for controlling malaria transmission. The successful application of rasRNAi in Anopheles suggests that it could also be applied to other organisms where RNAi is feasible to dissect complex phenotypes to the level of individual quantitative trait alleles.

Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium.

Plasmodium development within Anopheles mosquitoes is a vulnerable step in the parasite transmission cycle, and targeting this step represents a promising strategy for malaria control. The thioester-containing complement-like protein TEP1 and two leucine-rich repeat (LRR) proteins, LRIM1 and APL1, have been identified as major mosquito factors that regulate parasite loads. Here, we show that LRIM1 and APL1 are required for binding of TEP1 to parasites. RNAi silencing of the LRR-encoding genes results in deposition of TEP1 on Anopheles tissues, thereby depleting TEP1 from circulation in the hemolymph and impeding its binding to Plasmodium. LRIM1 and APL1 not only stabilize circulating TEP1, they also stabilize each other prior to their interaction with TEP1. Our results indicate that three major antiparasitic factors in mosquitoes jointly function as a complement-like system in parasite killing, and they reveal a role for LRR proteins as complement control factors.

Antimalarial responses in Anopheles gambiae: from a complement-like protein to a complement-like pathway.

Malaria transmission between humans depends on the ability of Anopheles mosquitoes to support Plasmodium development. New perspectives in vector control are emerging from understanding the mosquito immune system, which plays critical roles in parasite recognition and killing. A number of factors controlling this process have been recently identified, and key among them is TEP1, a homolog of human complement factor C3 whose binding to the parasite surface targets it for subsequent killing. Here, we review our current knowledge of mosquito factors that respond to Plasmodium infection and elaborate on the activity and mode of action of the TEP1 complement-like pathway.

Reverse genetics analysis of antiparasitic responses in the malaria vector, Anopheles gambiae.

Anopheles mosquitoes are the major vectors of human malaria parasites. Mosquito-parasite interactions are critical for disease transmission and therefore represent a potential target for malaria control strategies. Mosquitoes mount potent antiparasitic responses, and identification of mosquito factors that limit parasite development is one of the major objectives in the field. To address this question, we have developed a convenient reverse genetics approach by injection of double-stranded RNA (dsRNA) in adult mosquitoes, to evaluate the function of candidate genes in mosquito antiparasitic responses.

Phagocytosis in mosquito immune responses.

Anopheles mosquitoes are the only vectors of human malaria parasites. Mosquito-parasite interactions are critical for disease transmission and therefore are a potential target for malaria control strategies. Mosquitoes mount potent immune responses that efficiently limit proliferation of a variety of infectious agents, including microbial pathogens and malaria parasites. The recent completion of the Anopheles gambiae genome sequencing project combined with the development of the powerful RNA interference-based gene silencing helped to identify major players of the immune defenses and uncovered evolutionarily conserved mechanisms in the anti-bacterial and anti-Plasmodium responses. The anti-bacterial responses are based on phagocytosis at early steps of infections, followed, several hours later, by the synthesis of anti-microbial peptides. The principal regulators of anti-parasitic responses are predominantly synthesized by the mosquito blood cells; however, the exact molecular mechanisms of parasite killing remain unclear. Several regulators of phagocytosis are also required for efficient parasite killing. Here, we summarize our current knowledge of the anti-bacterial and anti-parasitic responses, with the particular emphasis on the role of phagocytosis in mosquito immunity.