CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts

The malarial parasite has two hosts in its life cycle, a vertebrate and a mosquito. We report here that malarial invasion into these hosts is mediated by a protein, designated cell-traversal protein for ookinetes and sporozoites (CelTOS), which is localized to micronemes that are organelles for parasite invasive motility. Targeted disruption of the CelTOS gene in Plasmodium berghei reduced parasite infectivity in the mosquito host approximately 200-fold. The disruption also reduced the sporozoite infectivity in the liver and almost abolished its cell-passage ability. Liver infectivity was restored in Kupffer cell-depleted rats, indicating that CelTOS is necessary for sporozoite passage from the circulatory system to hepatocytes through the liver sinusoidal cell layer. Electron microscopic analysis revealed that celtos-disrupted ookinetes invade the midgut epithelial cell by rupturing the cell membrane, but then fail to cross the cell, indicating that CelTOS is necessary for migration through the cytoplasm. These results suggest that conserved cell-passage mechanisms are used by both sporozoites and ookinetes to breach host cellular barriers. Elucidation of these mechanisms might lead to novel antimalarial strategies to block parasite's transmission.     

Motility and infectivity of Plasmodium berghei sporozoites expressing avian Plasmodium gallinaceum circumsporozoite protein

Avian and rodent malaria sporozoites selectively invade different vertebrate cell types, namely macrophages and hepatocytes, and develop in distantly related vector species. To investigate the role of the circumsporozoite (CS) protein in determining parasite survival in different vector species and vertebrate host cell types, we replaced the endogenous CS protein gene of the rodent malaria parasite Plasmodium berghei with that of the avian parasite P. gallinaceum and control rodent parasite P. yoelii. In anopheline mosquitoes, P. berghei parasites carrying P. gallinaceum and rodent parasite P. yoelii CS protein gene developed into oocysts and sporozoites. Plasmodium gallinaceum CS expressing transgenic sporozoites, although motile, failed to invade mosquito salivary glands and to infect mice, which suggests that motility alone is not sufficient for invasion. Notably, a percentage of infected Anopheles stephensi mosquitoes showed melanotic encapsulation of late stage oocysts. This was not observed in control infections or in A. gambiae infections. These findings shed new light on the role of the CS protein in the interaction of the parasite with both the mosquito vector and the rodent host.     

The complex interplay between mosquito positive and negative regulators of Plasmodium development

The malaria parasite, Plasmodium, requires sexual development in the mosquito before it can be transmitted to the vertebrate host. Mosquito genes are able to substantially modulate this process, which can result in major decreases in parasite numbers. Even in susceptible mosquitoes, haemolymph proteins implicated in systemic immune reactions, together with local epithelial responses, cause lysis of more than 80% of the ookinetes that cross the mosquito midgut. In a refractory mosquito strain, immune responses lead to melanisation of virtually all parasites. Conversely, certain mosquito genes have an opposite effect: they are used by the parasite to evade defence reactions. Detailed understanding of the interplay between positive and negative regulators of parasite development could lead to the generation of novel approaches for malaria control through the vector.     

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.     

Plasmodium yoelii: axenic development of the parasite mosquito stages

Study of the parasite mosquito stages of Plasmodium and its use in the production of sporozoite vaccines against malaria has been hampered by the technical difficulties of in vitro development. Here, we show the complete axenic development of the parasite mosquito stages of Plasmodium yoelii. While we demonstrate that matrigel is not required for parasite development, soluble factors produced and secreted by Drosophila melanogaster S2 cells appear to be crucial for the ookinete to oocyst transition. Parasites cultured axenically are both morphologically and biologically similar to mosquito-derived ookinetes, oocysts, and sporozoites. Axenically derived sporozoites were capable of producing an infection in mice as determined by RT-PCR; however, the parasitemia was significantly much less than that produced by mosquito-derived sporozoites. Our cell free system for development of the mosquito stages of P. yoelii provides a simplified approach to generate sporozoites that may be for biological assays and genetic manipulations.     

Rhoptry neck protein 11 has crucial roles during malaria parasite sporozoite invasion of salivary glands and hepatocytes

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells – mosquito salivary glands and mammalian hepatocytes – via involvement in sporozoite motility.     

PbSR is synthesized in macrogametocytes and involved in formation of the malaria crystalloids

Crystalloids are transient organelles that form in developing malaria ookinetes and disappear after ookinete-to-oocyst transition. Their origins and functions remain poorly understood. The Plasmodium berghei scavenger receptor-like protein PbSR is essential for mosquito-to-host transmission of the parasite: PbSR knockout parasites produce normal numbers of oocysts that fail to form sporozoites, pointing to a role for PbSR in the oocyst during sporogony. Here, using fluorescent protein tagging and targeted gene disruption, we show that PbSR is synthesized in macrogametocytes, gets targeted to the crystalloids of developing ookinetes and is involved in crystalloid formation. While oocyst sporulation rates of PbSR knockout parasites are highly reduced in parasite-infected mosquitoes, sporulation rates in vitro are not adversely affected, supporting the view that mosquito factors could be involved in the PbSR loss-of-function phenotype. These findings are the first to identify a parasite protein involved with the crystalloid organelle, and suggest a novel protein-trafficking mechanism to deliver PbSR to the oocysts.     

Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host

The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes).     

Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.     

Getting infectious: formation and maturation of Plasmodium sporozoites in the Anopheles vector

Research on Plasmodium sporozoite biology aims at understanding the developmental program steering the formation of mature infectious sporozoites - the transmission stage of the malaria parasite. The recent identification of genes that are vital for sporozoite egress from oocysts and subsequent targeting and transmigration of the mosquito salivary glands allows the identification of mosquito factors required for life cycle completion. Mature sporozoites appear to be equipped with the entire molecular repertoire for successful transmission and subsequent initiation of liver stage development. Innovative malaria intervention strategies that target the early, non-pathogenic phases of the life cycle will crucially depend on our insights into sporozoite biology and the underlying molecular mechanisms that lead the parasite from the mosquito midgut to the liver.     

Plasmodium falciparum gametocytes: still many secrets of a hidden life

Sexual differentiation and parasite transmission are intimately linked in the life cycle of malaria parasites. The specialized cells providing this crucial link are the Plasmodium gametocytes. These are formed in the vertebrate host and are programmed to mature into gametes emerging from the erythrocytes in the midgut of a blood-feeding mosquito. The ensuing fusion into a zygote establishes parasite infection in the insect vector. Although key mechanisms of gametogenesis and fertilization are becoming progressively clear, the fundamental biology of gametocyte formation still presents open questions, some of which are specific to the human malaria parasite Plasmodium falciparum. Developmental commitment to sexual differentiation, regulation of stage-specific gene expression, the profound molecular and cellular changes accompanying gametocyte specialization, the requirement for tissue-specific sequestration in P. falciparum gametocytogenesis are proposed here as areas for future investigation. The epidemiological relevance of parasite transmission from humans to mosquito in the spread of malaria and of Plasmodium drug resistance genes indicates that understanding molecular mechanisms of gametocyte formation is highly relevant to design strategies able to interfere with the transmission of this disease.     

The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum,and their transmission by Anopheles stephensi,Anopheles gambiae and Aedes aegypti

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.     

Plasmodium yoelii: semiquantitative analyses of circumsporozoite protein gene expression during the sporogonic development of P. y. yoelii and P. y. nigeriensis in the mosquito vector Anopheles stephensi

Malaria infection in the mosquito vector can be modulated by the vertebrate host, mosquito factors, and interactions between different parasite populations. Modulation of parasite development can be assessed through the study of gene expression. The present report describes a specific, sensitive, and nonradioactive method that permits assessment of parasite load and quantification of circumsporozoite protein gene expression during the sporogonic stages of Plasmodium yoelii yoelii and P. y. nigeriensis. A decrease in parasite load was observed when comparing DNA of oocysts on day 7 postinfection with that of oocysts and sporozoites on day 19. On day 7, parasites (oocysts) showed a marked increase of circumsporozoite protein expression when compared with that (sporozoites and oocysts) on day 19. The method developed in this work can be a valuable tool to understand parasite interaction mechanisms that are involved in mosquito malaria infections.     

Reverse genetics screen identifies six proteins important for malaria development in the mosquito

Transmission from the vertebrate host to the mosquito vector represents a major population bottleneck in the malaria life cycle that can successfully be targeted by intervention strategies. However, to date only about 25 parasite proteins expressed during this critical phase have been functionally analysed by gene disruption. We describe the first systematic, larger scale generation and phenotypic analysis of Plasmodium berghei knockout (KO) lines, characterizing 20 genes encoding putatively secreted proteins expressed by the ookinete, the parasite stage responsible for invasion of the mosquito midgut. Of 12 KO lines that were generated, six showed significant reductions in parasite numbers during development in the mosquito, resulting in a block in transmission of five KOs. While expression data, time point of essential function and mutant phenotype correlate well in three KOs defective in midgut invasion, in three KOs that fail at sporulation, maternal inheritance of the mutant phenotype suggests that essential function occurs during ookinete formation and thus precedes morphological abnormalities by several days.     

In vivo imaging of malaria parasites--recent advances and future directions

A new view into the life of malaria parasites is now possible owing to recent advances in imaging techniques and to the generation of tagged parasites. Insights into how parasites interact with their insect vectors and mammalian hosts have been gained by the study of various parasitic forms in their natural environment. Quantitative analysis of Plasmodium ookinete motility has revealed different modes of motility in parasite invasion of the mosquito gut and the extrusion of invaded gut cells from the epithelium. Similar analysis with Plasmodium sporozoites has revealed the importance of parasite motility in transmission from the mosquito vector to the mammalian host.