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 vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

Plasmodium falciparum: release of circumsporozoite protein by sporozoites in the mosquito vector.

The release of circumsporozoite (CS) protein by Plasmodium falciparum sporozoites was investigated to identify factors regulating this process within infected Anopheles gambiae mosquitoes. The potential for sporozoites to release CS protein in vitro was not dependent upon their site-specific developmental stage (i.e., mature oocysts, hemolymph, salivary glands), their duration in the vector, or their exposure to mosquito-derived components such as salivary glands or hemolymph. The capacity of sporozoites to release CS protein was depressed by mosquito blood feeding during periods of sporozoite migration to the salivary glands, but the effect was only temporary and those sporozoites already in the glands were not affected. Free CS protein in the salivary glands was present in 93.3% of 45 infective mosquitoes. Sporozoites from these same, individual mosquitoes were also tested in vitro for CS protein release. In both cases, the amount of soluble CS protein increased as a function of sporozoite density but the total amount of CS protein per sporozoite became progressively less with increasing numbers of sporozoites. Further experiments showed that sporozoite contact with increasing amounts of soluble CS protein caused a down-regulation of CS protein release. Thus, a primary factor regulating the production and release of CS protein by sporozoites is their contact with soluble CS protein within the mosquito.     

Thrombospondin-related adhesive protein (TRAP) of Plasmodium falciparum: expression during sporozoite ontogeny and binding to human hepatocytes.

Plasmodium sporozoites collected from oocysts, haemocoel and salivary glands of the mosquito show profound differences in their biological properties such as motility, ability to induce protective immune response and infectivity for vertebrate host cells. Sporozoites from salivary glands are much more infectious than those from oocysts and haemocoel. Differential expression of proteins, such as the circumsporozoite (CS) protein and the thrombospondin-related adhesive protein (TRAP), implicated in sporozoite recognition and entry into hepatocytes may account for the development of infectivity during ontogeny. We have carried out a series of experiments to: (i) analyse the expression and localization of TRAP in P.falciparum sporozoites during development in the mosquito; and (ii) elucidate the biochemical and adhesive properties of recombinant TRAP. Our data indicate that TRAP is not expressed in oocysts, whereas variable amounts of CS protein are found in this parasite developmental stage. Hemocoel sporozoites display the distinct phenotypes TRAP- CS protein+ and TRAP+ CS protein+ at a frequency of 98.5 and 1.5% respectively. Salivary gland sporozoites are all TRAP+ CS protein+. We also provide experimental evidence showing that recombinant TRAP binds to the basolateral cell membrane of hepatocytes in the Disse's space and that sulfated glycoconjugates function as TRAP ligands on human hepatocytes.     

The parasite invasion marker SRPN6 reduces sporozoite numbers in salivary glands of Anopheles gambiae

For malaria transmission to occur, Plasmodium sporozoites must infect the salivary glands of their mosquito vectors. This study reports that Anopheles gambiae SRPN6 participates in a local salivary gland epithelial response against the rodent malaria parasite, Plasmodium berghei . We showed previously that SRPN6, an immune inducible midgut invasion marker, influences ookinete development. Here we report that SRPN6 is also specifically induced in salivary glands with the onset of sporozoite invasion. The protein is located in the basal region of epithelial cells in proximity to invading sporozoites. Knockdown of SRPN6 during the late phase of sporogony by RNAi has no effect on oocyst rupture but significantly increases the number of sporozoites present in salivary glands. Despite several differences between the passage of Plasmodium through the midgut and the salivary glands, this study identifies a striking overlap in the molecular responses of these two epithelia to parasite invasion.     

A role for heparan sulfate proteoglycans in Plasmodium falciparum sporozoite invasion of anopheline mosquito salivary glands

HS (heparan sulfate) has been shown to be an important mediator of Plasmodium sporozoite homing and invasion of the liver, but the role of this glycosaminoglycan in mosquito vector host-sporozoite interactions is unknown. We have biochemically characterized the function of AgOXT1 (Anopheles gambiae peptide-O-xylosyltransferase 1) and confirmed that AgOXT1 can modify peptides representing model HS and chondroitin sulfate proteoglycans in vitro. Moreover, we also demonstrated that the mosquito salivary gland basal lamina proteoglycans are modified by HS. We used RNA interference-mediated knockdown of HS biosynthesis in A. gambiae salivary glands to determine whether Plasmodium falciparum sporozoites that are released from mosquito midgut oocysts use salivary gland HS as a receptor for tissue invasion. Our results suggest that salivary gland basal lamina HS glycosaminoglycans only partially mediate midgut sporozoite invasion of this tissue, and that in the absence of HS, the presence of other surface co-receptors is sufficient to facilitate parasite entry.     

The transmembrane isoform of Plasmodium falciparum MAEBL is essential for the invasion of Anopheles salivary glands

Malaria transmission depends on infective stages in the mosquito salivary glands. Plasmodium sporozoites that mature in midgut oocysts must traverse the hemocoel and invade the mosquito salivary glands in a process thought to be mediated by parasite ligands. MAEBL, a homologue of the transmembrane EBP ligands essential in merozoite invasion, is expressed abundantly in midgut sporozoites. Alternative splicing generates different MAEBL isoforms and so it is unclear what form is functionally essential. To identify the MAEBL isoform required for P. falciparum (NF54) sporozoite invasion of salivary glands, we created knockout and allelic replacements each carrying CDS of a single MAEBL isoform. Only the transmembrane form of MAEBL is essential and is the first P. falciparum ligand validated as essential for invasion of Anopheles salivary glands. MAEBL is the first P. falciparum ligand experimentally determined to be essential for this important step in the life cycle where the vector becomes infectious for transmitting sporozoites to people. With an increasing emphasis on advancing vector-based transgenic methods for suppression of malaria, it is important that this type of study, using modern molecular genetic tools, is done with the agent of the human disease. Understanding what P. falciparum sporozoite ligands are critical for mosquito transmission will help validate targets for vector-based transmission-blocking strategies.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.     

Molecular mechanisms of malaria sporozoite motility and invasion of host cells.

Malaria sporozoites have the unique capacity to invade two entirely different types of target cell in the mosquito vector and the vertebrate host during the course of the parasite's life cycle. Although little is known about the specific interaction of the sporozoite with its target cells, two sporozoite proteins, circumsporozoite (CS) and thrombospondin-related adhesive protein (TRAP), have been shown to play important roles in the invasion of both cell types. CS protein is a multifunctional protein involved in sporogony, invasion of the salivary glands, the specific arrest of sporozoites in the liver sinusoid, gliding motility of the sporozoite, and hepatocyte recognition and entry. TRAP has been shown to be critical for sporozoite infection of the mosquito salivary glands and liver cells, and is essential for sporozoite gliding motility. This review will focus on the involvement of these molecules in sporozoite motility and the invasion of host cells.     

Quantitation of malaria sporozoites transmitted in vitro during salivation by wild Afrotropical Anopheles

Abstract. The malaria transmission potential of wild, infective Anopheles from western Kenya was evaluated by determining the number of sporozoites transmitted in vitro by salivation when their mouthparts were inserted into capillary tubes containing either sucrose or blood. With sucrose, 86.6% of 102 infective Anopheles transmitted a geometric mean (GM) of 3.84 sporozoites (range 1–34). With blood, 23.1% of 104 infective Anopheles , tested on the day of collection, transmitted a GM of 2.30 sporozoites (range 1–117). For Anopheles held 5 days postcapture before testing with blood, 53.6% of 56 transmitted a GM of 6.04 sporozoites (range 1–420). Transmitting Anopheles contained significantly more salivary gland sporozoites than non-transmitters. No significant differences were detected between Anopheles gambiae Giles sensu lato and Anopheles funestus Giles in sporozoite transmission by individuals with sporozoites in their salivary glands.
Sporozoites were detected microscopically in the salivary duct from heads in 80.3% of 117 infective Anopheles (GM=11.2, range 1–71). Sporozoite detection in mosquito heads by ELISA was 25% less efficient than microscopic detection.
Over 98% of the infective Anopheles transmitted less than twenty-five sporozoites. Transmitted sporozoites represented only about 3% of the total sporozoites in the salivary glands suggesting that sporozoite transmission may be restricted to sporozoites in the salivary duct at the time of feeding. Results are discussed in relation to anti-sporozoite vaccine development.     

Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes

Previous studies have shown that mosquitoes inject Plasmodium sporozoites into avascular portions of the skin of their rodent host rather than directly into the blood circulation. Then, over time, these sporozoites move into the circulation, from where they reach the liver to initiate a malaria infection. By use of intravital microscopy of the skin, we present direct morphological evidence of mosquito probing that introduces sporozoites into avascular tissue, of the migration of these sporozoites through the dermis and into blood vessels, and of the role of anti-sporozoite antibodies in blocking sporozoite invasion of these dermal blood vessels.     

The Journey of Malaria Sporozoites in the Mosquito Salivary Gland

The life cycle of malaria parasites in the mosquito vector is completed when the sporozoites infect the salivary gland and are ready to be injected into the vertebrate host. This paper describes the fine structure of the invasive process of mosquito salivary glands by malaria parasites. Plasmodium gallinaceum sporozoites start the invasion process by attaching to and crossing the basal lamina and then penetrating the host plasma membrane of the salivary cells. The penetration process appears to involve the formation of membrane junctions. Once inside the host cells, the sporozoites are seen within vacuoles attached by their anterior end to the vacuolar membrane. Mitochondria surround, and are closely associated with, the invading sporozoites. After the disruption of the membrane vacuole, the parasites traverse the cytoplasm, attach to, and invade the secretory cavity through the apical plasma membrane of the cells. Inside the secretory cavity, sporozoites are seen again inside vacuoles. Upon escaping from these vacuoles, sporozoites are positioned in parallel arrays forming large bundles attached by multilammelar membrane junctions. Several sporozoites are seen around and inside the secretory duct. Except for the penetration of the chitinous salivary duct, our observations have morphologically characterized the entire process of sporozoite passage through the salivary gland.     

A mosquito-specific protein family includes candidate receptors for malaria sporozoite invasion of salivary glands

We describe a previously unrecognized protein family from Aedes and Anopheles mosquitoes, here named SGS proteins. There are no SGS homologues in Drosophila or other eukaryotes, but SGS presence in two mosquito genera suggests that the protein family is widespread among mosquitoes. Ae. aegypti aaSGS1 mRNA and protein are salivary gland specific, and protein is localized in the basal lamina covering the anatomical regions that are preferentially invaded by malaria sporozoites. Anti-aaSGS1 antibodies inhibited sporozoite invasion into the salivary glands in vivo, confirming aaSGS1 as a candidate sporozoite receptor. By homology to aaSGS1 we identified the complete complement of four SGS genes in An. gambiae, which were not recognized in the genome annotation. Two An. gambiae SGS genes display salivary gland specific expression like aaSGS1. Bioinformatic analysis predicts that SGS proteins possess heparin-binding domains, and have among the highest density of tyrosine sulphation sites of all An. gambiae proteins. The major sporozoite surface proteins (CS and TRAP) also bind heparin, and interact with sulphoconjugates during liver cell invasion. Thus, we speculate that sporozoite invasion of mosquito salivary glands and subsequently the vertebrate liver may share similar mechanisms based on sulphation. Phylogenomic analysis suggests that an SGS ancestor was involved in a lateral gene transfer.     

Function of region I and II adhesive motifs of Plasmodium falciparum circumsporozoite protein in sporozoite motility and infectivity

The circumsporozoite protein of Plasmodium falciparum contains two conserved motifs (regions I and II) that have been proposed to interact with mosquito and vertebrate host molecules in the process of sporozoite invasion of salivary glands and hepatocytes, respectively. To study the function of this protein we have replaced the endogenous circumsporozoite protein gene of Plasmodium berghei with that of P. falciparum and with versions lacking either region I or region II. We show here that P. falciparum circumsporozoite protein functions in rodent parasite and that P. berghei sporozoites carrying the P. falciparum CS gene develop normally, are motile, invade mosquito salivary glands, and infect the vertebrate host. Region I-deficient sporozoites showed no impairment of motility or infectivity in either vector or vertebrate host. Disruption of region II abolished sporozoite motility and dramatically impaired their ability to invade mosquito salivary glands and infect the vertebrate host. These data shed new light on the role of the CS protein in sporozoite motility and infectivity.     

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.     

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).     

The A-domain and the thrombospondin-related motif of Plasmodium falciparum TRAP are implicated in the invasion process of mosquito salivary glands.

Sporozoites from all Plasmodium species analysed so far express the thrombospondin-related adhesive protein (TRAP), which contains two distinct adhesive domains. These domains share sequence and structural homology with von Willebrand factor type A-domain and the type I repeat of human thrombospondin (TSP). Increasing experimental evidence indicates that the adhesive domains bind to vertebrate host ligands and that TRAP is involved, through an as yet unknown mechanism, in the process of sporozoite motility and invasion of both mosquito salivary gland and host hepatocytes. We have generated transgenic P.berghei parasites in which the endogenous TRAP gene has been replaced by either P.falciparum TRAP (PfTRAP) or mutated versions of PfTRAP carrying amino acid substitutions or deletions in the adhesive domains. Plasmodium berghei sporozoites carrying the PfTRAP gene develop normally, are motile, invade mosquito salivary glands and infect the vertebrate host. A substitution in a conserved residue of the A-domain or a deletion in the TSP motif of PfTRAP impairs the sporozoites' ability to invade mosquito salivary glands. Notably, midgut sporozoites from these transgenic parasites are still able to infect mice. Midgut sporozoites carrying a mutation in the A-domain of PfTRAP are motile, while no gliding motility could be detected in sporozoites with a TSP motif deletion.     

Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes

Background

The population dynamics of Plasmodium sporogony within mosquitoes consists of an early phase where parasite abundance decreases during the transition from gametocyte to oocyst, an intermediate phase where parasite abundance remains static as oocysts, and a later phase where parasite abundance increases during the release of progeny sporozoites from oocysts. Sporogonic development is complete when sporozoites invade the mosquito salivary glands. The dynamics and efficiency of this developmental sequence were determined in laboratory strains of Anopheles dirus, Anopheles minimus and Anopheles sawadwongporni mosquitoes for Plasmodium vivax parasites circulating naturally in western Thailand.

Methods

Mosquitoes were fed blood from 20 symptomatic Thai adults via membrane feeders. Absolute densities were estimated for macrogametocytes, round stages (= female gametes/zygotes), ookinetes, oocysts, haemolymph sporozoites and salivary gland sporozoites. From these census data, five aspects of population dynamics were analysed; 1) changes in life-stage prevalence during early sporogony, 2) kinetics of life-stage formation, 3) efficiency of life-stage transitions, 4) density relationships between successive life-stages, and 5) parasite aggregation patterns.

Results

There was no difference among the three mosquito species tested in total losses incurred by P. vivax populations during early sporogony. Averaged across all infections, parasite populations incurred a 68-fold loss in abundance, with losses of ca. 19-fold, 2-fold and 2-fold at the first (= gametogenesis/fertilization), second (= round stage transformation), and third (= ookinete migration) life-stage transitions, respectively. However, total losses varied widely among infections, ranging from 6-fold to over 2,000-fold loss. Losses during gametogenesis/fertilization accounted for most of this variability, indicating that gametocytes originating from some volunteers were more fertile than those from other volunteers. Although reasons for such variability were not determined, gametocyte fertility was not correlated with blood haematocrit, asexual parasitaemia, gametocyte density or gametocyte sex ratio. Round stages and ookinetes were present in mosquito midguts for up to 48 hours and development was asynchronous. Parasite losses during fertilization and round stage differentiation were more influenced by factors intrinsic to the parasite and/or factors in the blood, whereas ookinete losses were more strongly influenced by mosquito factors. Oocysts released sporozoites on days 12 to 14, but even by day 22 many oocysts were still present on the midgut. The per capita production was estimated to be approximately 500 sporozoites per oocyst and approximately 75% of the sporozoites released into the haemocoel successfully invaded the salivary glands.

Conclusion

The major developmental bottleneck in early sporogony occurred during the transition from macrogametocyte to round stage. Sporozoite invasion into the salivary glands was very efficient. Information on the natural population dynamics of sporogony within malaria-endemic areas may benefit intervention strategies that target early sporogony (e.g., transmission blocking vaccines, transgenic mosquitoes).