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 journey of the malaria sporozoite through its hosts: two parasite proteins lead the way

Malaria is transmitted to a mammalian host when the sporozoite stage of the Plasmodium parasite is injected by a mosquito vector. Sporozoites are unique in being able to interact with both hosts. Formed and released in the mosquito midgut, sporozoites bind to the salivary glands and invade their secretory cells. Once injected into the mammalian host, they home to the liver and invade hepatocytes. Recent work has shown that two sporozoite surface proteins, CS and TRAP, act in both hosts, perform multiple functions, and are each essential for the parasite at more than one step of its life cycle.     

Plasmodium sporozoite invasion into insect and mammalian cells is directed by the same dual binding system

Plasmodium sporozoites, the transmission form of the malaria parasite, successively invade salivary glands in the mosquito vector and the liver in the mammalian host. Sporozoite capacity to invade host cells is mechanistically related to their ability to glide on solid substrates, both activities depending on the transmembrane protein TRAP. Here, we show that loss-of- function mutations in two adhesive modules of the TRAP ectodomain, an integrin-like A-domain and a thrombospondin type I repeat, specifically decrease sporozoite invasion of host cells but do not affect sporozoite gliding and adhesion to cells. Irrespective of the target cell, i.e. in mosquitoes, rodents and cultured human or hamster cells, sporozoites bearing mutations in one module are less invasive, while those bearing mutations in both modules are non-invasive. In Chinese hamster ovary cells, the TRAP modules interact with distinct cell receptors during sporozoite invasion, and thus act as independently active pass keys. As these modules are also present in other members of the TRAP family of proteins in Apicomplexa, they may account for the capacity of these parasites to enter many cell types of phylogenetically distant origins.     

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.     

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.     

Functional characterization of a redundant Plasmodium TRAP family invasin, TRAP-like protein, by aldolase binding and a genetic complementation test

Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.     

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.     

Malaria sporozoite antigen‐directed genome‐wide response in transgenic Drosophila

Malaria kills a million people annually. Understanding the relationship between a causative parasite, Plasmodium falciparum, and the mosquito vector might suggest novel prevention approaches. We created and transformed into Drosophila two genes encoding, thrombospondin‐related adhesive protein (TRAP) and circumsporozoite protein (CSP), found on the cell surface of Plasmodium sporozoites. To understand a model insect's response, we induced these proteins separately and together, performing whole genome microarray analysis measuring gene expression changes. Gene ontology classification of responding genes reveals that TRAP and CSP strongly and differentially influence Drosophila genes involved with cell motility and gene regulation, respectively; however, the most striking effects are on the immune system. While immune‐related genes are but modestly elevated compared with responses to sepsis, there is a marked repression of the Toll pathway. This suggests: (1) how Plasmodium infection of the mosquito might use TRAP and CSP to modulate the host insect's physiology to promote sporozoite survival and transmission to man and (2) that approaches to elevate expression of the mosquito's Toll pathway might lead to novel methods of malaria prevention. genesis 47:196–203, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species

Plasmodium sporozoites can enter host cells by two distinct pathways, either through disruption of the plasma membrane followed by parasite transmigration through cells, or by formation of a parasitophorous vacuole (PV) where the parasite further differentiates into a replicative exo-erythrocytic form (EEF). We now provide evidence that following invasion without PV formation, transmigrating Plasmodium falciparum and Plasmodium yoelii sporozoites can partially develop into EEFs inside hepatocarcinoma cell nuclei. We also found that rodent P. yoelii sporozoites can infect both mouse and human hepatocytes, while human P. falciparum sporozoites infect human but not mouse hepatocytes. We have previously reported that the host tetraspanin CD81 is required for PV formation by P. falciparum and P. yoelii sporozoites. Here we show that expression of human CD81 in CD81-knockout mouse hepatocytes is sufficient to confer susceptibility to P. yoelii but not P. falciparum sporozoite infection, showing that the narrow P. falciparum host tropism does not rely on CD81 only. Also, expression of CD81 in a human hepatocarcinoma cell line is sufficient to promote the formation of a PV by P. yoelii but not P. falciparum sporozoites. These results highlight critical differences between P. yoelii and P. falciparum sporozoite infection, and suggest that in addition to CD81, other molecules are specifically required for PV formation during infection by the human malaria parasite.     

Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.     

Initiation of Plasmodium sporozoite motility by albumin is associated with induction of intracellular signalling

Malaria infection is initiated when a mosquito injects Plasmodium sporozoites into a mammalian host. Sporozoites exhibit gliding motility both in vitro and in vivo. This motility is associated with the secretion of at least two proteins, circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Both derive from micronemes, which are organelles that empty out of the apical end of the sporozoite. Sporozoite motility can be initiated in vitro by albumin added to the medium. To investigate how albumin functions in this process, we studied second messenger signalling within the sporozoite. Using pharmacological activators and inhibitors, we have concluded that gliding motility is initiated when albumin interacts with the surface of the sporozoite and that this leads to a signal transduction cascade within the sporozoite, including the elevation of intracellular cAMP, the modulation of sporozoite motility by Ca²⁺ and the release of microneme proteins.