Malaria sporozoites release circumsporozoite protein from their apical end and translocate it along their surface.

Plasmodium sporozoites, the causative agents of malaria, release circumsporozoite (CS) protein into medium when under conditions simulating those that the parasites encounter in the bloodstream of the vertebrate host. CS protein of the rodent parasite, Plasmodium berghei, is released as the lower molecular weight form, Pb44. This release is substratum- and antibody-independent. Previous studies show that CS protein is released at the trailing, posterior end of motile sporozoites. Video and electron microscopic studies now demonstrate that CS protein is released at the apical end of cytochalasin b-immobilized sporozoites. We propose that CS protein released from the apical end, the leading end of gliding sporozoites, adheres to the sporozoite surface and is translocated posteriorly by a cytochalasin-sensitive and apparently actin-mediated surface motor, which drives gliding motility. This model explains the mechanism of both the circumsporozoite precipitation (CSP) reaction and formation of the CS protein trail by gliding sporozoites.     

Malaria sporozoites leave behind trails of circumsporozoite protein during gliding motility

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveness did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS protein trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.     

Malaria Sporozoites Leave Behind Trails of Circumsporozoite Protein During Gliding Motility1

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveaess did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS prolem trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.     

Electron Microscopic Analysis of Circumsporozoite Protein Trail Formation by Gliding Malaria Sporozoites

Immunoelectron microscopic techniques were utilized to characterize the morphology of circumsporozoite protein-containing trails deposited on various substrates by gliding Plasmodium berghei and Plasmodium falciparum sporozoites. The basic components of the trails are beadlike particles, 25 to 90 nm in diameter, which are devoid of unit membrane and have an electronlucent center. Trails were captured on formvar-covered grids coated with anticircumsporozoite protein monoclonal antibodies and compared with trails produced on uncoated formvar; the results suggest that material containing circumsporozoite protein forms the matrix within which the particles are embedded. The trails exhibit morphological features similar to those displayed by circumsporozoite precipitation reactions; of note is the demonstration of sheaths of circumsporozoite protein-containing material that emanate from sporozoites prior to their gliding. The sheaths narrow into accumulations of electron-dense material, which eventually taper to form typical trails. The structural manifestation of sheaths and other morphological details of the formed trails enables us to correlate sporozoite behavior during trail formation with the motile actions of gliding sporozoites observed by light microscopy.     

Electron microscopic analysis of circumsporozoite protein trail formation by gliding malaria sporozoites.

Immunoelectron microscopic techniques were utilized to characterize the morphology of circumsporozoite protein-containing trails deposited on various substrates by gliding Plasmodium berghei and Plasmodium falciparum sporozoites. The basic components of the trails are beadlike particles, 25 to 90 nm in diameter, which are devoid of unit membrane and have an electron-lucent center. Trails were captured on formvar-covered grids coated with anticircumsporozoite protein monoclonal antibodies and compared with trails produced on uncoated formvar; the results suggest that material containing circumsporozoite protein forms the matrix within which the particles are embedded. The trails exhibit morphological features similar to those displayed by circumsporozoite precipitation reactions; of note is the demonstration of sheaths of circumsporozoite protein-containing material that emanate from sporozoites prior to their gliding. The sheaths narrow into accumulations of electron-dense material, which eventually taper to form typical trails. The structural manifestation of sheaths and other morphological details of the formed trails enables us to correlate sporozoite behavior during trail formation with the motile actions of gliding sporozoites observed by light microscopy.     

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.     

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.     

Exit of Plasmodium sporozoites from oocysts is an active process that involves the circumsporozoite protein

Plasmodium sporozoites develop within oocysts residing in the mosquito midgut. Mature sporozoites exit the oocysts, enter the hemolymph, and invade the salivary glands. The circumsporozoite (CS) protein is the major surface protein of salivary gland and oocyst sporozoites. It is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. CS protein contains a conserved motif of positively charged amino acids: region II-plus, which has been implicated in the initial stages of sporozoite invasion of hepatocytes. We investigated the function of region II-plus by generating mutant parasites in which the region had been substituted with alanines. Mutant parasites produced normal numbers of sporozoites in the oocysts, but the sporozoites were unable to exit the oocysts. In in vitro as well, there was a profound delay, upon trypsin treatment, in the release of mutant sporozoites from oocysts. We conclude that the exit of sporozoites from oocysts is an active process that involves the region II-plus of CS protein. In addition, the mutant sporozoites were not infective to young rats. These findings provide a new target for developing reagents that interfere with the transmission of malaria.     

Conserved group-specific epitopes of the circumsporozoite proteins revealed by antibodies to synthetic peptides

The immunogenic properties of sporozoites are associated mainly with the circumsporozoite (CS) protein that covers the surface of mature sporozoites. This stage-specific protein has an immunodominant region with repetitive epitopes. Rabbits that are repeatedly immunized with sporozoites of Plasmodium knowlesi, a monkey malaria parasite, also recognize two synthetic peptides (N2 and C2) representing other polar domains of the CS protein. We show in this report that antibodies to the N2 and C2 synthetic peptides react not only with P. knowlesi but also with conserved regions of the surface membrane of other human, monkey, and rodent (but not avian) malaria sporozoites. Moreover, antibodies to N2 partially neutralize the infectivity of sporozoites of P. berghei, a rodent malaria parasite. In contrast, antibodies to synthetic peptides representing the repetitive epitope of P. knowlesi were strictly species specific.     

Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage.     

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.     

Sporozoites of Rodent and Simian Malaria, Purified by Anion Exchangers, Retain their Immunogenicity and Infectivity

SYNOPSIS. Sporozoites of rodent malaria, Plasmodium berghei , and simian malaria, Plasmodium knowlesi and Plasmodium cynomolgi , were partially separated from mosquito debris and microbial contaminants by passage of Anopheles material through a DEAE-cellulcse column. In addition to eliminating most of the contaminants (80–90%), this simple technic has made it possible to recover rapidly large numbers of viable sporozoites (55–75% yield), which have retained their infectivity, immunogenicity, and capacity to react with known antisera. Mice injected with varying doses of column-purified sporozoites (CS) of P. berghei produced infections which paralleled those seen in the controls. Total protection against challenge with a potentially lethal dose of viable sporozoites was acquired by mice inoculated twice with irradiated CS of P. berghei. CS of P. berghei and P. cynomolgi gave positive circumsporozoite precipitation (CSP) reactions, upon inoculation with the respective immune sera. The preservation of the surface antigens of CS was documented by immunofluorescence.
It was shown that differences in elution behavior exist among sporozoites of certain species of Plasmodium as well as among sporozoites of the same species derived from different organs of the mosquito. These results may be attributed to differences in the surface charge of the sporozoites or conditions in sample media.
Purified sporozoites obtained by the method described in this report provide an adequate source of parasites for a variety of in vitro studies.     

Binding and invasion of liver cells by Plasmodium falciparum sporozoites. Essential involvement of the amino terminus of circumsporozoite protein.

Plasmodium sporozoites display circumsporozoite (CS) protein on their surface, which is involved in the attachment of sporozoites to liver cells. CS protein is a member of the thrombospondin type I repeat (TSR) domain family and possess a single copy of TSR domain toward its carboxyl terminus. We show by a direct measurement the correlation between the binding activity of various segments of the CS protein and their ability to inhibit the invasion of liver cells by the sporozoites. We made eight truncated versions of Plasmodium falciparum CS protein to elucidate the role of various regions in the binding and invasion process. Deletion of the TSR domain actually enhanced binding activity by 2-3-fold without the loss of receptor specificity, indicating that TSR may not be the only domain in defining the specificity of binding. These same deletions blocked invasion of live sporozoites more efficiently than proteins that include the TSR domain. Deletion of as little as six amino acids from amino terminus of the protein, however, renders it incapable of binding to liver cells and as an inhibitor of sporozoite invasion. Hence, the binding of CS protein to liver cells and its ability to inhibit the invasion process are affected in a parallel manner, both positively and negatively, by sequence changes in the encoded CS gene. This indicates that both assays are measuring interrelated phenomenon and points to the essential involvement for the amino-terminal portion of the CS protein in these processes.     

Mutational analysis of the GPI-anchor addition sequence from the circumsporozoite protein of Plasmodium

The plasma membrane of Plasmodium sporozoites is uniformly covered by the glycosylphosphatidylinositol (GPI)-anchored circumsporozoite (CS) protein. Sporozoites form in the mosquito midgut through a budding process that occurs within a multinucleate oocyst underneath the basal lamina of the gut. Earlier genetic studies established that normal sporozoite development requires CS. Mutant parasites lacking CS [CS (-)] do not form sporozoites. Ultrastructural analysis of the oocysts from these parasites revealed that there is an early block in the cytokinesis that occurs within the multinucleate oocysts to generate individual sporozoites. Parasites that are hypomorphic for CS expression gave rise to sporozoites with abnormal morphology. These results proved that CS plays a direct role in the maturation of oocysts and in the normal budding of sporozoites. In this article, we examined if the membrane localization of CS via a GPI-anchor, is crucial for its function during sporozoite formation. We generated three mutants in Plasmodium berghei CS, CS-DeltaGPI, CS-TM1 and CS-TM2. In CS-DeltaGPI, we deleted the signal sequence required for the addition of a GPI-anchor to CS. The resulting protein was found only in the cytoplasm of the oocyst. In CS-TM1 and CS-TM2, the GPI-anchor addition sequence of CS was substituted by the transmembrane domain and truncated (to different degrees) cytoplasmic tail of Plasmodium thrombospondin-related anonymous protein (TRAP). The resulting CS protein was detected on the plasma membrane of the oocysts. The amount of CS in the mutants was similar to that of wild type. The sporozoite budding and development were abrogated in both CS-DeltaGPI and CS-TM mutants. The ultrastructure of the mutant oocysts was indistinguishable from that of the CS (-) parasites. Our results suggest that the GPI-anchor of the CS protein is required for sporogenesis.     

Immune response gene regulation of immunity to Plasmodium berghei sporozoites and circumsporozoite protein vaccines. Overcoming genetic restriction with whole organism and subunit vaccines

We conducted a series of experiments to define Ir gene regulation of the immune response to Plasmodium berghei sporozoites and circumsporozoite (CS) protein-derived subunit vaccines. The studies demonstrated that there is no apparent genetic restriction of the capacity to develop protective immunity against a large sporozoite challenge after immunization with irradiation-attenuated P. berghei sporozoites; that the Th response to (Asp-Pro-Ala-Pro-Pro-Asn-Ala-Asn)n, the predominant protective B epitope on the P. berghei CS protein, is genetically restricted and regulated by Class II genes (I-Ab) and by genes in the Class I region (H-2Dk) or telomeric to this region; and that this restriction can be overcome by immunization with a r protein including the entire P. berghei CS protein. The results support the development of full length human CS protein vaccines to take advantage of all potential T epitopes on this protein.     

Sporozoites of rodent and simian malaria, purified by anion exchangers, retain their immunogenicity and infectivity.

Sporozoites of rodent malaria, Plasmodium berghei, and simian malaria, Plasmodium knowlesi and Plasmodium cynomolgi, were partially separated from mosquito debris and microbial contaminants by passage of Anopheles material through a DEAE-cellulose column. In addition to eliminating most of the contaminants (80-90%), this simple technic has made it possible to recover rapidly large numbers of viable sporozoites (55-75% yield), which have retained their infectivity, immunogenicity, and capacity to react with known antisera. Mice injected with varying doses of column-purified sporozoites (CS) of P. berghei produced infections which paralleled those seen in the controls. Total protection against challenge with a potentially lethal dose of viable sporozoites was acquired by mice inoculated twice with irradiated CS of P. berghei CS of P. berghei and P. cynomolgi gave positive circumsporozoite precipitation (CSP) reactions, upon inoculation with the respective immune sera. The preservation of the surface antigens of CS was documented by immunofluorescence. It was shown that differences in elution behavior exist among sporozoites of certain species of Plasmodium as well as among sporozoiters of the same species derived from different organs of the mosquito. These results may be attributed to differences in the surface charge of the sporozoites or conditions in sample media. Purified sporozoites obtained by the method described in this report provide an adequate source of parasites for a variety of in vitro studies.     

Plasmodium malariae: distribution of circumsporozoite protein in midgut oocysts and salivary gland sporozoites

The distribution of the circumsporozoite protein within developing Plasmodium malariae oocysts and salivary gland sporozoites was examined by immunoelectron microscopy using protein A-gold and a monoclonal antibody specific for the CS protein of P. malariae. Gold particles were found along the capsule of immature oocysts but rarely within the cytoplasm. Gold label was detected on the inner surface of peripheral vacuoles during oocyst maturation and the plasma membrane of the sporoblast. Salivary gland sporozoites and budding sporozoites in mature oocysts were labeled uniformly on the outer surface of their plasma membranes. The surface of sporozoites that ruptured into midgut epithelial cells were entirely covered with gold particles. No label was seen on the surface of sporozoites which ruptured into the midgut lumen. In addition, a rabbit polyclonal antibody against repeat a region of P. brasilianum CS protein reacted with P. malariae sporozoites.     

Waiting for the vaccine: sporozoite vaccine research entails important progress in malaria epidemiology.

The research efforts aimed at developing a vaccine against malaria, although failing thus far in their main objective, have produced molecular tools of great utility for epidemiological studies. For example, monoclonal antibodies directed against the repeats of Plasmodium circumsporozoite (CS) protein allowed the 2-site assay for detecting sporozoites in mosquitoes to be established. This immunoassay is advantageous compared with the conventional method of salivary gland dissection and microscopic examination, for it makes the identification of the sporozoite species possible, thanks to species-specific aminoacid sequences of the CS repeats. Other examples of vaccine research-derived tools are synthetic peptides reproducing the repetitive part of the CS protein, which allow antibodies to sporozoites, in individuals exposed to malaria, to be detected. Antibodies to the CS repeats of Plasmodium (Laverania) falciparum proved to be a reliable indicator of the intensity of malaria transmission and, therefore, were suitable for monitoring the impact of malaria control programmes. Finally, a project is outlined that, relying on the application of these tools, will aim at characterizing the transmission of Plasmodium (Plasmodium) malariae and at unveiling the possible relationship among different species thriving in the same distribution area, an issue which may become of relevance in view of the likely introduction of a vaccine directed against a single species.     

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.     

Malaria circumsporozoite protein inhibits protein synthesis in mammalian cells.

Native Plasmodium circumsporozoite (CS) protein, translocated by sporozoites into the cytosol of host cells, as well as recombinant CS constructs introduced into the cytoplasm by liposome fusion or transient transfection, all lead to inhibition of protein synthesis in mammalian cells. The following findings suggest that this inhibition of translation is caused by a binding of the CS protein to ribosomes. (i) The distribution of native CS protein translocated by sporozoites into the cytoplasm as well as microinjected recombinant CS protein suggests association with ribosomes. (ii) Recombinant CS protein binds to RNase-sensitive sites on rough microsomes. (iii) Synthetic peptides representing the conserved regions I and II-plus of the P.falciparum CS protein displace recombinant CS protein from rough microsomes with dissociation constants in the nanomolar range. (iv) Synthetic peptides representing region I from the P.falciparum CS protein and region II-plus from the P.falciparum, P.berghei or P.vivax CS protein inhibit in vitro translation. We propose that Plasmodium manipulates hepatocyte protein synthesis to meet the requirements of a rapidly developing schizont. Since macrophages appear to be particularly sensitive to the presence of CS protein in the cytosol, inhibition of translation may represent a novel immune evasion mechanism of Plasmodium.