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.     

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.     

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 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.     

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.     

Common Epitopes In the Circumsporozoite Proteins of Plasmodium Berghei and Plasmodium Gallinaceum Identified By Monoclonal Antibodies to the P. Gallinaceum Circumsporozoite Protein

ABSTRACT. Monoclonal antibodies that react with the circumsporozoite protein of the avian malaria Plasmodium gallinaceum sporozoites also reacted with circumsporozoite protein of the rodent malaria Plasmodium berghei. Two types of reactivity were identified: 1) two monoclonal antibodies reacted with P. berghei sporozoite protein by enzyme-linked immunosorbent assay, Western blot and indirect immunofluorescence antibody, 2) six other monoclonal antibodies reacted with P. berghei sporozoites by ELISA and Western blot only. We studied whether these differences could be explained by reactivity in enzyme-linked immunosorbent assay with different P. berghei circumsporozoite peptides. Although all P. gallinaceum monoclonal antibodies reacted with the P. berghei repeats, the first group reacted with a conserved peptide sequence, N1, whereas the second group did not. These results suggest that circumsporozoite proteins from P. gallinaceum and P. berghei share common epitopes. the biological significance of our finding is not yet clear. Indeed, the cross-reactive monoclonal antibodies giving a positive indirect immunofluorescence antibody with the P. berghei sporozoites only caused a borderline effect on the living P. berghei parasites in vitro as measured by inhibition of sporozoite infectivity.     

Interactions of Plasmodium berghei sporozoites and murine Kupffer cells in vitro

Malaria sporozoites must leave the bloodstream and cross a layer of sinusoidal lining cells in order to infect hepatocytes and undergo exoerythrocytic schizogony. To determine whether Kupffer cells (KC) derived from this layer interact with sporozoites, murine KC were isolated from perfused livers of BALB/cJ mice and incubated in vitro with Plasmodium berghei sporozoites. Isolated KC had characteristic macrophage surface Ag and were phagocytic, ingesting both latex particles and Leishmania major amastigotes. In the absence of immune serum, sporozoites associated with fewer than 10% of these KC. By 30 min, 10% of the cell-associated sporozoites were completely ingested, 30% were in the process of being ingested, and 60% were attached to the surface of the cells. Opsonization of sporozoites with monoclonal or polyclonal antibodies directed against P. berghei circumsporozoite protein markedly enhanced sporozoite association with KC. Up to 40% of cells exposed to opsonized sporozoites had parasites inside or attached to their surfaces. Sporozoites attached to or ingested by KC were uniformly destroyed within 240 min in all cultures; there was no evidence of conversion of sporozoites to the exoerythrocytic stage within KC by light microscopy, and there was no evidence of residual sporozoites, either inside or outside of cells, by either light or electron microscopy. These data suggest that under nonimmune conditions, KC play a minor role in resistance to infection by malaria sporozoites. However, when sporozoites are opsonized by circumsporozoite antibodies, phagocytosis by KC may be an important immune mechanism that prevents parasitization of hepatocytes.     

Immunological relationship of Plasmodium inui with two other quartan malaria parasites, P. malariae and P. brasilianum

A series of monoclonal antibodies was produced against sporozoites of the OS strain of Plasmodium inui, a simian quartan malaria parasite, and used to characterize the circumsporozoite protein of this parasite. The results confirm that the immunodominant epitope of the circumsporozoite protein of P. inui is immunologically distinct from those of 2 other quartan parasites, the human P. malariae and simian P. brasilianum, which are identical.     

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.     

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.     

Plasmodium falciparum: sporozoite boosting of immunity due to a T-cell epitope on a sporozoite vaccine

Plasmodium falciparum: Sporozoite boosting of immunity due to a T-cell epitope on a sporozoite vaccine. Experimental Parasitology 64, 64-70. The impact of a malaria sporozoite vaccine may be enhanced if protective immunity elicited by the vaccine is boosted by natural exposure to sporozoites. For this to occur, a helper T lymphocyte epitope present on the vaccine must be shared by sporozoites. These studies show that T cells from mice immunized with R32tet32, the Plasmodium falciparum sporozoite vaccine candidate, recognize an epitope of less than or equal to 7 amino acids derived from the circumsporozoite protein repeat region of R32tet32, as well as an epitope on the tet32 fusion protein tail of R32tet32. Exposure of R32tet32 immunized animals to P. falciparum sporozoites elicits a significant secondary antibody response which suggests that humans who are immunized and respond to this vaccine may be boosted by field exposure to sporozoite infected mosquitoes.     

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.

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.     

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.     

Experimental vaccination of chicks with Plasmodium gallinaceum sporozoites. I. Circumsporozoite proteins are expressed by sporozoites recovered from both salivary glands and midguts of mosquitoes

Immunogenicity of Plasmodium gallinaceum sporozoites for chicks and their in vitro reactivity with normal and specific immune sera were studied. Two sporozoite populations recovered from experimentally infected Aedes fluviatilis were used: sporozoites from salivary glands and sporozoites from midgut oocysts. Populations seven to nine days old of sporozoites recovered from salivary glands were infective for all chicks until the chicks were three weeks old; however, sporozoites recovered from midguts containing oocysts infected these chicks only if isolated on days 8-9, but not on day 7 after the mosquitoes' infective blood meal. Infectivity of the sporozoites was lost after exposure to ultraviolet (UV) light (30 min) or X-rays (13 krad). Inactivated sporozoites from both sources proved highly immunogenic to chicks that were immunized by several intravenous or intramuscular injections. These parasites elicited a strong humoral immune response in the chicks, as measured by the circumsporozoite precipitation (CSP) reaction. The levels of the CSP antibodies were similar with sporozoites from both sources, there being no detectable differences in the percentage of reactive sporozoites or the intensity of the CSP reaction with sera containing antibodies to either sporozoites from salivary glands or sporozoites from oocysts. These results provide the first evidence that avian malaria sporozoites express the circumsporozoite protein that has been extensively characterized in mammalian malaria (rodent, simian, human sporozoites). Furthermore, we observed that the yields of sporozoites obtained from mosquito midguts, on days 8 and 9 of the P. gallinaceum infection, were at least twice as great as those obtained by salivary gland dissection, even 20 days after a blood meal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.     

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.     

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.     

Experimental Vaccination of Chicks with Plasmodium gallinaceum Sporozoites. I. Circumsporozoite Proteins Are Expressed by Sporozoites Recovered from Both Salivary Glands and Midguts of Mosquitoes1

Immunogenicity of Plasmodium gallinaceum Sporozoites for chicks and their in vitro reactivity with normal and specific immune sera were studied. Two sporozoite populations recovered from experimentally infected Aedes fluviatilis were used: sporozoites from salivary glands and sporozoites from midgut oocysts. Populations seven to nine days old of sporozoites recovered from salivary glands were infective for all chicks until the chicks were three weeks old; however, sporozoites recovered from midguts containing oocysts infected these chicks only if isolated on days 8–9, but not on day 7 after the mosquitoes' infective blood meal. Infectivity of the sporozoites was lost after exposure to ultraviolet (UV) light (30 min) or X-rays (13 krad). Inactivated sporozoites from both sources proved highly immunogenic to chicks that were immunized by several intravenous or intramuscular injections. These parasites elicited a strong humoral immune response in the chicks, as measured by the circumsporozoite precipitation (CSP) reaction. The levels of the CSP antibodies were similar with sporozoites from both sources, there being no detectable differences in the percentage of reactive sporozoites or the intensity of the CSP reaction with sera containing antibodies to either sporozoites from salivary glands or sporozoites from oocysts. These results provide the first evidence that avian malaria sporozoites express the circumsporozoite protein that has been extensively characterized in mammalian malaria (rodent, simian, human sporozoites). Furthermore, we observed that the yields of sporozoites obtained from mosquito midguts, on days 8 and 9 of the P. gallinaceum infection, were at least twice as great as those obtained by salivary gland dissection, even 20 days after a blood meal. This is an advantage since obtaining the midguts is less tedious, as well as more efficient and faster.