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.     

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.     

Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection

Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as specific SR-BI-blocking antibodies, we demonstrate that SR-BI significantly boosts hepatocyte permissiveness to P. falciparum, P. yoelii, and P. berghei entry and promotes parasite development. We show that SR-BI, but not the low-density lipoprotein receptor, acts as a major cholesterol provider that enhances Plasmodium infection. SR-BI regulates the organization of CD81 at the plasma membrane, mediating an arrangement that is highly permissive to penetration by sporozoites. Concomitantly, SR-BI upregulates the expression of the liver fatty-acid carrier L-FABP, a protein implicated in Plasmodium liver-stage maturation. These findings establish the mechanistic basis of the CD81-dependent Plasmodium sporozoite invasion pathway.     

Monoclonal, but not polyclonal, antibodies protect against Plasmodium yoelii sporozoites

One of the primary strategies for malaria vaccine development has been to design subunit vaccines that induce protective levels of antibodies against the circumsporozoite (CS) protein of malaria sporozoites. In the Plasmodium yoelii mouse model system such vaccines have been uniformly unsuccessful in protecting against sporozoite-induced malaria. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of Navy yoelii sporozoite 1 (NYS1), an IgG3 mAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, a synthetic peptide derived from the repeat region of the P. yoelii CS protein, indicating that the epitope on sporozoites recognized by this mAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by indirect fluorescent antibody test and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing (QGPGAP)2 but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not absolute concentration could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective mAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. Although the results of the conventional antibody assays did not correlate with protection, sera from the protected animals inhibited sporozoite development in mouse hepatocyte cultures significantly more than did the sera from the unprotected, subunit vaccine-immunized animals, correlating with protection. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective mAb and nonprotective polyclonal antibodies will be important in the further development of malaria vaccines.     

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.     

Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection

Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases. PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACalpha and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.     

Plasmodium yoelii: experimental evidences for the conserved epitopes between mouse and human malaria parasite, Plasmodium falciparum

Bioinformatic analyses of gene homologues have revealed functionally conserved epitopes between human and rodent malaria parasites. Here, we present experimental evidence for the presence of functionally and antigenically conserved domains between Plasmodium falciparum and Plasmodium yoelii asexual blood-stages. Merozoite released soluble proteins (MRSPs) from both P. falciparum and P. yoelii bound to heterologous mouse or human red blood cells, respectively. The presence of conserved antigenic epitopes between the two species of parasites was evident by the inhibitory effect of antibodies, developed against P. yoelii in convalescent mice, on P. falciparum growth and merozoite reinvasion in vitro. Furthermore, mice immunized with P. falciparum MRSPs were protected from infection by a P. yoelii challenge. These data indicate that different species of Plasmodium contain antigenically conserved interspecies domains, which are immunogenic and, thus constitute a potential novel antigen source for vaccine development and testing using a mouse model.     

A protective monoclonal antibody recognizes a variant-specific epitope in the precursor of the major merozoite surface antigen of the rodent malarial parasite Plasmodium yoelii

The precursor of the major merozoite surface Ag (PMMSA) represents one of the principal molecules of the erythrocytic stages of malarial parasites. Previously we reported that mAb 302 recognizing the 230-kDa PMMSA of Plasmodium yoelii provided passive protection to mice challenged with this parasite. We now report that the protective capacity of mAb 302 is variant specific, affording protection against infection with only three of five P. yoelii lines. Immunoprecipitation analyses of their PMMSA revealed that the expression of the epitope recognized by mAb 302 also varied and correlated completely with the results of the passive protection studies. Although this specific determinant was not present on the merozoite Ag of all P. yoelii lines, the common expression of other B cell epitopes was noted by the demonstration of serologic cross-reactivity between these molecules. Furthermore, the relatedness of the genes encoding the PMMSA of several murine plasmodial strains and species was clearly shown in nucleic acid hybridization studies. Although strain-common and strain-variable epitopes have been observed in the PMMSA of the human parasite, Plasmodium falciparum, little is known concerning the variability of its biologically relevant epitopes. The current studies using the P. yoelii model system demonstrate that the epitope recognized by a protective mAb is strain variable. Because of the similarities between these antigens of P. falciparum and P. yoelii, this information may impact on the construction of an effective blood-stage malarial vaccine.     

A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection.     

A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins.

An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.     

Host scavenger receptor SR-BI plays a dual role in the establishment of malaria parasite liver infection

An obligatory step of malaria parasite infection is Plasmodium sporozoite invasion of host hepatocytes, and host lipoprotein clearance pathways have been linked to Plasmodium liver infection. By using RNA interference to screen lipoprotein-related host factors, we show here that the class B, type I scavenger receptor (SR-BI) is the strongest regulator of Plasmodium infection among these factors. Inhibition of SR-BI function reduced P. berghei infection in Huh7 cells, and overexpression of SR-BI led to increased infection. In vivo silencing of liver SR-BI expression in mice and inhibition of SR-BI activity in human primary hepatocytes reduced infection by P. berghei and by P. falciparum, respectively. Heterozygous SR-BI(+/-) mice displayed reduced P. berghei infection rates correlating with liver SR-BI expression levels. Additional analyses revealed that SR-BI plays a dual role in Plasmodium infection, affecting both sporozoite invasion and intracellular parasite development, and may therefore constitute a good target for malaria prophylaxis.     

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum binds specifically to sulfated glycoconjugates and to HepG2 hepatoma cells suggesting a role for this molecule in sporozoite invasion of hepatocytes.

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum contains an amino acid motif based around the sequence WSPCSVTCG which is also found in region II of the circumsporozoite (CS) proteins of different species of Plasmodium. This amino acid motif confers on the CS protein the ability to bind specifically to sulfated glycoconjugates and to hepatocytes. This suggests that the interaction of CS protein with sulfated glycoconjugates on the surface of the hepatocytes may represent the first molecular event of sporozoite invasion of liver cells. Experimental evidence indicates that TRAP is localized both on the micronemes and on the surface of P. falciparum sporozoites implying that TRAP with its putative sulfated glycoconjugate binding motif may also be involved in recognition and/or entry of hepatocytes by the sporozoite. We show here that different TRAP constructs expressed in Escherichia coli bind to sulfogalactosyl-cerebrosides (sulfatides) and to the surface of HepG2 cells. These interactions are dependent on the presence of the conserved amino acid motif WSPCSVTCG within the sequences of the constructs and are completely inhibited by several sulfated glycoconjugates as well as by suramin, a polysulfonated drug with anti-protozoan activity. Moreover, sporozoite invasion of HepG2 cells is inhibited by antisera raised against these different TRAP constructs and by the presence of low concentrations of suramin. We concluded that TRAP may be one of the parasite encoded molecules in the host-parasite interaction that results in sporozoite invasion of hepatocytes.     

Alternative invasion pathways for Plasmodium berghei sporozoites

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a natural malaria infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that infection by Plasmodium falciparum and Plasmodium yoelii sporozoites depends on CD81 and cholesterol-dependent tetraspanin-enriched microdomains (TEMs) on the hepatocyte surface. Here we have analyzed the role of CD81 and TEMs during infection by sporozoites from the rodent parasite Plasmodium berghei. We found that depending on the host cell type, P. berghei sporozoites can use several distinct pathways for invasion. Infection of human HepG2, HuH7 and HeLa cells by P. berghei does not depend on CD81 or host membrane cholesterol, whereas both CD81 and cholesterol are required for infection of mouse hepatoma Hepa1-6 cells. In primary mouse hepatocytes, both CD81-dependent and -independent mechanisms participate in P. berghei infection and the relative contribution of the different pathways varies, depending on mouse genetic background. The existence of distinct invasion pathways may explain why P. berghei sporozoites are capable of infecting a wide range of host cell types in vitro. It could also provide a means for human parasites to escape immune responses and face polymorphisms of host receptors. This may have implications for the development of an anti-malarial vaccine targeting sporozoites.     

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.     

Antigenic relationship between Plasmodium falciparum and Babesia bovis: reactivity with antibodies to culture-derived soluble exoantigens

Antigenic similarities between Plasmodium and Babesia parasites of the phylum Apicomplexa have been previously demonstrated primarily by the serological cross reactivity observed in the indirect fluorescent antibody (IFA) test. We have now studied the antigenic relationship between the human malaria parasite, Plasmodium falciparum, and the hemoparasitic agent of cattle, Babesia bovis, using rabbit monospecific antibodies produced against individual culture-derived P. falciparum polypeptides and bovine polyspecific antibodies to B. bovis exoantigens. These respective antibodies were found to be distinctly cross reactive in the IFA test using infected erythrocytes (squirrel monkey--P. falciparum; bovine--B. bovis) as antigen substrates. Immunofluorescence was shown to be highly specific for parasite surfaces. Additionally, the degree of reactivity with soluble exoantigens contained in Plasmodium and Babesia culture supernatants was monitored by a two-site enzyme immunoassay employing the cross-reactive antibodies. Further evidence for antigenic cross reactivity between P. falciparum and B. bovis parasites was shown with the in vitro inhibition assay. Antibodies to P. falciparum and B. bovis were found to be highly inhibitory for the in vitro growth of P. falciparum in human erythrocytes.     

AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver

The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA‐like protein, merozoite apical erythrocyte‐binding ligand (MAEBL). MAEBL‐deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages.     

Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured cells; an in vitro assay of protective antibodies

Plasmodium falciparum and P. vivax sporozoites were observed to invade cultured human hepatoma cells in vitro. Monoclonal antibodies to the circumsporozoite (CS) protein of each of these malarial species blocked invasion. Inhibition was species-specific, but was independent of the geographic origin of each strain. Because these monoclonal antibodies have been shown to diminish or abolish sporozoite infectivity to susceptible primate hosts, it is suggested that inhibition of invasion of sporozoites (ISI) into cultured cells may represent in in vitro assay for protective antibodies. This was confirmed by the finding that serum taken from volunteers immune to sporozoite challenge also totally blocked sporozoite invasion. The ISI assay also detected naturally acquired invasive-neutralizing antibodies in areas endemic for malaria. This ISI assay may therefore be useful in determining the incidence of inhibitory anti-sporozoite antibodies in general populations, and allow the monitoring of the effect of an anti-malarial vaccine using sporozoite-derived antigens.     

Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue

Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.     

Hepatocyte permissiveness to Plasmodium infection is conveyed by a short and structurally conserved region of the CD81 large extracellular domain

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein.