Molecular Mechanism of Host Specificity in Plasmodium falciparum Infection

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. 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. Malaria infection in humans is initiated with the bite of an infectious female mosquito, which inject sporozoites of Plasmodium species into the circulation. These sporozoites rapidly bind and invade liver cells and undergo rapid multiplication, leading to the release of thousands of infective merozoites (1Cerami C. Frevert U. Sinnis P. Takacs B. Clavijo P. Santos M.J. Nussenzweig V. Cell. 1992; 70: 1021-1033Abstract Full Text PDF PubMed Scopus (311) Google Scholar). Out of more than 20 well documented and characterized species of Plasmodium that cause malaria in various vertebrates, only four species viz., P. falciparum, P. vivax, P. malariae, and P. ovale infect humans. It is intriguing that, although numerous parasite surface antigens involved in infectivity and pathogenesis possess inter-species homology and have been identified in numerous humans, rodents, and simian Plasmodium species (2Dame J.B. Williams J.L. McCutchan T.F. Weber J.L. Wirtz R.A. Hockmeyer W.T. Maloy W.L. Haynes J.D. Schneider I. Roberts D. Sanders G.S. Reddy E.P. Diggs C.L. Miller L.H. Science. 1984; 225: 593-599Crossref PubMed Scopus (563) Google Scholar, 3McCutchan T.F. Kissinger J.C. Touray M.G. Rogers M.J. Li J. Sullivan M. Braga E.M. Krettli A.U. Miller L.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11889-11894Crossref PubMed Scopus (127) Google Scholar, 4Robson K.J. Hall J.R. Jennings M.W. Harris T.J. Marsh K. Newbold C.I. Tate V.E. Weatherall D.J. Nature. 1988; 335: 79-82Crossref PubMed Scopus (341) Google Scholar, 5Templeton T.J. Kaslow D.C. Mol. Biochem. Parasitol. 1997; 84: 13-24Crossref PubMed Scopus (69) Google Scholar), the parasite maintains its host specificity and non-natural host infections are rare. In laboratory conditions, it is possible to infect Aotus monkeys with human parasites (6Collins W.E. Miller L.H. Glew R.H. Contacos P.G. Howard W.A. Wyler D.J. J. Parasitol. 1973; 59: 855-858Crossref PubMed Scopus (7) Google Scholar, 7Schmidt L.H. Am. J. Trop. Med. Hyg. 1978; 27: 671-702Crossref PubMed Scopus (50) Google Scholar). Most of these infections have been induced by inoculating erythrocytic stage parasites or salivary gland-isolated sporozoites in splenectomized animals and with a parasite load not seen in malaria endemic areas (7Schmidt L.H. Am. J. Trop. Med. Hyg. 1978; 27: 671-702Crossref PubMed Scopus (50) Google Scholar, 8Collins W.E. Galland G.G. Sullivan J.S. Morris C.L. Am. J. Trop. Med. Hyg. 1994; 51: 224-232Crossref PubMed Scopus (35) Google Scholar). Initiation of a human malaria infection by sporozoites in Aotus monkeys through the natural course (bites of infected mosquitoes) has not been successful. Maintenance of host specificity is an important aspect of pathogenicity and sustenance of an infection. It generally involves a complex interplay between pathogen and host factors, which are influenced by various evolutionary and genetic determinants. Understanding which parasite components play a role in host specificity can provide information about the pathogenesis and can also help in disease control. Plasmodium sporozoites express CSP 1The abbreviations used are: CSP, circumsporozoite protein; PfCSP, P. falciparum CSP; PgCSP, P. gallinaceum CSP; PkCSP, P. knowlesi CSP; PyCSP, P. yoelii CSP; HSPG, heparan sulfate proteoglycans; SPR, surface plasmon resonance; TSR, thrombospondin type I repeat; MOPS, 4-morpholinepropanesulfonic acid; RU, response unit. on the surface of all the Plasmodium species. CSP is an important molecule for the parasite, because it is involved in the development of infectious sporozoites in mosquitoes (9Menard R. Sultan A.A. Cortes C. Altszuler R. van Dijk M.R. Janse C.J. Waters A.P. Nussenzweig R.S. Nussenzweig V. Nature. 1997; 385: 336-340Crossref PubMed Scopus (260) Google Scholar), plays a role in the invasion of salivary gland, and is essential to the binding and invasion of liver cells in the vertebrate host (1Cerami C. Frevert U. Sinnis P. Takacs B. Clavijo P. Santos M.J. Nussenzweig V. Cell. 1992; 70: 1021-1033Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 10Rathore D. Sacci J.B. de la Vega P. McCutchan T.F. J. Biol. Chem. 2002; 277: 7092-7098Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). It is also a malaria vaccine candidate undergoing clinical trails (11Stoute J.A. Slaoui M. Heppner D.G. Momin P. Kester K.E. Desmons P. Wellde B.T. Garcon N. Krzych U. Marchand M. N. Engl. J. Med. 1997; 336: 86-91Crossref PubMed Scopus (784) Google Scholar, 12Wang R. Doolan D.L. Le T.P. Hedstrom R.C. Coonan K.M. Charoenvit Y. Jones T.R. Hobart P. Margalith M. Ng J. Weiss W.R. Sedegah M. de Taisne C. Norman J.A. Hoffman S.L. Science. 1998; 282: 476-480Crossref PubMed Scopus (694) Google Scholar, 13Nardin E.H. Oliveira G.A. Calvo-Calle J.M. Castro Z.R. Nussenzweig R.S. Schmeckpeper B. Hall B.F. Diggs C. Bodison S. Edelman R. J. Infect. Dis. 2000; 182: 1486-1496Crossref PubMed Scopus (143) Google Scholar). The general structural organization of CSP is conserved across the species. The protein can be divided into three regions of roughly equal sizes; the amino terminus, a central repeat segment, and a carboxyl terminus region containing a conserved TSR domain (schematic in Fig. 1). The TSR domain is present in a large number of proteins and performs diverse functions (14Naitza S. Spano F. Robson K.J.H. Crisanti A. Parasitol. Today. 1998; 14: 479-484Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Apweiler R. Attwood T.K. Bairoch A. Bateman A. Birney E. Biswas M. Bucher P. Cerutti L. Corpet F. Croning M.D. Durbin R. Falquet L. Fleischmann W. Gouzy J. Hermjakob H. Hulo N. Jonassen I. Kahn D. Kanapin A. Karavidopoulou Y. Lopez R. Marx B. Mulder N.J. Oinn T.M. Pagni M. Servant F. Nucleic Acids Res. 2001; 29: 37-40Crossref PubMed Scopus (838) Google Scholar). In contrast, the central repeat region is a species-specific, low complexity segment with no known function. We have recently demonstrated that in P. falciparum amino terminus of the CSP is involved in the binding and invasion process of the sporozoites. The amino terminus has regions of homology that are conserved across the species (3McCutchan T.F. Kissinger J.C. Touray M.G. Rogers M.J. Li J. Sullivan M. Braga E.M. Krettli A.U. Miller L.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11889-11894Crossref PubMed Scopus (127) Google Scholar). In the present study we have investigated the role of CSP in maintaining the host specificity during the invasion of liver cells by the P. falciparum parasite, a critical step in initiating the malaria infection in humans. The sensor chip used in the SPR studies was a Pioneer Chip C1, which was obtained from Biacore (Biacore AB, Uppsala, Sweden) along with N-hydroxysuccinimide, N-ethyl-N-(dimethyaminopropyl)carbodiimide, and the sodium acetate immobilization buffer. Bovine serum albumin was from Amresco (Solon, OH). The albumin-heparin conjugate, HEPES, NaCl, Tween 20, EDTA, glycine, and guanidine were acquired through Sigma-Aldrich (St. Louis, MO). Cloning, expression, and purification of recombinant CS protein of P. falciparum (16Rathore D. McCutchan T.F. Infect. Immun. 2000; 68: 740-743Crossref PubMed Scopus (15) Google Scholar) and P. knowlesi (17Rogers W.O. Weiss W.R. Kumar A. Aguiar J.C. Tine J.A. Gwadz R. Harre J.G. Gowda K. Rathore D. Kumar S. Hoffman S.L. Infect. Immun. 2002; 70: 4329-4335Crossref PubMed Scopus (76) Google Scholar) has been described previously. DNA encoding P. gallinaceum mature CS protein was amplified as a NdeI-EcoRI fragment by PCR and cloned in pET11a, a T7 promoterbased Escherichia coli expression vector. Protein expression was done using E. coli strain BL21(λDE3). Cells were transformed and grown in super broth (pH 7.2), containing 100 μg/ml ampicillin, at 37 °C with shaking. At an A 600 of 2.0, the cells were induced with 2 mm isopropyl-1-thio-β-d-galactopyranoside, and 4 h later, they were harvested by centrifugation at 4000 × g for 20 min. Total cell pellet was resuspended in washing buffer (50 mm Tris, pH 7.5, 20 mm EDTA) containing lysozyme at a concentration of 0.5 mg/ml. Cell suspension was incubated at room temperature, for 1 h with intermittent shaking. NaCl and Triton X-100 were added to obtain a final concentration of 0.5 m and 2.5%, respectively, and the suspension was further incubated at room temperature for 30 min, with vigorous shaking. This suspension was then spun at 13,000 × g at 4 °C, for 50 min, and the resultant pellet was resuspended in washing buffer containing 1% Triton X-100, using a Tissuemizer, and spun at 13,000 × g for 50 min. The pellet was washed repeatedly in washing buffer, without Triton X-100. After four washings, the pellet containing inclusion bodies was dissolved in 6 m guanidine hydrochloride and incubated for 2 h at room temperature followed by centrifugation at 50,000 × g at 4 °C, for 30 min. Supernatant, containing denatured protein, was collected, and the protein concentration was adjusted to 10 mg/ml with 6 m guanidine hydrochloride. Denatured protein was reduced by adding dithioerythritol, to a final concentration of 65 mm, and incubating at room temperature for 2 h. Protein was renatured by diluting 100-fold, in refolding buffer (100 mm Tris, pH 8.0, 0.5 ml-arginine-HCl, 2 mm EDTA, and 0.9 mm oxidized glutathione) (18Rathore D. Nayak S.K. Batra J.K. FEBS Lett. 1996; 392: 259-262Crossref PubMed Scopus (21) Google Scholar). After incubating at 10 °C for 36 h, renatured material was dialyzed against 20 mm Tris, pH 8.0, containing 100 mm urea. The renatured protein was loaded onto a Q-Sepharose column, and eluted by a salt gradient using a fast-protein liquid chromatography system. Relevant fractions were pooled and purified to homogeneity by gel filtration chromatography on a TSK 3000 column. DNA encoding PyCSP was cloned in a Yeast expression with a at its The was in cell line The protein was purified on a using followed by was to the of recombinant of recombinant CSP in 10 mm sodium buffer, pH were in the at °C, using a This was using Scopus Google Scholar). cell line in containing 2 mm and serum was and the was as described (1Cerami C. Frevert U. Sinnis P. Takacs B. Clavijo P. Santos M.J. Nussenzweig V. Cell. 1992; 70: 1021-1033Abstract Full Text PDF PubMed Scopus (311) Google Scholar). cells at a of cells well were with followed by with containing 1% serum were incubated with cells for 1 h followed by for and for 30 min. for protein is a of of these an of 1 in an was used for the 1 mm was used as and was in a with at and at HepG2 cells were with of I or buffer (100 mm MOPS, pH 10 mm for 2 h at 37 The cells were washed with followed by of 50 recombinant protein and at 37 °C for 1 h. was as described HepG2 cells were collected, and resuspended in 50,000 cells were added to well of and incubated at 37 °C to to P. falciparum sporozoites were from infected sporozoites were added to the HepG2 cells followed by the of concentration of recombinant CS protein and incubated for h. Cells were washed and with P. falciparum CS region was added followed by to the sporozoites. sporozoites were identified and and the inhibition of invasion was with the × plasmon were using a 3000 which was with Biacore were at were and to in of the was to the sensor surface using a F. M. R. Biochem. 2002; PubMed Scopus Google Scholar). the sensor chip surface was with an of followed by the immobilization of albumin-heparin and of with The albumin-heparin was in of which response of material and the Bovine serum albumin was to the sensor surface in a to as a control. The sensor surface in the cell was and then and it as the surface for changes in the and CS protein were with buffer m HEPES, mm NaCl, mm EDTA, Tween 20, pH from to were the sensor surface at a of 50 for 1 to 2 min. The sensor surface was using a mm glycine, pH and an of 2 m was the sensor chip three in to prevent in the were at three to of binding of CS protein to heparin was using D.G. J. Mol. PubMed Scopus Google Scholar). Although the protein were 2 the were further for of the The and and the affinity were by to the and to a stage using of a for the was on the to for the for the of surface albumin-heparin the to The were by the of the affinity and the are the obtained from Cloning, and of CS CSP from four species of Plasmodium representing human, simian, and malaria species were recombinantly expressed, and the proteins were purified to homogeneity. DNA encoding PfCSP, PkCSP, and were in pET11a, a T7 E. coli expression vector. and were into the which was used as a to obtain the purified protein, as described (16Rathore D. McCutchan T.F. Infect. Immun. 2000; 68: 740-743Crossref PubMed Scopus (15) Google Scholar, W.O. Weiss W.R. Kumar A. Aguiar J.C. Tine J.A. Gwadz R. Harre J.G. Gowda K. Rathore D. Kumar S. Hoffman S.L. Infect. Immun. 2002; 70: 4329-4335Crossref PubMed Scopus (76) Google Scholar). the proteins were purified on a affinity followed by gel filtration to obtain the purified protein was present in the as inclusion bodies not bodies were from the using guanidine hydrochloride and reduced by adding The protein was renatured in in a using oxidized (18Rathore D. Nayak S.K. Batra J.K. FEBS Lett. 1996; 392: 259-262Crossref PubMed Scopus (21) Google Scholar). The protein was purified to homogeneity by chromatography on and gel filtration PyCSP was purified on a followed by gel filtration of CS of the recombinant proteins was evaluated by The of purified CS proteins was obtained and four recombinant CS proteins had with a at a to the predominant of in the protein of the that the all the four CS proteins have than of of the protein has a the was into and have been obtained by the CS from species K. 29: PubMed Scopus Google Scholar, B. P. Hoffman S.L. U. Lopez J.A. F. H. N. Mol. PubMed Scopus Google Scholar), which that the proteins are to obtain a of CS protein from in a of CS on HepG2 binding of the four CS proteins was evaluated on HepG2 cells. concentration of four CS proteins was added onto the HepG2 cells followed by the of was then to HepG2 cells in a P. falciparum CSP, PgCSP, the CS protein from malaria parasite P. gallinaceum, also showed binding onto these although its was lower in comparison to In contrast, and PyCSP from simian and rodent malaria respectively, showed reduced binding at the concentration of to HepG2 with on liver cells S. Hoffman S.L. J. Cell Biol. 1992; PubMed Scopus Google Scholar, U. Sinnis P. C. W. Takacs B. Nussenzweig V. J. Med. PubMed Scopus Google Scholar). possible for the binding of to HepG2 cells be the of a cell surface that by can as a on HepG2 cells for its binding was on liver cells of HepG2 cells were with an that heparin and in heparan and the binding of and was of and was on HepG2 that the receptor specificity is PfCSP, binding was also through liver cells of by the differences in binding capacity of the CSP from four species the human liver cells is a of maintaining the the of these proteins to for and prevent the invasion of HepG2 cells by P. falciparum sporozoites was P. falciparum sporozoites were from A. mosquitoes and were incubated with HepG2 cells in the or of of of the four HepG2 cells the in because sporozoites are to invade and of the sporozoite invasion by at the concentration and showed inhibition at the concentration the of the binding and showed significant sporozoite invasion inhibition which was to the invasion inhibition of In contrast, and PyCSP showed inhibition at the These that a difference in the liver cell invasion of CSP is directly involved in maintaining the species specificity of infection. of CS binding and invasion of liver sporozoites on liver cells. We have recently the binding affinity of and that the protein has the affinity for heparin D. McCutchan T.F. M.J. 2001; PubMed Scopus Google Scholar). The differences in the binding and sporozoite invasion inhibition of the four CSP proteins be to differences in binding affinity We investigated and the of CSP with heparin by surface plasmon albumin-heparin chip was by the amino in albumin to the carboxyl on the to a cell surface showed the affinity with significant binding at a concentration of At a concentration of 100 the general of the binding which that a significant to were for of the heparin and to the from The for was as and demonstrated a affinity for heparin with an of and of with heparin was only at with a binding affinity of PyCSP showed with heparin and its binding affinity was This that the differences in the binding affinity of these proteins the liver cell receptor are maintaining the specificity of malaria affinity of CSP from species to 10 in a Malaria sporozoite into a human by the bite of an infected the sporozoite stage is the in the parasite because a of the parasite into the its to the host liver and with a host receptor for invasion and that process of invasion in a natural to an infection. We investigated the role of CSP from four well characterized species of Plasmodium genus P. falciparum P. gallinaceum P. knowlesi and P. yoelii in the host specificity of malaria infection in P. falciparum The proteins were purified to and evaluated for their to bind to HepG2 a human cell and the invasion of P. falciparum sporozoites. PfCSP, a protein, to HepG2 cells and P. falciparum sporozoites from invading PgCSP, although an CSP, also to HepG2 cells and P. falciparum sporozoites from invading these cells with an to the these cells using cell surface a receptor specificity to In contrast, and PyCSP were in binding to these cells and showed low in sporozoite invasion inhibition Although these differences in binding and invasion inhibition were in on a cell they a in as non-natural host infections are rare. We the binding affinity of of the CSP heparin by SPR The the of binding and invasion because binding affinity of and PyCSP was 4,790- and respectively, in comparison to are of the CSP and and on the We have recently that the CSP to the liver cells through its amino terminus, and the of the binding domain is important for its D. Sacci J.B. de la Vega P. McCutchan T.F. J. Biol. Chem. 2002; 277: 7092-7098Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). the Plasmodium the amino terminus of the CSP (3McCutchan T.F. Kissinger J.C. Touray M.G. Rogers M.J. Li J. Sullivan M. Braga E.M. Krettli A.U. Miller L.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11889-11894Crossref PubMed Scopus (127) Google Scholar), although all the four CSP have a and a structural in heparin binding proteins J. A. M. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), we that changes during the course of have in significant differences in the of the binding without significant in the These changes have differences in binding the receptor to provide a mechanism for maintaining species specificity of infection. This mechanism to at the as of P. CSP with P. falciparum CSP in P. sporozoites in a in the salivary sporozoite in comparison to the type parasites R. R. F. A.A. Crisanti A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). using in with numerous parasite DNA and protein we and have that P. falciparum and P. gallinaceum parasites have from a the rodent and simian parasites have and (3McCutchan T.F. Kissinger J.C. Touray M.G. Rogers M.J. Li J. Sullivan M. Braga E.M. Krettli A.U. Miller L.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11889-11894Crossref PubMed Scopus (127) Google Scholar, A.P. D.G. McCutchan T.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A.A. W.E. A.A. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, D. Sullivan M. McCutchan T.F. Mol. Biochem. Parasitol. 2001; PubMed Scopus Google Scholar). The from study not only provide the of the between the P. gallinaceum and P. falciparum it also a to the of in the Plasmodium changes in the CSP between species in maintaining the the on its also be by maintaining differences in the of the liver cell receptor across species. It is known that of a species has a heparin from and the in the and Biochem. Biochem. PubMed Scopus Google Scholar, U. and Scholar). The of and parasite for maintaining species specificity along of the parasite has not been We of for help with We of for and with

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