Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11889-11894, October 1996 Medical Sciences Comparison of circumsporozoite proteins from avian and mammalian malarias: Biological and phylogenetic implications (plasmodium/apicomplexa/evolution/sporozoite invasion/cell adhesion motif) THOMAS F. MCCUTCHAN*t, JESSICA C. KISSINGER*, MUSA G. TOURAY*4, M. JOHN ROGERS*, JUN LI*, MARGERY SULLIVAN*, ERIKA M. BRAGA§, ANTONIANA U. KRETrLI§, AND Louis H. MILLER* *Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425; and §Centro de Pesquisas Rend Rachou, FIO Cruz and Federal University of Minas Gerais, Belo Horizonte 30 190-002, Minas Gerais, Brazil Contributed by Louis H. Miller, July 31, 1996 ABSTRACT The circumsporozoite (CS) protein of ma- among Plasmodium species, there are a few fundamental laria parasites (Plasmodium) covers the surface of sporozoites differences (7). For example, sporozoites of avian malaria that invade hepatocytes in mammalian hosts and macro- parasites develop predominantly in culicine mosquitoes, while phages in avian hosts. CS genes have been characterized from the primate malarias use anopheline vectors. Sporozoites of many Plasmodium that infect mammals; two domains of the avian parasites infect macrophages; sporozoites of primate corresponding proteins, identified initially by their conserva- malarias infect hepatocytes. These biological differences may tion (region I and region II), have been implicated in binding be reflected in differences in the functional domains of the CS to hepatocytes. The CS gene from the avian parasite Plasmo- protein, an important ligand on the sporozoite surface. dium gallinaceum was characterized to compare these func- The sequence of the CS gene has been determined for a tional domains to those ofmammalian Plasmodium and for the number of species that infect mammals but no sequence has study ofPlasmodium evolution. The P. gallinaceum protein has been determined for an avian malaria parasite. Herein we the characteristics ofCS proteins, including a secretory signal compare the sequence of the CS protein gene of an avian sequence, central repeat region, regions of charged amino parasite, Plasmodium gallinaceum, with corresponding se- acids, and an anchor sequence. Comparison with CS signal quences from mammalian parasites. Analysis of the CS protein sequences reveals four distinct groupings, with P. gaUinaceum genes supports the evolutionary relationship between avian most closely related to the human malaria Plasmodium fakci- parasites and P. falciparum, despite similarity of the biology of parum. The 5-amino acid sequence designated region I, which P. falciparum sporozoites to other mammalian Plasmodium is identical in all mammalian CS and implicated in hepatocyte species. In addition we focus on comparisons of regions I and invasion, is different in the avian protein. The P. gaUlinaceum II that have been implicated in hepatocyte invasion for mam- repeat region consists of 9-amino acid repeats with the malian Plasmodium species. consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region 1I-plus, which is associated with MATERIALS AND METHODS targeting the invasion of liver cells, is also conserved in the Parasites. P. gallinaceum was maintained in chickens and avian protein. Phylogenetic analysis of the aligned Plasmo- infected Aedes aegypti maintained at 28°C and 80% relative dium CS sequences yields a tree with a topology similar to the humidity. The infection of the mosquitoes was checked at day one obtained using sequence data from the small subunit 5 for the presence of oocysts and at day 10 for the presence of rRNA gene. The phylogeny using the CS gene supports the sporozoites in the salivary gland. proposal that the human malaria P.falciparum is significantly Preparation of RNA and DNA. RNA was isolated from 107 more related to avian parasites than to other parasites infect- sporozoites purified from the salivary glands from approxi- ing mammals, although the biology of sporozoite invasion is mately 1000 P. gallinaceum-infected Ae. aegypti mosquitoes as different between the avian and mammalian species. described (8). Total RNA was purified from the sporozoites by organic extraction (9). DNA was purified from 50 ml of P. Comparisons of homologous proteins from distantly related gallinaceum-infected chicken blood that had a parasitemia of Plasmodium species have suggested domains retained for 70%. Total nucleic acids were isolated by organic extraction of function. Two examples are conserved regions of the circum- the lysate of P. gallinaceum-infected erythrocytes produced by sporozoite (CS) protein of sporozoites (1) and the conserved SDS/proteinase K treatment (9). P. gallinaceum DNA was regions of a family of erythrocyte binding proteins on mero- separated from host DNA and other nucleic acids by Hoechst zoites (2). Sporozoites are the infective form of malaria dye/CsCl gradient centrifugation as described (9). parasites that are inoculated by mosquitoes into the vertebrate Competitive Indirect Immunofluorescence (IIF). The synthetic host. The merozoite is the parasite stage that invades eryth- peptide 3893 (GGVQPAGGNGGVQPAGGNGGVQPAGGN- rocytes. A comparison of CS protein sequences from Plasmo- amide) corresponding to a portion of the repeat region of the CS dium falciparum and the distantly related parasites, Plasmo- of P. gallinaceum was used at different concentrations (1 ,ug/ml, dium knowlesi and Plasmodium vivax, identified domains (re- 10 ,ug/ml, 25 ,ug/ml, 50 ,g/ml, and 100 ,ug/ml) to compete for gions I and II) that were shown to be conserved (1) and there binding of the mAbs N2H1D5 and NSG3H6 to P. gallinaceum is evidence suggesting that these domains function in sporo- zoite binding to hepatocytes (3, 4). A similar approach iden- Abbreviation: CS, circumsporozoite. tified domains on a family of merozoite proteins that were Data deposition: The sequence reported in this paper has been demonstrated to bind erythrocytes (5, 6). deposited in the GenBank data base (accession no. U65959). Although the processes whereby the sporozoite develops tTo whom reprint requests should be addressed at: Growth and and infects the invertebrate and vertebrate hosts are similar Development Section, Laboratory of Parasitic Diseases Building 4, Room B1-28, 9000 Rockville Pike, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD The publication costs of this article were defrayed in part by page charge 20892-0425. e-mail: [email protected]. payment. This article must therefore be hereby marked "advertisement" in tPresent address: Department of Rheumatology, University Hospital accordance with 18 U.S.C. §1734 solely to indicate this fact. of Lausanne, 1011 Lausanne, Switzerland. 11889 Downloaded by guest on September 29, 2021 11890 Medical Sciences: McCutchan et al. Proc. Natl. Acad. Sci. USA 93 (1996) sporozoites (10). A nonrelated peptide (NANPNVDPNANP) repeat sequence of the CS protein of P. gallinaceum. After corresponding to the repeat of the CS of P. falciparum was used incubation, the antibody did not bind to sporozoites as de- as a control in the competition experiments at a concentration of tected by the IIF test. A control peptide derived from the 100 ,ug/ml. Dilutions of the mAbs were incubated with the repeat of the CS protein of P. falciparum did not inhibit IIF at different concentrations of peptides at various dilutions for 3 h at the same concentration. The finding that a peptide derived 37°C. The mixture was incubated with air dried sporozoites on from the cloned repeat sequence blocks the binding of an slides for 30 min at 37°C. Slides were washed and incubated with anti-CS mAb to P. gallinaceum sporozoites is consistent with fluorescein-conjugated anti-mouse IgG (<-chain specific, Sigma) fact that the repeat is from a portion of the CS gene. for 30 min at 37°C. The washed slides were examined for Isolation of the Complete CS Protein Gene from P. galli- immunofluorescence. Each dilution and antigen concentration naceum. The CS protein gene was isolated from a genomic was coded and read blind. library of P. gallinaceum DNA. Genomic DNA used for the Isolation and Sequence Analysis of the CS Protein Gene. construction of the P. gallinaceum library was purified from cDNA was made from RNA isolated from sporozoites using total DNA isolated from P. gallinaceum-infected erythrocytes the oligo(dT) priming as described (11). A recombinant cDNA from a chicken. Most of this material was chicken DNA library was made in A-ZAP II (Stratagene) as described by the because avian red blood cells are nucleated. Parasite DNA was manufacturer. The mAb-specific for the P. gallinaceum cir- separated from the host DNA by CsCl centrifugation in the cumsporozoite protein N5G3H6 (10) was used to screen the presence of Hoechst dye as described (9). DNA fragments, cDNA library for phage containing the CS protein gene (12). averaging approximately 10 kb in size, that resulted from A genomic library in A ZAP Express (Stratagene) was pre- partial digestion of parasite DNA with the restriction enzyme pared as described by the manufacturer, carrying fragments of Sau3AI were used to construct a genomic library. The library DNA obtained from a partial digest of P. gallinaceum DNA with was screened for CS protein gene-related fragments, using a the restriction endonuclease Sau3AI. Recombinant phage that radiolabeled probe homologous to the repeated