Targeting Malaria Virulence and Remodeling Proteins to the Host Erythrocyte Matthias Marti,* Robert T. Good,* Melanie Rug, Ellen Knuepfer, Alan F. Cowman. To establish infection in the host, malaria parasites export remodeling and virulence proteins into the erythrocyte. These proteins can traverse a series of membranes, including the parasite membrane, the parasitophorous vacuole membrane, and the erythrocyte membrane. We show that a con- served pentameric sequence plays a central role in protein export into the host cell and predict the exported proteome in Plasmodium falciparum. We identified 400 putative erythrocyte-targeted proteins corresponding to È8% of all predicted genes, with 225 virulence proteins and a further 160 proteins likely to be involved in remodeling of the host erythrocyte. The conservation of this signal across Plasmodium species has implications for the development of new antimalarials. Species of the genus Plasmodium are obli- gate intracellular parasites of the phylum Apicomplexa that switch between an arthro- pod vector and a vertebrate host, where they undergo cycles of asexual reproduction in blood cells. Each year, several hundred mil- lion people become infected with P. falcipa- rum, which causes the most severe form of malaria in humans, and 1 to 2 million die from complications. Once the parasite is in the blood, its continuous asexual multiplica- tion inside erythrocytes is responsible for clinical symptoms of malaria and the associ- ated morbidity and mortality. Asexual stages reside in a parasitophorous vacuole, and they elaborate a membranous network in the erythrocyte cytoplasm, including Maurer_s clefts. Moreover, the surface of the infected erythrocyte membrane is also remodeled with electron-dense elevations called knobs. The 85- to 110-kD knob-associated histidine- rich protein (KAHRP) is present on the cyto- plasmic side of the knob structure (1), and the 200- to 300-kD antigen PfEMP1 is lo- cated on the exterior surface (2). PfEMP1 is encoded by È60 var genes per parasite (3), and monoallelic expression of variant forms of the protein is responsible for antigenic variation (4). The primary structure of PfEMP1 consists of a large N-terminal ecto- Fig. 1. Identification of a novel motif in exported P. falciparum proteins. Multiple sequence align- ment of the N-terminal portion of 10 exported P. falciparum proteins shows that gene struc- ture is conserved with a hydrophobic signal se- quence (see bracket; hy- drophobic amino acids are highlighted in blue) encoded in exon 1 (upper panel) and a highly vari- able sequence encoded in exon 2 (lower panel). However, a conserved pentameric motif is pres- ent 16 to 24 amino acids into exon 2, as highlighted in color. The sequence logo shows the conservation of this motif in 158 predicted P. falciparum proteins encoded by genes with the same structure, excluding the multiple gene families of var, rif, and Stevor (fig. S1A). The height of each letter is proportional to the frequency of amino acids in each position. The total letter height indicates the amount of information (bits) contained in the amino acids at that position. Amino acid color coding for both the alignment and sequence logo: red, basic; purple, acidic; blue, hydrophobic; green, polar amino acids. Numbers at the C terminus of the alignment (lower panel) indicate the extra amino acids not shown in the alignment. Abbrevia- tions for amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia. *These authors contributed equally to this work. .To whom correspondence should be addressed. E-mail: cowman@wehi.edu.au R EPORTS 10 DECEMBER 2004 VOL 306 SCIENCE www.sciencemag.org 1930