Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites

Abstract

Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.

Figure 1

TRAP cytoplasmic tail mutants and gene targeting strategy. (A) Schematic representation of the TRAP protein and amino acid sequences (one letter code), of the cytoplasmic tails of Plasmodium berghei TRAP and TRAP recombinants. CoTRAP indicates the residues conserved in at least five of six plasmodial TRAP sequenced to date (+ indicates E or D). In the TRAP recombinants shown below, the amino acid substitutions or heterologous exchange are underlined. Hatched boxes represent the leader sequence and the transmembrane domain. (B) Generation of the TRAP mutations by insertion mutagenesis in P. berghei. The wild-type (Wt), single-copy TRAP is targeted with an insertion plasmid whose targeting sequence contains the deletion/mutation (*) and is linearized upstream from the mutation (crossover); thin lines, TRAP untranslated region; open box, TRAP coding region; thick lines, bacterial plasmid and DHFR-TS resistance cassette. The recombinant locus (Rec. locus) expected to result from plasmid integration that preserves the mutation is shown. Below are the restriction maps of the 3′ end of the TRAP gene in the first duplicate of the recombinant clones. The nucleotide and the amino acid sequences tagging the mutations are indicated, and the corresponding restriction sites italicized in the sequence and the map. P, PstI; Pa, PacI; X, XbaI; B, BamHI; N, NheI. (C) The first TRAP duplicate of recombinant parasites were amplified by PCR using primer O1 and T7, which annealed upstream from the region of homology and to the vector sequence, respectively, and digested with restriction enzymes. See the restriction maps and mutation-tagging restriction sites in B.

Figure 2

TRAP truncates are correctly expressed and targeted to the sporozoite surface. (A) Western blot analysis of midgut sporozoite extracts. Sporozoites were collected from midguts of mosquitoes dissected at day 16 postfeeding. Crude extracts from ∼10⁵ sporozoites of each population were separated by SDS-PAGE and transferred to a membrane that was probed successively with polyclonal antibodies to the TRAP repeats (Rep), the TRAP cytoplasmic tail (Tail), and Mabs 3D11 to the repeats of the circumsporozoite protein (CS). (B) Immunofluorescence assays of sporozoites collected from the hemolymph of infected mosquitoes. Mosquitoes infected with WT P. berghei, the INCO, TΔS, or TΔL clones, or the TRAP knockout (KO) REP line (Sultan et al. 1997) were dissected at days 14–16 postfeeding, permeabilized or processed live, and stained using antibodies to the TRAP repeats (Rep.) or cytoplasmic tail (Tail). Typical fluorescence patterns are shown. A small proportion of live WT, INCO, TΔS, and TΔL sporozoites displayed a cap-like fluorescence frequently limited to one sporozoite pole. WT live sporozoites occasionally displayed a bright, ring-like pattern around a portion of the sporozoite body. (C) Immunolocalization of WT TRAP and of the TΔL truncate on ultrathin sections of the corresponding sporozoites using antibodies to the TRAP repeats (Rep.) or cytoplasmic tail (Tail). Using TRAP repeat antibodies, full-length TRAP and the TΔL truncate showed an identical distribution, frequently over most of the sporozoite length and associated with electron-dense micronemes.

Figure 3

Gliding phenotypes of wild-type and mutant sporozoites. (A) Time-lapse micrographs of sporozoites gliding on uncoated glass slides. Sporozoites were collected from the hemolymph of infected mosquitoes at days 14–16 postfeeding and kept 2 h in 3% BSA at 4°C before microscopic examination. The parasite population and a schematic representation of the TRAP cytoplasmic tail are shown at left. Wild-type, INCO, and TMIC gliding sporozoites described circular patterns and completed one circle in ∼20 s. ACID, TRYP, and TΔS gliding sporozoites described a “pendulum” movement covering one third of a circle and going back to the starting position, repeated several times (shown here with an ACID sporozoite). Numbers indicate seconds. (B) Immunofluorescence using TRAP antirepeat antibodies of trails left behind WT sporozoites gliding over glass slides. Note in the top panels (phase + immunofluorescence at left) the presence of a “ring” of TRAP around the middle portion of the sporozoite. Unlike the glycosylphosphatidylinositol-anchored CS protein of Plasmodium sporozoites that is uniformly deposited in the trail (Stewart and Vanderberg 1988), TRAP found in the trail displays a periodic intensity pattern reminiscent of the nonuniform expression of TRAP on the sporozoite surface (see also Fig. 2 B).