MAEBL is essential for malarial sporozoite infection of the mosquito salivary gland

Abstract

Malarial sporozoites mature in the oocysts formed in the mosquito midgut wall and then selectively invade the salivary glands, where they wait to be transmitted to the vertebrate host via mosquito bite. Invasion into the salivary gland has been thought to be mediated by specific ligand-receptor interactions, but the molecules involved in these interactions remain unknown. MAEBL is a single transmembrane-like protein that is structurally related to merozoite adhesive proteins. We found MAEBL of the rodent malaria parasite, Plasmodium berghei, to be specifically produced by the sporozoites in the oocyst and localized in their micronemes, which are secretory organelles involved in malarial parasite invasion into the host cell. A targeted disruption experiment of the P. berghei MAEBL gene revealed that it was essential for sporozoite infection of the salivary gland and was involved in the attachment to the salivary gland surface. In contrast, the disruption of the MAEBL gene did not affect sporozoite motility in vitro nor infectivity to the vertebrate host. These results suggest that P. berghei MAEBL is a sporozoite attachment protein that participates in specific binding to and infection of the mosquito salivary gland.

Figure 1.

Stage-specific production of PbMAEBL and localization in the microneme. (A) PbMAEBL is specifically produced by sporozoites in the oocyst. Immunofluorescent staining was performed in sporozoites collected from the midgut (left), sporozoites collected from the salivary glands (middle), and schizonts (right). Parasites were fixed with acetone for 2 min, incubated with polyclonal rabbit antibodies against a synthetic peptide corresponding to the amino acid sequence of the repeat region of PbMAEBL, and then conjugated with FITC with second antibodies. Oocyst sporozoites were stained mainly in the anterior half, which is a typical pattern for microneme staining. The corresponding phase-contrast image or DAPI staining images are shown below. (B) PbMAEBL is localized in micronemes of oocyst sporozoites. Immunoelectron microscopy was performed in the infected mosquito midgut (left) and cultured mature merozoites (right). The midgut was dissected 18 d after an infective blood meal. Merozoites were purified from infected blood after culture for 16 h in vitro. They were fixed and embedded in LR Gold resin. Ultrathin sections were incubated with the same antibodies as used in A and then conjugated with gold particles (15 nm) with second antibodies. Antibodies were attached to the micronemes of oocyst sporozoites but not to the rhoptories (R) and showed no specific affinities to merozoites. D, dense body; bars, 0.5 μm. (C) PbMAEBL was detected as an ∼200-kD protein only in the sporozoites. Western blot analysis of PbMAEBL was performed in lysates of sporozoites in the oocysts (Sp) and merozoites (Me). Sporozoites (10⁶) and merozoites (3 × 10⁶) were lysed in the SDS-PAGE sample buffer containing 0.5% SDS, separated on a 8% acrylamide gel, and analyzed by immunoblotting with the same antibodies as in (A). PbMAEBL was detected as a major ∼200-kD protein band (closed arrowhead) with a minor band of its putative degraded product (open arrowhead).

Figure 2.

Targeted disruption of the PbMAEBL and PbTRAP gene. (A) Schematic representation of the targeted disruption procedure of the PbMAEBL gene. The targeting vector (top) containing a selectable marker gene is integrated into the PbMAEBL gene locus (middle) by double crossover. This recombination event results in the disruption of the PbMAEBL gene locus (bottom) and confers pyrimethamine resistance to PbMAEBL gene-disrupted parasites. The probe used for Southern blot analysis (C, left) is indicated by a solid bar. The recognition sites for EcoRV and the fragment size excised by this restriction enzyme are indicated. (B) Schematic representation of the targeted disruption of the PbTRAP gene. The procedure of TRAP gene disruption is the same as in A. The probe used for Southern blot analysis in C (right) is indicated by a solid bar. The recognition sites for EcoT22i and the fragment size excised by this restriction enzyme are indicated. (C) Southern blot analysis of PbMAEBL (left) and PbTRAP gene-disrupted (right) parasites. DNAs were isolated from the intraerythrocytic stages of respective parasite populations as indicated over each lane. They were digested with the restriction enzyme that is indicated over each panel and hybridized with the probe to the targeted gene that is indicated under each panel. By the integration of the maebl targeting construct, the size of the detected restricted fragments increased from 0.7 to 5.0 kbp (left). By the integration of the trap targeting construct, the size of the detected restricted fragments increased from 1.6 to 1.9 kbp (right). (D) Immunofluorescence microscopy of the wild-type maebl(+) 1 (WT, left) and the PbMAEBL gene-disrupted maebl(−) 1 (KO, right) sporozoite. Sporozoites were collected from mosquito midguts 18 d after an infective blood meal. They were fixed with acetone for 2 min, incubated with polyclonal antibodies against the PbMAEBL repeat region, and then conjugated with FITC with second antibodies. maebl(−) 1 sporozoites were not stained. The same results were obtained in all parasite populations. Arrowheads, the apical end of each sporozoite; ×200. (E) Western blot analysis of PbMAEBL in maebl(+) 1 (WT) and maebl(−) 1 (KO) sporozoites. The immunoblot analysis was performed with the same antibodies as used in D. Protein bands in the wild-type parasites disappeared in the knockout parasite.

Figure 3.

PbMAEBL gene-disrupted parasites, in contrast to PbTRAP-disrupted parasites, showed normal gliding motility in vitro. Sporozoites were collected from the hemolymph of mosquitoes infected with the respective parasite populations 24 d after an infective blood meal. They were kept for 2 h in 3% BSA at 4°C and then gliding on an uncoated microscope slide was observed under a phase-contrast microscope. Time lapse micrographs were recorded at 4-s intervals. ×200.