Anopheles stephensi salivary glands bear receptors for region I of the circumsporozoite protein of Plasmodium falciparum

Abstract

In the mosquito, Plasmodium sporozoites rupture from oocysts found on the midgut wall, circulate in the hemolymph and invade salivary glands where they wait to be injected into a vertebrate host during a bloodmeal. The mechanisms by which sporozoites specifically attach to and invade salivary glands are not known but evidence suggests that it is a receptor-mediated process. Here we show that the major surface protein of sporozoites, the circumsporozoite protein (CS), binds preferentially to salivary glands when compared to other organs exposed to the circulating hemolymph. In addition, we show that a peptide encompassing region I, a highly conserved sequence found in all rodent and primate Plasmodium CS proteins, inhibits binding of CS to mosquito salivary glands.

Fig. 1

(A) Schematic representation of CS showing the centrally located species-specific repeats; N-terminal and C-terminal to the repeats are region I and region II-plus, respectively. These regions contain motifs that are highly conserved in CS proteins from all species of Plasmodium. The amino acid sequences of these conserved regions from P. falciparum CS are shown. (B) Peptides used in this study were chosen to represent the three regions of P. falciparum CS shown above, namely the repeats, region I and region II-plus. Overlapping peptides from the region immediately N-terminal to the repeats were used to determine which residues from this region were required for inhibition of CS binding to salivary glands. The control peptide represents a region 40 residues upstream from region I. Bold letters indicate the positively-charged amino acids.

Fig. 2

CS binds to mosquito salivary glands and not to other organs exposed to the hemolymph. Organs are dissected from mosquitoes, fixed and stained with recombinant CS protein (A, C, D, E) or Pfs25 (B). The organs are washed and binding of the protein is revealed with a mAb specific for either CS or Pfs25, followed by anti-mouse Ig conjugated to β-galactosidase and X-gal. Protein binding is indicated by a blue color. As shown, salivary glands (A) bind CS where as the midgut (C), Malpighian tubules (D) and ovary (E) do not. Salivary glands incubated with a control protein, Pfs25 (B) do not turn blue. This experiment was repeated 6 times and a representative experiment is shown. Bar=0.1 mm.

Fig. 3

Male salivary glands bind CS. Salivary glands were dissected from male and female (not shown) mosquitoes, fixed and incubated with CS. Binding was revealed with mAb 2A10 followed by anti-mouse Ig conjugated to β-galactosidase and X-gal. Shown is a representative male salivary gland. Two experiments were performed, each with four male salivary glands, with identical results. Bar =0.1 mm.

Fig. 4

CS binds specifically to salivary glands after injection into the mosquito's hemocoele. Approximately 0.9 μg of CS was injected into the hemocoele of ten mosquitoes. After 1 h the organs were dissected from the mosquitoes, mounted, fixed and incubated with mAb 2A10 followed by anti-mouse Ig conjugated to β-galactosidase and X-gal. CS binding is indicated by the blue color. Salivary glands (A) bind CS where as midguts and Malpighian tubules (B) and ovaries (C) do not. The blue in panel C is on the external cuticle of the mosquito and probably represents nonspecific binding of one of the antibodies. Shown are representative organs from one experiment. This experiment was repeated with identical results. Bar=0.1 mm.

Fig. 5

Peptides encompassing region I inhibit CS binding to salivary glands. Mosquito salivary glands were fixed, blocked and preincubated with either no peptide (A), repeat peptide (B), control peptide (C), region II-plus (D) or region I-pep3 (E). The peptide solution was removed and the glands were then incubated with CS in the presence of the peptide, washed and CS binding was revealed as stated above. Bar=0.1 mm.