Members of the salivary gland surface protein (SGS) family are major immunogenic components of mosquito saliva

Abstract

Mosquitoes transmit Plasmodium and certain arboviruses during blood feeding, when they are injected along with saliva. Mosquito saliva interferes with the host's hemostasis and inflammation response and influences the transmission success of some pathogens. One family of mosquito salivary gland proteins, named SGS, is composed of large bacterial-type proteins that in Aedes aegypti were implicated as receptors for Plasmodium on the basal salivary gland surface. Here, we characterize the biology of two SGSs in the malaria mosquito, Anopheles gambiae, and demonstrate their involvement in blood feeding. Western blots and RT-PCR showed that Sgs4 and Sgs5 are produced exclusively in female salivary glands, that expression increases with age and after blood feeding, and that protein levels fluctuate in a circadian manner. Immunohistochemistry showed that SGSs are present in the acinar cells of the distal lateral lobes and in the salivary ducts of the proximal lobes. SDS-PAGE, Western blots, bite blots, and immunization via mosquito bites showed that SGSs are highly immunogenic and form major components of mosquito saliva. Last, Western and bioinformatic analyses suggest that SGSs are secreted via a non-classical pathway that involves cleavage into a 300-kDa soluble fragment and a smaller membrane-bound fragment. Combined, these data strongly suggest that SGSs play an important role in blood feeding. Together with their role in malaria transmission, we propose that SGSs could be used as markers of human exposure to mosquito bites and in the development of disease control strategies.

FIGURE 1.

Sgs4 and Sgs5 are produced specifically in the salivary glands of adult female A. gambiae, and their production is associated with blood feeding. A and B, conventional PCR (A) and real-time quantitative PCR (B) comparing sgs transcript levels in tissues from 5-day-old adult mosquitoes. sgs4 and sgs5 are only transcribed in the salivary glands. C and D, Western blots of tissue samples from 5-day-old adult A. gambiae, showing that Sgs4 and Sgs5 are only present in the salivary glands of female mosquitoes. E, Western blot of A. gambiae salivary glands at different times posteclosion, showing that SGS levels are minimal in newly emerged adults and peak around 10 days of age. F, Western blot of A. gambiae salivary glands at different times following blood feeding (bf) along with non-blood fed controls (nbf). Time 0 represents non-blood-fed 5-day-old mosquitoes, and these data show that blood feeding enhances SGS production. G, the same lane of a Western blot stripped and reprobed with each of the antibodies produced against recombinant SGSs, showing their binding specificities and that Sgs5 is slightly more massive than Sgs4. Western blots in C–F were probed using α-SGS_RHS (which recognizes both Sgs4 and Sgs5) and were performed after electrophoresing samples in 4% Tris/glycine SDS-polyacrylamide gels. For an explanation of SGS mass size, see Fig. 6.

FIGURE 2.

Sgs4 and Sgs5 are present in the distal-lateral acinar cells and the salivary ducts. A–I, immunohistochemistry of female salivary glands using α-Sgs4 (A–C), α-Sgs5 (D–F), or preimmune antibodies (G–I; from a rabbit later immunized with Sgs4). Serial micrographs show imaging through differential interference contrast (DIC) (A, D, and G), Texas Red (B, E, and H; antibody labeling), and Hoechst 33342 (C, F, and I; DNA stain) channels. J, close-up of E showing labeling of the acinar cells of the distal lateral lobes and the salivary ducts of the proximal lobes. K, Western blot of disarticulated salivary glands, confirming that Sgs4 (bottom bands) and Sgs5 (top bands) are present only in the lateral lobes and showing that both are slightly less massive in the proximal lateral lobes than they are in the distal lateral lobes (4% Tris/glycine SDS-PAGE).

FIGURE 3.

Sgs4 and Sgs5 are the only prevalent proteins in their size range found in mosquito salivary glands and form major constituents of mosquito saliva. A, saliva collection in A. gambiae (top) and A. aegypti (bottom). Droplet formation can be seen in both images, with the A. aegypti image showing saliva being ejected from the hypopharynx (arrowhead). B, Western blot of A. gambiae saliva collected following pilocarpine treatment, showing that Sgs4 and Sgs5 are a component of mosquito saliva. C, bite blots probed with α-SGS_RHS (top) or 3 times the amount of preimmune serum (bottom), showing that SGSs are released with the saliva during probing. D, 4% Tris/glycine SDS-polyacrylamide gel stained with zinc-imidazole, showing that Sgs4 and Sgs5 are the only proteins in their size range present in mosquito salivary glands. Note that when glands were immediately immersed in denaturing LDS buffer, their size was ∼10 kDa more massive than when they were incubated in Nonidet P-40 detergent prior to denaturation. Sgs4 and -5 in the saliva match the smaller SGS forms seen in whole salivary glands initially soaked in non-denaturing buffer. E, Coomassie-stained 4–12% SDS-PAGE of salivary glands and saliva from A. gambiae and A. aegypti. The combined SGS bands are marked by an arrowhead. F, densitogram of select lanes from the gel in D, showing relative protein content. In both salivary glands and saliva, SGSs form one of three major peaks in A. gambiae and form the major peak in A. aegypti. Peak identities, other than SGS, are inferred using data from a previous study (45).

FIGURE 4.

SGSs are one of two major immunogenic components of A. gambiae saliva. Shown are Western blots of salivary glands (sal glands) and carcasses excluding salivary glands (carcass) electrophoresed in 4–12% BisTris gels and probed with the following (from left to right): sera from non-immune mice (non-Imm; 1:80 dilution), α-SGS_RHS, sera from mice exposed to weekly mosquito bites for 6 weeks (Imm 1–3; 1:500 dilution), and sera from mice exposed to weekly mosquito bites for 3 weeks (Imm 4–5; 1:500 dilution). Results are shown for extracts run under denaturing (D only) and denaturing plus reducing (D + R) conditions (for sera shown only under denaturing conditions, reducing yielded identical results). Overall, the major immunogenic components of mosquito saliva are SGSs (arrowhead) and another protein that migrates at 110 or 55 kDa, depending on whether it has been reduced.

FIGURE 5.

SGS levels fluctuate within 24-h periods and are most prevalent during the late afternoon and early evening. A, surface plot of relative combined band intensities of both Sgs4 and Sgs5 from Western (top) and Coomassie (bottom) analyses of salivary glands from mosquitoes of the same cohort, at different ages, collected at +1 h into the light and dark cycles. B and C, Western blots of salivary glands (SG) extracted from 1–2- and 5–6-day-old mosquitoes showing SGS levels over a 24-h period.

FIGURE 6.

The masses of Sgs4 and Sgs5, along with bioinformatic analyses, suggest that they are cleaved into a 300-kDa soluble N-terminal fragment and a smaller, membrane-bound fragment. A, Western blots of 10 female salivary glands run on 4–12% BisTris (left) or 3–8% Tris/acetate (right) SDS-polyacrylamide gels and probed with α-SGS_RHS. A single band is seen at around 300 kDa that has been shown to correspond to a large fragment of Sgs4 and -5 (one band seen here due to lack of resolution on these gels). A doublet of ∼47 kDa is also observed. B, plot of observed protein migration distances versus two sets of standards on various gel types. Gray symbols represent SGS band migration distances, whereas black symbols and trend lines represent the observed migration of different standards. In all cases, SGS mass is calculated to be 300 ± 10 kDa. C and D, graphical representation of Sgs4 (C) and Sgs5 (D) showing the locations of the putative RHS domains, transmembrane regions (TM), PAPs, caspase-1 cleavage sites (Casp-1), and the regions that the anti-SGS antibodies recognize. Below these gene diagrams, hydrophobicity plots show that cleavage at the PAP sites would result in an N-terminal fragment of ∼300 kDa and a highly hydrophobic membrane-bound fragment of ∼85 kDa (higher values represent higher hydrophobicity).