Anopheles salivary gland proteomes from major malaria vectors

Abstract

Background: Antibody responses against Anopheles salivary proteins can indicate individual exposure to bites of malaria vectors. The extent to which these salivary proteins are species-specific is not entirely resolved. Thus, a better knowledge of the diversity among salivary protein repertoires from various malaria vector species is necessary to select relevant genus-, subgenus- and/or species-specific salivary antigens. Such antigens could be used for quantitative (mosquito density) and qualitative (mosquito species) immunological evaluation of malaria vectors/host contact. In this study, salivary gland protein repertoires (sialomes) from several Anopheles species were compared using in silico analysis and proteomics. The antigenic diversity of salivary gland proteins among different Anopheles species was also examined.

Results: In silico analysis of secreted salivary gland protein sequences retrieved from an NCBInr database of six Anopheles species belonging to the Cellia subgenus (An. gambiae, An. arabiensis, An. stephensi and An. funestus) and Nyssorhynchus subgenus (An. albimanus and An. darlingi) displayed a higher degree of similarity compared to salivary proteins from closely related Anopheles species. Additionally, computational hierarchical clustering allowed identification of genus-, subgenus- and species-specific salivary proteins. Proteomic and immunoblot analyses performed on salivary gland extracts from four Anopheles species (An. gambiae, An. arabiensis, An. stephensi and An. albimanus) indicated that heterogeneity of the salivary proteome and antigenic proteins was lower among closely related anopheline species and increased with phylogenetic distance.

Conclusion: This is the first report on the diversity of the salivary protein repertoire among species from the Anopheles genus at the protein level. This work demonstrates that a molecular diversity is exhibited among salivary proteins from closely related species despite their common pharmacological activities. The involvement of these proteins as antigenic candidates for genus-, subgenus- or species-specific immunological evaluation of individual exposure to Anopheles bites is discussed.

Figure 1

Salivary protein sequence comparisons among six anopheline species. (A) Phylogenetic relationships among six Anopheles species using the cytochrome oxidase subunit II (COII) protein sequences. Evolutionary analyses were conducted in MEGA5 [62]. The Aedes aegypti sequence was taken as an outgroup. The tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. (B) Clustal alignment. The numbers into brackets in the sequence titles indicate the NCBI accession number. The level of sequence identity is graphically represented above sequences alignment. (C) Average normalised BLAST scores ± standard deviations (numbers in bold into square brackets) and percentage identities (numbers in italic into brackets) between local alignments of secreted salivary proteins pertaining to sialomes from different Anopheles species. Pairwise protein-protein sequence comparisons were performed using “BLAST 2 Sequences” [63] (q.v. Additional file 1). This analysis of divergence among secreted salivary protein repertoires was carried out using all protein sequences from each Anopheles species matching at least one other salivary protein in another species at 40% identity (q.v. Additional file 2). The number of secreted salivary proteins used in each species is indicated into brackets.

Figure 2

Hierarchical clustering of secreted salivary protein sequences from Anopheles. Three clustering steps were performed sequentially at different similarity thresholds (≥ 90%, ≥ 70% and ≥ 40% identity), producing a hierarchical structure. The repartition of proteins from the Anopheles species into clusters of more than 2 protein sequences are proportionally represented by stacked bars and non-redundant (NR) protein sequences (i.e., sequences that were not clustered with other sequences over a specified similarity threshold) by pie charts. The cluster numbers indicated on the left side of the stacked bars correspond to protein clusters listed in Additional file 2. A total of 71, 5, 44, 30, 5 and 117 secreted salivary protein sequences were recovered from the NCBInr online database for An. gambiae, An. arabiensis, An. stephensi, An. funestus, An. albimanus and An. darlingi, respectively. The correspondence between the number of proteins in a cluster and length of stacked bars is indicated as well as the correspondence between the colours and each Anopheles species.

Figure 3

Salivary gland protein profiles among four Anopheles species. Salivary gland proteins collected from An. gambiae, An. arabiensis, An. stephensi and An. albimanus were separated on 12% SDS-PAGE gels and stained with Sypro Ruby. The Anopheles species, corresponding to each protein track, are indicated at the top of the gel. Standard molecular masses are indicated on the left side. (B) Densitometric protein profiles of salivary gland proteins from the four Anopheles species are presented. Species are indicated by the same colour at the top of each immunoblot profile. MW, molecular weight; kDa, kiloDalton; A.U., arbitrary units; Rf, relative front of migration.

Figure 4

Venn diagrams indicating the amount of secreted salivary proteins identified in four Anopheles species. The amount of putative secreted proteins identified by MS in An. gambiae, An. arabiensis, An. stephensi and An. albimanus SGEs was represented at each taxonomic level (q.v., Figure 1A, 1B). The percentage of proteins identified is indicated in bold with the corresponding number of protein in brackets.

Figure 5

Singularity of IgG immune profiles among the Anopheles species. Fifteen micrograms of salivary gland extracts from An. gambiae (1), An. arabiensis (2), An. stephensi (3) and An. albimanus (4) labelled with Cyanine 5, were loaded and separated by 12% SDS-PAGE. (A) IgG immune profiles from pooled sera from 5 Senegalese individuals exposed to An. gambiae s.l. and An. funestus were tested by immunoblotting experiments. (B) Normalised densitometric IgG profiles were represented for the four Anopheles species. Species are indicated by the same colour at the top of each immunoblot profile. Molecular weights of the antigenic bands are indicated and corresponding gel bands are presented into brackets. (C) Protein profiles of whole protein present in SGEs from each mosquito species were scanned at the Cy5 wavelength before blotting. The numbers correspond to antigenic protein bands excised for mass spectrometry identification (Additional file 5). MW, molecular weight; kDa, kiloDalton; Rf, relative front of migration; A.U., Arbitrary Unit.

Figure 6

Schematic representation of the identified antigenic protein bands in salivary gland extracts from four Anopheles species. Secreted salivary proteins identified in antigenic bands (q.v., Additional file 5) are indicated with their corresponding species into squared brackets and their molecular weights. No protein was identified in antigenic bands represented by dotted lines. The percentage identity between two protein sequences was either retrieved from the in silico analysis (Additional file 2) or from analysis of “BLAST 2 Sequences”. Coloured numbers correspond to protein bands from the gel from the Figure 5C. MW, molecular weight; AGA, An. gambiae; AAR. An. arabiensis, AST, An. stephensi; AAL, An. albimanus .