An insight into the sialome of Glossina morsitans morsitans

Abstract

Background: Blood feeding evolved independently in worms, arthropods and mammals. Among the adaptations to this peculiar diet, these animals developed an armament of salivary molecules that disarm their host's anti-bleeding defenses (hemostasis), inflammatory and immune reactions. Recent sialotranscriptome analyses (from the Greek sialo = saliva) of blood feeding insects and ticks have revealed that the saliva contains hundreds of polypeptides, many unique to their genus or family. Adult tsetse flies feed exclusively on vertebrate blood and are important vectors of human and animal diseases. Thus far, only limited information exists regarding the Glossina sialome, or any other fly belonging to the Hippoboscidae.

Results: As part of the effort to sequence the genome of Glossina morsitans morsitans, several organ specific, high quality normalized cDNA libraries have been constructed, from which over 20,000 ESTs from an adult salivary gland library were sequenced. These ESTs have been assembled using previously described ESTs from the fat body and midgut libraries of the same fly, thus totaling 62,251 ESTs, which have been assembled into 16,743 clusters (8,506 of which had one or more EST from the salivary gland library). Coding sequences were obtained for 2,509 novel proteins, 1,792 of which had at least one EST expressed in the salivary glands. Despite library normalization, 59 transcripts were overrepresented in the salivary library indicating high levels of expression. This work presents a detailed analysis of the salivary protein families identified. Protein expression was confirmed by 2D gel electrophoresis, enzymatic digestion and mass spectrometry. Concurrently, an initial attempt to determine the immunogenic properties of selected salivary proteins was undertaken.

Conclusions: The sialome of G. m. morsitans contains over 250 proteins that are possibly associated with blood feeding. This set includes alleles of previously described gene products, reveals new evidence that several salivary proteins are multigenic and identifies at least seven new polypeptide families unique to Glossina. Most of these proteins have no known function and thus, provide a discovery platform for the identification of novel pharmacologically active compounds, innovative vector-based vaccine targets, and immunological markers of vector exposure.

Figure 1

Dendrogram of the Glossina morsitans morsitans salivary endonuclease-like proteins with the Culex quinquefasciatus salivary endonuclease included as an outgroup. The G. m. morsitans sequences are indicated by GM-X where X is the number shown in additional file 2. The remaining sequences derived from the National Center for Biotechnology Information (NCBI) are represented by five letters followed by the NCBI gi| accession number. The five letters are taken from the first three letters of the genus and the first two letters from the species name. The protein sequences were aligned by the Clustal program [176], and the dendrogram was done with the Mega package [178] after 10,000 bootstraps with the neighbor joining (NJ) algorithm. Bootstrap values above 75% are shown in the nodes. The bar at the bottom represents 20% amino acid substitution. The roman numerals indicate clades discussed in the text.

Figure 2

Alignment of the active center region of Culex quinquefasciatus endonuclease with Glossina morsitans morsitans proteins of the same family. The ten amino acids making contact to substrate, based on the Serratia marcescens crystal structure, are shown in turquoise background for the Culex and Glossina sequences. Note the absence of three conserved residues in the tsetse proteins. Other conserved residues are marked in yellow background.

Figure 3

Alignment of members of the 5' nucleotidase family from D. pseudoobscura, Bos taurus, Rattus rattus, Glossina morsitans morsitans and the horse fly Chrysops spp. The numbers following the species abbreviations indicate the NCBI accession number for each protein. Notice, in the haematophagous dipteran sequences, the absence of the carboxyterminal region where the glycophophatidylinositol anchor normally attaches (indicated by the blue box). Symbols above the alignment indicate standard ClustalW nomenclature: (*) identity, (:) high conservation and (.) conservation.

Figure 4

Alignment of Drosophila melanogaster NOS protein (gi|6707649) with three deduced fragments of the NOS from Glossina morsitans morsitans. The G. m. morsitans sequences are indicated by GM-X where X is the number shown in additional file 2.

Figure 5

Differential splicing of the nitric oxide synthase gene product in the salivary glands of Glossina morsitans morsitans. The blue lines are above regions of differential splicing.

Figure 6

Normalized blast scores of Glossina morsitans morsitans proteins compared to Drosophila melanogaster, and three species of mosquitoes: Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. Symbols and bars represent the average and standard error of the mean. The number in parenthesis indicates the number of sequences compared for each functional classification.

Figure 7

2D gel electrophoresis of Glossina morsitans morsitans salivary gland homogenates. Numbers on the left indicate the molecular mass marker positions in the gel. The + and - symbols indicate the anode or cathode side of the isoelectrophocusing first dimension, which incorporates a pI range of 3.0 - 10.0. (A) Unmarked gel. (B) Protein spots identified (following tryptic digestion and mass spectrometry) are labeled on the gel. In some cases, several spots were identified as the same protein. For experimental details, see Materials and Methods.

Figure 8

Western blot analysis of four recombinant proteins found in tsetse saliva. Purified His-tagged peptides were resolved using a 12.5% SDS-PAGE gel and subjected to Western blot using an anti-tsetse saliva antiserum. The order of peptides loaded on the gel is: His₆Tag-5 (positive control, lane 1), His₆-GMsg-06 h03 (2), His₆-GMsg-15f12 (3), and His₆-GMsg-45 g06 (4). Molecular weights from Protein Markers are indicated on the left.