Signal sequence analysis of expressed sequence tags from the nematode Nippostrongylus brasiliensis and the evolution of secreted proteins in parasites

Abstract

Background: Parasitism is a highly successful mode of life and one that requires suites of gene adaptations to permit survival within a potentially hostile host. Among such adaptations is the secretion of proteins capable of modifying or manipulating the host environment. Nippostrongylus brasiliensis is a well-studied model nematode parasite of rodents, which secretes products known to modulate host immunity.

Results: Taking a genomic approach to characterize potential secreted products, we analyzed expressed sequence tag (EST) sequences for putative amino-terminal secretory signals. We sequenced ESTs from a cDNA library constructed by oligo-capping to select full-length cDNAs, as well as from conventional cDNA libraries. SignalP analysis was applied to predicted open reading frames, to identify potential signal peptides and anchors. Among 1,234 ESTs, 197 (~16%) contain predicted 5' signal sequences, with 176 classified as conventional signal peptides and 21 as signal anchors. ESTs cluster into 742 distinct genes, of which 135 (18%) bear predicted signal-sequence coding regions. Comparisons of clusters with homologs from Caenorhabditis elegans and more distantly related organisms reveal that the majority (65% at P < e-10) of signal peptide-bearing sequences from N. brasiliensis show no similarity to previously reported genes, and less than 10% align to conserved genes recorded outside the phylum Nematoda. Of all novel sequences identified, 32% contained predicted signal peptides, whereas this was the case for only 3.4% of conserved genes with sequence homologies beyond the Nematoda.

Conclusions: These results indicate that secreted proteins may be undergoing accelerated evolution, either because of relaxed functional constraints, or in response to stronger selective pressure from host immunity.

Figure 1

Similarity of N. brasiliensis ESTs to sequences from other nematodes. SimiTri [54] was used to plot 736 N. brasiliensis EST clusters against related species database entries. For each consensus sequence associated with the 736 Nippo clusters, a BLAST was performed against a series of different databases. Each tile in the graphic represents a unique consensus sequence and its relative position is computed from the raw BLAST scores derived above (with a cutoff of ≥ 50). Hence each tile's position shows its degree of sequence similarity to each of the three selected databases. Sequences showing similarity to only one database are not shown. Sequences showing sequence similarity to only two databases appear on the lines joining the two databases. Tiles are colored by their highest TBLASTX score to each of the databases: red ≥ 300; yellow ≥ 200; green ≥ 150, blue ≥ 100 and purple < 100. (a) SimiTri plot showing sequence similarity relationships between N. brasiliensis consensus sequences and database entries of Ancylostoma caninum/duodenale ESTs (20,177 entries, 386 hits), Haemonchus contortus ESTs (22,337 entries, 384 hits) and Teladorsagia circumcincta ESTs (5,300 entries, 264 hits). Database comparisons were performed using TBLASTX. (b) SimiTri plot showing sequence similarity relationships between N. brasiliensis consensus sequences and database entries of Necator americanus ESTs (4,821 entries, 244 hits), Teladorsagia circumcincta ESTs (5,300 entries, 264 hits), and C. elegans wormpep (21,600 entries, 466 hits). Database comparisons were performed using TBLASTX for N. americanus and T. circumcincta, while C. elegans wormpep comparions used BLASTX.

Figure 2

Proportion of ESTs predicted to encode signal sequences. (a) EST sequences were classified as conserved (similarities to non-nematode database entries), nematode-specific (similarities only to C. elegans or other nematode sequences), or novel (no similarities to existing entries), using a cutoff score of 80 in BLASTX (P < e^-10). The number of ESTs bearing potential signal sequences was then calculated and the results are shown here. (b) Effects of relaxing cutoff scores on distribution of signal peptide-containing predicted gene products among conserved, nematode-specific and novel categories. Numbers of clusters in each category are given for cutoffs of 80 (P <^e-10), as used in (a), and 50 (P <^e-6).