Background
A central tenet of parasitology is that parasites must secrete biologically active mediators that modify or customize their niche within the host in order to survive immune attack. Such secretions have long been the focus of biochemical and immunological analyses 1234. With larger-scale genomic approaches now possible, a screen can be designed in which the characteristic signal sequences, necessary for proteins to exit the eukaryotic cell via the secretory pathway, can be identified by bioinformatic methods 56789. We describe here an analysis of this nature, applied to a widely used model system, Nippostrongylus brasiliensis, the gastrointestinal nematode of rats 101112.
N. brasiliensis biology encapsulates many key aspects of parasite infection and immunology. It is a multicellular metazoan belonging to the phylum Nematoda, which together with the platyhelminth groups (Cestoda and Trematoda) are collectively known as helminths. Helminth infections are typically accompanied by a polarized type-2 (Th2) immune response, characterized by IgE antibody production, eosinophilia and mastocytosis 131415. N. brasilensis drives extremely strong Th2 responses 16, and this bias can be reproduced with secreted proteins collected from parasites in vitro 17. More than 100 secreted proteins have been found by two-dimensional SDS-PAGE analysis (Y.H. and R.M.M., unpublished work), and among those experimentally verified are acetylcholinesterases 181920, cysteine proteases 2122, and a hydrolase that degrades an important host inflammatory mediator, platelet activating factor 2324.
The molecular biological analysis of N. brasiliensis genes and gene products is at a very early stage. Secreted and intracellular globins have been characterized 25, and genes for both secretory 2627 and neuronal 28 acetylcholinesterases cloned. A recombinant cystatin (cysteine protease inhibitor) has been shown functionally to inhibit host antigen-processing pathways 29. Structural genes for both tubulin 30 and a keratin-like protein 31 have been described, and an α-crystallin-like small heat-shock protein (Hsp20) has been reported 32. However, these studies on individual genes have yet to be complemented by higher-throughput molecular analyses. The potential of N. brasiliensis as an experimental system for functional genomics has been greatly enhanced by the demonstration of successful RNAi knockdown in this species 33.
The genomes of parasitic nematode species are between 60 and 250 megabases (Mb) in size 34, and there are more than 20 species of medical, veterinary and scientific importance 35. Over the past decade, the most tractable way of applying genomics to this group of organisms has been by expressed sequence tag (EST) projects 36. Large-scale EST sequencing of the human filarial parasite Brugia malayi 3738 has been followed by similar studies in the sheep intestinal worm Haemonchus contortus 39, human hookworms 40, the river-blindness parasite Onchocerca volvulus 41, and important plant-parasitic species such as Meloidogyne incognita 42. Smaller projects have added Litomosoides sigmodontis 43, Toxocara canis 44 and many other related species to the available database of parasitic nematode sequences 36. In designing a study on N. brasiliensis, we wished to focus on the potential for secreted proteins that may interact with the host immune system. We therefore conducted an EST project that included a cDNA library specifically enriched for full-length inserts 45, allowing analysis of amino-terminal signal peptides to be carried out.
The evolutionary history of secreted immunomodulators is likely to be that of recent adaptation from ancestral genes which fulfilled other functions in free-living ancestors. Comparative studies on nematodes can take advantage of full-genome information available for the free-living species Caenorhabditis elegans 46 and C. briggsae 47, which are quite closely related to N. brasiliensis 48. If rapid evolution of secreted gene products was required for efficient parasitism, this may be evident in greater diversity among signal peptide-bearing sequences than among genes coding for non-secreted proteins. We report here our results that support this hypothesis.
Results and discussion
A high proportion of N. brasiliensis ESTs encode proteins with predicted signal sequences
A total of 1,234 ESTs were collected from adult N. brasiliensis cDNA libraries constructed either by conventional means or by an oligo-capping method to select full-length cDNAs 45. A full analysis of these has been posted on our website 49. ESTs were then analyzed by SignalP, which predicted that 16.0% of total ESTs (197/1,234) contained either 5' signal peptide sequences (176/1,234) or signal anchors (21/1,234, Table 1). The oligo-capped cDNA library yielded a notably higher proportion of sequences with predicted signal peptides (20.4%) than did conventional cDNA libraries (10.1%).
<p>Table 1</p>
Analysis of transcripts represented in conventional and oligo-capped cDNA libraries
Conventional cDNA libraries
Oligo-capped cDNA library
Total sequences providing peptide predictions
734
500
In-frame ATG followed by ≥ 99-nucleotide open reading frame (ORF)
567 (77.2%)
430 (86.0%)
Predicted ORF length (average)
114.6
101.5
% Signal peptide or signal anchor
SP: 74 (10.1%)
SP: 102 (20.4%)
SA: 16 (2.2%)
SA: 5 (1.0%)
% Spliced leader
0
37 (7.4%)
The dataset was then clustered to account for multiple ESTs from highly expressed genes, and ESTs were assigned to 742 clusters, including 567 singletons. The proportion of clusters bearing potential signal sequences remained high (135/742; 18.2%), confirming that the dataset is not skewed by over-representation of a few abundant transcripts. The overall proportion of cDNAs encoding predicted signal peptides is within the 15-25% range estimated by analyis of whole-genome sequence data 50. Of all predicted signal-sequence-bearing clones or clusters from N. brasiliensis, around 90% were classified as conventional signal peptides associated with export and secretion into the extracellular environment. The remaining approximately 10% were identified as potential signal anchors, in which the hydrophobic amino-terminal segment is retained, without cleavage, as a transmembrane domain for type II plasma membrane proteins 7.
Presence of trans-spliced leaders in N. brasiliensis
All nematodes undergo trans-splicing at the 5' end of a proportion of their mRNA transcripts; a short leader sequence is added upstream of the initiation codon. The leader is normally a 22-nucleotide sequence termed SL1 51. The precise SL1 sequence is highly conserved throughout the phylum, although the degree to which transcripts are trans-spliced varies between different nematode species 52. To evaluate the prominence of SL1-trans-splicing in N. brasiliensis, we searched the 1,234 ESTs with the 3' 14 nucleotides of SL1, to allow for any minor truncation of cDNAs. Only 37 matches were found, all from the oligo-capped cDNA library (from 500 ESTs, giving a frequency of 7.4%); a few clones from the conventional libraries had 10 or fewer nucleotides identical to the SL1 sequence at their 5' termini. Although the overall frequency of trans-splicing in N.brasiliensis is not yet known, this level is well below those of other species, such as C. elegans. Moreover, transcripts bearing the spliced leader (and its unique tri-methylguanosine cap) are, in certain species, under-represented by the method we used to selectively amplify full-length mRNAs 45. Hence the true extent of trans-splicing may be higher than the proportion evident in the current dataset.
N. brasiliensis sequences show closest similarity to those of other trichostrongyles
N. brasiliensis is a stronglylid nematode, closely related to veterinary parasites such as Haemonchus contortus and Teladorsagia (previously Ostertagia) circumcincta in the Superfamily Trichostrongyloidea, and within the Order Strongylida which includes human hookworm pathogens Ancylostoma duodenale and Necator americanus 53. The closest free-living taxa to the Strongylida are members of the Rhabditina, including C. elegans, and both are grouped in Clade V of the Nematoda, on the basis of small subunit rRNA sequence analysis 48.
A more objective technique for visualizing the evolutionary relationships between species for which large datasets are available is to use SimiTri, which plots in two-dimensional space the relative similarities of gene sequences between one species (N. brasiliensis) and three comparators 54. As shown in Figure 1a, N. brasiliensis sequences group slightly closer to Haemonchus than to Ancylostoma, consistent with the relationship described above. Likewise, in Figure 1b, N. brasiliensis sequences group more towards Teladorsargia than Necator.
<p>Figure 1</p>
Similarity of N. brasiliensis ESTs to sequences from other nematodes
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.
![]()
A compilation of the N. brasiliensis clusters, for which assigned homologs exist in protein databases, is presented in Table 2. Many sequences with high similarities to biosynthetic, structural, signaling and regulatory pathway proteins can readily be identified, corresponding to predicted nuclear or cytoplasmic proteins. Interestingly, multiple clusters encode categories of genes which are prominent in other nematode parasites, such as the five clusters encoding homologs of Ancylostoma secreted protein 2, five clusters of C-type and S-type lectins 55 and seven clusters for cysteine proteinases 56.
<p>Table 2</p>
ESTs from adult cDNAs with known homologs, classified by function
Cluster number
Conventional cDNAs
Oligo-capped cDNAs
P
Accession
Description
Proteases/proteosome/ubiquitin
NBC00018
2
0
1e-33
S66528
26S proteinase regulatory complex, non-ATPase chain (Drosophila melanogaster)
NBC00030
2
0
8e-56
U41556
Cysteine protease CPR-6 (Caenorhabditis elegans)
NBC00086
1
0
3e-29
A48454
Cathepsin B-like cysteine proteinase (Ostertagia ostertagi)
5e-28
D48435
Cysteine proteinase AC-3 (Haemonchus contortus)
NBC00168
1
0
2e-42
NM_065563
Calpain thiol protease (Caenorhabditis elegans)
NBC00198
1
0
7e-60
NM_073736
Cysteine protease (legumain, asparaginyl endopeptidase) (Caenorhabditis elegans)
NBC00204
3
0
2e-32
NM_072733
Protease (aspartic) (Caenorhabditis elegans)
NBC00231
2
0
5e-90
NM_064106
Serine carboxypeptidase (Caenorhabditis elegans)
NBC00307
1
0
2e-32
NM_015277
Ubiquitin-protein ligase NEDD4-like; neural precursor (Homo sapiens)
NBC00311
1
0
5e-31
NM_073736
Cysteine protease (legumain, asparaginyl endopeptidase) (Caenorhabditis elegans)
NBC00352
2
0
6e-31
NM_065253
Ubiquitin (Caenorhabditis elegans)
NBC00348
1
0
2e-83
A48145
Ubiquitin-conjugating enzyme, UBC-2 (Caenorhabditis elegans)
NBC00362
1
0
1e-76
S17521
Multicatalytic endopeptidase complex (proteasome) zeta chain (Caenorhabditis elegans)
NBC00368
1
0
9e-13
LCE_ORYLA
Low choriolytic enzyme precursor (zinc metalloprotease) (Oryzias latipes)
NBC00377
1
0
3e-75
PSA4_CAEEL
Proteasome subunit, alpha type 4, PAS-3 (Caenorhabditis elegans)
NBC00459
2
1
2e-26
NM_072733
Protease (aspartic) (Caenorhabditis elegans)
NBC00469
1
0
7e-17
NM_060215
Zinc metalloprotease (Caenorhabditis elegans)
NBC00509
1
1
4e-71
AL161503
Polyubiquitin, UBQ10 (Arabidopsis thaliana)
NBC00664
0
1
5e-09
NM_074798
Cathepsin-like (cysteine) protease (Caenorhabditis elegans)
NBC00670
0
1
3e-18
S17435
Polyubiquitin 6 (Helianthus annuus)
NBC00772
0
1
4e-24
NM_003352
Sentrin, ubiquitin-like small protein (Gallus gallus)
NBC00783
0
1
2e-89
U41556
Cysteine protease CPR-6 (Caenorhabditis elegans)
NBC00828
0
1
9e-63
NC_003424
Pad1 protein; 26S proteasome subunit (Schizosaccharomyces pombe)
Enzymes (other than proteases)
NBC00045
2
0
2e-92
NM_065870
Fructose-biphosphate aldolase (Caenorhabditis elegans)
NBC00049
1
0
9e-50
NM_070783
Lipase (Caenorhabditis elegans)
NBC00066
2
1
7e-76
NM_074348
Peptidyl-prolyl cis-trans isomerase (Caenorhabditis elegans)
NBC00079
1
0
2e-35
NM_058712
Helicase (Caenorhabditis elegans)
NBC00102
1
0
7e-37
NM_074031
Peroxidase-like (Caenorhabditis elegans)
NBC00139
1
0
8e-29
NM_060074
Hexokinase (Caenorhabditis elegans)
NBC00143
1
0
4e-66
ADHX_MYXGL
Alcohol dehydrogenase class III (Caenorhabditis elegans)
NBC00147
1
0
6e-19
XM_087230
Similar to Uridine phosphorylase (UDRPase) (Homo sapiens)
NBC00157
1
0
3e-13
XM_058660
Similar to Protein tyrosine phosphatase 1E (Homo sapiens)
NBC00173
1
0
5e-72
AJ440747
Protein disulphide isomerase 1 (Ostertagia ostertagi)
NBC00183
1
0
3e-56
T46280
Isocitrate dehydrogenase, NADP+, cytosolic (Homo sapiens)
NBC00189
1
0
1e-21
XM_129069
Similar to Acetyltransferase (GNAT) family (Mus musculus)
NBC00212
1
0
6e-57
NM_016100
N-terminal acetyltransferase complex ard1 subunit (Homo sapiens)
NBC00283
1
0
4e-27
NM_012088
6-phosphogluconolactonase (Homo sapiens)
NBC00285
1
0
2e-47
LDHA_ANGRO
L-lactate dehydrogenase A chain (Anguilla rostrata)
NBC00290
1
0
3e-17
I55976
Dihydrolipoamide S-acetyltransferase (Rattus norvegicus)
NBC00292
1
0
1e-40
NM_006223
Peptidyl-prolyl cis/trans isomerase (Homo sapiens)
NBC00304
1
0
4e-12
NM_073341
Glucose-1-dehydrogenase (Caenorhabditis elegans)
NBC00309
1
0
1e-18
NM_066225
Hydroxymethylglutaryl-coA reductase (Caenorhabditis elegans)
NBC00326
1
0
1e-65
NM_065761
Protein phosphatase 2A (Caenorhabditis elegans)
NBC00337
1
0
2e-60
GMD1_CAEEL
Probable GDP-mannose 4,6 dehydratase 1 (Caenorhabditis elegans)
NBC00353
1
0
2e-56
NM_065537
ATP synthase B chain (Caenorhabditis elegans)
NBC00378
1
0
2e-43
NM_073253
Acetyltransferase (GNAT) family (Caenorhabditis elegans)
NBC00382
1
0
4e-49
NM_063827
Phospholipase A2 (Caenorhabditis elegans)
NBC00389
2
0
1e-48
NM_058626
Phosphotransferase (Caenorhabditis elegans)
NBC00404
1
0
2e-76
NM_064078
Glucosamine-fructose-6-phosphate aminotransferase (Caenorhabditis elegans)
NBC00413
1
0
6e-22
NM_078324
AMP-activated protein kinase (Caenorhabditis elegans)
NBC00427
1
0
2e-20
NC_003423
3-oxoacyl-(acyl-carrier-protein)-synthase (Schizosaccharomyces pombe)
NBC00475
1
0
3e-42
NM_065313
Serine/threonine protein phosphatase (Caenorhabditis elegans)
NBC00483
1
0
4e-25
NM_059984
Phospholipase, similar to ADRAB-b (Caenorhabditis elegans)
NBC00504
1
0
7e-65
AF292096
Protein kinase AIRK2 (Xenopus laevis)
NBC00508
1
2
5e-64
PPCK_HAECO
Phosphoenolpyruvate carboxykinase (Haemonchus contortus)
NBC00528
1
0
5e-66
PPCK_HAECO
Phosphoenolpyruvate carboxykinase (Haemonchus contortus)
NBC00561
0
7
1e-54
NDKB_RAT
Nucleoside diphosphate kinase B (Rattus norvegicus)
NBC00713
0
1
1e-08
XM_140038
Similar to tau-tubulin kinase (Mus musculus)
NBC00729
0
2
4e-21
NM_079041
Flap endonuclease 1 (Drosophila melanogaster)
NBC00743
0
1
3e-64
G3P_BRUMA
Glyceraldehyde 3-phosphate dehydrogenase (Brugia malayi)
NBC00745
0
1
4e-13
NM_068436
Casein kinase (Caenorhabditis elegans)
NBC00689
0
3
2e-17
CLYC_CAEEL
Serine hydroxymethyltransferase MEL-32 (Caenorhabditis elegans)
NBC00696
0
2
2e-15
NM_000414
Hydroxysteroid (17-beta) dehydrogenase 4 (Homo sapiens)
NBC00770
0
1
3e-45
NM_066907
Serine/threonine kinase, casein kinase-like (Caenorhabditis elegans)
NBC00777
0
1
8e-21
OAZ_PRIPA
Ornithine decarboxylase antizyme (Pristionchus pacificus)
NBC00796
0
1
8e-52
XM_125017)
Putative lysophosphatidic acid acyltransferase (Mus musculus)
NBC00802
0
1
4e-49
NM_078623
Enoyl Coenzyme A hydratase, short chain 1 (Rattus norvegicus)
Structural
NBC00056
1
0
4e-58
NM_071024
Actin depolymerizing factor (Caenorhabditis elegans)
NBC00062
1
0
1e-11
NM_006400
Dynactin 2; dynactin complex 50 kD subunit; dynamitin (Homo sapiens)
NBC00078
2
0
0
NM_059538
Calponin (Caenorhabditis elegans)
NBC00097
1
0
1e-42
MLR1_CAEEL
Myosin regulatory light chain 1 (Caenorhabditis elegans)
NBC00142
1
0
2e-76
S53776
Beta-tubulin isotype I (Haemonchus contortus)
NBC00172
2
0
0
NM_073416
Actin (Caenorhabditis elegans)
NBC00224
1
0
2e-40
NM_063850
Troponin C (Caenorhabditis elegans)
NBC00239
4
1
2e-39
NM_077559
Collagen (Caenorhabditis elegans)
NBC00241
2
0
2e-47
NM_069715
Collagen (Caenorhabditis elegans)
6e-47
NM_077291
Cuticular collagen (Caenorhabditis elegans)
NBC00246
1
1
3e-19
NM_077087
Troponin I (Caenorhabditis elegans)
NBC00287
2
0
2e-61
MLR1_CAEEL
Myosin regulatory light chain 1 (Caenorhabditis elegans)
NBC00360
1
1
3e-30
NM_145671
Actinfilin (Rattus norvegicus)
NBC00396
1
0
2e-67
MYSP_CAEEL
Paramyosin (Caenorhabditis elegans)
NBC00403
1
0
3e-32
NM_077291
Cuticular collagen (Caenorhabditis elegans)
NBC00418
1
0
6e-27
NM058881
Calponin (Caenorhabditis elegans)
NBC00430
1
0
3e-11
NM_011722
Dynactin 6; p27 dynactin subunit (Mus musculus)
NBC00526
1
0
2e-44
NM_060857
Profilin (Caenorhabditis elegans)
NBC00552
0
1
9e-47
MYSP_CAEEL
Paramyosin (Caenorhabditis elegans)
NBC00569
0
1
1e-23
NM_060369
Alpha crystallin B chain (Caenorhabditis elegans)
NBC00749
0
1
3e-43
NM_060857
Profilin (Caenorhabditis elegans)
Embryo/egg/mating etc
NBC00068
3
0
1e-25
VIT5_CAEEL
Vitellogenin 5 precursor (Caenorhabditis elegans)
NBC00161
1
0
2e-15
VIT5_CAEEL
Vitellogenin 5 precursor (Caenorhabditis elegans)
NBC00397
1
9
7e-61
MS10_CAEEL
Major Sperm Protein 10 (Caenorhabditis elegans)
NBC00523
1
0
4e-69
XM_038960
Similar to preimplantation protein 3 (Homo sapiens)
NBC00585
0
5
2e-30
NM_076467
Vitellogenin (Caenorhabditis elegans)
NBC00611
0
1
1e-25
NM_060189
Placental protein 11 (Caenorhabditis elegans)
Transporters/receptors/lectins and other binding proteins
NBC00027
2
0
9e-17
NM_062882
Lectin, C-type (Caenorhabditis elegans)
5e-15
NM_076712
Asialoglycoprotein receptor (C-type lectin) (Caenorhabditis elegans)
NBC00110
1
0
4e-17
NC_001263
Acyl-CoA-binding protein (Deinococcus radiodurans)
NBC00118
1
0
4e-41
T31073
Multidrug resistance P-glycoprotein (Haemonchus contortus)
NBC00128
3
0
1e-92
NM_067381
ADP/ATP carrier protein/translocase (Caenorhabditis elegans)
NBC00167
1
0
2e-12
NM_130415
Lysosomal amino acid transporter 1 (Rattus norvegicus)
NBC00175
1
0
7e-15
A48925
Mannose receptor (C-type lectin), macrophage (Mus musculus)
NBC00319
1
0
8e-15
NXT2_HUMAN
NTF2-related export protein 2 (p15-2 protein) (Homo sapiens)
NBC00324
2
0
7e-15
AJ243873
Galectin (S-type lectin) (Haemonchus contortus)
NBC00340
1
0
2e-61
NM_077246
Galectin (S-type lectin) LEC-10 (Caenorhabditis elegans)
NBC00355
1
0
8e-21
NM_059527
Fatty acid-binding protein LBP-6 (Caenorhabditis elegans)
NBC00363
1
0
6e-48
NM_016208
Vacuolar protein sorting 28 homolog (Homo sapiens)
NBC00583
0
5
4e-35
NM_065836
Low density lipoprotein receptor (Caenorhabditis elegans)
NBC00593
0
2
2e-26
NM_059525
Fatty acid-binding protein LBP-6 (Caenorhabditis elegans)
NBC00752
0
1
3e-08
NM_059071
Acetylcholine receptor UNV-38 (Caenorhabditis elegans)
NBC00766
0
1
7e-44
POR2_MELGA
Voltage-dependent anion-selective channel protein 2 (VDAC-2) (Meleagris gallopavo)
NBC00808
0
1
6e-53
NM_072174
Calreticulin precursor (Caenorhabditis elegans)
NBC00838
0
1
1e-78
NM_063349
T-complex protein, delta subunit (cytosolic chaperonin CCT-4) (Caenorhabditis elegans)
Signaling
NBC00207
1
0
0
RAB2_LYMST
RAS-Related protein RAB-2 (Lymnea stagnalis)
NBC00252
1
0
8e-97
NM_070558
RAS-like GTP-binding protein RhoA (Caenorhabditis elegans)
NBC00297
1
0
4e-17
NM_009106
Rhotekin (Mus musculus)
NBC00312
1
0
4e-46
A35350
Protein kinase C inhibitor (Bos bovis)
NBC00269
1
0
1e-43
NM_058274
RAS-related protein RAB-11 (Caenorhabditis elegans)
NBC00282
1
0
9e-25
NP_741191
A kinase anchor protein 1 (Caenorhabditis elegans)
NBC00395
1
0
2e-29
NM_07328
RAS-like GTP-binding protein (cdc42-like) (Caenorhabditis elegans)
NBC00436
1
0
2e-44
NM_070985
Calmodulin (Caenorhabditis elegans)
NBC00462
1
0
2e-13
SSRP_DROME
Single-strand recognition protein (SSRP) (Chorion-factor 5) (Drosophila melanogaster)
NBC00409
1
0
1e-16
NM_019746
Programmed cell death 5/TFAR19 protein (Mus musculus)
NBC00440
1
0
3e-72
S43599
SNF5 homolog R07E5.3 (Caenorhabditis elegans)
NBC00510
1
0
2e-28
XM_129572
Calcyclin (S100 family) binding protein (Mus musculus)
NBC00629
0
1
1e-20
NM_026297
RAB (RAS oncogene family-like 3) (Mus musculus)
NBC00648
0
1
3e-20
NM_002624
Prefoldin 5 isoform alpha; myc modulator-1; c-myc binding protein (Homo sapiens)
NBC00727
0
1
3e-17
AB091687
TGF-beta induced apotosis protein 3 (Mus musculus)
NBC00768
0
1
3e-18
NM_078471
TGF-beta-1 induced anti-apoptotic factor 1 isoform 1 (Homo sapiens)
NBC00829
0
1
1e-42
A49146
Developmental regulator WNT-4 (Xenopus laevis)
NBC00841
0
1
1e-31
NM_012453
Transducin (beta)-like 2, isoform 1 (Homo sapiens)
DNA-related/transcription/DNA binding/regulation
NBC00024
1
0
1e-37
NM_003752
Eukaryotic translation initiation factor 3, subunit 8 (Homo sapiens)
NBC00048
1
0
1e-28
NM_069150
Glycine-rich RNA-binding protein (Caenorhabditis elegans)
5e-21
NM_007007
Cleavage and polyadenylation specific factor 6 (Homo sapiens)
NBC00050
1
0
2e-12
HEXP_LEIMA
DNA-binding protein HEXBP (Hexamer-binding protein) (Leishmania major)
NBC00055
1
1
2e-24
NM_060622
RNA recognition motif (RRM, RBD, or RNP domain) (Caenorhabditis elegans)
NBC00090
2
1
0
NM_066119
Elongation factor 1-alpha (Caenorhabditis elegans)
NBC00099
1
0
2e-30
NM_067248
Splicing factor (Caenorhabditis elegans)
NBC00170
1
0
2e-56
NM_011304
RuvB DNA helicase -like protein 2 (Mus musculus)
NBC00181
1
0
4e-13
NM_001698
AU RNA-binding protein/enoyl-Coenzyme A hydratase (Homo sapiens)
NBC00192
1
0
2e-26
NM_060622
RNA recognition motif (RRM, RBD, or RNP domain) (Caenorhabditis elegans)
NBC00210
1
0
3e-15
NM_018403
Transcription factor (SMIF gene) (Homo sapiens)
NBC00267
1
0
4e-20
T2EB_XENLA
Transcription initiation factor IIE, beta subunit (Xenopus laevis)
NBC00321
1
0
1e-16
NM_033224
Purine-rich element binding protein B (Homo sapiens)
NBC00280
1
0
3e-58
NM_006578
Guanine nucleotide-binding protein, beta-5 subunit (Homo sapiens)
NBC00350
1
0
6e-40
DPOD_DROME
DNA polymerase delta catalytic subunit (Drosophila melanogaster)
NBC00366
2
0
6e-79
NM_066119
Elongation factor 1-alpha (Caenorhabditis elegans)
NBC00370
1
0
1e-17
NM_031992
Eukaryotic translation initiation factor 4H, isoform 2 (Homo sapiens)
NBC00374
1
2
2e-53
NM_070415
Elongation factor 1-beta/delta chain (Caenorhabditis elegans)
NBC00480
1
0
3e-21
NM_061014
Regulator of chromosome condensation, RCC1 (Caenorhabditis elegans)
NBC00543
0
2
5e-23
NM_065536
Zinc finger, C3HC4 type (RING finger) (Caenorhabditis elegans)
NBC00577
0
7
2e-31
NP_872244
Translation elongation factor EFT-4 (Caenorhabditis elegans)
NBC00600
0
1
3e-74
NM_063406
Initiation factor 5A (Caenorhabditis elegans)
NBC00630
0
1
9e-39
SFR4_MOUSE
Splicing factor, arginine/serine-rich 4 (Mus musculus)
NBC00764
0
1
4e-16
XM_132357
Similar to Translation Initiation factor EIF-2B alpha (Mus musculus)
NBC00776
0
1
6e-27
SN2L_CAEEL
Potential global transcription activator SNF2L (Caenorhabditis elegans)
NBC00791
0
1
5e-38
NM_001207
Basic transcription factor 3 (Homo sapiens)
NBC00816
0
1
2e-24
S3B2_HUMAN
Splicing factor 3B subunit 2 (Spliceosome associated protein 145) (Homo sapiens)
Other homologs of interest
NBC00025
1
0
3e-16
AF352714
HC40 putative secretory protein precursor (ASP homolog) (Haemonchus contortus)
NBC00065
1
0
6e-20
AA063577
Secreted protein 5 precursor (ASP homolog) (Ancylostoma caninum)
NBC00095
1
0
8e-59
GLB2_NIPBR
Myoglobin (body wall isoform globin) (Nippostrongylus brasiliensis)
NBC00103
1
0
9e-12
DIM1_CAEEL
Protein dim-1 (2D-page protein spot 8) (Caenorhabditis elegans)
NBC00029
1
0
5e-17
NM_001545
Immature colon carcinoma transcript 1 (Homo sapiens)
NBC00141
1
0
2e-35
NM_018984
Slingshot 1 (Homo sapiens)
NBC00160
1
0
5e-12
NM_053810
Synaptosomal-associated protein, 29kD (Rattus norvegicus)
NBC00199
1
0
9e-39
AF278538
Nucleosome assembly protein 1 (Xenopus laevis)
NBC00256
2
0
2e-09
NM_075227
Transthyretin-like family (Caenorhabditis elegans)
NBC00293
1
0
7e-08
NC_003424
F-box protein (Schizosaccharomyces pombe)
NBC00399
1
0
2e-22
NM_076443
Calumenin, calcium-binding protein (Caenorhabditis elegans)
NBC00429
1
0
4e-14
XM_122362
Chromobox homolog 2 (Drosophila Pc class) (Mus musculus)
NBC00491
1
0
3e-21
NM_076885
Thrombospondin (Caenorhabditis elegans)
NBC00518
1
0
3e-73
T37461
Mago nashi-like protein (Caenorhabditis elegans)
NBC00544
0
1
2e-45
NM_061213
Alpha-2-macroglobulin family (Caenorhabditis elegans)
NBC00560
0
1
1e-35
NM_021305
SEC61, alpha subunit 2 (Saccharomyces cerevisiae)
NBC00705
0
1
3e-31
DVA1_DICVI
DVA-1 nematode polyprotein allergen precursor (NPA) (Dictyocaulus viviparus)
2e-12
ABA1_ASCSU
ABA-1 nematode polyprotein allergen precursor (Body fluid allergen-1) (Ascaris suum)
NBC00753
0
1
4e-10
AF089728
Ancylostoma-secreted protein 2 precursor, ASP-2 (Ancylostoma caninum)
NBC00755
0
1
2e-40
TCPB_CAEEL
T-complex protein 1, beta subunit (CCT-beta) (Caenorhabditis elegans)
NBC00757
0
1
2e-68
1432_SCHMA
14-3-3 Protein homolog 2 (14-3-3-2) (Schistosoma mansoni)
NBC00803
0
1
3e-09
ASP_ANCCA
Ancylostoma secreted protein (ASP-1) precursor (Ancylostoma caninum)
3e-09
AF079521
Ancylostoma-secreted protein 1 precursor (ASP-1 homolog) (Necator americanus)
NBC00827
0
1
3e-14
NM_070108
Testis-specific protein TPX-1 like (ASP homolog) (Caenorhabditis elegans)
The table gives, for each numbered cluster, the highest homolog with a functional description where available; in a number of cases a C. elegans homolog exists with a higher similarity, but has no description. Similarities to entries described as 'hypothetical proteins' are excluded, as are heat-shock proteins, cytochromes, mitochondrial and ribosomal products. Where C. elegans protein description is ambiguous (for example, protease, lectin), further descriptors added manually are italicized. Different clusters may derive from a single gene if sequences are non-overlapping; for example, NBC00198 and NBC00311 align to different segments of the C. elegans protease gene NM_073736. This table does not include N. brasiliensis gene products discovered previously and/or reported by other laboratories. All entries for this species are aggregated on the NEMBASE website.
Proteins bearing signal sequences are less evolutionarily conserved
The set of 742 clusters was then divided into three categories according to their similarity to existing database sequences. 'Conserved' genes were defined as those with similarities to any non-nematode database entry above a given cutoff score; 'nematode-specific' genes were similar only to sequences from C. elegans or other nematode species, and 'novel' showed no similarity to any existing entry. BLASTX cutoff scores of 50 (P < e-6) and 80 (P < e-10) were both used to define these categories at different levels. Using the more stringent criterion, roughly one third (27-37%) of clusters fell into each category (Figure 2a), while the lower cutoff resulted in approximately half (48%) being classified as conserved, with the remainder evenly divided between nematode-specific (25%) and novel (27%).
<p>Figure 2</p>
Proportion of ESTs predicted to encode signal sequences
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).
![]()
The distribution of clusters containing signal sequences was, however, remarkably skewed towards the novel category. Because the primary classification of 92 novel genes was based on 5' EST sequences, all clusters initially designated as novel signal-sequence positive were further scrutinized. In 72 cases, clusters read through to a 3' poly(A) tail (either single reads from clones of 700 or fewer nucleotides or overlapping ESTs with at least one poly(A) tail present); in 20 cases, where no poly(A) tail was observed, 3' sequencing was carried out. Of these, three showed database homologies from 3' sequence and were reclassified as conserved, and two showed no poly(A) tail and were excluded from further analysis as presumed internal fragments. The remaining 15 clusters showed overlap between 3' and 5' cluster reads, without revealing any additional similarities. Thus, a total of 87 clusters were verified as novel signal-sequence positive.
Taking this more rigorously defined subset, some 65% (87/133) of sequences are predicted to encode either signal peptides or signal anchors when classified as novel at the higher cutoff (49% at the lower level), and only 4% were found in the conserved category (7% at the lower cutoff). Moreover, 32% of all novel sequences contained a signal peptide or anchor, compared to 18% of nematode-specific and only 3.4% of conserved.
Although the latter category will include many structural and housekeeping proteins for which secretion is unlikely to confer a selective advantage, the data suggest that nematode secreted proteins have diversified more rapidly than those that do not enter the secretory pathway.
This association between signal peptides and novel proteins may be falsely amplified where, for example, conserved domains are sufficiently distant from the amino terminus to have been omitted from EST sequences. Equally, some clones will have been sequenced from truncated transcripts, and a proportion of those erroneously classified as encoding non-signal sequence bearing proteins. However, neither of these considerations seems likely to account for the very large disparity in signal sequence frequency between the three categories we describe. A more general caveat with these analyses is that SignalP is a fallible prediction tool, with an accuracy of 70% or less when applied to non-mammalian sequences 6. There is no reason, however, to expect that false-positive assignations would occur disproportionately in the novel group rather than the conserved, and the conclusion drawn here would remain valid over a wide range of prediction accuracies.
Has there been evolutionary acquisition of signal peptides?
The subset of signal-peptide-encoding N. brasiliensis clusters with similarity to predicted genes from C. elegans with either assigned function or of no known function was then identified. Examples of each category are given in Table 3. Some nine clusters were identified as bearing signal-peptide sequences, where in each case the C. elegans homologs appear not to possess a signal-pepide motif. Five of these clusters represent globins, which have previously been noted to possess signal peptides in N. brasiliensis even though the C. elegans paralogs do not 2557. One cluster (NBC00028) is almost identical to the recorded cuticular isoform precursor (P51536), but four additional clusters represent new members of this family in N. brasiliensis bearing signal peptides. In contrast, a distinct globin (NBC00095) closely related to the known body-wall isoform (P51535) lacks a predicted signal peptide. Hence, gene duplication may have predated the development in some globin forms, of a secretory function.
<p>Table 3</p>
ESTs from adult cDNAs with predicted amino-terminal signal peptides and with homologs in C. elegans
Cluster
Score
P
Conventional cDNAs
Oligo-capped cDNAs
Wormpep ID
SignalP criteria
SignalP scores
Signal in C. elegans?
Description of C. elegans gene
C-p
Amino acids
SP-p
SP?
(a) Signal peptides predicted in both N. brasiliensis and C. elegans
NBC00012
86
6e-18
4
0
CE20223
YYYYS
0.533
16
1.000
Y
Y
Unknown (similar to NBC00237)
NBC00031
80
3e-16
2
2
CE17924
YYYYS
0.932
18
0.999
Y
Y
Unknown
NBC00237
84
5e-17
1
2
CE20223
YYYYS
0.671
19
1.000
Y
Y
Unknown (similar to NBC00012)
NBC00258
145
1e-35
1
0
CE00133
YYYYS
0.524
19
0.999
Y
Y
FAR-1 fatty acid/retinol-binding protein
NBC00266
129
6e-31
1
0
CE19630
YYYYS
0.662
20
1.000
Y
Y
Unknown
NBC00314
147
3e-36
1
1
CE03639
YYYYS
0.708
19
0.987
Y
Y
Transthyretin-like family
NBC00327
94
2e-20
1
0
CE00906
YYYYS
0.542
25
0.998
Y
Y
Unknown
NBC00336
138
2e-33
1
0
CE23545
YYYYS
0.903
17
1.000
Y
Y
Unknown
NBC00354
91
4e-21
4
0
CE16530
YYYYS
0.511
17
0.943
Y
Y
Unknown
NBC00472
215
8e-57
1
0
CE04886
YYYYS
0.319
15
0.999
Y
Y
Signal sequence receptor
NBC00487
55
7e-09
1
0
CE05972
YYYYS
0.979
21
0.988
Y
Y
Unknown
NBC00495
51
3e-07
1
1
CE13171
YYYYS
0.566
19
0.999
Y
Y
Transthyretin-like family
NBC00502
176
3e-45
1
0
CE32298
YYYYS
0.634
20
1.000
Y
Y
Ectonucleotide pyrophosphatase/phosphodiesterase
NBC00592
80
1e-15
0
3
CE17924
YYYYS
0.920
16
1.000
Y
Y
Unknown
NBC00606
81
4e-16
0
2
CE02454
YYYYS
0.399
20
1.000
Y
Y
Similar to O. volvulus hypodermal antigen Ov-17
NBC00615
207
3e-54
0
1
CE04533
YYYYS
0.995
18
1.000
Y
Y
LBP-1 fatty acid-binding protein
NBC00616
61
3e-10
0
1
CE20257
YYYYS
0.754
19
0.993
Y
Y
Unknown
NBC00633
153
4e-38
0
1
CE03639
YYYYS
0.450
17
1.000
Y
Y
Transthyretin-like family
NBC00641
145
1e -35
0
1
CE33289
YYYYS
0.219
19
0.930
Y
Y
Unknown
NBC00643
102
2e-22
0
2
CE27850
YYYYS
0.961
17
0.999
Y
Y
Unknown
NBC00706
50
9e-07
0
1
CE06014
YYYYS
0.466
20
1.000
Y
Y
Unknown
NBC00720
12
3e-30
0
1
CE16958
YYYYS
0.967
19
0.998
Y
Y
NLP-13 neuropeptide
NBC00742
60
3e-10
0
1
CE16731
YYYYS
0.880
21
0.993
Y
Y
Unknown
NBC00748
50
4e-07
0
1
CE02932
YYYYS
0.804
17
0.998
Y
Y
Transthyretin-like family
NBC00767
79
7e-16
0
1
CE31662
YYYYS
0.559
17
1.000
Y
Y
Unknown
(b) Signal peptides predicted in N. brasiliensis but not C. elegans
NBC00028
104
1e-23
1
1
CE00431
YYYYS
0.731
18
0.999
Y
N
Globin
NBC00124
128
8e-31
1
1
CE00431
YYYYS
0.731
18
0.999
Y
N
Globin
NBC00144
195
7e-51
1
0
CE29663
YYNYS
0.866
19
0.963
Y
N
Transport-secretion protein
NBC00197
143
8e-35
3
6
CE00431
YYYYS
0.557
16
1.000
Y
N
Globin
NBC00272
144
2e-35
1
0
CE32475
YYNYS
0.262
22
0.513
Y
N
Unknown
NBC00328
147
4e-36
3
4
CE00431
YYYYS
0.523
17
0.999
Y
N
Globin
NBC00581
122
7e-29
0
1
CE00431
YYYYS
0.404
21
0.998
Y
N
Globin
NBC00601
93
5e-20
0
1
CE30218
YYYYS
0.535
34
0.944
Y
N
Unknown
NBC00607
159
4e-40
0
1
CE29597
YYNYS
0.529
18
0.786
Y
N
Unknown
Entries in table do not match numbers in Figure 2, which includes predicted signal anchors. SignalP criteria are C-score (raw cleavage site score); S-score (signal peptide score); Y-score (combined cleavage site score); mean S score; and assignation as signal peptide (S as in all entries above; otherwise A for signal anchor or N for neither). SignalP scores are as follows: C-p: probability of predicted cleavage site being correct; amino acids: length of predicted signal peptide in amino acids; SP-p: probability of existence of signal peptide; SP?: overall prediction for signal peptide. Note that NBC00028 is almost identical to the cuticular globin of N. brasiliensis (P51536), and NBC00197 and NBC00328 are closely related, whereas NBC0124 and NBC00581 are more similar to, but not identical to, the body-wall form of globin (P51535).
In these cases, and in the four additional examples given in Table 3, it is possible that pre-existing genes have been adapted for secretion or membrane expression in order to promote parasitism. Acquisition of secretory signals may not, in evolutionary terms, be demanding, in view of the report that approximately 20% of protein-coding fragments from Saccharomyces cerevisiae can function as a signal peptide 58. In the case of the globins, conversion to the secretory pathway (as well as gene multiplication) may be interpreted as a physiological adaptation to the environment within the mammalian gastrointestinal tract 57. Whether any of the four remaining genes in this category might have undergone a similar evolutionary process to counter immune attack is unknown at this stage.
Similar findings have previously been reported in individual genes from other nematode parasites. In B. malayi, the microfilarial secreted serpin gene (Bm-spn-2) is homologous to eight C. elegans genes, none of which encodes a signal peptide 59. Likewise, the extracellular glutathione-S-transferase gene, Ov-gst-1, of Onchocerca volvulus has acquired a signal-peptide sequence 60, as has a gene for keratin-like protein (KLP) in N. brasiliensis itself 31. Hence, conversion of key gene products to secretory function may be a common adaptive strategy for parasitic organisms.
Conclusions
Our study raises both methodological and evolutionary questions. First, it remains to be determined how valid is the assumption that signal sequences reflect secretion into the parasite environment. Clearly, this notion must be qualified in a metazoan parasite, because many such proteins will remain on the cell surface or be sorted to extracellular and extracytosolic compartments within the worm. However, the extent to which signal-peptide-bearing proteins are truly exported by these multicellular organisms will be clarified by current proteomic analyses on proteins secreted by the same adult-stage parasites as were used to construct the cDNA libraries. The same studies will answer a further methodological caveat: proteins can be secreted by non-signal-sequence-dependent pathways, and we have no information on the extent to which parasites may avail themselves of this possibility. One example already exists, of the macrophage migration inhibitory factor homolog of B. malayi which is exported despite lacking a signal peptide 6162.
On a broader platform, we have addressed the question of whether secreted proteins of parasitic nematodes show accelerated evolution, and our results indicate that this is the case. The predominance of predicted secreted proteins in the novel class prevents us, at this stage, from discerning whether rapid evolution was consequent upon acquiring secretory status, or if the more divergent gene products were those most advantageous to co-opt into secretion. Parallel studies on other parasitic nematodes would now clarify these and additional issues. Have genes for parasite secreted proteins indeed acquired signal peptides, or have free-living lineages lost these motifs in the genes in question? Is more rapid diversification of secreted proteins a specific feature of parasitic nematodes, or can a similar phenomenon be observed in comparisons between divergent free-living organisms (such as C. elegans and C. briggsae)? These questions are now under study.
Materials and methods
Parasite material
N. brasiliensis was maintained in Sprague-Dawley rats as previously described 1063. For cDNA synthesis, adult worms were recovered from gastrointestinal contents 5 or 6 days following subcutaneous injection of 3,000 infective L3 larvae. Adults were recovered by Baermannization in saline at 37°C, washed 6 × in saline and 6 × in RPMI1640 containing 100 μg/ml penicillin and 100 U/ml streptomycin. Worms were incubated with 10% gentamicin for 20 min and then washed a further 6 × in RPMI1640 with antibiotics before immersion in Trizol for mRNA preparation.
cDNA libraries
Conventional libraries were constructed in Uni-Zap (Stratagene) and propagated in pBluescript SK+ from mixed adult worm mRNA as previously described 27. To construct an oligo-capped cDNA library, the technique of Fernández 45 was followed. mRNA was isolated from 1 ml of packed adult N. brasiliensis (approximately 10,000 worms) homogenized in 10 ml Trizol (Gibco Life Technologies). The homogenate was centrifuged (12,000g, 10 min), and the supernatant extracted with chloroform before isopropanol precipitation of RNA from the aqueous phase. mRNA was then purified with PolyA Purist oligo-dT cellulose (Ambion). Following dephosphorylation with calf intestinal phosphatase, mRNA was treated with tobacco acid pyrophosphatase to remove the 7-methylguanosine terminal cap on full-length mRNAs, leaving these with a reactive phosphate group. These were then adducted with the GeneRacer oligonucleotide (Invitrogen). Reverse transcription of mRNA was primed with a tagged oligo-dT (NotI primer-adapter). In this way, full-length transcripts contained specific extension sequences (5' Gene Racer and 3' oligo-dT tag) amenable to PCR amplification. Following PCR, products were ligated at both ends to SalI adapters, so that subsequent digestion with NotI provided inserts with cohesive ends to be directionally cloned into NotI/SalI-digested pSPORT1 vector.
EST sequencing
The library was used to transform DH10B Escherichia coli by electroporation, plated on ampicillin agar petri dishes, and colonies picked for sequencing. All colonies picked were grown overnight in 96-well plates, which were used to provide template samples for PCR before being directly archived. PCR reactions used M13 forward and reverse primers, and following shrimp alkaline phosphatase/exonuclease I treatment, products were directly sequenced with T7 primer on ABI automated sequencers. Archived clones are available on request from R.M.M. Where 3' sequencing was required, T3 primer was used.
Bioinformatics
Raw sequence trace data were processed to screen out vector and linking sequence, to remove low-quality sequence, and to trim poly(dA) tails using an in-house software solution. The resulting sequences were annotated with similarity information and library details and submitted to dbEST. To identify the nonredundant set of putative gene objects, sequences were clustered on the basis of sequence similarity using the CLOBB program 64. Consensus sequences representing the putative gene objects were then generated from clusters containing more than one sequence using the assembly program phrap (Phil Green, University of Washington; available from 65). Clusters containing only a single sequence ('singletons') and the consensuses generated from clusters containing more than one sequence ('clusters') were then subjected to the following BLAST analyses: BLASTN against a nonredundant DNA database (GenBank); BLASTX against a nonredundant protein database (SwissProt-trEMBL) and BLASTN against dbEST. Results from these analyses are available from our online database - NEMBASE 49. Peptide predictions were performed on individual sequences using the program DEcoder 66. Where DEcoder was unable to predict a peptide, ESTscan 67 was used. SignalP V2.0 6 was used to predict the presence of secretory signal peptides and signal anchors for each of the predicted proteins. Peptides were defined as bearing a signal peptide if both the hidden Markov model (HMM) predicted the presence of a secretory leader and three of the four parameters defined by the neural network model (C-score, Y-score, S-score and S-mean, as described in legend to Table 3) were fulfilled. Signal anchors were predicted if both the HMM predicted a signal anchor and two of the four criteria specified by the neural network model were fulfilled. Selected clones were subject to comparative analysis with database entries from C. elegans and other species. Alignments were made using Clustal X within MacVector 7.0 (Oxford Molecular) and the SignalP V2.0 web server 68 was used to chart hydrophobicity and potential cleavage sites in predicted protein sequences.
Cross-taxon similarity analysis
The relative similarity between N. brasiliensis EST sequences and those from the related parasitic nematodes Ancylostoma caninum/duodenale, Haemonchus contortus and Teladorsagia circumcincta were plotted with the SimiTri program 54, downloadable from 69.