Open Access

Use of expressed sequence tags as an alternative approach for the identification of Taenia solium metacestode excretion/secretion proteins

  • Bjorn Victor1Email author,
  • Pierre Dorny1,
  • Kirezi Kanobana2,
  • Katja Polman2,
  • Johan Lindh3, 4,
  • André M Deelder5,
  • Magnus Palmblad5 and
  • Sarah Gabriël1
BMC Research Notes20136:224

DOI: 10.1186/1756-0500-6-224

Received: 25 February 2013

Accepted: 3 June 2013

Published: 6 June 2013

Abstract

Background

Taenia solium taeniasis/cysticercosis is a zoonotic helminth infection mainly found in rural regions of Africa, Asia and Latin America. In endemic areas, diagnosis of cysticercosis largely depends on serology, but these methods have their drawbacks and require improvement. This implies better knowledge of the proteins secreted and excreted by the parasite. In a previous study, we used a custom protein database containing protein sequences from related helminths to identify T. solium metacestode excretion/secretion proteins. An alternative or complementary approach would be to use expressed sequence tags combined with BLAST and protein mapping to supercontigs of Echinococcus granulosus, a closely related cestode. In this study, we evaluate this approach and compare the results to those obtained in the previous study.

Findings

We report 297 proteins organized in 106 protein groups based on homology. Additional classification was done using Gene Ontology information on biological process and molecular function. Of the 106 protein groups, 58 groups were newly identified, while 48 groups confirmed previous findings. Blast2GO analysis revealed that the majority of the proteins were involved in catalytic activities and binding.

Conclusions

In this study, we used translated expressed sequence tags combined with BLAST and mapping strategies to both confirm and complement previous research. Our findings are comparable to recent studies on other helminth genera like Echinococcus, Schistosoma and Clonorchis, indicating similarities between helminth excretion/secretion proteomes.

Keywords

Expressed sequence tag Excretion/secretion proteins Taenia solium Proteomics

Findings

Introduction

Taenia solium taeniasis/cysticercosis is a zoonotic helminth infection mainly found in poor and rural regions of Africa, Asia and Latin America where it has a large impact on public health [13]. The adult tapeworm develops in the small intestine of humans (taeniasis). Mature proglottids full of eggs break off from the distal end of the worm and leave the body with the stool. Both humans and pigs can act as intermediate hosts when the infective larval stages (oncospheres) inside the eggs are ingested and liberated in the stomach. The oncospheres then enter the blood flow through the intestinal mucosa. Cysticercosis is caused when oncospheres lodge themselves in the subcutaneous and muscle tissues and the central nervous system, where they develop into metacestode larval stages (cysts). In humans, epilepsy and other neurological symptoms can be provoked by immunological reactions against degenerating cysts that have developed in the central nervous system (neurocysticercosis).

Diagnosis of porcine and human (neuro) cysticercosis largely depends on antigen and/or antibody detection, but these serological methods also have their specific drawbacks [4]. Improving current diagnostic assays automatically implies better knowledge of the proteins secreted and excreted by the metacestodes.

Proteomic experiments involving liquid chromatography and tandem mass spectrometry (LC-MS/MS) typically attempt to match the generated experimental spectra to in silico spectra from a (target) protein database. Ideally, this database contains every protein likely to be in the sample, but obtaining such an all-including protein database proves difficult when there is little to no genomic information available, as was the case for T. solium until recently [5]. In our previous study, we bypassed this limitation by using a custom database with known proteins from related helminths (Taenia, Echinococcus, Schistosoma and Trichinella) as a target database in the LC-MS/MS experiments [6]. We deliberately did not use translated expressed sequence tags (ESTs), because we wanted to investigate to usefulness of a target database made up of protein sequences originating mostly from (closely) related helminths.

The usefulness of ESTs for the identification of helminth proteins has already been described for e.g. Haemonchus contortus[7, 8] and Echinococcus granulosus[9]. In the case of T. solium, ESTs from different parasite stages have been made available by different research groups, both published [10, 11] and unpublished (Huang J. et al., Analysis of Taenia solium and Taenia saginata adult gene expression profile, 2009 and Aguilar-Diaz H. et al., Taenia solium larva/adult ESTs, 2007). In this study, we use T. solium ESTs combined with the Basic Local Alignment Search Tool (BLAST) and protein mapping to supercontigs of E. granulosus (a member of the Taeniidae family) to investigate whether we could increase the number of T. solium metacestode excretion/secretion protein identifications from the previous study.

Materials and methods

Generation of the data set

The in vitro production of the T. solium metacestode excretion/secretion proteins from Peru and Zambia at 24h and 48h and the generation of line spectra mzXML files have been previously described [6].

Database design and data analysis

To construct the target database, 30,700 expressed sequence tags were downloaded from the National Center for Biotechnology Information (NCBI) website in April 2012 and a six frame translation was performed using transeq [12]. A Sus scrofa database with 1,388 Swiss-Prot sequences (http://www.uniprot.org/) and the common Repository of Adventitious Proteins database (112 protein sequences; http://ftp.thegpm.org/fasta/cRAP/crap.fasta) were also included to assist detection of host proteins and accidental contaminations, respectively. A decoy database with 185,700 reversed sequences was created using decoyfasta. These databases were fused into one final database. Database searching with X!Tandem (2010.10.01.1) [13] and subsequent analyses with PeptideProphet [14, 15], iProphet [16] and ProteinProphet [17] were also performed as previously described [6]. All above mentioned tools, except transeq, are included with the Trans-Proteomic Pipeline v4.5 RAPTURE rev 2 [18]. The identified translated ESTs were further filtered to a false discovery rate of < 1% and ESTs with an individual probability of zero were discarded. The remaining ESTs were blasted against the NCBI nonredundant database (E-value < 10 −10) and for each recognized EST, the best matching protein was retained. The resulting proteins were then screened by mapping the proteins to the E. granulosus supercontigs using TBLASTN (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/Echinococcus). Identifications with a Score > 200 were considered valid. Identifications with a lower score were manually evaluated and proteins originating from T. solium were retained. This step also helped to filter out host contaminations. Finally, proteins were grouped based on homology. All proteins that could not be grouped and were identified by only one EST were also discarded. Finally, Blast2GO was used for Gene Ontology (GO) annotations (biological process, molecular function and cellular component) and the construction of level 2 pie charts [19]. In order to gain more specific information, the largest categories were analyzed to levels 3 and 4.

Results and discussion

Identified proteins and gene ontology annotation

In this study, 297 proteins (from 1,787 translated ESTs) were identified and organized in 106 protein groups based on homology (Additional file 1). For simplicity, each protein group is represented by one protein. The groups were further organized by Gene Ontology annotation information on biological process and molecular function. A total of 48 protein groups are labelled with an asterisk, indicating that they were also identified in the previous study (Additional file 2) [6]. For brevity, Table 1 shows only the 58 newly identified protein groups. For a number of proteins/protein groups, no Gene Ontology information was available. Nonetheless, many of them, like the 8 kDa protein family [20], have been extensively studied and used in diagnostic assays.
Table 1

Protein groups ( n = 58) newly identified in Taenia solium metacestode excretion/secretion proteins, organized by Gene Ontology annotation information on biological processes and molecular functions

Gene ontology classification

Closest organism

gi code

Proteins a)

ESTs b)

 

Protein group

    

1) No Gene Ontology classification

 

Major egg antigen

Clonorchis sinensis

358336515

1

2

 

ES1 protein homolog

Multiplec)

-

3

5

 

Phosphoglyceride transfer protein

Taenia asiatica

124782980

1

7

 

Alpha-2-macroglobulin-like protein 1

Clonorchis sinensis

358333571

1

5

 

Aldose 1-epimerase

Clonorchis sinensis

358334888

1

2

 

SJCHGC02626 protein

Schistosoma japonicum

-

3

7

 

Hypothetical protein

Schistosoma mansoni

256079415

1

4

 

TSP1

Echinococcus multilocularis

209967595

1

4

 

Putative major vault protein

Echinococcus granulosus

62178032

1

2

2) Binding (miscellaneous)

 
 

Filamin

Multiple

-

3

7

 

Methionyl-tRNA synthetase cytoplasmic

Clonorchis sinensis

358255967

1

5

 

SJCHGC09631 protein

Schistosoma spp.

-

2

2

 

four and a half LIM domains protein 3

Clonorchis sinensis

358341124

1

7

 

Alpha-actinin isoform B

Taenia asiatica

124783372

1

3

 

Calumenin

Taenia asiatica

124784033

1

2

 

Calcium-binding protein

Schistosoma mansoni

256071353

1

2

 

Lysyl oxidase-like

Schistosoma mansoni

256072781

1

2

 

Porphobilinogen synthase

Multiple

-

3

3

 

Phosphoglucomutase-1

Clonorchis sinensis

358337844

1

2

 

Fibrillar collagen

Multiple

-

9

16

3) Glycolysis/Metabolic processes (miscellaneous)

 
 

Adenylosuccinate synthetase

Schistosoma mansoni

387912858

1

4

 

Adenylate kinase

Multiple

-

2

4

 

UDP-glucose pyrophosphorylase 2

Schistosoma spp.

-

2

2

 

Hypothetical protein SINV_09109

Solenopsis invicta

322793762

1

2

 

Aspartate aminotransferase

Multiple

-

3

5

 

Lactate dehydrogenase A

Taenia solium

318054471

1

6

 

SJCHGC05968 protein

Multiple

-

2

2

 

Methylthioadenosine phosphorylase

Multiple

-

2

2

 

Ornithine aminotransferase

Multiple

-

3

3

 

Endoglycoceramidase

Multiple

-

3

3

 

Aminoacylase

Multiple

-

2

2

 

Glucose-6-phosphate 1-dehydrogenase-like

Sus scrofa

350595984

1

2

 

Phosphoglycerate mutase

Multiple

-

3

7

4) (Endo)peptidase activity

 
 

Calpain

Multiple

-

5

5

 

UDP-glucose 4-epimerase

Multiple

-

2

2

 

Dipeptidyl-peptidase

Multiple

-

2

3

 

Glutamate carboxypeptidase 2

Clonorchis sinensis

358331956

1

3

5) Endopeptidase inhibitor activity

 

Kunitz protein 8

Multiple

-

2

3

6) Cell redox homeostasis/Oxidation-reduction related

 
 

Carbonyl reductase

Schistosoma spp.

-

3

3

 

Methionine sulfoxide reductase

Multiple

-

2

4

 

procollagen-lysine, 2-oxoglutarate

Multiple

-

3

6

 

5-dioxygenase 3

    

7) Transport

 
 

Charged multivesicular body protein

Multiplec)

-

3

4

 

SJCHGC06082 protein

Multiple

-

2

8

 

Glycolipid transfer protein-like protein

Taenia asiatica

124782916

1

2

 

Gamma-soluble NSF attachment protein

Multiple

-

4

10

 

Sodium/glucose cotransporter

Multiple

-

2

7

8) Motor activity/Cytoskeleton and Microtubule related

 
 

Tubulin polymerization-promoting protein

Multiple

-

2

2

 

Myophilin

Multiple

-

2

19

9) Miscellaneous Gene Ontology classification

 
 

Translation initiation factor 5A

Multiple

-

2

4

 

Ubiquitin-conjugating enzyme

Multiple

-

4

10

 

Protein-l-isoaspartate o-methyltransferase

Schistosoma mansoni

256081696

1

2

 

Protein DJ-1-like

Multiple

-

2

4

 

6-phosphogluconolactonase

Multiple

-

2

4

 

SJCHGC02435 protein

Schistosoma japonicum

56756018

1

5

 

Family T2 unassigned peptidase

Schistosoma mansoni

256088374

1

4

 

3’(2’), 5’-bisphosphate nucleotidase

Multiple

-

2

2

 

RAB GDP dissociation inhibitor alpha

Multiple

-

2

3

 

Laminin

Multiple

-

2

2

a) The number of proteins in each protein group.

b) The number of expressed sequence tags that were matched to proteins in this protein group.

c) ’Multiple’ indicates that different (helminth) genera have identified proteins in that protein group.

For simplicity, all protein groups are represented by one protein.

Most of the identified protein groups could be categorized in miscellaneous binding activities (e.g. Actin binding, calcium binding and metal ion binding), various metabolic processes, gluconeogenesis (Triosephosphate isomerase, Enolase, Phosphoenolpyruvate carboxykinase and Phosphoglucose isomerase), glycolysis (Glyceraldehyde-3-phosphate dehydrogenase, Phosphoglycerate kinase, Phosphoglycerate mutase and Fructosebisphosphate aldolase) and proteins with (endo) peptidase activity, including cysteine-type (Calpain, UDP-glucose 4-epimerase and Cathepsin), threonine-type (Proteasome subunits) and serine-type endopeptidase activity (Trypsin-like protein). Endopeptidase inhibitors with both serine-type (Kunitz protein 8 and Leukocyte elastase inhibitor) and cysteine-type endopeptidase inhibitor activity (Immunogenic protein Ts11) and components of the enzymatic antioxidant system of Taeniidae (Cu/Zn Superoxide dismutase, Glutathione S-transferase and Peroxiredoxin) were also identified [21].

Gene Ontology level 2 pie charts were created for biological process (Figure 1A), molecular function (Figure 1B) and cellular component (Figure 1C). To avoid overly busy charts, the sequence filter was set to 10. The two largest categories of the biological process chart were cellular and metabolic processes. Others included biological regulation, response to stimulus, multicellular organismal processes and cellular component organization or biogenesis. Further investigation of the general cellular and metabolic processes revealed primary and cellular metabolic processes at level 3 and protein, cellular macromolecule and cellular nitrogen compound metabolic processes at level 4 (Additional file 3, tab 1). Molecular function was clearly divided between binding and catalytic activity. GO level 3 showed protein binding and hydrolase activity while level 4 entailed mostly nucleotide binding, hydrolase activity (acting on acid anhydrides), cation binding, peptidase activity, cytoskeletal and identical protein binding (Additional file 3, tab 2). The level 2 pie chart for the cellular component indicated cell and organelle as the largest categories. Further analyses showed mostly cell part and membrane-bound organelle, and intracellular (part) GO terms at levels 3 and 4, respectively (Additional file 3, tab 3). Human Keratin and porcine Trypsin were identified in all samples. As Keratin is a common contamination and Trypsin was deliberately added during the LC-MS/MS experiments, both were omitted from the final results.
https://static-content.springer.com/image/art%3A10.1186%2F1756-0500-6-224/MediaObjects/13104_2013_Article_2252_Fig1_HTML.jpg
Figure 1

Gene Ontology level 2 pie charts displaying the biological processes (A), the molecular functions (B) and the cellular components (C) of the 297 proteins that were identified in the Taenia solium metacestode excretion/secretion proteins. Values within parentheses are the number of sequences associated with each Gene Ontology term. The biological processes are mostly metabolic and cellular processes, while the molecular functions are predominantly catalytic activity and binding. The cellular components reveal a number of intercellular proteins. All charts were created using Blast2GO with the sequence filter set to 10.

The presence of intracellular/non-secreted proteins in the ESPs is interesting and has been observed in other ESP studies before [22, 23]. Although it is highly likely that the majority of those proteins are indeed excreted or secreted by the parasite, the possibility that they are the result of leakage due to cyst damage or death should not be excluded.

In general, the findings reported in this study are comparable to recent studies on other helminth genera like Echinococcus[23], Schistosoma[24] and Clonorchis[25], indicating that excretion/secretion proteomes are not very different between helminth genera/species.

Comparison between the two studies

When comparing the level 2 GO terms identified in both studies (Table 2), all GO terms from the previous study were identified here as well. Additionally, we identified 6 new GO terms with the EST analyses: rhythmic process (GO:0048511), antioxidant activity (GO:0016209), molecular transducer activity (GO:0060089), protein binding transcription factor activity (GO:0000988), receptor activity (GO:0004872) and synapse (GO:0045202). Although a direct comparison between numbers should be avoided (due to proteins having multiple GOs and the presence of homologous proteins in the proteins groups, especially in the previous study where it is a logical result of the target database construction), the general levels of abundance (= proteins in each GO term) are largely comparable between the two studies e.g. in both studies, cellular process, metabolic process and biological stimulation are the largest groups for ‘biological process’ while binding and catalytic activity are the largest groups for ‘molecular function’ and cell and organelle are the largest groups for ‘cellular component’. The 6 new GO terms were identified by a very small number of proteins and may be a result of proteins being linked to multiple GO terms. This is supported by the fact that the proteins linked to these GO terms are homologous to other proteins identified in both studies, so none of these GO terms was identified by a ’new’ protein group.
Table 2

Gene Ontology level 2 annotations identified in this study alongside the ones identified in the previous study

Gene Ontology information

Current

Previous

  

(EST) study

study [[6]]

Biological process

  

cellular process

GO:0009987

185

162

metabolic process

GO:0008152

182

150

biological regulation

GO:0065007

91

153

response to stimulus

GO:0050896

82

147

multicellular organismal process

GO:0032501

75

107

cellular component organization or biogenesis

GO:0071840

74

92

developmental process

GO:0032502

68

85

localization

GO:0051179

60

88

signaling

GO:0023052

32

55

death

GO:0016265

32

65

immune system process

GO:0002376

20

59

locomotion

GO:0040011

20

29

growth

GO:0040007

18

28

multi-organism process

GO:0051704

14

65

reproduction

GO:0000003

10

36

biological adhesion

GO:0022610

9

13

viral reproduction

GO:0016032

8

12

cell proliferation

GO:0008283

5

24

cell killing

GO:0001906

2

21

rhythmic process

GO:0048511

2

-

Molecular function

  

binding

GO:0005488

176

168

catalytic activity

GO:0003824

175

129

structural molecule activity

GO:0005198

26

20

enzyme regulator activity

GO:0030234

11

21

electron carrier activity

GO:0009055

7

13

antioxidant activity

GO:0016209

7

-

transporter activity

GO:0005215

6

14

molecular transducer activity

GO:0060089

4

-

protein binding transcription factor activity

GO:0000988

3

-

nucleic acid binding transcription factor activity

GO:0001071

2

17

receptor activity

GO:0004872

1

-

Cellular component

  

cell

GO:0005623

182

168

organelle

GO:0043226

120

155

macromolecular complex

GO:0032991

72

79

membrane

GO:0016020

53

76

membrane-enclosed lumen

GO:0031974

27

63

extracellular region

GO:0005576

26

71

extracellular matrix

GO:0031012

10

11

synapse

GO:0045202

5

-

cell junction

GO:0030054

2

12

Although a direct comparison between numbers should be avoided (due to proteins having multiple GOs and the presence of homologous proteins in the proteins groups, especially in the previous study where it is a logical result of the target database construction), the general levels of abundance are largely comparable between the two studies. Additionally, this study revealed 6 new GO annotations.

Concluding remarks

In this study, we have used a library of translated ESTs combined with BLAST and mapping strategies not only to confirm previously identified T. solium metacestode excretion/secretion proteins, but to identify several new proteins as well, thereby effectively increasing the overall number of protein identifications.

The larger and more complete the EST database, the better proteomic coverage likely obtained. No ESTs from other Taeniidae were used in this study, since the available T. solium ESTs were already a merge of EST submissions by different groups and were therefore likely to offer decent proteome coverage. However, in cases where only a small EST library is available with low coverage, one could also include protein sequences and/or ESTs from related organisms in a combined database. This may be particularly advantageous in proteomic studies on less studied, unsequenced, organisms. It should be noted that research on non-sequenced organisms mostly relies on homology to already existing proteins from other (preferably closely related) organisms. Therefore, there is no possibility of finding unique proteins, unless (i) de novo sequencing is performed on the good quality unmatched experimental spectra or (ii) ESTs that were identified by spectra but remained unmatched during BLAST are further investigated.

Finally, it is important to realize that, although the mapping to the E. granulosus supercontigs helped to remove S. scrofa host proteins (e.g. Albumin, Protegrin and Hemopexin), some may still be present. Heat shock protein 70, for example, is identified both in S. scrofa and E. granulosus.

In future T. solium work, it is sensible to make use of the T. solium genome sequence that was recently published [5]. However, since no curated protein database or convenient mapping solution is currently available and, for many other helminths, no complete genome sequence is available, the method described here is still valid.

Availability of supporting data

The data sets supporting the results of this article are available in the PRIDE repository at http://www.ebi.ac.uk/pridewith accession numbers 19232 – 19267.

Abbreviations

BLAST: 

Basic Local Alignment Search Tool

ESPs: 

Excretion/Secretion Proteins

ESTs: 

Expressed Sequence Tags

GO: 

Gene Ontology

LC-MS/MS: 

Liquid Chromatography and tandem Mass Spectrometry

NCBI: 

National Center for Biotechnology Information.

Declarations

Acknowledgements

The authors thank the University of Zambia, School of Veterinary Medicine (Zambia) and the Universidad National Mayor de San Marcos (Lima, Peru) for assistance and use of facilities, and Hans Dalebout (LUMC, Leiden, The Netherlands) for technical support.

The research leading to these results has received funding from The Research Foundation - Flanders (FWO) (project number: G.0192.10N) and the European Union’s Seventh Framework Program (FP7/2007-2013) under grant agreement no. 221948 (ICONZ). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission.

Authors’ Affiliations

(1)
Veterinary Helminthology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine
(2)
Medical Helminthology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine
(3)
Swedish Institute for Communicable Disease Control
(4)
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet
(5)
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center

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