Acta Tropica 176 (2017) 224–227

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Short communication

Proteomic analysis of Taenia hydatigena cyst fluid reveals unique internal microenvironment Yadong Zhenga,b,

MARK

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a State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, CAAS, Lanzhou 730046, China b Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Taenia hydatigena Echinococcus granulosus Metacestode Cyst fluid

Taenia hydatigena is a parasitic flatworm that is widely distributed around the world. Using MS/MS, the proteome of T. hydatigena cyst fluid (CF) was profiled and a total of 520 proteins were identified, 430 of which were of sheep origin. T. hydatigena shared 37 parasite-origin and 109 host-origin CF proteins with Echinococcus granulosus. Compared with E. granulosus, T. hydatigena had much more CF proteins associated with amino acid synthesis and complement cascades. In addition, glutamate metabolism and anti-oxidative reactions were identified as relatively more important events. These results suggest that T. hydatigena metacestodes have internal microenvironment with special immune and oxidative conditions.

1. Introduction Infection by Taenia hydatigena, a cestode that is mainly transmitted between dogs and livestock, is occurred worldwide and normally asymptomatic. Although there are no public health issues concerned, T. hydatigena encystment in pigs causes difficulties in serological diagnosis of cysticercosis by Taenia solium due to strong cross-reactions, a pathogen responsible for human neurocysticercosis (Nguyen et al., 2016). T. hydatigena resides in the intestine of canines and other carnivores, such as polar bears and cats. Eggs are expelled with faeces into environment and intermediate hosts, including pigs, cattle, sheep and wild animals, get infected after consumption of egg-contaminated food or water. In the digestive tract, oncospheres are released from eggs and activated. Then activated oncospheres reach the liver with blood stream, in which parasites undergo migration from the liver parenchyma to the surface and eventually into the abdomen, causing acute hepatitis. After nearly three months, metacestodes become matured and infective, which are characterized by a milk-white bell-shaped cyst filled with transparent or slightly yellow fluid. Matured metacestodes mostly encyst onto the omentum and mesenterium, occasionally on the liver surface or in the chest cavity, and develop into adult worms when definitive hosts eat cyst-containing visceral offal, thus finishing its lifecycle. Cyst fluid (CF) is composed of a variety of molecules, such as sucrose, uric acid, enzymes and proteins with antigenic properties. The protein components of CF are dynamically changed with a parasitic

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Corresponding author at: 1 Xujiaping, Yanchangbu, Lanzhou 730046, Gansu, China. E-mail address: [email protected].

http://dx.doi.org/10.1016/j.actatropica.2017.08.015 Received 4 June 2017; Received in revised form 8 August 2017; Accepted 16 August 2017 0001-706X/ © 2017 Elsevier B.V. All rights reserved.

environment, and seem to be likely associated with the reproductivity of Echinococcus granulosus (Aziz et al., 2011; Santos et al., 2016), a zoonotic cestode that causes cystic echinococcosis in humans and animals. Moreover, CF contains a number of host-derived proteins and is considered as a protein reservoir that influences the outcome of parasite-host interactions (Santos et al., 2016). These ideas are further evidenced by a recent study that CF protein profiles were various with their location and fertility status of the parasite (Zeghir-Bouteldja et al., 2017). Therefore, the protein profiling of CF is useful for us to understand T. hydatigena biology. It was herein shown the proteome of T. hydatigena CF by high performance liquid chromatography-coupled tandem mass spectrometry (LC–MS/MS), and the results revealed its unique features in amino acid metabolism, immune responses and oxidative stresses. 2. Materials and methods Fresh and intact T. hydatigena cyst was dissected from the liver surface of slaughtered sheep in an abattoir, Qinghai Province, China. After five washes in ice-cold PBS, the CF was collected and then centrifuged at 10,000g for 10 min at 4 °C, followed by addition of protease inhibitor cocktail (Sigma). Approximately 2 mg of CF proteins were used for preparation of peptides, and peptide sequencing was conducted using LC–MS/MS (ThermoFisher Scientific) as described previously (Zheng et al., 2017). Due to the unavailability of T. hydatigena full genome, the raw data were searched using Mascot (Matrix Science,

Acta Tropica 176 (2017) 224–227

Y. Zheng

Fig 1. Proteins identified in T. hydatigena CF. (A) Comparison of parasite-origin proteins between T. hydatigena CF and E. granulosus fertile CF (FCF) and infertile CF (ICF). 183 FCF and 63 ICF proteins give a nonredundant dataset of 204 proteins. (B) Comparison of host-origin proteins between T. hydatigena CF and E. granulosus FCF and ICF. 44 FCF and 282 ICF proteins give a nonredundant dataset of 294 proteins. (C) Top 20 pathways identified for sheep-origin proteins. The number of proteins involved in each of pathways is shown beside individual columns.

The presence of the enzyme indicates the tight regulation of glutamate homeostasis in T. hydatigena CF, of which the fluctuations may be harmful to parasites. Consistent with this idea, the content of ornithine aminotransferase significantly increased in E. granulosus ICF compared with FCF (Santos et al., 2016). Additionally, a number of parasite enzymes involved in glycolmetabolism, such as phosphoenolpyruvate carboxykinase, enolase and phosphoglycerate kinase 1, were also abundant in T. hydatigena CF, but some of them were absent in E. granulosus CF (Table 1), suggesting the divergence in glycol metabolism between two parasites. But this explanation may be premature because protein profiles of E. granulosus CF are various with an encystment site and fertile status of the parasite (Zeghir-Bouteldja et al., 2017). Surprisingly, nearly 83% (430/520) of the CF proteins identified were of sheep origin (Fig. 1B and Table S2), much higher than 44 of cattle proteins in E. granulosus FCF (Santos et al., 2016). Similarly, GO analysis also returned catalytic activity (42.1%) and binding (41.9%) as main molecular function terms, and metabolic pathways were most frequently represented (Fig. 1C and Table S2). Of 430 sheep proteins, the most proteins seemed to be specific for T. hydatigena CF, only with 25.3% (109/430) shared with E. granulosus ICF and FCF (Fig. 1B). It was worthy to mention that the amino acid biosynthesis was annotated as one of the frequently represented pathways and 31 sheep proteins were found to be involved in, only 13 of which existed in E. granulosus CF (Fig. 1C), suggesting a more important role of amino acid metabolism in the growth and development of T. hydatigena larvae. It is well known that cestodes have no digestive system and obtain various nutrients by tegument. Unlike E. granulosus metacestodes that mostly live in the liver parenchyma, T. hydatigena metacestodes reside onto the omentum and mesenterium or the liver surface and have less intimate contacts with host tissues/organs, giving rise to the limitation of nutrient intake. The existence of a plethora of proteins involved in amino acid biosynthesis may compensate this limitation to satisfy with parasite nutrient needs. Another interesting finding was that a total of 25 sheep proteins identified were associated with complement and coagulation cascades,

version 2.3.02) against T. solium protein database (12,329 sequences) retrieved from GeneDB (http://www.genedb.org/Homepage/Tsolium) with parameters as did before (Zheng et al., 2017). Then the filtered data were searched against sheep protein database (34,806 sequences) retrieved from NCBI (https://www.ncbi.nlm.nih.gov/genome/?term= sheep) for identification of host-origin proteins. To minimize false peptide identification, peptides were counted if they had a score no less than 20 at the 99% confidence interval by Mascot probability analysis. Protein identification was accepted as reported elsewhere (Aziz et al., 2011), and all the identified proteins contained at least two unique peptides. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for the identified proteins were annotated. 3. Results and discussion A total of 25,282 spectra were generated, and 443 and 3679 unique peptides were obtained for T. hydatigena and sheep, respectively. In total, 520 proteins were identified. Of them, 90 proteins were of parasite origin (Fig. 1A and Table S1), less than 183 in E. granulosus fertile CF (FCF) (Santos et al., 2016). The majority of T. hydatigena proteins were annotated as catalytic activity (52.8%) and binding (37.5%) terms of molecular function, and 33.3% of them (30/90) seemed to be involved in metabolic pathways (Table S1). 37 parasite proteins were commonly shared by both T. hydatigena CF and E. granulosus FCF and infertile CF (ICF), but 53 were only present in T. hydatigena CF (Fig. 1A and Table S1). It was found that ornithine aminotransferase, an enzyme essential for glutamate metabolism, was enriched in T. hydatigena CF (Table 1). This enzyme is highly conserved in all eukaryotic organisms and primarily expressed in the liver and kidney. Using ornithine as a substrate, ornithine aminotransferase produces glutamate (Ginguay et al., 2017). Although the content of free ornithine and glutamate in CF changes in different hosts, it seems to keep stable in a given host even after drug treatment (Yao et al., 1994). 225

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Table 1 Top 20 parasite proteins abundant in T. hydatigena CF. Protein

Size (kDa)

Unique peptides

Unique spectra

E. granulosusa

Metabolic enzymes Phosphoenolpyruvate carboxykinase [GTP] Enolase Malate dehydrogenase, cytoplasmic Glucose-6-phosphate isomerase Fructose 1, 6 bisphosphate aldolase Glyceraldehyde-3-phosphate dehydrogenase Ornithine aminotransferase Aldo keto reductase family 1, member B4 Delta-aminolevulinic acid dehydratase Phosphoglycerate kinase 1

70 47 37 62 40 36 27 34 39 42

25 18 12 11 11 10 8 8 7 7

31 19 16 14 19 13 11 8 9 8

Y Y Y Y Y Y Y N N N

Protein binding Heat shock 70 kDa protein 4 14-3-3 protein beta:alpha 14-3-3 protein

71 28 28

20 9 8

25 11 8

Y Y Y

Cytoskeletal/membrane Actin Basement membrane specific heparan sulfate Basement membrane specific heparan sulfate

42 93 882

15 10 7

19 13 7

Y Y Y

Stress response/signaling N-myc downstream regulated Delta protein 4

45 134

8 7

12 8

N N

Other Ag5 Gynecophoral canal protein

54 95

8 11

14 12

Y Y

a

Y: a protein is also present in E. granulosus CF; N: a protein is absent in E. granulosus CF (Aziz et al., 2011; Santos et al., 2016).

survive immune attacks by peritoneal immune cells, especially small peritoneal macrophages. In parallel, more than 10 different glutathione S-transferases, an enzyme involved in oxidative stresses (Board and Menon, 2013), were also identified in T. hydatigena CF (Table S2), but only one in E. granulosus ICF (Santos et al., 2016). Taken together, these results suggest that T. hydatigena metacestodes have internal microenvironment with special immune and oxidative conditions.

which were also another frequently represented pathway. At least 8 different complement-related factors, including complement I, B, C2, C3, C3-like, C4, C5 and C9, existed in T. hydatigena CF, whereas only one, complement C4, in E. granulosus ICF (Table S2). In mammals, complement proteins are mainly secreted by hepatocytes and macrophages, possibly involved in inflammatory responses against Taenia crassiceps infection (Ramirez-Aquino et al., 2011). Complement-induced responses may also be detrimental to E. granulosus, as indirectly evidenced by the presence of several complement inhibitors in the cyst wall (Irigoin et al., 2008). Although their bioactivities and role remains unclear, the existence of many complement components suggests the predominance of complement-mediated immune responses in T. hydatigena CF. In accord with the previous studies (Aziz et al., 2011; Monteiro et al., 2010; Santos et al., 2016), sheep serum albumin was also identified as the most abundant protein in T. hydatigena CF (Table 2). However, immunoglobulins were scarce in T. hydatigena CF, which were abundant in E. granulosus CF (Monteiro et al., 2010; ZeghirBouteldja et al., 2017). Interestingly, some enriched proteins such as catalase and dihydrodiol dehydrogenase 3 were present in T. hydatigenaCF. In mammals, catalase is ubiquitously distributed in all major organs and predominantly enriched in peroxisomes. In addition to the dismutation of hydrogen peroxide, it can also catalyze nitric oxide (NO) to form nitrogen dioxide (Glorieux et al., 2015). During Echinococcus species infection, NO is detrimental and involved in regulation of immune responses (Amri and Touil-Boukoffa, 2015; Touil-Boukoffa et al., 1998; Zheng, 2013). Accordingly, parasites have evolved multiple mechanisms to modulate NO production (Zheng, 2013; Zheng et al., 2016). In peritoneal cavity, there exist many types of specialized immune cells, including eosinophils and macrophages that are divided into two subpopulations: large peritoneal macrophages and small peritoneal macrophages. Under steady state conditions, these peritoneal macrophages account for 30%–35% of all peritoneal cells, but small peritoneal macrophages swiftly proliferate and produce highlevel NO during infections (Cassado Ados et al., 2015). Therefore, abundant catalase is supposed to help T. hydatigena metacestodes

4. Conclusion Comparatively, T. hydatigena had much more CF proteins associated with amino acid synthesis and complement cascades than E. granulosus did, and T. hydatigena CF had unique immune and oxidative conditions.

Conflict of interest statement The author has declared that no competing interest exists.

Acknowledgements The author is indebted to the editor and anonymous reviewers for their constructive comments. The study was financially supported by grants from the National Key Basic Research Program (973 program) of China (2015CB150300), the National Natural Science Foundation of China (31201900 and 31472185) and Central State Public-interest Scientific Institution Basal Research Fund (1610312016017). The funders had no role in study design, data collection and analysis, decision to publish and preparation of the manuscript.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actatropica.2017.08. 015. 226

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Table 2 Top 20 sheep proteins abundant in T. hydatigena CF. Size (kDa)

Unique peptides

Unique spectra

E. granulosusa

55 37

34 26

57 54

Y N

99

50

64

N

48 98

28 35

52 49

Y Y

62

26

43

Y

40 45

21 27

43 42

N N

63

21

39

Y

37

22

38

Y

36

19

37

Y

60 75 52 119

34 30 22 31

77 44 39 37

N N Y Y

Transport Serum albumin precursor Serotransferrin isoform X1 Selenium-binding protein 1 isoform X1

69 110 53

68 60 28

126 95 53

Y Y Y

Protein binding/other Regucalcin Heat shock cognate 71 kDa protein isoform X1

33 71

24 27

63 36

Y Y

Protein

Metabolic enzymes Retinal dehydrogenase 1 Dihydrodiol dehydrogenase 3 Cytosolic 10formyltetrahydrofolate dehydrogenase isoform X1 Alpha-enolase isoform X3 Cytoplasmic aconitate hydratase Phosphoglucomutase-1 isoform X2 Alcohol dehydrogenase 1C 4-hydroxyphenylpyruvate dioxygenase Glucose-6-phosphate isomerase Fructose-1,6bisphosphatase 1 Prostaglandin reductase 1 Immune response Catalase isoform X1 Complement C3-like Hemopexin isoform X1 Complement C3 isoform X1

a Y: a protein is also present in E. granulosus CF; N: a protein is absent in E. granulosus CF (Aziz et al., 2011; Santos et al., 2016).

References Amri, M., Touil-Boukoffa, C., 2015. A protective effect of the laminated layer on

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