Proteomic Analysis by Shotgun LC-MS/MS of Cyst Wall Protein Components From the Protozoan Parasite Cryptocaryon irritans, the Causative Agent of White Spot Disease in Marine Fish

Honghua Wang1,2, Summia Perveen1,2, Shucheng Shao1,2, Kun Wang1,2 and Fei Yin1,2,*

1Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Collaborative Innovation Centre for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo 315211, People’s Republic of China

2School of Marine Sciences, Ningbo University, 169 South Qixing Road, Ningbo 315832, People’s Republic of China

ABSTRACT

To illustrate the protein composition of the Cryptocaryon irritans cyst wall, a shotgun-liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique was applied and all identified proteins were categorised using a bioinformatics approach. The results showed that 3,472 proteins were identified with a theoretical molecular weight of 11.53–610.90 kDa and an isoelectric point of 4.13–11.77. A total of 63.07% or 50.09% proteins were annotated by Kyoto Encyclopaedia of Genes and Genomes (KEGG) or Gene Ontology (GO) terms, respectively. These results highlight that mass material is transported and catabolised and growth- and death-related proteins are highly expressed, including a number of vesicle-mediated transport, cytoskeletal and extracellular secretion-related proteins. These results provide a platform to study tomont cell characteristics and provide a further understanding of cyst wall formation and mechanisms.

Article Information

Received 07 June 2019

Revised 12 July 2019

Accepted 19 July 2019

Available online 27 January 2020

Authors’ Contribution

HW and FY conceived and designed the study. HW, SP, SS, KW performed experiments, analyzed the sequencing data and interpreted the data. HW wrote the manuscript. FY helped in interpreted the data and wrote the paper.

Key words

Cryptocaryon irritans, Protozoan parasite, Cyst wall, Proteomics, LC-MS/MS.

DOI: https://dx.doi.org/10.17582/journal.pjz/20190707110736

* Corresponding author: feige895@gmail.com

0030-9923/2020/0002-0649 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Introduction

Cryptocaryon irritans, a parasitic ciliate that penetrates the epithelium of the gills, skin, and fins of marine fish, causes acute mortality in cultured fish. The tomont of C. irritans is an important stage for cell division and proliferation. Therefore, it is necessary to cut off the life cycle before the parasites proliferate and reinfect the host.

The cyst wall is the cell’s ‘armour’, which prevents a large number of drugs from penetrating the cell, which may otherwise cause damage to the protoplasm (Dan, 2006; Zahid et al., 2018). Ultrastructural observations show that the thickness of the C. irritans cyst wall is about 4 μm, including a multi-layered structure composed of three regions with different densities but with similar forms (Ma et al., 2017). The chemical composition of the C. irritans cyst wall, however, is not known. The chemical composition of the protozoan cyst wall is mainly protein and polysaccharides (Calvo et al., 2003). Furthermore, as the structural and regulatory proteins of the cyst wall of parasitic protozoa are often used as targets for screening and designing new anti-parasite drugs (Jarroll and Sener, 2003; Aguilar-Díaz et al., 2011), identifying the C. irritans cyst wall proteins will be helpful to screen and develop drugs that inhibit cyst wall formation.

Shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a technique used to determine proteins in complex systems. This method increases the probability that a protein of low abundance is identified. This technique has been widely used in many fields of life science (Aggarwal et al., 2006), especially proteomics, including for large-scale screening of different proteins in Pseudourostyla cristata (Gao et al., 2015). In the present study, shotgun- LC-MS/MS and bioinformatics analysis techniques were used to identify the protein components of the C. irritans cyst wall.

 

Materials and methods

Cryptocaryon irritans cyst wall

C. irritans were isolated from naturally infected Larimichthys crocea, and the same species of fish with an average body mass of 100 g were used as animal models to establish the passage system (Yin et al., 2018). A large number of tomonts adhered to the bottom of the aquaria (1062-L, Фbottom=130 cm ×H=80 cm) four days after the infection. The fish were transferred to another clean aquarium, and residual tomonts were collected by carefully discarding the debris. The tomonts were incubated in a one liter beaker. The theronts were released about -four days later, and the empty cyst wall was harvested under a microscope. Approximately 1.5×104 empty cyst walls were gathered and combined in a 1.5 ml centrifuge tube. The empty cyst walls were washed with three times in fresh seawater and clean empty cyst walls (Fig. 1) were stored at −80°C before the proteomic analysis.

Seawater salinity and temperature for aquaculture were 29%–31% and 28±1°C, respectively.

 

Protein extraction

The frozen clean empty cyst walls were transferred to tubes and ground with SDT lysis buffer (pH 8.5, 4% SDS, 100 mM Tris-HCl and 100 mM DL-dithiothreitol (DTT) using the MP FastPrep-24 5G homogeniser (MP Biomedical, Santa Ana, CA, USA). The homogenised cyst wall samples were incubated in a buffer of six M urea containing 2% SDS and 5 % β-mercaptoethanol at 100°C for 10 min to extract the cyst wall proteins. Subsequently, the cyst wall proteins were concentrated using an Ultrafree-0.5 centrifugal filter device with a high-flux Biomax-100 membrane (Millipore, Bedford, MA, USA). First, 500 μl of solubilised protein sample was added to the tube and centrifuged at 14,000×g for 30 min; the column was inserted into a new tube and centrifuged at 1,000×g for two min. Finally, 20 μl (20 mg/ml) of concentrated protein was obtained (Wang et al., 2017). The protein content in the supernatant was quantified by the Bradford method (Bradford, 1976). Aliquots were stored at −80°C until use.

Cyst wall protein enzymolysis

The filter-aided proteome preparation method was used for enzymolysis of the cyst wall proteins. Total-protein samples (200 μg) diluted in 100 mM DTT solution to a concentration of 100 mg/mL were boiled for five min. The samples were left to cool to 25°C and were then loaded into a 10 kDa ultrafiltration tube (Sartorius, Goettingen Germany), followed by adding 200 μL UA buffer (8 M urea and 150 mM Tris-HCl pH 8.0) to the tube and centrifugation at 14,000×g for 15 min, followed by a wash in UA buffer. Then, 100 μL iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues, and the samples were incubated for 30 min in the dark. The filters were washed three times with 100 μL UA buffer and then twice with 100 μL 25 mM NH4HCO3 buffer. Finally, the protein suspensions were digested with 4 μg trypsin (Promega, Madison, WI, USA) in 40 μL of 25 mM NH4HCO3 buffer overnight at 37°C, and the resulting peptides were collected as the filtrate. The peptides in each sample were desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL; Sigma, St. Louis, MO, USA), concentrated by vacuum centrifugation, and reconstituted in 40 µL of 0.1% (v/v) formic acid. Finally, the concentration of the resulting peptide was analysed at OD280, and aliquots of the samples were freeze-dried for the proteomic analysis.

LC-MS/MS analysis

Peptide mixtures from the above extraction were separated by reverse phase-high-performance liquid chromatography (RP-HPLC) followed by tandem MS analysis. RP-HPLC was carried out using a surveyor EASY-nLC system (Thermo Finnigan, San Jose, CA, USA). The fractions were thawed and dissolved in buffer A (0.1% v/v formic acid in Milli-Q water). The column was equilibrated for 20 min with 95% (v/v) buffer A. The peptide mixtures were separated using a Thermo Scientific EASY column (2 cm × 100 μm 5 μm-C18) and a Thermo Scientific EASY column (75 μm × 100 mm 3 μm-C18) at a flow rate of 300 nL/min. The peptides were separated with solvent B [acetonitrile with 0.1% (v/v) formic acid] using a segmented gradient from 4–9% (v/v) in one min, from 9–50% in 54 min, from 50–90% (v/v) in one min and then at 90% (v/v) for 20 min.

A Q-Exactive (Thermo Finnigan) mass spectrometer was set to perform data acquisition in the positive ion mode, with a selected mass range of 300–1,800 mass/charge (m/z). Resolving power for the Q-Exactive was set to 70,000 for the MS scan and 17,500 for the MS/MS scan at m/z 200. MS/MS data were collected using the top 20 most abundant precursor ions with charge ≥ 2 as determined from the MS scan. The maximum ion injection times for the survey scan and the MS/MS scans were 50 and 60 ms, respectively, and the automatic gain control target values for both scan modes were set to 3 × 106. These parameters were selected with an isolation window of 2 m/z and fragmented by higher-energy collisional dissociation with normalised collision energy of 27 eV. Dynamic exclusion for selected precursor ions was set to 60 s and the under ratio on the Q-Exactive was 0.1%.

Mass spectrometry data analysis and functional annotation of the proteins

Raw files were searched using MASCOT 2.2 software for database retrieval against the protein database (uniprot_Ciliophora_169050_20171127.Fasta, http://www.uniprot.org). The following search parameters were set: trypsin as the enzyme; missed cleavage sites were set to 2; fixed modification was carbamidomethyl (C); dynamic modification of setting oxidation (M). Blast2Go (https://www.blast2go.com/) software was used to screen the differentially expressed proteins in the Gene Ontology (GO) functional annotations. The expression levels of the proteins were annotated using a KO analysis (http://www.genome.jp/kegg/tool/map_pathway2.html). Kyoto Encyclopaedia of Genes and Genomes automatic annotation server (KASS) was used for the default parameters to perform the pathway annotation.

Results and discussion

Cyst wall proteins tandem mass spectrometric analysis

C. irritans cyst wall proteins were excised, digested and analysed by shotgun LC-MS/MS. The C. irritans cyst wall proteins were identified by searching the Ciliophora database at the NCBI; 3,472 proteins were identified with mascot scores ≥ 20, of which 1,194 proteins had a unique peptide value ≥ 2 (Supplementary Table I). All 3,472 proteins were distributed in a range of molecular weights of 5.72–998.30 kDa and isoelectric points of 4.13–11.77, according to the spectrometric analysis (Figs. 2, 3; Supplementary Table I).

 

 

GO and KEGG categories of C. irritans cyst wall proteins

In this study, a total of 1,739 proteins (50.09 %) (Table I) were assigned predicted GO terms (Fig. 4, Supplementary Table  II). These terms were summarized into 2167 sub-categories under three GO terms corresponding to the biological process (BP) category (1248), cellular component (CC) category (299), and molecular function (MF) category (620). In the BP category, the top 20 sub-categories were above 35 %, which proteins most enriched, included cellular process, several metabolic process, organism process, macromolecule modification, localization, and phosphorylation. In the MF category, catalytic activity, cyclic compound binding, ion binding, small molecular binding, nucleotide binding, and hydrolase activity were most enriched sub-categories, were above 52% of this part. In the CC category, the cell, memebrane, intracellular, organelle, cytoplasm, macromolecular complex, and cytoskeleton were the most enriched parts, were above 65%.

 

Table I.- Annotation of proteins of proteomic profiles of Cryptocaryon irritans cyst wall.

Database	Number of annotated proteins	Percentage of annotated proteins in protein group
KO	2,499	71.98%
GO	1,739	50.09%
KEGG	2,190	63.07%
Protein group (Total)	3,472	100.00%

 

The proteins were also categorized using the KEGG database to identify the biological pathways in C. irritans cyst wall proteins. A total of 2,190 proteins (63.07 %) (Table I) were further annotated by KEGG and classified into six categories with 47 subclasses (290 known KEGG pathways) (Fig. 5).

Studies on the components of the cyst wall have deepened. As early as the 1960s, researchers used cytochemical labelling to suggest that the Entamoeba cyst wall is composed of a carbohydrate-protein complex (McConnachie, 1969). In 1980, biochemical analysis, fluorescence labelling, ultrastructural observations and X-ray diffraction were combined to confirm that chitin is the main component of the E. invadens cyst wall (Arroyo-Begovich et al., 1980). In recent years, studies have demonstrated that the cyst wall of protozoa is mainly composed of proteins and polysaccharides (Jarroll and Sener, 2003), and the protein components of the cyst walls of a variety of protozoa have been identified. For example, SDS-PAGE has been used to isolate small molecular proteins (Rios et al., 2012); MS analysis of purified E. invadens cyst walls revealed chitinase and two lectins (Samuelson and Robbins, 2011); eight SDS-PAGE strips were analysed by MALDI-TOF MS, and 42 proteins were found in the E. encysticus cyst wall (Wang et al., 2017). In the present study, 3,472 proteins were identified by LC–MS/MS.

 

 

Table II.- The top 1% confident proteins in turn following unique peptide count.

	Reference	Unique pep count
1	Agglutination/immobilization antigen OS=Cryptocaryon irritans PE=2 SV=1	22
2	Agglutination/immobilization antigen OS=Cryptocaryon irritans PE=2 SV=1	20
3	Surface antigen OS=Cryptocaryon irritans PE=2 SV=1	19
4	Immobilization antigen (Fragment) OS=Cryptocaryon irritans PE=2 SV=1	17
5	Agglutination/immobilization antigen OS=Cryptocaryon irritans GN=iAg PE=4 SV=1	14
6	ADP/ATP carrier OS=Cryptocaryon irritans PE=2 SV=1	14
7	Tubulin beta chain OS=Tetrahymena thermophila PE=1 SV=1	13
8	Agglutination/immobilization antigen (Fragment) OS=Cryptocaryon irritans PE=2 SV=1	12
9	Tubulin beta chain OS=Tetrahymena thermophila GN=BTU1 PE=1 SV=1	12
10	Tubulin beta chain OS=Euplotes focardii PE=3 SV=1	11
11	Immobilization antigen (Fragment) OS=Cryptocaryon irritans PE=2 SV=1	10
12	Tubulin beta chain (Fragment) OS=Sorogena stoianovitchae GN=sbt PE=2 SV=1	10
13	Tubulin beta chain OS=Euplotes crassus PE=3 SV=1	10
14	Actin (Fragment) OS=Metopus es PE=3 SV=1	10
15	Actin (Fragment) OS=Heterometopus palaeformis PE=3 SV=1	10
16	Actin (Fragment) OS=Metopus es PE=3 SV=1	10
17	Agglutination/immobilization antigen isoform 1 OS=Cryptocaryon irritans PE=2 SV=1	9
18	Agglutination/immobilization antigen isoform 3 (Fragment) OS=Cryptocaryon irritans PE=2 SV=1	9
19	Agglutination/immobilization antigen isoform 5 (Fragment) OS=Cryptocaryon irritans PE=2 SV=1	9
20	Uncharacterized protein OS=Paramecium tetraurelia GN=GSPATT00035237001 PE=4 SV=1	9
21	Tubulin beta chain (Fragment) OS=Chilodonella uncinata GN=btub PE=3 SV=1	9
22	Tubulin beta chain (Fragment) OS=Colpoda sp. PE=3 SV=1	9
23	Tubulin beta chain OS=Stylonychia lemnae GN=TUBB1 PE=3 SV=1	9
24	Tubulin beta chain OS=Gastrostyla steinii PE=3 SV=1	9
25	ATP synthase subunit alpha OS=Stentor coeruleus GN=SteCoe_10017 PE=3 SV=1	9
26	ATP synthase subunit alpha OS=Paramecium tetraurelia GN=GSPATT00039376001 PE=3 SV=1	9
27	Tubulin alpha chain OS=Tetrahymena thermophila PE=1 SV=1	9
28	Tubulin alpha chain (Fragment) OS=Frontonia sp. PE=3 SV=1	9
29	Tubulin alpha chain OS=Paramecium tetraurelia GN=alphaPT4 PE=3 SV=1	9
30	ATP synthase subunit beta OS=Stylonychia lemnae GN=Contig4879.g5215 PE=3 SV=1	9
31	Tubulin beta chain OS=Stentor coeruleus GN=BTU01 PE=3 SV=1	8
32	Tubulin beta chain OS=Stentor coeruleus GN=BTU05 PE=3 SV=1	8
33	Tubulin beta chain (Fragment) OS=Myrionecta rubra PE=2 SV=1	8
34	Tubulin beta chain (Fragment) OS=Nyctotherus ovalis PE=3 SV=1	8
35	Tubulin beta chain (Fragment) OS=Chilodonella uncinata GN=Btub PE=3 SV=1	8

 

If the unique peptide count was considered a reference indicator for the confidence of correctly identifying the proteins, the top 1% (35 in 3,472) of proteins were antigens, ATP/ADP transfer/creation-related proteins, and cytoskeleton-related proteins (tubulins and actin) (Table II). In addition, the top 100 proteins also included glyceraldehyde-3-phosphate dehydrogenase, heat shock proteins, ubiquitin, elongation factor (EF) 1-α, p-loop containing nucleoside triphosphate hydrolase, histone H4, calmodulin 1, fat domain-containing protein, transitional endoplasmic reticulum ATPase, histone variant H3.5 and preprotein translocase subunit-like protein (Supplementary Table I). It proved that the cytoskeleton-related proteins comprise the major components and protect against adverse environmental conditions in the resting cyst wall of E. encysticus (Wang et al., 2017). Agglutination/ immobilisation and surface antigens have been identified in all stages of the C. irritans life cycle (Huang et al., 2012; Lin et al., 2013). Fish immunised with C. irritans tomonts with a cyst wall produce specific antibody (Bai et al., 2008), suggesting that antigens are present in the cyst wall. Heat shock proteins are important housekeeping genes and are involved in encystation of parasitic protozoa when they encounter a hostile environment (Wang et al., 2012). ADP glycosylation factor-ARFs in Pseudvurostyla cristata cells are related to intracellular transport and extracellular polymerisation of cyst wall materials (Gao et al., 2015). The cyst wall protein-EF-Tu of C. cucullus is related to the formation of a fibrous or network-like structure of the scale layer of its cyst wall (Funadani et al., 2016). Feeding RNAi to silence the EF-1α gene suppresses the formation of resting cysts in C. cucullus (Sogame et al., 2016).

Structural and regulatory proteins of the cyst wall are often targets for screening and designing new drugs (Jarroll and Sener, 2003). For example, the formation of a cyst wall requires the synthesis of a large number of proteins, and puromycin is a protein synthesis inhibitor; a 50 μg mL−1 dose can cause failure of the C. cucullus cyst wall to form (Funadani et al., 2016). β-1,3-glucan is a component of the oocyst wall of Toxoplasma and Eimeria, and biosynthesis of glucans is regulated by glucan-hydrolase/synthase. This enzyme is also a target of echinocandins, which inhibit formation of the cyst wall by interfering with glucan-hydrolase/synthase activity (Bushkin et al., 2012). Synthesis of E. invadens cysts relies on chitin synthase and chitinase, while inhibitors (poly D, nikkomycin and allosamidin) of these enzymes lead to failed cyst formation (Ghosh et al., 1999). Protein kinases, as an important drug target, are also widely regarded in the control of parasitic protozoa, and different protein kinases inhibitors (staurosporine, chelerythrine chloride and Calphostin C) exhibit an inhibitory effect on encystation (Samanta et al., 2018). Human Glrac has been considered a target for designing anticancer drugs (Aznar and Lacal, 2001). Because Glrac regulates the trafficking and secretion of cyst wall proteins in the protozoa Giardia, it has also been used as a potential target to develop new anti-parasite drugs (Krtková et al., 2016). In this study, eight nuclear transport-related proteins (40S ribosomal protein SA, GTP-binding nuclear protein, transmembrane proteins, N-acetyltransferase B complex non-catalytic subunit, GTP-binding nuclear protein ran and nucleoporin nup155) were found in the C. irritans cyst wall. Our previous study confirmed that the nuclear transporter inhibitor leptomycin B effectively induces C. irritans theronts and tomontsdeath (Yin et al., 2016). In addition, a number of cyst wall formation-related proteins, particularly some proteins peculiar to the tomonts of the C. irritans, were identified (Supplementary Table II) (Mai et al., 2015). These proteins will provide a basis for researchers to develop anti-C. irritans drugs.

 

Table III.- Vesicle-mediated transport-related proteins.

	Reference	MW (kDa)	PI
1	Dynein heavy chain 1, axonemal protein OS=Tetrahymena thermophila (strain SB210) GN=TTHERM_00558640 PE=4 SV=2	487.70	5.79
2	WD40-repeat-containing domain OS=Pseudocohnilembus persalinus GN=PPERSA_00474 PE=4 SV=1	222.44	8.9
3	Zinc-binding dehydrogenase family oxidoreductase OS=Tetrahymena thermophila (strain SB210) GN= TTHERM_00384890 PE=4 SV=2	201.13	6.07
4	Intraflagellar transport protein, putative OS=Ichthyophthirius multifiliis (strain G5) GN=IMG5_148350 PE=4 SV=1	198.15	6.55
5	Clathrin heavy chain OS=Ichthyophthirius multifiliis (strain G5) GN=IMG5_187840 PE=3 SV=1	198.04	5.97
6	Uncharacterized protein OS=Paramecium tetraurelia GN=GSPATT00020489001 PE=4 SV=1	133.07	7.81
7	Transmembrane protein, putative OS=Tetrahymena thermophila (strain SB210) GN=TTHERM_00923100 PE=4 SV=3	113.30	8.2
8	Adaptin amine-terminal region family protein OS=Tetrahymena thermophila (strain SB210) GN=TTHERM_00155580 PE=4 SV=3	113.00	5.73
9	Phospholipase OS=Paramecium tetraurelia GN=GSPATT00019109001 PE=3 SV=1	112.86	8.67
10	Coatomer subunit gamma OS=Tetrahymena thermophila (strain SB210) GN=TTHERM_00444320 PE=3 SV=1	105.39	5.47
11	Adaptin amine-terminal region family protein OS=Tetrahymena thermophila (strain SB210) GN=TTHERM_00287890 PE=4 SV=2	100.45	5.8
12	Coatomer subunit beta' OS=Stentor coeruleus GN=SteCoe_76 PE=3 SV=1	97.81	5.08
13	Coatomer subunit beta OS=Pseudocohnilembus persalinus GN=PPERSA_03040 PE=4 SV=1	97.32	5.92
14	Uncharacterized protein OS=Pseudocohnilembus persalinus GN=PPERSA_05656 PE=4 SV=1	91.97	5.3
15	Phospholipid-transporting ATPase OS=Ichthyophthirius multifiliis (strain G5) GN=IMG5_078220 PE=3 SV=1	91.56	8.21
16	Transport protein OS=Stylonychia lemnae GN=Contig1948.g2110 PE=4 SV=1	86.28	5.74
17	Sec1-like protein OS=Pseudocohnilembus persalinus GN=PPERSA_07320 PE=3 SV=1	69.11	6.2
18	Uncharacterized protein OS=Stentor coeruleus GN=SteCoe_14443 PE=3 SV=1	62.67	5.33
19	Uncharacterized protein OS=Paramecium tetraurelia GN=GSPATT00026361001 PE=3 SV=1	50.81	8.54
20	Mu1 adaptin OS=Oxytricha trifallax GN=OXYTRI_04462 PE=3 SV=1	50.28	8.65
21	Uncharacterized protein OS=Paramecium tetraurelia GN=GSPATT00015008001 PE=4 SV=1	48.82	6.45
22	Uncharacterized protein OS=Stentor coeruleus GN=SteCoe_5626 PE=4 SV=1	23.96	9.1

 

Acknowledgements

The authors gratefully acknowledge funding support from the Natural Science Foundation of Shanghai (Grant No. 17ZR1439600), the Natural Science Foundation of Zhejiang Province (Grant No. Y18C190005), and the K.C. Wong Magna Fund in Ningbo University.

 

Supplementary material

There is supplementary material (Tables I and II) associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20190707110736

 

Statement of conflict of interest

The authors declare no conflict of interest.

 

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