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Peihao Zhang, Wei Cai, Isabelle Vallee, Yonghua Zhou, et al.
To cite this version:
Yong Yang, Mingwei Tong, Xue Bai, Xiaolei Liu, Xuepeng Cai, et al.. Comprehensive proteomic
analysis of lysine acetylation in the foodborne pathogen Trichinella spiralis. Frontiers in Microbiology,
Frontiers Media, 2018, 8, pp.1-15. �10.3389/fmicb.2017.02674�. �hal-02621878�
doi: 10.3389/fmicb.2017.02674
Edited by:
Maria Schirone, Università di Teramo, Italy
Reviewed by:
Zhao Chen, University of California, Davis, United States M. Guadalupe Ortega-Pierres, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico
*Correspondence:
Mingyuan Liu liumy36@163.com Yonghua Zhou toxo2001@163.com
†
These authors have contributed equally to this work.
Specialty section:
This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology
Received: 01 August 2017 Accepted: 21 December 2017 Published: 11 January 2018
Citation:
Yang Y, Tong M, Bai X, Liu X, Cai X, Luo X, Zhang P, Cai W, Vallée I, Zhou Y and Liu M (2018) Comprehensive Proteomic Analysis of Lysine Acetylation in the Foodborne Pathogen Trichinella spiralis.
Front. Microbiol. 8:2674.
doi: 10.3389/fmicb.2017.02674
Comprehensive Proteomic Analysis of Lysine Acetylation in the Foodborne Pathogen Trichinella spiralis
Yong Yang
1,2†, Mingwei Tong
3†, Xue Bai
1†, Xiaolei Liu
1, Xuepeng Cai
4,5, Xuenong Luo
5, Peihao Zhang
2, Wei Cai
2, Isabelle Vallée
6, Yonghua Zhou
7* and Mingyuan Liu
1*
1
Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis/College of Veterinary Medicine, Jilin University, Changchun, China,
2Wu Xi Medical School, Jiangnan University, Affiliated Hospital of Jiangnan University, Wuxi, China,
3State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, China,
4China Institute of Veterinary Drug Control, Beijing, China,
5State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China,
6JRU BIPAR, ANSES, École Nationale Vétérinaire d’Alfort, INRA, Université Paris-Est, Animal Health Laboratory, Maisons-Alfort, France,
7Jiangsu Institute of Parasitic Disease, Wuxi, China
Lysine acetylation is a dynamic and highly conserved post-translational modification that plays a critical role in regulating diverse cellular processes. Trichinella spiralis is a foodborne parasite with a considerable socio-economic impact. However, to date, little is known regarding the role of lysine acetylation in this parasitic nematode. In this study, we utilized a proteomic approach involving anti-acetyl lysine-based enrichment and highly sensitive mass spectrometry to identify the global acetylated proteome and investigate lysine acetylation in T. spiralis. In total, 3872 lysine modification sites were identified in 1592 proteins that are involved in a wide variety of biological processes.
Consistent with the results of previous studies, a large number of the acetylated proteins appear to be involved in metabolic and biosynthetic processes. Interestingly, according to the functional enrichment analysis, 29 acetylated proteins were associated with phagocytosis, suggesting an important role of lysine acetylation in this process. Among the identified proteins, 15 putative acetylation motifs were detected. The presence of serine downstream of the lysine acetylation site was commonly observed in the regions surrounding the sites. Moreover, protein interaction network analysis revealed that various interactions are regulated by protein acetylation. These data represent the first report of the acetylome of T. spiralis and provide an important resource for further explorations of the role of lysine acetylation in this foodborne pathogen.
Keywords: lysine acetylation, post-translational modification, lysine acetylation motif, interaction network, Trichinella spiralis
INTRODUCTION
Trichinellosis is a common food-borne parasitic zoonosis worldwide. Infection occurs by consuming raw or inadequately cooked meat containing muscle larvae of the Trichinella parasite.
This parasite can infect a wide variety of hosts, including humans. Trichinella infection has been
reported in 66 countries around the world. Trichinella spiralis is the major causative agent of
trichinellosis. Outbreaks of trichinellosis in humans have been regularly reported worldwide, and serological analysis has indicated that more than 11 million people may be infected with Trichinella (Murrell and Pozio, 2011). Trichinellosis is regarded as an emerging or re-emerging infectious disease, particularly in developing countries, where cooking habits, poor sanitary conditions, and a lack of veterinary inspection facilitate infection (Gottstein et al., 2009). This zoonosis is both a public health hazard and an economic issue in terms of the porcine breeding industry and food safety (Bai et al., 2017). Therefore, in order to control and eradicate this disease, it is necessary to develop novel diagnostic and therapeutic methods for trichinellosis, which will require a comprehensive understanding of the parasite’s biology.
In recent years, the genome, transcriptome, epigenome, and proteome of T. spiralis have been reported and released in public databases, providing a greater understanding of the molecular basis of T. spiralis biology, host–parasite interactions, and pathogenesis (Mitreva et al., 2011; Gao et al., 2012; Liu et al., 2012, 2016). However, to the best of our knowledge, no post-translational modifications (PTMs), which occur during or after protein biosynthesis, have been described in T. spiralis, limiting further functional studies. PTMs regulate diverse protein properties, including folding, stability, localization, binding affinities, and activity, and they play important roles in a variety of cellular pathways and processes. Because of their substantial effects on protein functions and cellular processes, studies of protein PTMs have become more important in the post-genomic era (Walsh et al., 2005; Witze et al., 2007). To date, more than 461 different PTMs, such as phosphorylation, methylation, ubiquitination, malonylation, succinylation, glycosylation, and acetylation, have been identified to play a role in the functional regulation of many prokaryotic and eukaryotic proteins. These PTMs form a complex regulatory system for the dynamic control of cellular processes under various conditions (Yang and Seto, 2008).
Among these PTMs, lysine acetylation (Kac) is a highly dynamic and regulated modification that is defined as the reversible transfer of an acetyl group from the acetyl donor acetyl-CoA to the ε -amino group of a lysine residue of a protein molecule. The Kac process is regulated by lysine acetyltransferases and deacetylases (KDACs), resulting in a regulatory range that is extensive and comparable with those of other major PTMs (Choudhary et al., 2009; Drazic et al., 2016).
KDACs have been identified as drug target to develop novel anti- parasite treatments, and some selective KDAC ligands showed the potential for the treatment of parasitic diseases (Wang et al., 2015).
Recent studies have indicated that non-enzymatic chemical acetylation occurs broadly in eukaryotic cells, suggesting that this PTM may have various roles in protein function and cellular processes (Wagner and Hirschey, 2014; Olia et al., 2015). Kac was first identified in eukaryotic histones, where it is involved in regulating chromatin structure and transcription. In addition to its role in histones, Kac were found in a number of non-nuclear proteins which are located in almost every compartment of the cell (Friedmann and Marmorstein, 2013; Drazic et al., 2016).
Protein acetylation outside the nucleus participates in diverse
cellular physiological processes, including cell-cycle regulation, cytoskeletal dynamics, apoptosis, autophagy, and metabolism (Kim et al., 2006; Close et al., 2010; Wang et al., 2010; Zhao et al., 2010; Sun et al., 2015). In addition, acetylation has also been implicated to influence the persistence, virulence and antibiotic resistance of pathogenic bacteria (Xie et al., 2015).
Recent advances in antibody-based affinity purification of acetylated peptides and highly sensitive mass spectrometry (MS) have made significant contributions to the study of Kac at the whole-proteome level. Global Kac has been characterized in a number of eukaryotic and prokaryotic organisms (Jeffers and Sullivan, 2012; Zhang et al., 2013; Pan et al., 2014; Nie et al., 2015; Xie et al., 2015). These proteome-wide analyses of Kac have mapped large datasets of lysine-acetylated proteins and have confirmed that Kac is involved in various cellular events, especially in modulating metabolic processes. Based on these studies, it is proposed that the functional roles of acetylation in these processes are evolutionarily conserved in both eukaryotes and prokaryotes, and acetylation may thus rival phosphorylation.
Although Kac has been characterized in many organisms, the study of the lysine acetylome in helminths has been relatively limited, with only one nematode species (Schistosoma japonicum) being previously characterized (Hong et al., 2016). Given the general use of acetylation as a regulatory mechanism in different organisms, it can be speculated that T. spiralis also employs acetylation as a modification to modulate various aspects of its biology. Lending evidence to this hypothesis, T. spiralis appears to contain a great number of acetyltransferases and deacetylases in its genome (Mitreva et al., 2011).
To bridge this knowledge gap, in this study, proteomics was used in combination with immunoprecipitation in the first large-scale analysis of lysine-acetylated proteins in T. spiralis, identifying 3872 Kac sites in 1592 proteins. These lysine- acetylated proteins are involved in a variety of biological functions and are localized to multiple cellular compartments.
Bioinformatics analysis showed that acetylation in T. spiralis modulates a wide range of cell processes. This study provides a global picture of the T. spiralis acetylome, which not only greatly expands our knowledge of helminth protein acetylation but also provides important information for the design of new effective drugs or vaccines for controlling trichinellosis.
MATERIALS AND METHODS Parasite Collection and Protein Extraction
Animal experiments were performed according to the guidelines
of the National Institute of Health (publication No. 85–23,
revised 1996) and Animal Experimentation Guidelines of Jilin
University and were approved by the Animal Experimental
Ethics Committee of Jilin University, China. T. spiralis (ISS534)
parasites were maintained by serial passage in female BALB/c
mice. T. spiralis muscle larvae (ML) were recovered from infected
BALB/c mice at 35 days post-infection (dpi) using a standard
HCl-pepsin digestion method. ML were washed manually in
phosphate-buffered saline (PBS) at 37 ◦ C to remove any residual host proteins and were then collected for further lysine acetylome analysis.
Total proteins were extracted as previously described (Zhou et al., 2016) with minor modifications. In brief, parasites were lysed with lysis buffer containing 8 M urea, 10 mM dithiothreitol (DTT) and 0.1% protease inhibitor cocktail, followed by sonication. Cellular debris was then removed by centrifugation at 20,000 × g and 4 ◦ C for 10 min. Proteins contained in the supernatant were precipitated with cold 15% trichloroacetic acid (TCA) for 2 h at − 20 ◦ C. After centrifugation, the pellet was washed three times with cold acetone, followed by dissolution in buffer containing 8 M urea and 100 mM NH 4 CO 3 (pH 8.0).
Protein concentration was measured using a 2-D Quant kit (GE Healthcare) according to the manufacturer’s instructions.
Trypsin Digestion and High-Performance Liquid Chromatography (HPLC)
Fractionation
Proteins were reduced with 10 mM DTT, alkylated with 20 mM iodoacetamide (IAA), and digested with trypsin (Promega) overnight at a 1:50 trypsin:protein mass ratio. To ensure complete digestion, additional trypsin was added at a 1:100 trypsin:protein mass ratio for 4 h for a second digestion. Tryptic peptides were separated into six fractions using high-pH reverse-phase HPLC, as previously described (Zhou et al., 2016). Separated peptides were then dried completely in a SpeedVac (Thermo Scientific) and stored at − 80 ◦ C for further enrichment of acetylated peptides.
Affinity Enrichment and Liquid
Chromatography Electrospray Ionization Tandem Mass Spectrometry
(LC-ESI–MS/MS) Analysis
Fractionated peptides were dissolved in NETN buffer containing 100 mM NaCl, 1 mM EDTA, 50 mM Tris–HCl, and 0.5%
NP-40 (pH 8.0) and incubated with anti-acetyl lysine antibody beads (PTM Biolabs) at 4 ◦ C overnight with gentle shaking.
After washing with NETN buffer and deionized H 2 O, the bound peptides were eluted with 0.1% trifluoroacetic acid, dried in a SpeedVac, and cleaned using C18 ZipTips (Millipore) according to the manufacturer’s instructions.
The obtained peptides were then analyzed by LC-ESI–
MS/MS on a Q Exactive TM Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled online to the EASY-nLC 1000 ultra-performance liquid chromatography (UPLC) system. The resulting MS/MS data were processed using MaxQuant with an integrated Andromeda search engine (v.1.5).
Tandem mass spectra were queried against the UniProt T. spiralis database using the following parameters (Zhou et al., 2016):
trypsin/P was specified as the cleavage enzyme and allowed up to four missed cleavages, five modifications per peptide, and five charges. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as a fixed modification, and oxidation on Met, acetylation on Lys, and acetylation on the protein N-terminus
were specified as variable modifications. False discovery rate (FDR) thresholds for the protein, peptide, and modification sites were set at 1%. The minimum peptide length was set at 7. The site localization probability was set as > 0.75.
Bioinformatics Analysis
Gene Ontology (GO) annotation of the proteome was derived from the UniProt-GOA database
1according to Zhou et al.
(2016). All identified acetylated proteins were classified using three categories of GO annotations: biological process, cellular component, and molecular function. The subcellular localization predication program, Wolfpsort (Horton et al., 2007), was used to analyze cellular localization data, and the InterPro database
2and InterProScan were used to compile functional descriptions of protein domains. Protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa et al., 2004). Amino acid sequence motifs in identified protein were analyzed using Soft motif-x, and a position- specific heat map was generated using WebLogo 3.4
3. Secondary structures of all acetylated proteins were predicted using NetSurfP software. Fisher’s exact test was used to analyze GO and KEGG pathway and domain enrichment. Terms with adjusted p-values < 0.05 were considered significantly enriched.
The Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database
4was used for analysis of the protein–
protein interaction (PPI) network. The interaction network form STRING was visualized using Cytoscape (version 3.0) software (Bauer-Mehren, 2013).
RESULTS AND DISCUSSION Identification and Analysis of
Lysine-Acetylated Proteins in T. spiralis
Sequencing of the T. spiralis genome revealed the existence of a number of acetyltransferase and deacetylase orthologs, suggesting that Kac may play a vital role in T. spiralis development and metabolism (Mitreva et al., 2011). To comprehensively characterize protein acetylation in T. spiralis, analysis of Kac was carried out at the proteome level using tryptic digestion, anti-acetyl lysine antibody enrichment, and high- resolution LC-MS/MS. Generally, the distribution of mass errors was near zero, and most were ≤ 10 ppm (Figure 1A). Moreover, all lysine-acetylated peptides were between 7 and 37 amino acids in length, which is consistent with tryptic peptides (Figure 1B).
These results indicated that the sample preparation method was adequate and that the modified peptide data obtained from MS was highly accurate.
In total, 3872 Kac sites distributed across 1592 acetylated proteins were identified (Supplementary Table S1). This exceeds the level of Kac that has so far been reported in S. japonicum (Hong et al., 2016), indicating the essential role of Kac in
1
http://www.ebi.ac.uk/GOA/
2
http://www.ebi.ac.uk/interpro/
3
http://weblogo.threeplusone.com/create.cgi
4