Accepted Manuscript Proteomic analysis of macrophage in response to Edwardsiella tarda-infection Lei Qin, Fuhou Li, Xingqiang Wang, Yuying Sun, Keran Bi, Yingli Gao PII:

S0882-4010(17)30453-9

DOI:

10.1016/j.micpath.2017.08.028

Reference:

YMPAT 2417

To appear in:

Microbial Pathogenesis

Received Date: 25 April 2017 Revised Date:

15 August 2017

Accepted Date: 16 August 2017

Please cite this article as: Qin L, Li F, Wang X, Sun Y, Bi K, Gao Y, Proteomic analysis of macrophage in response to Edwardsiella tarda-infection, Microbial Pathogenesis (2017), doi: 10.1016/ j.micpath.2017.08.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Proteomic analysis of macrophage in response to Edwardsiella tarda-infection Lei Qina,b*, Fuhou Lia, Xingqiang Wanga, Yuying Suna, Keran Bia, Yingli Gaoa a

Jiangsu Key Laboratory of Marine Biotechnology/ College of Marine Life and Fisheries, Huaihai Institute of

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Technology, Lianyungang, China Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Lianyungang, China

ABSTRACT

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Edwardsiella tarda is an important facultative intracellular pathogen infecting a wide range of host from fish to humans. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. E. tarda -macrophage interaction is vital in determining the outcome of edwardsiellasis. To fully elucidate the pathogenesis of E. tarda, the differential proteomes of RAW264.7 cells in response to E. tarda-infection, were analyzed at different time points with two-dimensional gel electrophoresis (2-DE) followed by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) identification. 26 altered proteins (18 up-regulated and 8 down-regulated proteins) were successfully identified, which are mainly involved in formation of phagosomes, macrophage microbicidal activity and anti-apoptosis of macrophage. Moreover, 6 corresponding genes of the differentially expressed proteins were quantified by quantitative real-time PCR (qPCR) to examine the transcriptional profiles. Western blot analysis further confirmed the differential expression of 5 proteins in the proteomic profiles. Based on these findings, we hypothesize that these differentially expressed proteins likely play a pivotal role in determining the course of E. tarda-infection. The result suggested that E. tarda could develop some strategies to achieve a successful intracellular lifestyle, including modulation of phagosome biogenesis, resistance to macrophage microbicidal agent and anti-apoptosis of macrophages. Thus, this work effectively provides useful and novel protein-related information to further understand the underlying pathogenesis of E. tarda-infection. Keywords: Edwardsiella tarda Macrophage Proteomic analysis LC-MS/MS 1. Introduction E. tarda is an important facultative intracellular bacterium infecting a wide range of hosts from fish to humans [1-3]. Many studies showed that the role of macrophages is essential for host defense against E. tarda- infection. Occurrence of the increased numbers of macrophages in diseased fish was the most prominent pathological changes evoked by E. tarda. Meanwhile, numerous E. tarda could be observed within macrophages [4-8]. E. tarda prefers to live inside macrophages in order to replication and escapes the defense of innate immunity, eventually resulting *

Corresponding author. E-mail address: [email protected]

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in upgrading the local infection to a systemic infection [2]. These findings indicated that macrophage could play very important roles in the defense mechanism of host against invading E. tarda. The macrophage-pathogen interaction is thought to be vital in determining the outcome of infection [9]. Given the prominent roles of macrophage in the host immune response to E. tarda, further exploration of the E. tarda -macrophage interactions is needed. Proper understanding of this mechanism could help us to understand the strategies used by E. tarda to survive and how intracellular survival leads to disease manifestation, thereby contributing to develop suitable strategies to overcome disease caused by this bacterium. Primary macrophages are terminally differentiated cells, which present several barriers to investigation, including their scarcity, heterogeneity and resistance to gene transduction, making many experiments difficult to interpret [10-12]. Therefore, macrophages cell lines are generally selected for study on macrophage-mediated immunity. For Mus musculus, entire genome sequence and abundant protein information are available at present. Furthermore, a large number of commercially available detective systems based on gene and protein of mouse could also be used, resulting in more efficiency and accuracy in carrying out the study involved in pathogen pathogenesis using mouse macrophage lines [1, 13-15]. Likewise, mouse macrophage lines have been widely used for pathogenesis-related study on E. tarda [1, 16-21]. With this in mind, we selected the RAW264.7 cells (a mouse macrophage line) as a model system to investigate the differential proteomes of macrophages in response to E. tarda-infection because of its biochemical and physiological resemblance to primary macrophages. To date, how E. tarda interact with macrophages has not been fully elucidated, although a variety of virulence factors have been implicated in this bacterium, including T3SS, T6SS, two-component system, dermatotoxin, hemolysins, OMPs, etc. [22]. Proteins are the actual functional molecules in the cell. The macrophages proteins will be altered greatly in response to E. tarda infection. By applying comparative proteomics techniques, the expression profile of protein alteration in E. tarda-infected macrophage can be probed. Therefore, proteomics analysis may provide more direct insights into the mechanism of interactions between macrophage and E. tarda. In this study, we performed 2-DE followed by LC-MS/MS identification, qPCR and western blot analysis to analyze the disparity in proteomic profiles between E. tarda-infected and uninfected macrophages at different time points, which may help to further elucidate the underlying pathogenesis of E. tarda-infection. 2. Materials and Methods 2.1 Cell culture and bacterial strains RAW264.7 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum at 37°C in 5% CO2 atmosphere. E. tarda strain QL-S (GenBank no. DQ233654) were stored at -70°C with 15% glycerol.

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2.2 E. tarda infection and sample preparation RAW264.7 cells were inoculated with E. tarda at a multiplicity of infection (MOI) of 10:1 or with medium (control), and incubated for 2 h to reach an adequate infection level. Three biological replicates were performed. After removal of the supernatant, fresh medium with 100 µg/mL gentamicin was added. The cells were then allowed to incubate for 1 h to ensure that all extracellular bacteria were killed. The infected cells were washed with PBS three times and incubated for extended 0 h (3 h post infection), 3 h (6 h post infection) and 6 h (9 h post infection), respectively. Then, cells were harvested by centrifugation at 1500×g for 10 min three times. The sediment were lysed in a buffer (7 M urea, 4% (w/v) CHAPS, 65 mM DTT, 2 M thiourea, 1 mM PMSF) by freezing and thawing in liquid nitrogen three times. The protein lysate were added with DNase (200 µg/mL)/RNase (50 µg/mL) at 4°C for 15 min. Then, the supernatant was collected by centrifugation at 40,000×g for 30 min at 4°C and stored at -70°C. The protein concentration was determined using the 2-D Quant kit (GE Healthcare, Piscataway, NJ). 2.3 2-DE and image analyses The 2-DE assay was performed as described by Cheng et al. [23] with some modifications. Isoelectric focusing (IEF) was performed using the Ettan IPGphor Ш system (GE Healthcare), with 18-cm IPG strips (linear, pH 3–10, GE Healthcare). The prepared proteins (700 µg/strip) were mixed with a total volume of 350 µl rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 50 mM DTT, 0.8% (v/v) IPG buffer). Three technical replicates were performed for each group. The protein samples were focused for a total of 70 kV·h (VHr). Post-IEF, the isoelectric-focused strips were incubated for 15 min in an equilibration buffer (6 M urea, 2% (w/v) SDS, 20% (v/v) glycerol, 0.375 M Tris pH 8.8) containing 2% DTT and then incubated for 15 min in an equilibration buffer containing 2.5% iodoacetamide. After equilibration, IPG strips were electrophoresed on 10% SDS-PAGE gels at a constant current of 5 mA/gel for 40 min and then at 30 mA/gel until the dye front reached the bottom of the gels using the Ettan DALTsix Electrophoresis unit (GE Healthcare). The proteins were visualised by colloidal Coomassie stain and scanned using a Powerlook 2100XL-USB scanner (UMAX, Taiwan). Then protein spots were analyzed using ImageMaster 2D platinum 5.0 (GE Healthcare). The normalization percentage volumes (vol.%) of protein spots between E. tarda-infected and uninfected cells was performed with Student’s t-test, which involved three biological and technical replicates for each group. Differentially expressed protein spots (P < 0.05) with at least 3-fold intensity changes both up and down were selected and subjected to identification by LC-MS/MS. 2.4 Protein extraction and LC-MS/MS Protein spots were excised from the gel and destained by incubation two times with 25 mM ammonium hydrogen bicarbonate (NH4HCO3)/50% acetonitrile (ACN) (v/v) for 10 min and shrunk by dehydration in 100% ACN, which was then removed in a vacuum centrifuge. 10 mM dithiotreitol (DTT) was added and the proteins were reduced for 1 h at 56°C. Then the DTT solution was replaced with 55mM

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iodoacetamide. After 45 min incubation in the dark, the gel pieces were washed with 25 mM NH4HCO3 for 10 min and dehydrated by addition of acetonitrile. The resulting gel pieces were dried and digested overnight at 37°C using 12.5 ng/µL trypsin dissolved in 25 mM NH4HCO3. After trypsin digestion, the peptide mixtures were mixed with 0.1% formic acid prior to LC-MS/MS analysis as described previously [24]. A prominence Nano LC-2D system (Shimadzu) interfaced with a MicrOTOF-QII (Bruker Daltonics) mass spectrometer was used for LC-MS/MS. 10µL of each sample were injected. The chromatographic separation was performed on a reversed phase C18 column (5 µm, 150 Å, Eprogen) at a flow rate of 400 nL/min with a 60 min gradient of 5 to 80% acetonitrile in 0.1% formic acid. The capillary voltage was maintained at 1500V and the temperature of the heated capillary was set at 150°C. The ion activation was achieved by utilizing argon at normalized collision energy. The positive ion mode was employed. The scan range of each full MS scan was m/z 50-2200. Mass spectrometry data were obtained and analyzed by Data acquisition software (Bruker Daltonics micrOTOFcontrol). 2.5 LC-MS/MS data analysis Data were analyzed using Data Analysis Software (Bruker Daltonics). The obtained MS/MS spectra were searched against the mouse subset of the Uniprot and NCBInr database in the Mascot server, version 2.3.01(Matrix Science) by using the following search parameters: fixed modifications: Carbamidomethylation (C), variable modifications: Oxidation (M) and Gln→Pyro-Glu (N-term Q), trypsin with one miscleavages, peptide mass tolerance: 0.05 Da, fragment mass tolerance: 0.1 Da and instrument type: ESI-QUAD-TOF. Only protein identifications with a score equal to or greater than the 95% confidence limit threshold estimated by Mascot were accepted. The classification of molecular function and biological process for the identified proteins with altered expressional levels were determined using GOSlimViewer tool (http://www.agbase.msstate.edu/) at Agbase database. In addition, the protein interaction network were analyzed using the STRING version 10.0 (http://string.embl.de/). 2.6 Quantitative real-time PCR (qPCR) validation RAW264.7 cells were infected with E. tarda as described above for the proteomic analysis. After the incubation for the indicated period time, total RNA was isolated from the cells with Trizol reagent (Invitrogen, USA). 6 protein genes of interest were selected for further examination with respect to expression alterations. According to the corresponding gene sequences of MS/MS identified proteins, specific primers were designed (Table 1). Total RNA was reverse transcribed using a PrimeScript™ RT reagent Kit with gDNA Eraser (Tarara), according to the manufacturer's protocol. Quantitative real-time PCR was performed in Stepone plus real-time PCR system (ABI) with the SYBR® Premix Ex Taq (Tarara) in a total volume of 20 µL. The amplification conditions were 10 min at 95˚C, 40 cycles of 5 s at 95˚C and 1 min at 60˚C, followed by a one-cycle reaction for melting curve: 95˚C for 15 s; 60˚C for 1 min and 95˚C for 15 s. All samples were performed in triplicate.

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Data were normalized to GAPDH and relative mRNA expression was determined using the 2−∆∆CT method. 2.7 Western blot validation Western blot of individual samples was performed to validate proteomic quantitation of 5 selected candidate proteins, including vimentin, β-actin, moesin, ATP Synthase beta and HSP60. Infections were also performed as described above for the proteomic analysis. Whole cell lysates of three independent experiments (50 µg each) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Non-specific binding sites were blocked with 5% milk powder in TBST for 2 h. The membranes were then incubated overnight with the specific primary antibodies (Abcam) at 4°C. Membranes were washed with TBST and incubated for 2h with goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma) at RT for 2 h. After washing steps, signals were visualized by enhanced chemiluminescence (ECL, Pierce). GAPDH antibody (Abcam) was used to ensure equal loading of protein on the gel. The mean anti-GAPDH normalized signal intensities were used to identify significant differences of protein amount between control and E. tarda-infected cells (Student’s t-test P < 0.05). Table 1 The specific primers based on the corresponding gene sequences of the identified proteins. Differentially expressed protein

Forward primer (5′–3′)

Reverse primer (5′–3′)

AGAGAAATTGCAGGAGGAGATG

Calreticulin

TGGAGGATGATTGGGACTTTC

TCGATCTTGGCTCGTTCATC

Transketolase

CACAGGGATTGAAGACAAGGAG

GCTCTGAACCTGGCTGTAAAT

Cis-aconitate decarboxylase

TE D

Vimentin

GTCAAGACGTGCCAGAGAAG

GTGCCTTCTATGCCAACTACTC

GTGCCGGGAAGCTCTTAAA

Protein disulfide-isomerase A3

CAAGGACTTACTCACCGCTTAC

CACCATCATGACCCTGTTTCT

Moesin

GAGGATGCTGTCCTGGAATATC

CCATAGCTCTGAGCCTTTCTT

CCAGTATGACTCCACTCACG

GACTCCACGACATACTCAGC

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GAPDH

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3. Results 3.1 2-DE profiles of E. tarda- infected RAW264.7 cells Proteins were extracted from E. tarda -infected and uninfected RAW264.7 cells at 3, 6, and 9 h post infection for 2-DE analysis. Spot matching, quantitative intensity analysis and statistics revealed 29 differentially expressed protein spots with a threshold greater than 3-fold ≥3(p < 0.05), including 19 significantly up-regulated spots and 10 significantly down-regulated spots. Of the up-regulated proteins in infected RAW264.7 cells, 4 protein spots were up-regulated at 6 h p.i. and 15 protein spots were up-regulated at 9 h p.i. Among the down-regulated proteins, 2 protein spots were down-regulated at 6 h p.i., 6 protein spots displayed a decrease at 9 h p.i. and 2 protein spots were only detected in uninfected RAW264.7 cells. In general, the majority of differentially expressed proteins appeared at 9 h after infection (Fig.1). No significant changes could be detected in expressed proteins at 3 h after infection.

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Fig. 1. Representative 2-DE gels of the protein expression profiles obtained from E. tarda -infected and uninfected RAW264.7 cells. (A) Protein gel from uninfected RAW264.7 cells at 9 hpi. (B) Protein gel from E. tarda -infected RAW264.7 cells at 9 hpi. The proteins of the samples were separated on a pH 3–10 liner IPG strip, followed by 10% SDS-polyacrylamide gel and colloidal Coomassie stain. The protein spots were analyzed using ImageMaster 2D platinum and the number allocated by the software indicate spots with at least 3-fold intensity changes both up and down in intensity (p < 0.05).

3.2 Mass spectral identification of the differentially expressed proteins To identify the differentially expressed proteins in 2-DE gels, the differentially

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expressed spots were excised and subjected to LC-MS/MS identification. 29 protein spots were all successfully identified corresponding to 26 proteins (Table 2). Among them, 3 different protein spots including 2 protein spots that only detected in uninfected RAW264.7 cells were all identified as vimentin. 2 protein spots were identified to be beta-actin. Their details, including identities, percentages of sequence coverage, isoelectric points, molecular weights, score and identified peptides, are summarized in Table 2. According to go annotations from AgBase, ontology revealed that the identified proteins were mainly associated with protein binding, nucleic acid binding, hydrolase activity and various other molecular functions (Fig. 2A). The differential cellular proteins were mainly involved in cellular process, metabolic processes, regulation of biological processes, response to stimulus and other activities (Fig. 2B).

Fig. 2. Gene ontology analysis of significantly expressed proteins in E. tarda -infected and uninfected RAW264.7 cells according to their molecular function (A) and biological process (B). This classification was produced based on an analysis using the GOSlimViewer tool at Agbase database (http://www.agbase.msstate.edu/).

We employed the STRING tool to analyze the interactions between identified proteins, Casp3 and Bcl2, the latter two play a vital role in cell apoptosis. Most of the differentially expressed proteins were highly connected as shown in Fig.3. The

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Protein ID

Abbr.

Swiss-Prot

NCBI

no.

no.

score

Protein a

Up- regulated proteins

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Table 2 Summary of differentially expressed proteins in E. tarda- infected RAW264.7 cells. MW(kDa) / PI

b

(%)

c

Peptides Identified

d

ACOD1

P54987

gi|119364598

2052

Serine/arginine-rich splicing factor 1

SRSFL

Q6PDM2

gi|118582269

3224

Rho GDP-dissociation inhibitor 1

ARHGDIA

Q99PT1

gi|31982030

1997

Histone H2B type 1-C/E/G

HIST1H2BC

Q6ZWY9

gi|594542582

1760

13.9/10.3

57

AMGIMNSFVNDIFER

60kDa heat shock protein

HSPD1

P63038

gi|183396771

2377

61.1/5.9

48

ALMLQGVDLLADAVAVTMGPK

Stress-70 protein

HSPA9

P38647

gi|162461907

5961

73.5/6.1

60

TTPSVVAFTADGER

Transketolase

TKT

P40142

gi|6678359

2736

68.2/7.2

55

QAFTDVATGSLGQGLGAACGMAYTGK

Protein disulfide-isomerase A3

PDIA3

P27773

gi|112293264

4236

57.1/5.9

65

MDATANDVPSPYEVK

Peroxiredoxin-1

PRDX1

P35700

gi|6754976

2089

22.4/8.3

73

KQGGLGPMNIPLISDPK

ACTB

P60710

gi|6671509

2073

42.1/5.3

54

QEYDESGPSIVHR

Moesin

MSN

P26041

gi|70778915

2345

67.8/6.2

63

KTQEQLASEMAELTAR

Ubiquitin-40S ribosomal protein S27a

RPS27a

P62983

gi|13195690

893

18.3/9.7

33

TITLEVEPSDTIENVK

ATP synthase subunit beta

ATP5b

P56480

gi|23272966

56265

56.3/5.3

37

FTQAGSEVSALLGR

ATP synthase subunit alpha

ATP5a

Nucleoside diphosphate kinase B

NME2

Nucleoside diphosphate kinase A

NME1

Proliferating cell nuclear antigen

PCNA

Desmoplakin Down- regulated proteins Vimentin

f

T-complex protein 1 subunit beta

MILDSLGVGFLGTGTEVFHK

27.8/10.4

49

IYVGNLPPDIR

23.5/5.1

61

VAVSADPNVPNVIVTR

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5301

59.9/9.2

61

TGTAEMSSILEER

gi|6679078

1572

17.3/6.8

79

YMNSGPVVAMVWEGLNVVK

P15532

gi|37700232

1364

17.2/7.4

86

VMLGETNPADSKPGTIR

P17918

gi|7242171

2039

28.8/4.8

72

LSQTSNVDKEEEAVTIEMNEPVHLTFAL

DSP

E9Q557

gi|190194418

2785

33.3/6.8

30

GYFNEELSEILSDPSDDTK

VIM

P20152

gi|31982755

19917

53.7/5.1

93

LLQDSVDFSLADAINTEFK

CCT2

P80314

gi|126521835

9449

57.5/6.4

88

VQDDEVGDGTTSVTVLAAELLR

EP

Q03265

Q01768

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Beta-actin

54

SC

Cis-aconitate decarboxylase

e

53.8/7.5

Coverage

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CALR

P14211

gi|6680836

4201

48.1/4.3

76

DMHGDSEYNIMFGPDICGPGTK

Annexin A1

ANXAL

P10107

gi|124517663

2955

38.9/6.9

84

GAMKGLGTDEDTLIEILTTR

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Calreticulin

EF-hand domain-containing protein D2

EFHD2

Q9D8Y0

gi|31981086

1920

26.8/5

50

SMIQEVDEDFDSK

Tropomyosin alpha-3 chain

TPM3

P21107

gi| 658132466

2196

33/4.7

51

TIDDLEDELYAQK

Tubulin alpha-1B chain

TUBA1B

P05213

gi|34740335

16121

50.8/4.9

95

AFVHWYVGEGMEEGEFSEAR

Coronin-1A

COROLA

O89053

gi|6753492

3484

51.6/6.1

63

FMALICEASGGGAFLVLPLGK

a

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Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein families. Ions score is −10log(P), where P is the probability that the observed match is a random event. Based on the swissprot2015 database using the MASCOT searching program as MS/MS data. Individual ions scores > 25 indicate identity or extensive homology (p < 0.05). b Predicted MW and pI from sequence analysis. c Coverage (%) means the number of amino acids spanned by the assigned peptides divided by the protein sequence length. d The peptides identified by LC-MS/MS with the best ion score. e Data from representative beta-actin. f Data from representative vimentin.

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analysis revealed three main networks of protein interactions among phagosome proteins, immune response proteins and apoptosis proteins. Furthermore, the KEGG pathway-based analysis performed by STRING tool showed four pathways involved in the networks of protein interactions. The top two pathways identified were the tuberculosis (map05152, p < 0.01) and phagosome (hsa04145, p < 0.01).

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Fig. 3. Protein interaction network analysis of the significantly altered proteins in the E. tarda -infected RAW264.7 cells using the software tool STRING. Three networks of the associated proteins were found in identified proteins that were significantly altered in the E. tarda -infected RAW264.7 cells. These included the networks for phagosome proteins, immune response proteins and apoptosis proteins. Casp3 and Bcl2 are important apoptosis-associated proteins.

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3.3 qPCR validation To verify the results of differentially expressed proteins in 2-DE, the transcriptional alterations of 6 genes selected from the differentially expressed proteins were analyzed by quantifying the mRNA transcripts using GAPDH gene as a reference. The trends in the alterations of the mRNA level of these 6 genes were similar to the changes in the patterns of the corresponding proteins in the 2-DE gels. The mRNA abundance of cis-aconitate decarboxylase, protein disulfide-isomerase A3, transketolase and moesin were increased, whereas the vimentin and calreticulin gene were reduced (Fig.4). The results provided transcriptional information complementary to the differential protein expression detected by the proteomic analysis. 3.4 Western blot validation 5 differentially expressed proteins identified by proteomic analysis were randomly selected for subsequent confirmation by western blot analysis. As shown in Fig. 5, ATP synthase beta, mosein, Hsp60 and Beta-actin proteins showed apparent up-regulation in E. tarda-infected RAW264.7 cells. Meanwhile, a significant down-regulation of vimentin was observed. These data were consistent with the changes in expression observed in the 2-DE analysis. 4. Discussion In the present study, we obtained a dynamic overview of the altered protein expression of RAW264.7 cells responding to E. tarda-infection. Cells for proteomics analysis were harvested after 3, 6 and 9 h of exposure to E. tarda for RAW264.7 cells, respectively. The results showed that no significant alteration could be found

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Fig. 4. Transcript alterations of the differentially expressed proteins in E. tarda-infected RAW264.7 cells. The mRNA levels of detected genes were normalized to that of GAPDH, and the mRNA expression was expressed relative to the expression level of control cells.*, P < 0.05; **, P < 0.01

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Fig. 5. Validation of proteomic data by Western blot analyses. (A)The immunoblots of ATP synthase beta, mosein, Hsp60, beta-actin and vimentin from infected and uninfected RAW264.7 cells. Results demonstrated are representative of three independent experiments. (B) The intensity of the immunoblots. *, P < 0.05; **, P < 0.01

in expressed proteins at 3 h after infection. We speculated that there may be two reasons for explaining this finding. First, at short times scales post exposure, the cells were more likely to adapt via post translational modification rather than protein changes. Second, the altered proteins might not be detected because of their low expression level. No extended time longer than 9 h for proteomic analyses were chosen taking one primary factors into account that more cells would undergo apoptosis and degeneration at long incubation period. Finally, 26 differentially expressed cellular proteins were identified in E. tarda-infected RAW264.7 cells. Most interestingly, among the proteins that showed differential expression, those

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involved in formation of phagosomes, macrophage microbicidal activity and apoptosis of macrophage were predominantly affected. The most significant proteins and their potential biological importance are discussed as follows. 4.1 Proteins associated with formation of phagosomes in macrophages. We found that some differentially expressed proteins in E. tarda -infected macrophages were identified in phagosomes [25], indicating that phagosomes constitution might be affected in E. tarda-infected macrophages. It is worthwhile to note that most of these altered proteins associated with phagosomes were down-regulated, including T-complex protein 1 subunit beta, calreticulin, annexin A1, tubulin alpha-1B chain, tropomyosin alpha-3 chain and coronin-1A. Phagosomes are key organelles for macrophages to participate in restricting the spread of intracellular pathogens [25]. Macrophages could deliver pathogens into the phagosomes, which matures by sequential interactions with endocytic and lysosomal compartments. Phagosome maturation, a key event in pathogen digestion and process, is associated with changes in the composition of the phagosome membrane [26]. Hence we may speculate that alteration of these proteins involved in phagosome might be an important strategy employed by E. tarda, thereby arresting phagosome development to aid their survival. Coro1A, known as tryptophan aspartate containing coat protein (TACO), were also found to be associates with phagosomes in macrophages [25]. Although coro1A has been demonstrated to be the inhibitor of the phagosome maturation that allows intracellular survival of pathogenic Helicobacter pylori and mycobacterium [27-29], the exact role of coro1A in E. tarda -infected cells has not been well understood up to now. Another significantly altered protein involved in trafficking of E. tarda-laden phagosomes is β-actin. In addition to its roles as essential component of the cytoskeleton, β-actin has recently been implicated in performing other functions such as cell migration, division, and intracellular transport and phagocytosis [30]. It is important to note that β-actin is found around mature phagosomes in some cell types, as well as on isolated phagosomes [31]. Available evidence suggests that actin assembly is essential for the process of phagosomal maturation [32]. To date, many studies showed that β-actin levels were reduced in some virus and bacterial infection [33-35]. On the contrary, we found that β-actin was up-regulated in E. tarda-infected macrophages. Similar performance was also observed in macrophages treated with Piscirickettsia salmonis [36]. These data implicate that actin might play diverse roles in host cells confronting different pathogens. Intracellular bacteria were found to take advantage of the cytoskeleton as a scaffolding matrix to survive, and to facilitate their propagation [37]. Meanwhile, our previous study demonstrated that E. tarda could replicate within the vacuolar-like compartment in macrophages [21]. Hence, from the data in this study we speculate that the increased expression of β-actin might be involved in the vesicular traffic of macrophages that is crucial for the formation of phagosomes and other vacuolar-like compartment associated with survival of E. tarda within macrophages. The precise role of β-actin in E. tarda-infected macrophages will be explored in the future and ongoing work.

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Moesin, which belongs to the ERM family, was increased in E. tarda-infected macrophages. It has been found that moesin is associated with fully formed phagosomes, contributing to phagosome acidification by enabling actin assembly at the phagosome membrane [38]. Therefore, the increase in moesin might be in response to the increase of actin and related to enhanced phagocytosis and degradation of E. tarda in macrophages. 4.2 Proteins involved in macrophage microbicidal activity ROS, as powerful microbicidal agents against a variety of intracellular pathogens, play a paramount role in macrophage-mediate immunity, however, high levels of ROS pose a risk for the macrophage themselves [39]. Peroxiredoxins (Prdxs), a family of small antioxidant proteins, are thought to be involved in eliciting cellular defenses against ROS [40]. In the present study, increase in Prdx-1 expression was observed. These findings suggested that the up-regulation of Prx-1 probably may contribute to protecting macrophages against the ROS mediated cytotoxicity, and thereby promote the macrophage as a reservoir for E. tarda. RhoGDI is widely accepted for their roles in cell migration, gene transcription, cell cytokinesis, phagocytosis and vesicular traffic [41]. Furthermore, it has been suggested that RhoGDI may be involved in ROS production and act as an inhibitor of ROS-generating NADPH oxidase activation [42]. Therefore, to a certain degree, the elevation of levels of this protein might explain why highly virulent E. tarda caused lower level of ROS than low virulent strain [43]. Overall, our present study suggested that macrophages increased levels of Prdxs-1 and RhoGDI for the immune function and regulation of ROS stress in response to E. tarda-infection. Protein disulfide isomerase (PDI) plays an essential role in cell survival under various stress conditions. Some reports have determined PDI as an intracellular anti-inflammatory molecules, which could help macrophages dispose of toxic free radicals [44]. Meanwhile, PDIA3 were found to be beneficial for viral replication [33, 45]. Our study showed that PDIA3 were up-regulated in macrophages, which indicated that this protein might participate in immune response to E. tarda infection. Further studies should focus on the exact role of PDIA3 in facilitating or inhibiting the survival of E. tarda. Other antimicrobial proteins, such as cis-aconitate decarboxylase (IRG1) and serine/arginine-rich splicing factor 1(SRSF1) were also elevated in E. tarda-infected macrophages. IRG1, a link between cellular metabolisms with immune defense, could produce itaconic acid through the decarboxylation of cis-aconitate to inhibit bacterial growth and contribute to antimicrobial activity of macrophages [46]. Similar to IRG1, SRSF1 was demonstrated as a negative regulator of virus gene transcription and could inhibit virus replication [47, 48]. Therefore, these data suggest that induction of Irg1 and SRSF1 reflects the macrophage response to E. tarda, which might ultimately result in restriction to bacteria replication in host cells. 4.3 Apoptosis -associated proteins Vimentin is an intermediate filament protein. Apart from being a cytoskeletal protein, its roles involved in apoptosis are gaining more interest. Müller et al. [49] demonstrated that vimentin is cleaved into several fragments during apoptosis of

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macrophages. Caspase proteolysis of vimentin has been shown to disrupt intermediate filaments and could promote apoptosis [50]. In this study, 3 protein spots including 2 protein spots that were detected in uninfected cell but not in infected cell were all identified as vimentin, indicating that cleavage of vimentin might occur in E. tarda-infected macrophages. The specific cleavage of vimentin could be used as an apoptotic marker of both apical- and mitochondria-dependent caspase activation [51]. We have previously reported that E. tarda could trigger apoptosis of macrophages [21]. Our proteomic results seem to reinforce this observation. Vimentin has been shown to be up-regulated when cells are stressed in general [51, 52], whereas we identified that vimentin was down-regulated in E. tarda-infected macrophages. Similar observations were obtained in M. tuberculosis-infected macrophages showing that down-regulated vimentin expression was associated with down regulation of ROS by M. tuberculosis in stimulated macrophages [53]. This allows us to presume that significant down regulation of vimentin by E. tarda may be viewed as a modulatory mechanism in E. tarda-infected macrophages, although the mechanism of this is still unclear. Apoptosis is shown to contribute efficiently to intracellular pathogen removal by eliminating the favorable intracellular niche for survival. An invading pathogen and its host cells have developed a complex interaction in which the pathogen strives to survive and replicate [54]. Thus, it was not surprising that E. tarda develop mechanisms to block macrophages apoptosis. Anti-apoptotic mechanisms can be interpreted as an attempt by the intracellular pathogen to limit apoptosis to allow enough time for a change in phenotype to be complete before exiting the apoptotic cell [55]. The increased levels of HSP60, HSP70, Prx-1 and RhoGDI in the E. tarda-infected macrophages found in our study might be responsible for anti-apoptotic regulatory mechanisms. Heat-shock proteins (Hsps) are the most abundant and ubiquitous soluble intracellular proteins. They perform a multitude of housekeeping functions that are essential for cellular survival in response to various stress. Interestingly, available evidence suggest that Hsps are involved in the anti-apoptosis of cells. The Hsp60 are generally considered to be anti-apoptotic due to their role in preventing apoptosis by forming a complex with proteins responsible for apoptosis [56, 57]. Moreover, Hsp70 are demonstrated to suppress apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome [58]. In addition, aforementioned differentially expressed proteins including Prx-1 and RhoGDI were reported to be associated with anti-apoptotic effect [59, 60]. These data implicated that infection with the E. tarda might be accompanied by the triggering of anti-apoptotic mechanisms. Similar observations were reported in recent study by Okuda et al. [1] who demonstrated that E. tarda infection of J774 cells was accompanied by up-regulation of anti-apoptotic genes. We found that high virulent E. tarda could elicit less apoptosis than low virulent strain (unpublished results). Hence we may speculate that E. tarda could trigger apoptosis of macrophages, however, high virulent strain may have the capability to reduce apoptosis by developing anti-apoptotic mechanisms to protect their survival in macrophages. Additional studies to confirm this proposal are under way.

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5. Conclusions A set of the altered cellular proteins of macrophages in response to E. tarda-infection were identified. The information from these proteomic data showed that E. tarda-infection may extensively influence the expression of proteins associated with phagosomes formation, macrophage microbicidal activity and apoptosis of macrophages. To our knowledge, this study represents the first attempt to employ large-scale proteomic analysis to identify protein expression of macrophage in response to E. tarda infection. The data described here are expected to provide valuable information in understanding pathogenesis of E. tarda-infection. We propose that future studies are required to obtain evidence of whether these differentially expressed proteins can be used as novel markers and targets for the control of E. tarda-infection.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (NO. 31602188) and the Natural Science Foundation of Jiangsu Province (NO. BK20140442).

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E. tarda could survive and replicate in macrophages as an escape mechanism from the host defense.

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The differential proteomes of RAW264.7 cells in response to E.

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tarda-infection were analyzed at different time points with 2-DE followed by LC-MS/MS. •

The differentially expressed proteins were confirmed by qPCR and Western blot.

18 up-regulated and 8 down-regulated proteins were successfully identified.

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The identified proteins are mainly involved in formation of phagosomes,

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macrophage microbicidal activity and anti-apoptosis of macrophage.