Escherichia coli are a frequent cause of intestinal and extra intestinal infection in animals. E. coli infection in mammals is most a primary enteric disease, whereas in poultry is often extra intestinal infection [1,2]. Salpingitis-peritonitis has been reported in layer hens [3-5]. Since 1960's, this kind of disease was reported happening in layer geese and leading gross economic loss, and the pathogen of it were confirmed as E. coli [6]. But little was known about the pathogenesis of salpingitis-peritonitis E. coli in layer geese.

The purpose of this study was to explore the specific proteins associated with pathogenesis of salpingitis-peritonitis E. coli in layer geese. 2-D gel electrophoresis and MALDI TOF/TOF mass spectral analysis were done to analyze and search the specific proteins.

E. coli Strains

The E. coli strains G837, G803, G8107, E0056, E0060 used in this research were originally isolated in Ministry of Education Key Lab for Avian Preventive Medicine. E. coli strain G837, G803, G8107 were isolated from salpingitis-peritonitis samples in layer geese, E0056, E0060 were isolated from fibrinous pericarditis-perihepatitis-airsacculitis samples in geese. CMCC44251 was isolated from fibrinous pericarditis-perihepatitis-airsacculitis sample in chicken. All these strains were identified and reserved in CMCC.

Preparation of total cellular protein extracts from E. coli (Extraction of E. coli proteins)

All the E. coli strains were plated on Mac-Conkey agar plates and incubated at 37^° overnight. The following day, the clone of each strain was inoculated in 3 mL of LB broth. The cultures were grown overnight at 37^° with continuous shaking at 200 RPM.

E. coli cells were harvested from cultures by centrifugation at 10,000 g, 4^°C for 10 min, and washed three times in sterile phosphate buffer saline (PBS, 0.01 M, pH 7.2). The cells were then lysed in 2-D rehydration solution containing 0.0075 g dithiothreitol (DTT), 5 µl ampholytes. The bacterial cell suspension, in the rehydration solution, was incubated on ice for 30 min and sonicated using three bursts of 75 Watt, 30 s on an Ultrason 250 sonicator (LabPlant, UK). After removal of the insoluble debris at 11,000 g, 4^°for 90 min, the protein concentration of supernatant was determined using the Nano Drop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, U.S.A.) and was stored at 70°C or subsequent analysis.

Analysis of bacterial proteins by 2-D gel electrophoresis

The bacterial proteins were analysed by 2-DE as described previously and modified [7] For each sample, 350 µg of solubilized proteins was dissolved in 300 µl of rehydration solution, 8 M urea, 4% 3-(3-cholamidopropyl dimethylammonio)-1-propanesulfonic acid (CHAPS), 1% dithiothreitol (DTT), 0.5% ampholytes, trace bromophenol blue, and loaded onto a 17 cm immobilized pH gradient (IPG) linear pH 4-7 strip (Bio-Rad Laboratories, USA). The IPG strips were rehydrated at 20^°C for 14 h at 50 V. The rehydrated strips were automatically focused using the following parameters: 250 V, linear, 30 min; 500V, 30 min; 1000 V, linear, 2 h; 8000 V, linear, 5 h; 8000 V, rapid, 65,000 Vh. The current for each strip was limited to 50 A. Isoelectric Focusing (IEF) strips (Protean IEF Cells, Bio-Rad) were incubated for 15 min with gentle shaking in equilibration buffer (6 M urea, 20% glycerol, 2% SDS and 0.375 mM Tris-HC1, pH 8.8) containing 2% (w/v) DTT, followed by an equilibration for 15 min in equilibration buffer containing 2.5% (w/v) iodoacetamide instead of DTT. Equilibrated IPG strips were further resolved with 10% SDS-PAGE gels (1 mm gel thickness) at 50 V for 30 min, then 200 V until the bromophenol blue dye marker reached the bottom of the gel.

Protein visualization and image analysis

Gels were stained by blue silver staining and scanned at a resolution of 600 d.p.i. using a Powerlook 2100XL-USB scanner (UMAX, Taiwan) [8]. Spot detection, matching and quantitative intensity analysis were performed using PDQuest 2-D software, version 8.0.1 (Bio-Rad Laboratories, USA). The 2-DE was performed in salpingitis-peritonitis strains with fibrinous pericarditis-perihepatitis-airsacculitis strains. Each strain was repeated three times using total proteins. Finally, we selected representative gels from each strain, which reproducibility in the same stain was no less than 85%, which reflected the small variances between strains or running. Relative comparison between the two different types of strains. Only the protein spots which were showed expression only in salpingitis-peritonitis strains not in fibrinous pericarditis-perihepatitis-airsacculitis strains were selected and subjected to identification by MS.

Mass spectrometry (MS)

Spots of special proteins were manually cut from gels and sent to Southern Medical University (Guangzhou, China) to be identified by MALDI-TOF/TOF (ABI 4700 TOF-TOF Proteomics Analyzer, Applied Biosystems, USA).

Bioinformatics analysis of special expressed proteins

The data of those special expressed proteins showed in Goose salpingitis-peritonitis E. coli strains were searched and analysed by GPS Explorer using MASCOT (Matrix Science, London), Gene Ontology (GO) Analysis (http://www.geneontology.org/) and Pathway Analysis (http://www.genome.jp/kegg/).

Electrophoretic analysis of special proteins in salpingitis-peritonitis strains and fibrinous pericarditis- perihepatitis-airsacculitis strains from Geese

Total bacterial protein extracts of the E. coli strains were analysed by 2-DE as described in Section 2. Approximately 600-700 protein spots can be detected on each 17 cm 2-D gels (pI 4-7) loaded with 350 µg of total protein (Figure 1).

Figure 1: Representative 2-D protein profile of the total cellular proteins of E. coli isolate. A total protein extract from the salpingitis-peritonitis E. coli strains and fibrinous pericarditis-perihepatitis -airsacculitis E. coli strains was prepared and analysed as described in Section 2.

Comparing of multiple 2-DE gel images with PDQuest 2-D software (version 8.0.1), 42 protein spots were found that they were only showed in salpingitis-peritonitis strains not in fibrinous pericarditis- perihepatitis-airsacculitis strains (Figure 2). Twenty-nine proteins spots were selected for identification by MALDI-TOF/TOF (Figure 3). Twenty-one of them were successfully identified (Table 1).

Figure 2: PDQuest 2-D software (version 8.0.1) analysis indicated that 42 protein spots were only showed in salpingitis-peritonitis strains not in fibrinous pericarditis- perihepatitis-airsacculitis strains.

Figure 3: Twenty-nine protein spots were selected for identification by MALDI-TOF/TOF Protein spots identified as part of the current study are numbered automatically by the software. These proteins identified in salpingitis-peritonitis strains differentiation from fibrinous pericarditis-perihepatitis- airsacculitis E. coli strains and the locations have been mapped to the above profile using the PDQuest 2-D software package.

Table 1: List of the Special expressed protein spots in the salpingitis-peritonitis E. coli strains identified by MALDI-TOF/TOF

Analysis of all these special expressed proteins

According to annotations from the Uniprot Knowledgebase (Swiss-Prot/TrEMBL) and Gene Ontology Database, the identified proteins were classified based on the cellular component, molecular function and biological process (Figure 4 and Figure 6). By the bio-information analysis, the identified proteins were related with the synthesis of macromolecular material, Cellular metabolism, Cell proliferation and differentiation, Cytoskeleton composition and so on.

Figure 4: Proteins classification based on the cellular component

Figure 5: proteins classification based on the molecular function

Figure 6: proteins classification based on the biological process

Geese salpingitis-peritonitis was also named geese egg plague and goose Escherichia coli sex reproductive organs disease in China [6]. This kind of disease mainly happens in layer geese during egg peak period and leads serious economic losses. At present, few researches associated with geese salpingitis-peritonitis was reported. In this study, 2-D electrophoresis method was applied in the research of the proteomic heterogeneity of geese salpingitis-peritonitis E. coli strains to investigate the pathogenesis of geese salpingitis-peritonitis.

The data presented in this report showed the proteomic variation between geese salpingitis-peritonitis E. coli and pericarditis-perihepatitis-airsacculitis E. coli. Throughout this work, standard protein extraction and analytical protocols were used to compare the E. coli cellular proteomes. Bacterial cell proteins were prepared by sonication and lysis of the cells in a buffer compatible with 2-DE and then analysed using 2-D gel system.

In this work, 21 special expressed proteins in geese salpingitis-peritonitis E. coli were identified, and associated with the synthesis of macromolecular material, Cellular metabolism, Cell proliferation and differentiation, Cytoskeleton composition and so on.

In recent years, the research of related with the protein of E. coli, has become a hotspot research, especially in application of developing a new drug [9-13]. The protein found in this study can be used for resistance research and super bacteria with conditions research.

Thanks to Dr. Yassir Mohammed Eltahir for his modification. This work was supported by the grants (30800822) from NSFC, also sponsored by Qing Lan Project, Program for Changjiang Scholars and Innovative Research Team in University (IRT0978) and Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).