Jin Wen-Jie*, Zhang Yong-Pan, Qian Wen-Zheng, Zhang Di, Shao Hong-Xia, Qian Kun and Qin Ai-Jian
Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University, Jiangsu, China
Received date: February 26, 2014; Accepted date: August 28 2014; Published date: September 02, 2014
Citation: Wen-Jie J, Yong-Pan Z, Wen-Zheng Z, Di Z, Hong-Xia S, et al. (2014) Analysis of Escherichia Coli Inducing Salpingitis-Peritonitis in Layer Geese by Two-Dimensional Electrophoresis and MALDI-TOF/TOF. J Bacteriol Parasitol 5:198. doi: 10.4172/2155-9597.1000198
Copyright: © 2014 Wen-Jie J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Geese salpingitis-peritonitis mainly happens in layer geese during egg peak period and leads serious economic losses. This study was conducted in different resource E. coli inducing Geese salpingitis-peritonitis and Geese or chicken fibrinous pericarditis-perihepatitis-airsacculitis respectively. The molecular mechanism underlying pathogenicity of Geese salpingitis-peritonitis E. coli was investigated through analysis of the special protein of this E. coli using proteomics and bioinformatics techniques. Two-dimensional electrophoresis (2-DE) analysis showed a total of 42 protein spots only expressed in Geese salpingitis-peritonitis E. coli. A total of 21 proteins were identified by MALDI TOF/TOF mass spectral analysis. According to gene ontology (GO) and Pathway analysis, special expressed proteins in Geese salpingitis-peritonitis E. coli were mainly involved in the synthesis of macromolecular material, Cellular metabolism and differentiation, Cytoskeleton composition and other relevant Geese salpingitisperitonitis E. coli, 30S RPS6, KASwere possibly associated with its infection.
Geese salpingitis-peritonitis E. coli; Mass spectrum; Two-dimensional electrophoresis
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 . 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  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) . 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).
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 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.
|NoAccession No||Protein name||MW||pI|
|2||gi|20149796||Chain A, E. coli Cofactor-Dependent PhosphoglycerateMutaseComplexed With Vanadate||28408||5.86|
|3||gi|3318853||Chain A, The Structure Of Clpp At 2.3 Angstrom Resolution Suggests A Model For Atp-Dependent Proteolysis||21663||5.55|
|4||gi|15804334||F0F1 ATP synthase subunit alpha||55416||5.80|
|5||gi|16130258||3-oxoacyl-[acyl-carrier-protein] synthase I||42928||5.35|
|9||gi|290586581||30S ribosomal subunit protein S6||15694||4.93|
|10||gi|89107229||regulator protein that represses frmRAB operon||10425||5.84|
|12||gi|110591231||Chain A, Crystal Structure Of An N-Terminal Deletion Mutant||51547||5.27|
|13||gi|77416725||3-oxoacyl-[acyl-carrier-protein] synthase 2||43247||5.71|
|15||gi|84028790||Single-stranded DNA-binding protein||18963||5.44|
|16||gi|167008648||ATP-dependent Clp protease proteolytic subunit||23286||5.52|
|20||gi|84028666||Glutamine-binding periplasmic protein||27173||8.44|
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.
Geese salpingitis-peritonitis was also named geese egg plague and goose Escherichia coli sex reproductive organs disease in China . 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).