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Comparison of surface proteomes of enterotoxigenic (ETEC) and commensal Escherichia coli strains
Journal of Microbiological Methods 83 (2010) 13–19
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Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Comparison of surface proteomes of enterotoxigenic (ETEC) and commensal Escherichia coli strains Ulf Sommer a, Jørgen Petersen a, Michael Pfeiffer a, Petra Schrotz-King a, Christian Morsczeck a,b,⁎ a b
ACE BioSciences A/S, Roskildevej 12 C, 3400 Hillerød, Denmark Department of Operative Dentistry and Periodontology,University Hospital Regensburg,Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
a r t i c l e
i n f o
Article history: Received 11 May 2010 Received in revised form 5 July 2010 Accepted 6 July 2010 Available online 17 July 2010 Keywords: SILAC Escherichia coli Surface proteins Vaccine Proteomics
a b s t r a c t Pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections involves colonization of the small intestine mediated by cell-surface fimbriae (CS) or colonization fimbriae antigens (CFA). However, protection against reinfection of ETEC is also conferred by somatic antigens rather than by virulence factors. To discover ETEC specific somatic antigens, the surface proteome of the ETEC H10406 strain was compared with that of nonpathogenic E. coli K12 strains. In this study, we were using stable isotope labelling with amino acids in cell culture (SILAC) technology for the labelling and relative quantification of surface proteins in order to identify polypeptides that are specifically present on ETEC strains. Outer membrane proteins were isolated, separated by gel electrophoresis, and identified by mass spectrometry. Twenty-three differentially expressed cellsurface polypeptides of ETEC were identified and evaluated by bioinformatics for protein vaccine candidates. The combination of being surface-exposed and present differentially makes these polypeptides highly suitable as targets for antibodies and thus for use in passive or active immunisation/vaccination. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Escherichia coli is an adaptive bacterial species that is both a commensal resident of the intestine and a versatile pathogen of humans and animals. A panel of E. coli strains cause diarrhoea. ETEC is a major cause of diarrhoea in children in developing countries particularly under the age of five (Gaastra and Svennerholm, 1996). Diarrhoea is a condition characterised by a marked increase in the frequency of unformed bowel movements and is commonly accompanied by abdominal cramps, urgency, nausea, bloating, vomiting, fever, and malaise. Cases are caused after an infection with ETEC. During Operation Desert Shield, 25% of the troops stationed in Saudi Arabia experienced at least one episode of ETEC diarrhoea (Hyams et al., 1991). ETEC bacteria are not invasive and colonize the small intestine by attachment to the mucosa via colonization factors. Pathogenesis of ETEC infections involves colonization of the small intestine mediated by cellsurface fimbriae (CS) or colonization fimbriae antigens (CFA). At least 20 different types of CFA proteins were identified on diverse ETEC strains until today (Gaastra and Svennerholm, 1996). ETEC induce fluid and electrolyte secretion by the small intestinal epithelium in response to the production of heat-labile (LT) and/or heat-stabile enterotoxins (ST). The ETEC bacterium expresses at least one of these two toxins. LT is similar in structure and function to the known cholera toxin and severe
ETEC infections are comparable to infections with Vibrio cholerae (Steinsland et al., 2004; Gaastra and Svennerholm, 1996). Epidemiological evidence and results from experimental challenge studies in volunteers, clearly demonstrate that strain-specific immunity follows ETEC infection and that multiple infections with antigenically diverse ETEC strains lead to broad protection against ETEC diarrhoea (Steinsland et al., 2003, 2004). This indicates that a reliable ETEC vaccine is achievable. Recent efforts have been looking into the development of non-cellular candidates for vaccination, which are based for example on a patch of LT or CS6 (Frech et al., 2008). These are virulence factors defining an ETEC pathotype. However, an epidemiological study has demonstrated that protection conferred by ETEC infection against reinfection is conferred by somatic antigens defining a clone rather than by virulence factors defining a pathotype (Steinsland et al., 2004). Our study therefore intended to identify differentially expressed proteins on the surface of ETEC, which show lower expression on the surface of commensal E. coli strains (K12). ETEC specific surface proteins with a ratio ([ETEC]/[K12]) of 2 or greater were identified with 1D gels and 2D gels using the technology of stable isotope labelling with amino acids in cell culture (SILAC), which is mainly used for the characterization of eukaryotic cells (Ong et al., 2002). 2. Materials and methods 2.1. Bacterial strains
⁎ Corresponding author. Tel.: + 49 0 941 944 6161. E-mail address: [email protected] (C. Morsczeck). 0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2010.07.011
The ETEC strain D2166, original No.H10407, serotype O78:H11 LT and STp positive were derived from the Statens Serum Institut (SSI,
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Denmark). The commensal E. coli strain D2103 (MC1061) K12 strain serotype O-rough:H48 was derived from the SSI, and the BL21 (K12) strain purchased from Invitrogen. 2.2. Labelling of E. coli cells with SILAC and isolation of cell envelope proteins A 20-ml starter culture of E. coli strains BL21 or H10407 was inoculated in M9 minimal medium and cultured at 37 °C overnight. The following day cells were pelleted at 6000 g for 30 min and M9 medium was poured off. Cells were resuspended in 2 ml M9 medium, of which 1 ml of resuspended cells were inoculated in 98 ml. For SILAC labelling either heavy-Lys (10 mg L-lysine-13C6, 15N2·HCl (min 98+ Atom% 13C min 99+ atom% 15N) (Sigma-Aldrich)) or normal L-lysine (light-Lys) dissolved in 1 ml PBS were added to the cell solution to obtain a final concentration of 100 mg/l. The cultures were grown overnight at 37 °C under vigorous shaking at 200 rpm. Strain MC1061 was grown in parallel in 100 ml LB medium. The cell envelope fraction was isolated according to a protocol by Fountoulakis and Gasser (2003). Briefly, the overnight E. coli culture was pelleted and the bacteria were resuspended in buffer A (50 mM Tris (pH 7.6), 250 mM sucrose, 0.25 mM EDTA, 0.25 mg/ml lysozyme). The cell suspension was mixed by shaking for 1 h (4 °C), followed by a centrifugation step at 2800 g for 12 min. The new pellet was resuspended in buffer B (100 mM potassium phosphate (pH 7.6), 6 mM magnesium acetate, 0.1 mM DTT, 20% (v/v) glycerol + proteinase inhibitors). This solution was disrupted 3 times by sonication. To remove unbroken cells and debris, sonicated bacteria were centrifuged for 30 min at 12,000 g, and subsequently the supernatants were centrifuged at 41,000 rpm in a Sorvall A-641 rotor. The pelleted OM proteins were resuspended in buffer C (buffer A w/o lysozyme). 2.3. Surface protein identification Outer membrane proteins were isolated from bacteria cultures as described above and protein concentrations were estimated with the Bradford test (Bio-Rad). Protein samples for analysis were produced after mixing equal protein amounts from both E. coli strains. The complex mixture of proteins obtained after surface extraction was analysed by two complementary strategies based on mass spectrometry, a 2D gel and a 1D gel based strategy similar to those previously described (Morsczeck et al., 2008; Prokhorova et al., 2006). 2.3.1. 2-D gel based strategy (2D-gel MALDI-TOF/TOF) The first dimension run was performed on a pre-cast 13 cm IPG strip (pH 4–7) using the Ettan IPGphor isoelectric focusing system (Amersham Biosciences) according to the manufacturer's instructions, including 4% CHAPS. The second dimension was performed by inserting the strips on top of self-cast 12% SDS PAGE gels. Electrophoresis was performed on the Hoefer SE 600 PAGE system (Amersham Pharmacia), and the gels silver-stained (Mortz et al., 2001). Spots for mass spectrometric analysis were picked using the Ettan Spot Picker from Amersham according to the manufacturer instructions. Specific protein spots were spot-picked, and placed into punctured 96-well plates in Milli-Q water. Solvent was removed by spinning the plates at 2000 g. These gel plugs were washed in 50 mM NH4HCO3/ 50% ethanol and dehydrated by incubation in 96% ethanol. Reduction and alkylation was performed by incubating in reducing solution (10 mM DTT, 50 mM NH4HCO3) at 56 °C followed by room temperature incubation in alkylation solution (55 mM iodoacetamide, 50 mM NH4HCO3) in the dark. Two cycles of washing and dehydration were then performed prior to the addition of 5 μl trypsin solution (12.5 ng/ul Promega trypsin in 50 mM NH4HCO3, 10% acetonitrile). Then an additional amount of ammonium bicarbonate solution was added, and the digests were incubated overnight at 37 °C. Peptides
were extracted after acidification with trifluoroacetic acid to the overnight digest followed by incubation with shaking. Parts of the extract were spotted on anchorchip targets, using αcyano-4-hydroxy-cinnamic acid as the matrix, and submitted to automated MALDI-TOF peptide mass fingerprint and MALDI-TOF/TOF analysis (Ultraflex, Bruker Daltonics, Germany). Peak lists were created in FlexAnalysis and submitted to BioTools (both Bruker) for database searching against a specific in-house E. coli database. The Mascot search program and scoring algorithm (Matrix Science, UK) were used in database searching. Peptide mass tolerance was set to 60 ppm and 0.7 Da, respectively. Search parameters were adjusted to include oxidation of Met, the addition of Carbamidomethyl groups to Cys, and the SILAC modification (Lys-Silac + 8 (K)), and trypsin was allowed to miss one cleavage site per peptide. 2.3.2. 1D gel based strategy (GeLC-MS/MS) One-dimensional gel electrophoresis was performed on a Novex NuPage system (Invitrogen) according to the manual provided with the gel system. In brief we electrophoresed pre-cast 12% bis–tris gels (8 cm × 8 cm, × 1 mm, Invitrogen) at 200 V for 60 min in NuPageMOPS-SDS running buffer. Gels were Coomassie stained. Whole lanes were cut into gel slices with a razor blade. The gel slices were digested as described under the 2D gel strategy, but the amount of trypsin was a 20 μl trypsin solution (12.5 ng/μl Promega trypsin in 50 mM NH4HCO3, 10% Acetonitrile). The extracts were analysed by LC-MS/MS on a Waters Cap-LC and Micromass Ultima API QTOF mass spectrometer. Each sample was submitted to a 115 min LC-MS/MS analysis on a self-packed 12 cm, 75 m Zorbax-C18 column, using a trap column of the same material and including a gradient from 100% solvent A (5% acetonitrile, 0.1% formic acid in water) to 80% solvent B (5% water, 0.1% formic acid in acetonitrile) in data-dependent acquisition mode. The peak list (pkl) files generated by the MassLynx software were reduced in size by an in-house script for searching a specific in-house E. coli database. The Mascot Demon program and scoring algorithm (Matrix Science, UK) were used in database searching. Peptide mass tolerance was set to 200 ppm and 0.4 Da for fragment ions. Search parameters were adjusted to include oxidation of Met, the addition of Carbamidomethyl groups to Cys, and the SILAC modification (Lys-Silac + 8 (K)), and trypsin was allowed to miss one cleavage site per peptide. 2.3.3. Quantification Quantification of tryptic digested spots from either 1D gel or 2D gel was carried out by analyzing the ratio between the relative intensities of the peak pairs (light and heavy peptide). This was done manually using the FlexAnalysis software (Bruker) for MALDI data, and the ProteinLynx/MassLynx software (Micromass/Waters) for the ESI data. 2.4. Bioinformatics and protein numbering (AnrP) The ClustalV Alignment (MegAlign, DNA_Star) was used for the evaluation of homologies of identified proteins sequences. An internal protein database (ACE Biosciences) assigned AnrP (ACE non-
Table 1 Experiments. Experiment #
ETEC strain
K12 strain
Proteomics approach
Strain labelled with SILAC
1981 1986 1987 1989 1992 1993 2006
H10407 H10407 H10407 H10407 H10407 H10407 H10407
MC1061 MC1061 MC1061 BL21 BL21 BL21 BL21
1D 2D 2D 2D 2D 2D 1D
ETEC ETEC ETEC ETEC ETEC ETEC ETEC/BL21
gel + LC/MSMS gel + MALDI/TOFTOF gel + MALDI/TOFTOF gel + MALDI/TOFTOF gel + MALDI/TOFTOF gel + MALDI/TOFTOF gel + LC/MSMS
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Table 2 Proteins identified in cell envelope fractions from E. coli. Proteins were considered identified based on at least two identified peptides or one high-scoring peptide, and a protein M-Score considered significant by the Mascot program (p b 0.05). A high-scoring peptide is a fragmented peptide that identifies a protein with a score of above 50 after a GeLC-MS/MS strategy. AnrP#
Protein name
AnrP10213 AnrP104501 AnrP124628
(NC_002655) 30S ribosomal subunit protein S2 [Escherichia coli O157:H7 EDL933] (U70214) hypothetical protein [Escherichia coli] (NC_002655) 2-oxoglutarate dehydrogenase (dihydrolipoyltranssuccinase E2 component) [Escherichia coli O157:H7 EDL933] (NC_002655) pyruvate dehydrogenase (dihydrolipoyltransacetylase component) [Escherichia coli O157:H7 EDL933] (NC_002655) degrades sigma32, integral membrane peptidase, cell division protein [Escherichia coli O157:H7 EDL933] (NC_002655) phage lambda receptor protein; maltose high-affinity receptor [Escherichia coli O157:H7 EDL933] (M38305) RNA polymerase (rpoC) [Escherichia coli] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) orf, hypothetical protein [Escherichia coli O157:H7 EDL933] (NC_002695) PTS system, glucose-specific IIBC component [Escherichia coli O157:H7] NC_002695) PTS system, glucose-specific IIBC component [Escherichia coli O157:H7 EDL933] (J01594) ATP synthase alpha subunit (atp-6) [Escherichia coli] (NC_002655) putative lipoprotein [Escherichia coli O157:H7 EDL933] (NC_002655) putative lipoprotein [Escherichia coli O157:H7 EDL933] (NC_000913) maltodextrin phosphorylase [Escherichia coli K12] (NC_002655) outer membrane channel; specific tolerance to colicin E1 [Escherichia coli O157:H7 EDL933] (NC_002695) chaperonin GroEL [Escherichia coli O157:H7] (NC_000913) membrane-bound ATP synthase, F1 sector, alpha subunit [Escherichia coli K12] (AP002566) membrane-bound ATP synthase alpha subunit AtpA [Escherichia coli O157:H7] (X82400) phosphopyruvate hydratase; enolase [Escherichia coli] (D90701) Hypothetical protein 200 (entA 3′ region) [Escherichia coli] (NC_002695) CoA-linked acetaldehyde dehydrogenase/iron-dependent alcohol dehydrogenase [Escherichia coli O157:H7] (NC_002655) outer membrane protein 3a (II*;G;d) [Escherichia coli O157:H7 EDL933] (NC_002655) outer membrane protein 3a (II*;G;d) [Escherichia coli O157:H7 EDL933] NC_000913) PTS system, fructose-specific transport protein [Escherichia coli K12] putative ATPase subunit of translocase [Escherichia coli O157:H7 EDL933] (NC_002655) putative ATPase subunit of translocase [Escherichia coli O157:H7 EDL933] rpsA, protein S1 [Escherichia coli] D-methionine ABC-transporter, periplasmic D-methionine-binding lipoprotein [Escherichia coli 101-1] (NC_002695) succinate dehydrogenase flavoprotein subunit [Escherichia coli O157:H7] Ferrienterobactin receptor precursor [Escherichia coli CFT073] nmpC, outer membrane porin protein; locus of qsr prophage [Escherichia coli O157:H7 EDL933] (NC_002655) outer membrane protein 1a (Ia;b;F) [Escherichia coli O157:H7] (D90838) Adhesin AIDA-I precursor. [Escherichia coli] (NC_002655) lipoprotein-28 [Escherichia coli O157:H7 EDL933] (NC_000913) putative protease [Escherichia coli K12] (NC_002655) 50S ribosomal subunit protein L4 [Escherichia coli O157:H7] (NC_002655) 50S ribosomal subunit protein L4 [Escherichia coli O157:H7 EDL933] (NC_002655) membrane-bound ATP synthase, F1 sector, beta subunit [Escherichia coli O157:H7 EDL933] Chain A, Outer Membrane Cobalamin Transporter (Btub) From E. coli (NC_000913) L-lactate dehydrogenase [Escherichia coli K12] (NC_002695) hypothetical protein [Escherichia coli O157:H7] (AF037157) ATP synthase beta subunit [Yersinia enterocolitica] (NC_000913) membrane-bound ATP synthase, F0 sector, subunit b [Escherichia coli K12] (NC_002655) pyruvate dehydrogenase (decarboxylase component) [Escherichia coli O157:H7 EDL933] (NC_002655) acridine efflux pump [Escherichia coli O157:H7 EDL933] (NC_002655) 50S ribosomal subunit protein L3 [Escherichia coli O157:H7] (NC_000913) phosphoglycerate kinase [Escherichia coli K12] (X17440) hsg48 [Escherichia coli] (NC_002695) colicin I receptor precursor [Escherichia coli O157:H7] (Z19601) ORF, urf74.3. [Escherichia coli] protein L12 [Escherichia coli] (NC_002655) protease specific for phage lambda cII repressor [Escherichia coli O157:H7 EDL933] (X57402) lipoprotein-34 [Escherichia coli] (NC_000913) respiratory NADH dehydrogenase [Escherichia coli K12] (AJ243795) flagellin [Escherichia coli] (AJ243795) flagellin [Escherichia coli] Chain B, Crystal Structure Of The Long-Chain Fatty Acid Transporter Fadl (NC_002655) putative ATP-binding component of ABC transport system [Escherichia coli O157:H7 EDL933] (NC_003197) outer membrane porin, receptor for colicin I, requires TonB [Salmonella typhimurium LT2] (NC_000913) outer membrane receptor for ferric enterobactin (Escherichia coli str. K-12) (NC_002655) chaperone Hsp70; DNA biosynthesis; autoregulated heat shock protein 70 [Escherichia coli O157:H7 EDL933] (NC_002655) protease specific for phage lambda cII repressor [Escherichia coli O157:H7 EDL933] (NC_002655) putative transport protein [Escherichia coli O157:H7 EDL933] (NC_000913) outer membrane protein 1b (Ib;c) [Escherichia coli K12] (NC_000913) outer membrane protein 1a (Ia;b;F) [Escherichia coli K12] (NC_002655) protein chain elongation factor EF-Tu (duplicate of tufA) [Escherichia coli O157:H7 EDL933] Pyruvate dehydrogenase complex, dehydrogenase (E1) component [Escherichia coli B171]
AnrP12474 AnrP128059 AnrP140391 AnrP147873 AnrP17506 AnrP17506 AnrP17506 AnrP17506 AnrP17506 AnrP196226 AnrP204940 AnrP204940 AnrP229296 AnrP229453 AnrP229453 AnrP233606 AnrP238319 AnrP256328 AnrP26043 AnrP302876 AnrP311260 AnrP312095 AnrP314881 AnrP314881 AnrP318346 AnrP324273 AnrP324273 AnrP325807 AnrP3297020 AnrP33530 AnrP3555721 AnrP357746 AnrP362330 AnrP380051 AnrP380718 AnrP387792 AnrP390669 AnrP390669 AnrP39192 AnrP4116560 AnrP417078 AnrP421121 AnrP426538 AnrP431306 AnrP43799 AnrP442611 AnrP451981 AnrP465207 AnrP46819 AnrP47616 AnrP48657 AnrP493633 AnrP505092 AnrP510278 AnrP514889 AnrP553350 AnrP553350 AnrP5874704 AnrP617142 AnrP61933 AnrP619808 AnrP631052 AnrP659893 AnrP679551 AnrP693011 AnrP694356 AnrP69760 AnrP6992987
Peptides
M-score
7 5 3
308 152 74
4 9 3 3 8 11 2 1 2 5 4 4 17 3 3 2 5 2 6
156 345 135 160 255 363 126 56 86 156 109 80 742 134 134 76 178 124 214
7 2 9 7 5 5 2 2 8 7 3 5 10 5 2 5 1 2 5 22 11 5 2 8 3 10 5 4 6 1 6 2 13 9 7 4 13 5 4 1 3 17 3 6 5 3 11 15 3
205 115 298 226 115 199 96 68 225 240 167 75 339 141 112 141 91 82 210 627 134 223 63 316 144 325 137 162 251 54 240 92 349 292 260 155 648 248 258 90 79 727 99 243 230 80 281 477 148
(continued on next page)
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Table 2 (continued) AnrP#
Protein name
Peptides
M-score
AnrP6997368 AnrP702172 AnrP7083578 AnrP7083944 AnrP726813 AnrP728396 AnrP732180 AnrP744366 AnrP752782 AnrP768202 AnrP7963825 AnrP7963825 AnrP7963825 AnrP7963825 AnrP83888 AnrP84274 AnrP86905 AnrP86905 AnrP86905 AnrP886047 AnrP898114 AnrP899094 AnrP939220 AnrP954304 AnrP96882 AnrP98055
COG3203: Outer membrane protein (porin) [Escherichia coli F11] NC_002655) 50S ribosomal subunit protein L1 [Escherichia coli O157:H7 (COG0541) Signal recognition particle GTPase [Escherichia coli F11] COG4771: Outer membrane receptor for ferrienterochelin and colicins [Escherichia coli F11] (NC_002655) NADH dehydrogenase I chain F [Escherichia coli O157:H7 EDL933] (NC_002655) 50S ribosomal subunit protein L6 [Escherichia coli O157:H7] (NC_000913) outer membrane porin protein; locus of qsr prophage [Escherichia coli] Lipoprotein inner membrane ABC-transporter [Escherichia coli F11] (NC_002655) galactose-binding transport protein; receptor for galactose [Escherichia coli O157:H7 EDL933] (X67326) alcohol dehydrogenase [Escherichia coli] putative inner membrane ABC-transporter [Escherichia coli UTI89] putative inner membrane ABC-transporter [Escherichia coli UTI89] (putative inner membrane ABC-transporter [Escherichia coli UTI89] putative inner membrane ABC-transporter [Escherichia coli UTI89] (J04229) colicin I receptor [Escherichia coli] (NC_000913) NADH dehydrogenase I chain C, D [Escherichia coli K12] (NC_000913) pyridine nucleotide transhydrogenase, alpha subunit [Escherichia coli K12] (NC_000913) pyridine nucleotide transhydrogenase, alpha subunit [Escherichia coli K12] (NC_000913) pyridine nucleotide transhydrogenase, alpha subunit [Escherichia coli K12] (NC_002655) lipoamide dehydrogenase (NADH); [Escherichia coli O157:H7 EDL933] protein I,membrane [Escherichia coli] (NC_002655) periplasmic serine protease Do; heat shock protein HtrA [Escherichia coli O157:H7 EDL933] (NC_002655) putative function in exopolysaccharide production [Escherichia coli O157:H7] (NC_002655) yiaF gene product [Escherichia coli O157:H7 EDL933] (Z38065) FyuA precursor [Escherichia coli] (NC_002655) RNA polymerase, alpha subunit [Escherichia coli O157:H7 EDL933]
4 7 2 4 4 9 6 3 2 3 6 4 3 3 4 2 5 6 6 12 10 3 4 4 27 10
120 267 71 284 105 397 203 155 70 109 285 202 82 136 131 55 230 279 279 462 329 83 117 124 875 355
redundant proteins) numbers to individual E. coli proteins. This monthly-updated database contained all E. coli entries in the NCBI database found by certain key words, in addition to standard contaminant (keratins etc.). Relative ratios of proteins were estimated based on the height of the monoisotopic peaks of their peptide signals.
An in-house Basic Local Alignment Search Tool (BLAST) was used for the evaluation of homologies between selected bacterial proteins and the human proteome. This procedure is similar to a protein BLAST (BLASTp) at the NCBI (http://blast.ncbi.nlm.nih.gov/). The identification of identical 7-mers in the human proteome was done manually
Fig. 1. LC-MS survey scans of tryptic peptides were acquired as described in Materials and methods, and peptide ions were identified by MSMS fragmentation. Annotated are ions at m/z 916.44 for a light-Lys labelled peptide (residues 219–238), and m/z 920.20 for the heavy-Lys labelled peptide from flagellin. The ETEC specific peptide is labelled with heavy-Lys in the top and with light-Lys in the bottom of the figure. This figure shows that flagellin is up-regulated in the ETEC strain.
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Fig. 2. LC-MS survey scans, the first and third panel with spectra with the ETEC strain labelled with heavy lysine, the second and fourth panel with the commensal strain labelled. They show, exemplified by the ions at m/z 685.3/689.3, and 543.7/547.7, that Outer Membrane Protein 1a is down-regulated in the ETEC strain.
with the in-house BLAST program. For PSORT, a computer program for the prediction of protein localization sites in cells, following website were used: http://psort.ims.u-tokyo.ac.jp/. 3. Results and discussion H10407 and BL21 were grown either in M9 minimal medium containing heavy lysine or a light lysine M9 media (Table 1). The use of M9 medium enables the SILAC technology for E. coli, because prototroph E. coli bacteria utilize the 13C15 6 N2 modified L-lysine for protein biosynthesis, if supplemented in M9 medium. However, the MC1061 strain could only be cultivated in Luria Bertani broth or on blood agar plates, as it is an auxotroph strain and does not grow in M9 minimal media without additions; therefore, cell labelling with SILAC of MC1061 was not performed. In contrast, we were able to apply SILAC to H10407 and Bl21, and to produce cultures with either lysine version. Equal amounts of “normal” and “heavy” isolated OM proteins were mixed for a 1-D or 2-D PAGE analysis. From all three strains, we identified more than 80 different surface proteins of E. coli (Table 2) and compared quantities of surface
proteins between the ETEC strain and the BL21 strain. Distinction of ETEC or BL21 specific peptides was uncomplicated using normal and heavy L-lysine for cell labelling. The SILAC method enables the determination of relative protein expression levels from two different strains in one experiment. Table 1 displays different SILAC experiments in detail. For relative quantification of ETEC and K12 protein concentration ratios ([ETEC]/[K12]) peptide signal intensities derived from the ETEC protein and the K12 protein were compared (Figs. 1 and 2). Differentially expressed ETEC proteins with a ratio of 2 or greater are shown in Table 3. Comparison of the ETEC strain with MC1061 gave a much larger amount of differentially expressed proteins, but this could well be due to the different growth conditions (minimal vs. rich medium; Table 3B). We identified seven proteins (Table 3) that are differentially expressed in the ETEC strain (ratio [ETEC}[:BL21] N [2]:[1]). Protein weights of these up-regulated proteins are between 25 kDa and 85 kDa. Interestingly, the number of identified proteins was higher with the 1D gel based strategy (GeLCMS/MS) than with the 2D gel based strategy (2D-gel MALDI-TOF/ TOF). We had obtained similar results in our previous proteomics approach with Streptococcus pneumomiae (Morsczeck et al., 2008).
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Table 3 Differentially expressed proteins. Anrp#
Protein
Number of peptides
A) Differentially expressed ETEC surface proteins (H10407/BL21). Identified proteins with a lower [ETEC]/[K12] ratio than 2 are not shown. AnrP17506 (NC_002655) pyridine nucleotide transhydrogenase, beta subunit 8 [Escherichia coli O157:H7 EDL933] (GI:15802016) AnrP4116560 Chain A, Outer Membrane Cobalamin Transporter (Btub) From E. coli (GI:145439) 11 AnrP553350 (AJ243795) flagellin [Escherichia coli] ALSO found in D63 (GI:6706172) 13 AnrP6997368 COG3203: Outer membrane protein (porin) [Escherichia coli F11] (GI:193070759) 4 AnrP7083944 COG4771: Outer membrane receptor for ferrienterochelin and colicins [Escherichia coli F11] 11 (GI:191172891) AnrP7963825 Putative inner membrane ABC-transporter [Escherichia coli UTI89] (GI:91072771) 6 AnrP96882 (Z38065) FyuA precursor [Escherichia coli] (GI:557754) 27 B) Differentially expressed proteins identified in H10407 (ETEC) and MC1061 (K12). AnrP204940 NC_002695) PTS system, glucose-specific IIBC component [Escherichia coli O157:H7] AnrP256328 (NC_002695) chaperonin GroEL [Escherichia coli O157:H7] AnrP324273 Putative ATPase subunit of translocase [Escherichia coli O157:H7 EDL933] AnrP3297020 Putative lipoprotein [Shigella flexneri 2a str. 301] AnrP417078 (NC_000913) L-lactate dehydrogenase [Escherichia coli K12] AnrP47616 (NC_002695) colicin I receptor precursor [Escherichia coli O157:H7] AnrP514889 (NC_000913) respiratory NADH dehydrogenase [Escherichia coli K12] AnrP619808 (NC_000913) outer membrane receptor for ferric enterobactin (enteroche AnrP679551 (NC_002655) putative transport protein [Escherichia coli O157:H7 EDL93 AnrP7083578 COG0541: Signal recognition particle GTPase [Escherichia coli F11] AnrP744366 AJ132668) lipoprotein inner membrane ABC-transporter, Irp6 [Yersinia pestis KIM] AnrP83888 (J04229) colicin I receptor [Escherichia coli] AnrP86905 (NC_000913) pyridine nucleotide transhydrogenase, alpha subunit [Escherichia coli K12] AnrP886047 (NC_002655) lipoamide dehydrogenase (NADH); component of 2-oxodehydrog AnrP939220 (NC_002655) putative function in exopolysaccharide production [Escheri AnrP954304 (NC_002655) yiaF gene product [Escherichia coli O157:H7 EDL933]
4 2 2 7 5 6 4 17 5 2 3 4 5 12 4 4
M-score
Ratio (ETEC:K12)
255
N2
134 648 120 284
2 N3 2.5 3–5
285 875
N2 2
109 124 96 240 223 240 155 727 230 71 155 131 230 462 117 124
3 N5 N2 N2 N2 N4 N2 N5 3 N5 3? N2 3 N3 N4 2
expression of the putative vaccine target, can the localization be verified by FACS or similar techniques. While all cell-surface preparation methods will have to deal with the possibility of leakage, it has on the other hand also been reported that real cell-surface proteins have been predicted not to be such (Tjalsma et al., 2006; Rodríguez-Ortega et al., 2008). Putative vaccine candidates should have low identities to human proteins (b30%) and should have no 7-mers consisting of seven consecutive amino acids identities with human proteins. Four out of seven (or 17 out of 23) proteins were excluded as a good protein vaccine candidate because of more than 30% identity to human proteins. After these experiments, only three of the identified proteins of Table 3 fit all restrictive selection criteria for a new protein vaccine candidate: BtuB, FyuA and flagellin. BtuB is achain A of the outer membrane cobalamin transporter; FyuA is a siderophore and probably localized on a pathogenicity island originally derived from the “High-Pathogenicity Island” (HPI) of Yersinia enterocolitica (Okeke et al., 2004; Schubert et al., 1998). Here, approx. 21% of ETEC strains harbour fyuA on their genome. However, 90% of enteroaggregative E. coli (EAEC) are HPI positive (Schubert et al., 1998). The protein flagellin is a novel interesting candidate for ETEC vaccines, even though it may cause an inflammatory response via the activation of IL8 production (Steiner et al., 2000). Recently, it was shown that vaccination against the flagellin proteins protects against colonization
Some identified surface proteins were not differentially expressed in all experiments or were not identified in all approaches. However, experiment #2006 (Table 1) demonstrates that labelling of the commensal bacterial strain instead of the ETEC strain with heavy lysine allows to reproduce the results; for the measurement of differentially expressed surface proteins it was secondary which strain was labelled (Figs. 1 and 2). Although there are visible differences between the ratios in the two parts of experiment 2006, most prominent visible in Fig. 1, where the signal for the commensal strain is barely visible when labelled with heavy lysine, but at about ¼ of the ETEC signal's height when labelled with light lysine, the by far stronger expression in the ETEC strain is obvious. This variance in results might be due to incomplete incorporation or other technical issues, and proves the worth of performing these complementary sets as well as the necessity of the (commonly used) 2-fold ratio cutoff. We localized differentially expressed proteins (including those from MC1061) with Psort and searched for homologue human proteins or peptides with a BLAST search (Table 4). Most proteins are localized on the OM, the periplasm and the cytoplasmic membrane. However, only 2 proteins are localized in the cytoplasm (and none of those from the BL21 comparisons), which verifies the reliability of cell envelope preparation strategy. Obviously prediction tools like Psort do not prove that a molecule is really presented on the bacterial surface. Only at a later stage, after
Table 4 Human BLAST and Psort analysis (ND: not determined). Proteins are in italic and were excluded as putative ETEC vaccine candidates after bioinformatics. Protein
% Identity (human BLAST)
AnrP17506 (NC_002695) pyridine nucleotide transhydrogenase beta AnrP4116560 Chain A, Outer Membrane Cobalamin Transporter AnrP553350 (AJ243796) flagellin [Escherichia coli] (AJ243795) AnrP6997368 COG3203: Outer membrane protein (porin) AnrP7083944 COG4771: Outer membrane receptor AnrP7963825 putative inner membrane ABC-transporter [Escherichia coli UTI89] AnrP96882 (Z38065) FyuA precursor [Escherichia coli
52 20 27 48 39 34 25
Comments
7-mers
Localization with Psort
(Short peptide) (Short peptide) (Short peptide)
ND No 7-mers No 7-mers ND ND ND No 7-mers
Cytoplasmic membrane Outer membrane Extracellular Outer membrane Unknown Cytoplasmic membrane Outer membrane
U. Sommer et al. / Journal of Microbiological Methods 83 (2010) 13–19
with ETEC (Roy, et al., 2009a, b). Moreover, this protein is also important for the chemotaxis of E. coli, which is an important characteristic of pathogenic bacteria. Interestingly the flagellin protein of Campylobacter jejuni was also discussed for a protective C. jejuni protein vaccine (Lee et al., 1999). 4. Conclusion To our knowledge, this study is the first to use SILAC for the identification of outer membrane proteins differentially expressed in enterotoxigenic E. coli. This technology should prove generally applicable to the detection of differentially expressed bacterial surface proteins, and therewith help to improve current technologies for the development of protein vaccines. In our example, SILAC enabled the detection of two OM proteins, which may be interesting candidates for the development of an ETEC vaccine. References Fountoulakis, M., Gasser, R., 2003. Proteomic analysis of the cell envelope fraction of Escherichia coli. Amino Acids 24, 19–41. Frech, S.A., Dupont, H.L., Bourgeois, A.L., McKenzie, R., Belkind-Gerson, J., Figueroa, J.F., Okhuysen, P.C., Guerrero, N.H., Martinez-Sandoval, F.G., Meléndez-Romero, J.H., Jiang, Z.D., Asturias, E.J., Halpern, J., Torres, O.R., Hoffman, A.S., Villar, C.P., Kassem, R.N., Flyer, D.C., Andersen, B.H., Kazempour, K., Breisch, S.A., Glenn, G.M., 2008. Use of a patch containing heat-labile toxin from Escherichia coli against travellers' diarrhoea: a phase II, randomised, double-blind, placebo-controlled field trial. Lancet 371, 2019–2025. Gaastra, W., Svennerholm, A.M., 1996. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 4, 444–452. Hyams, K.C., Bourgeois, A.L., Merrell, B.R., Rozmajzl, P., Escamilla, J., Thornton, S.A., Wasserman, G.M., Burke, A., Echeverria, P., Green, K.Y., 1991. Diarrheal disease during Operation Desert Shield. N Engl J. Med. 325, 1423–1428. Lee, L.H., Burg, E., Baqar, S., Bourgeois, A.L., Burr, D.H., Ewing, C.P., Trust, T.J., Guerry, P., 1999. Evaluation of a truncated recombinant flagellin subunit vaccine against Campylobacter jejuni. Infect. Immun. 67, 5799–5805.
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Morsczeck, C., Prokhorova, T., Sigh, J., Pfeiffer, M., Bille-Nielsen, M., Petersen, J., Boysen, A., Kofoed, T., Frimodt-Møller, N., Nyborg-Nielsen, P., Schrotz-King, P., 2008. Streptococcus pneumoniae: proteomics of surface proteins for vaccine development. Clin. Microbiol. Infect. 14, 74–81. Mortz, E., Krogh, T.N., Vorum, H., Görg, A., 2001. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ ionization-time of flight analysis. Proteomics 1, 1359–1363. Okeke, I.N., Scaletsky, I.C.A., Soars, E.H., Macfarlane, L.R., Torres, A.G., 2004. Molecular epidemiology of the iron utilization genes of enteroaggregative Escherichia coli. J. Clin. Microbiol. 42, 36–44. Ong, S., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., Mann, M., 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386. Prokhorova, T.A., Nielsen, P.N., Petersen, J., Kofoed, T., Crawford, J.S., Morsczeck, C., Boysen, A., Schrotz-King, P., 2006. Novel surface polypeptides of Campylobacter jejuni as traveller's diarrhoea vaccine candidates discovered by proteomics. Vaccine 24, 6446–6455. Rodríguez-Ortega, M.J., Luque, I., Tarradas, C., Bárcena, J.A., 2008. Overcoming function annotation errors in the Gram-positive pathogen Streptococcus suis by a proteomics-driven approach. BMC Genomics 9, 588. Roy, K., Hamilton, D., Ostmann, M.M., Fleckenstein, J.M., 2009a. Vaccination with EtpA glycoprotein or flagellin protects against colonization with enterotoxigenic Escherichia coli in a murine model. Vaccine 27, 4601–4608. Roy, K., Hilliard, G.M., Hamilton, D.J., Luo, J., Ostmann, M.M., Fleckenstein, J.M., 2009b. Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells. Nature 457, 594–598. Schubert, S., Rakin, A., Karch, H., Carniel, E., Heesemann, J., 1998. Prevalence of the “high-pathogenicity island” of Yersinia species among Escherichia coli strains that are pathogenic to humans. Infect. Immun. 66, 480–485. Steiner, T.S., Nataro, J.P., Poteet-Smith, C.E., Smith, J.A., Guerrant, R.L., 2000. Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL8 release from intestinal epithelial cells. J. Clin. Invest. 105, 1769–1777. Steinsland, H., Valentiner-Branth, P., Gjessing, H.K., Aaby, P., Mølbak, K., Sommerfelt, H., 2003. Protection from natural infections with enterotoxigenic Escherichia coli: longitudinal study. Lancet 362, 286–291. Steinsland, H., Valentiner-Branth, P., Aaby, P., Mølbak, K., Sommerfelt, H., 2004. Clonal relatedness of enterotoxigenic Escherichia coli strains isolated from a cohort of young children in Guinea-Bissau. J. Clin. Microbiol. 42, 3100–3107. Tjalsma, H., Pluk, W., van den Heuvel, L.P., Peters, W.H.M., Roelofs, R., Swinkels, D.W., 2006. Proteomic inventory of “anchorless” proteins on the colon adenocarcinoma cell surface. Biochim. Biophys. Acta 1764, 1607–1617.
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Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Comparison of surface proteomes of enterotoxigenic (ETEC) and commensal Escherichia coli strains Ulf Sommer a, Jørgen Petersen a, Michael Pfeiffer a, Petra Schrotz-King a, Christian Morsczeck a,b,⁎ a b
ACE BioSciences A/S, Roskildevej 12 C, 3400 Hillerød, Denmark Department of Operative Dentistry and Periodontology,University Hospital Regensburg,Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany
a r t i c l e
i n f o
Article history: Received 11 May 2010 Received in revised form 5 July 2010 Accepted 6 July 2010 Available online 17 July 2010 Keywords: SILAC Escherichia coli Surface proteins Vaccine Proteomics
a b s t r a c t Pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections involves colonization of the small intestine mediated by cell-surface fimbriae (CS) or colonization fimbriae antigens (CFA). However, protection against reinfection of ETEC is also conferred by somatic antigens rather than by virulence factors. To discover ETEC specific somatic antigens, the surface proteome of the ETEC H10406 strain was compared with that of nonpathogenic E. coli K12 strains. In this study, we were using stable isotope labelling with amino acids in cell culture (SILAC) technology for the labelling and relative quantification of surface proteins in order to identify polypeptides that are specifically present on ETEC strains. Outer membrane proteins were isolated, separated by gel electrophoresis, and identified by mass spectrometry. Twenty-three differentially expressed cellsurface polypeptides of ETEC were identified and evaluated by bioinformatics for protein vaccine candidates. The combination of being surface-exposed and present differentially makes these polypeptides highly suitable as targets for antibodies and thus for use in passive or active immunisation/vaccination. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Escherichia coli is an adaptive bacterial species that is both a commensal resident of the intestine and a versatile pathogen of humans and animals. A panel of E. coli strains cause diarrhoea. ETEC is a major cause of diarrhoea in children in developing countries particularly under the age of five (Gaastra and Svennerholm, 1996). Diarrhoea is a condition characterised by a marked increase in the frequency of unformed bowel movements and is commonly accompanied by abdominal cramps, urgency, nausea, bloating, vomiting, fever, and malaise. Cases are caused after an infection with ETEC. During Operation Desert Shield, 25% of the troops stationed in Saudi Arabia experienced at least one episode of ETEC diarrhoea (Hyams et al., 1991). ETEC bacteria are not invasive and colonize the small intestine by attachment to the mucosa via colonization factors. Pathogenesis of ETEC infections involves colonization of the small intestine mediated by cellsurface fimbriae (CS) or colonization fimbriae antigens (CFA). At least 20 different types of CFA proteins were identified on diverse ETEC strains until today (Gaastra and Svennerholm, 1996). ETEC induce fluid and electrolyte secretion by the small intestinal epithelium in response to the production of heat-labile (LT) and/or heat-stabile enterotoxins (ST). The ETEC bacterium expresses at least one of these two toxins. LT is similar in structure and function to the known cholera toxin and severe
ETEC infections are comparable to infections with Vibrio cholerae (Steinsland et al., 2004; Gaastra and Svennerholm, 1996). Epidemiological evidence and results from experimental challenge studies in volunteers, clearly demonstrate that strain-specific immunity follows ETEC infection and that multiple infections with antigenically diverse ETEC strains lead to broad protection against ETEC diarrhoea (Steinsland et al., 2003, 2004). This indicates that a reliable ETEC vaccine is achievable. Recent efforts have been looking into the development of non-cellular candidates for vaccination, which are based for example on a patch of LT or CS6 (Frech et al., 2008). These are virulence factors defining an ETEC pathotype. However, an epidemiological study has demonstrated that protection conferred by ETEC infection against reinfection is conferred by somatic antigens defining a clone rather than by virulence factors defining a pathotype (Steinsland et al., 2004). Our study therefore intended to identify differentially expressed proteins on the surface of ETEC, which show lower expression on the surface of commensal E. coli strains (K12). ETEC specific surface proteins with a ratio ([ETEC]/[K12]) of 2 or greater were identified with 1D gels and 2D gels using the technology of stable isotope labelling with amino acids in cell culture (SILAC), which is mainly used for the characterization of eukaryotic cells (Ong et al., 2002). 2. Materials and methods 2.1. Bacterial strains
⁎ Corresponding author. Tel.: + 49 0 941 944 6161. E-mail address: [email protected] (C. Morsczeck). 0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2010.07.011
The ETEC strain D2166, original No.H10407, serotype O78:H11 LT and STp positive were derived from the Statens Serum Institut (SSI,
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Denmark). The commensal E. coli strain D2103 (MC1061) K12 strain serotype O-rough:H48 was derived from the SSI, and the BL21 (K12) strain purchased from Invitrogen. 2.2. Labelling of E. coli cells with SILAC and isolation of cell envelope proteins A 20-ml starter culture of E. coli strains BL21 or H10407 was inoculated in M9 minimal medium and cultured at 37 °C overnight. The following day cells were pelleted at 6000 g for 30 min and M9 medium was poured off. Cells were resuspended in 2 ml M9 medium, of which 1 ml of resuspended cells were inoculated in 98 ml. For SILAC labelling either heavy-Lys (10 mg L-lysine-13C6, 15N2·HCl (min 98+ Atom% 13C min 99+ atom% 15N) (Sigma-Aldrich)) or normal L-lysine (light-Lys) dissolved in 1 ml PBS were added to the cell solution to obtain a final concentration of 100 mg/l. The cultures were grown overnight at 37 °C under vigorous shaking at 200 rpm. Strain MC1061 was grown in parallel in 100 ml LB medium. The cell envelope fraction was isolated according to a protocol by Fountoulakis and Gasser (2003). Briefly, the overnight E. coli culture was pelleted and the bacteria were resuspended in buffer A (50 mM Tris (pH 7.6), 250 mM sucrose, 0.25 mM EDTA, 0.25 mg/ml lysozyme). The cell suspension was mixed by shaking for 1 h (4 °C), followed by a centrifugation step at 2800 g for 12 min. The new pellet was resuspended in buffer B (100 mM potassium phosphate (pH 7.6), 6 mM magnesium acetate, 0.1 mM DTT, 20% (v/v) glycerol + proteinase inhibitors). This solution was disrupted 3 times by sonication. To remove unbroken cells and debris, sonicated bacteria were centrifuged for 30 min at 12,000 g, and subsequently the supernatants were centrifuged at 41,000 rpm in a Sorvall A-641 rotor. The pelleted OM proteins were resuspended in buffer C (buffer A w/o lysozyme). 2.3. Surface protein identification Outer membrane proteins were isolated from bacteria cultures as described above and protein concentrations were estimated with the Bradford test (Bio-Rad). Protein samples for analysis were produced after mixing equal protein amounts from both E. coli strains. The complex mixture of proteins obtained after surface extraction was analysed by two complementary strategies based on mass spectrometry, a 2D gel and a 1D gel based strategy similar to those previously described (Morsczeck et al., 2008; Prokhorova et al., 2006). 2.3.1. 2-D gel based strategy (2D-gel MALDI-TOF/TOF) The first dimension run was performed on a pre-cast 13 cm IPG strip (pH 4–7) using the Ettan IPGphor isoelectric focusing system (Amersham Biosciences) according to the manufacturer's instructions, including 4% CHAPS. The second dimension was performed by inserting the strips on top of self-cast 12% SDS PAGE gels. Electrophoresis was performed on the Hoefer SE 600 PAGE system (Amersham Pharmacia), and the gels silver-stained (Mortz et al., 2001). Spots for mass spectrometric analysis were picked using the Ettan Spot Picker from Amersham according to the manufacturer instructions. Specific protein spots were spot-picked, and placed into punctured 96-well plates in Milli-Q water. Solvent was removed by spinning the plates at 2000 g. These gel plugs were washed in 50 mM NH4HCO3/ 50% ethanol and dehydrated by incubation in 96% ethanol. Reduction and alkylation was performed by incubating in reducing solution (10 mM DTT, 50 mM NH4HCO3) at 56 °C followed by room temperature incubation in alkylation solution (55 mM iodoacetamide, 50 mM NH4HCO3) in the dark. Two cycles of washing and dehydration were then performed prior to the addition of 5 μl trypsin solution (12.5 ng/ul Promega trypsin in 50 mM NH4HCO3, 10% acetonitrile). Then an additional amount of ammonium bicarbonate solution was added, and the digests were incubated overnight at 37 °C. Peptides
were extracted after acidification with trifluoroacetic acid to the overnight digest followed by incubation with shaking. Parts of the extract were spotted on anchorchip targets, using αcyano-4-hydroxy-cinnamic acid as the matrix, and submitted to automated MALDI-TOF peptide mass fingerprint and MALDI-TOF/TOF analysis (Ultraflex, Bruker Daltonics, Germany). Peak lists were created in FlexAnalysis and submitted to BioTools (both Bruker) for database searching against a specific in-house E. coli database. The Mascot search program and scoring algorithm (Matrix Science, UK) were used in database searching. Peptide mass tolerance was set to 60 ppm and 0.7 Da, respectively. Search parameters were adjusted to include oxidation of Met, the addition of Carbamidomethyl groups to Cys, and the SILAC modification (Lys-Silac + 8 (K)), and trypsin was allowed to miss one cleavage site per peptide. 2.3.2. 1D gel based strategy (GeLC-MS/MS) One-dimensional gel electrophoresis was performed on a Novex NuPage system (Invitrogen) according to the manual provided with the gel system. In brief we electrophoresed pre-cast 12% bis–tris gels (8 cm × 8 cm, × 1 mm, Invitrogen) at 200 V for 60 min in NuPageMOPS-SDS running buffer. Gels were Coomassie stained. Whole lanes were cut into gel slices with a razor blade. The gel slices were digested as described under the 2D gel strategy, but the amount of trypsin was a 20 μl trypsin solution (12.5 ng/μl Promega trypsin in 50 mM NH4HCO3, 10% Acetonitrile). The extracts were analysed by LC-MS/MS on a Waters Cap-LC and Micromass Ultima API QTOF mass spectrometer. Each sample was submitted to a 115 min LC-MS/MS analysis on a self-packed 12 cm, 75 m Zorbax-C18 column, using a trap column of the same material and including a gradient from 100% solvent A (5% acetonitrile, 0.1% formic acid in water) to 80% solvent B (5% water, 0.1% formic acid in acetonitrile) in data-dependent acquisition mode. The peak list (pkl) files generated by the MassLynx software were reduced in size by an in-house script for searching a specific in-house E. coli database. The Mascot Demon program and scoring algorithm (Matrix Science, UK) were used in database searching. Peptide mass tolerance was set to 200 ppm and 0.4 Da for fragment ions. Search parameters were adjusted to include oxidation of Met, the addition of Carbamidomethyl groups to Cys, and the SILAC modification (Lys-Silac + 8 (K)), and trypsin was allowed to miss one cleavage site per peptide. 2.3.3. Quantification Quantification of tryptic digested spots from either 1D gel or 2D gel was carried out by analyzing the ratio between the relative intensities of the peak pairs (light and heavy peptide). This was done manually using the FlexAnalysis software (Bruker) for MALDI data, and the ProteinLynx/MassLynx software (Micromass/Waters) for the ESI data. 2.4. Bioinformatics and protein numbering (AnrP) The ClustalV Alignment (MegAlign, DNA_Star) was used for the evaluation of homologies of identified proteins sequences. An internal protein database (ACE Biosciences) assigned AnrP (ACE non-
Table 1 Experiments. Experiment #
ETEC strain
K12 strain
Proteomics approach
Strain labelled with SILAC
1981 1986 1987 1989 1992 1993 2006
H10407 H10407 H10407 H10407 H10407 H10407 H10407
MC1061 MC1061 MC1061 BL21 BL21 BL21 BL21
1D 2D 2D 2D 2D 2D 1D
ETEC ETEC ETEC ETEC ETEC ETEC ETEC/BL21
gel + LC/MSMS gel + MALDI/TOFTOF gel + MALDI/TOFTOF gel + MALDI/TOFTOF gel + MALDI/TOFTOF gel + MALDI/TOFTOF gel + LC/MSMS
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15
Table 2 Proteins identified in cell envelope fractions from E. coli. Proteins were considered identified based on at least two identified peptides or one high-scoring peptide, and a protein M-Score considered significant by the Mascot program (p b 0.05). A high-scoring peptide is a fragmented peptide that identifies a protein with a score of above 50 after a GeLC-MS/MS strategy. AnrP#
Protein name
AnrP10213 AnrP104501 AnrP124628
(NC_002655) 30S ribosomal subunit protein S2 [Escherichia coli O157:H7 EDL933] (U70214) hypothetical protein [Escherichia coli] (NC_002655) 2-oxoglutarate dehydrogenase (dihydrolipoyltranssuccinase E2 component) [Escherichia coli O157:H7 EDL933] (NC_002655) pyruvate dehydrogenase (dihydrolipoyltransacetylase component) [Escherichia coli O157:H7 EDL933] (NC_002655) degrades sigma32, integral membrane peptidase, cell division protein [Escherichia coli O157:H7 EDL933] (NC_002655) phage lambda receptor protein; maltose high-affinity receptor [Escherichia coli O157:H7 EDL933] (M38305) RNA polymerase (rpoC) [Escherichia coli] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) pyridine nucleotide transhydrogenase, beta subunit [Escherichia coli O157:H7 EDL933] (NC_002655) orf, hypothetical protein [Escherichia coli O157:H7 EDL933] (NC_002695) PTS system, glucose-specific IIBC component [Escherichia coli O157:H7] NC_002695) PTS system, glucose-specific IIBC component [Escherichia coli O157:H7 EDL933] (J01594) ATP synthase alpha subunit (atp-6) [Escherichia coli] (NC_002655) putative lipoprotein [Escherichia coli O157:H7 EDL933] (NC_002655) putative lipoprotein [Escherichia coli O157:H7 EDL933] (NC_000913) maltodextrin phosphorylase [Escherichia coli K12] (NC_002655) outer membrane channel; specific tolerance to colicin E1 [Escherichia coli O157:H7 EDL933] (NC_002695) chaperonin GroEL [Escherichia coli O157:H7] (NC_000913) membrane-bound ATP synthase, F1 sector, alpha subunit [Escherichia coli K12] (AP002566) membrane-bound ATP synthase alpha subunit AtpA [Escherichia coli O157:H7] (X82400) phosphopyruvate hydratase; enolase [Escherichia coli] (D90701) Hypothetical protein 200 (entA 3′ region) [Escherichia coli] (NC_002695) CoA-linked acetaldehyde dehydrogenase/iron-dependent alcohol dehydrogenase [Escherichia coli O157:H7] (NC_002655) outer membrane protein 3a (II*;G;d) [Escherichia coli O157:H7 EDL933] (NC_002655) outer membrane protein 3a (II*;G;d) [Escherichia coli O157:H7 EDL933] NC_000913) PTS system, fructose-specific transport protein [Escherichia coli K12] putative ATPase subunit of translocase [Escherichia coli O157:H7 EDL933] (NC_002655) putative ATPase subunit of translocase [Escherichia coli O157:H7 EDL933] rpsA, protein S1 [Escherichia coli] D-methionine ABC-transporter, periplasmic D-methionine-binding lipoprotein [Escherichia coli 101-1] (NC_002695) succinate dehydrogenase flavoprotein subunit [Escherichia coli O157:H7] Ferrienterobactin receptor precursor [Escherichia coli CFT073] nmpC, outer membrane porin protein; locus of qsr prophage [Escherichia coli O157:H7 EDL933] (NC_002655) outer membrane protein 1a (Ia;b;F) [Escherichia coli O157:H7] (D90838) Adhesin AIDA-I precursor. [Escherichia coli] (NC_002655) lipoprotein-28 [Escherichia coli O157:H7 EDL933] (NC_000913) putative protease [Escherichia coli K12] (NC_002655) 50S ribosomal subunit protein L4 [Escherichia coli O157:H7] (NC_002655) 50S ribosomal subunit protein L4 [Escherichia coli O157:H7 EDL933] (NC_002655) membrane-bound ATP synthase, F1 sector, beta subunit [Escherichia coli O157:H7 EDL933] Chain A, Outer Membrane Cobalamin Transporter (Btub) From E. coli (NC_000913) L-lactate dehydrogenase [Escherichia coli K12] (NC_002695) hypothetical protein [Escherichia coli O157:H7] (AF037157) ATP synthase beta subunit [Yersinia enterocolitica] (NC_000913) membrane-bound ATP synthase, F0 sector, subunit b [Escherichia coli K12] (NC_002655) pyruvate dehydrogenase (decarboxylase component) [Escherichia coli O157:H7 EDL933] (NC_002655) acridine efflux pump [Escherichia coli O157:H7 EDL933] (NC_002655) 50S ribosomal subunit protein L3 [Escherichia coli O157:H7] (NC_000913) phosphoglycerate kinase [Escherichia coli K12] (X17440) hsg48 [Escherichia coli] (NC_002695) colicin I receptor precursor [Escherichia coli O157:H7] (Z19601) ORF, urf74.3. [Escherichia coli] protein L12 [Escherichia coli] (NC_002655) protease specific for phage lambda cII repressor [Escherichia coli O157:H7 EDL933] (X57402) lipoprotein-34 [Escherichia coli] (NC_000913) respiratory NADH dehydrogenase [Escherichia coli K12] (AJ243795) flagellin [Escherichia coli] (AJ243795) flagellin [Escherichia coli] Chain B, Crystal Structure Of The Long-Chain Fatty Acid Transporter Fadl (NC_002655) putative ATP-binding component of ABC transport system [Escherichia coli O157:H7 EDL933] (NC_003197) outer membrane porin, receptor for colicin I, requires TonB [Salmonella typhimurium LT2] (NC_000913) outer membrane receptor for ferric enterobactin (Escherichia coli str. K-12) (NC_002655) chaperone Hsp70; DNA biosynthesis; autoregulated heat shock protein 70 [Escherichia coli O157:H7 EDL933] (NC_002655) protease specific for phage lambda cII repressor [Escherichia coli O157:H7 EDL933] (NC_002655) putative transport protein [Escherichia coli O157:H7 EDL933] (NC_000913) outer membrane protein 1b (Ib;c) [Escherichia coli K12] (NC_000913) outer membrane protein 1a (Ia;b;F) [Escherichia coli K12] (NC_002655) protein chain elongation factor EF-Tu (duplicate of tufA) [Escherichia coli O157:H7 EDL933] Pyruvate dehydrogenase complex, dehydrogenase (E1) component [Escherichia coli B171]
AnrP12474 AnrP128059 AnrP140391 AnrP147873 AnrP17506 AnrP17506 AnrP17506 AnrP17506 AnrP17506 AnrP196226 AnrP204940 AnrP204940 AnrP229296 AnrP229453 AnrP229453 AnrP233606 AnrP238319 AnrP256328 AnrP26043 AnrP302876 AnrP311260 AnrP312095 AnrP314881 AnrP314881 AnrP318346 AnrP324273 AnrP324273 AnrP325807 AnrP3297020 AnrP33530 AnrP3555721 AnrP357746 AnrP362330 AnrP380051 AnrP380718 AnrP387792 AnrP390669 AnrP390669 AnrP39192 AnrP4116560 AnrP417078 AnrP421121 AnrP426538 AnrP431306 AnrP43799 AnrP442611 AnrP451981 AnrP465207 AnrP46819 AnrP47616 AnrP48657 AnrP493633 AnrP505092 AnrP510278 AnrP514889 AnrP553350 AnrP553350 AnrP5874704 AnrP617142 AnrP61933 AnrP619808 AnrP631052 AnrP659893 AnrP679551 AnrP693011 AnrP694356 AnrP69760 AnrP6992987
Peptides
M-score
7 5 3
308 152 74
4 9 3 3 8 11 2 1 2 5 4 4 17 3 3 2 5 2 6
156 345 135 160 255 363 126 56 86 156 109 80 742 134 134 76 178 124 214
7 2 9 7 5 5 2 2 8 7 3 5 10 5 2 5 1 2 5 22 11 5 2 8 3 10 5 4 6 1 6 2 13 9 7 4 13 5 4 1 3 17 3 6 5 3 11 15 3
205 115 298 226 115 199 96 68 225 240 167 75 339 141 112 141 91 82 210 627 134 223 63 316 144 325 137 162 251 54 240 92 349 292 260 155 648 248 258 90 79 727 99 243 230 80 281 477 148
(continued on next page)
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Table 2 (continued) AnrP#
Protein name
Peptides
M-score
AnrP6997368 AnrP702172 AnrP7083578 AnrP7083944 AnrP726813 AnrP728396 AnrP732180 AnrP744366 AnrP752782 AnrP768202 AnrP7963825 AnrP7963825 AnrP7963825 AnrP7963825 AnrP83888 AnrP84274 AnrP86905 AnrP86905 AnrP86905 AnrP886047 AnrP898114 AnrP899094 AnrP939220 AnrP954304 AnrP96882 AnrP98055
COG3203: Outer membrane protein (porin) [Escherichia coli F11] NC_002655) 50S ribosomal subunit protein L1 [Escherichia coli O157:H7 (COG0541) Signal recognition particle GTPase [Escherichia coli F11] COG4771: Outer membrane receptor for ferrienterochelin and colicins [Escherichia coli F11] (NC_002655) NADH dehydrogenase I chain F [Escherichia coli O157:H7 EDL933] (NC_002655) 50S ribosomal subunit protein L6 [Escherichia coli O157:H7] (NC_000913) outer membrane porin protein; locus of qsr prophage [Escherichia coli] Lipoprotein inner membrane ABC-transporter [Escherichia coli F11] (NC_002655) galactose-binding transport protein; receptor for galactose [Escherichia coli O157:H7 EDL933] (X67326) alcohol dehydrogenase [Escherichia coli] putative inner membrane ABC-transporter [Escherichia coli UTI89] putative inner membrane ABC-transporter [Escherichia coli UTI89] (putative inner membrane ABC-transporter [Escherichia coli UTI89] putative inner membrane ABC-transporter [Escherichia coli UTI89] (J04229) colicin I receptor [Escherichia coli] (NC_000913) NADH dehydrogenase I chain C, D [Escherichia coli K12] (NC_000913) pyridine nucleotide transhydrogenase, alpha subunit [Escherichia coli K12] (NC_000913) pyridine nucleotide transhydrogenase, alpha subunit [Escherichia coli K12] (NC_000913) pyridine nucleotide transhydrogenase, alpha subunit [Escherichia coli K12] (NC_002655) lipoamide dehydrogenase (NADH); [Escherichia coli O157:H7 EDL933] protein I,membrane [Escherichia coli] (NC_002655) periplasmic serine protease Do; heat shock protein HtrA [Escherichia coli O157:H7 EDL933] (NC_002655) putative function in exopolysaccharide production [Escherichia coli O157:H7] (NC_002655) yiaF gene product [Escherichia coli O157:H7 EDL933] (Z38065) FyuA precursor [Escherichia coli] (NC_002655) RNA polymerase, alpha subunit [Escherichia coli O157:H7 EDL933]
4 7 2 4 4 9 6 3 2 3 6 4 3 3 4 2 5 6 6 12 10 3 4 4 27 10
120 267 71 284 105 397 203 155 70 109 285 202 82 136 131 55 230 279 279 462 329 83 117 124 875 355
redundant proteins) numbers to individual E. coli proteins. This monthly-updated database contained all E. coli entries in the NCBI database found by certain key words, in addition to standard contaminant (keratins etc.). Relative ratios of proteins were estimated based on the height of the monoisotopic peaks of their peptide signals.
An in-house Basic Local Alignment Search Tool (BLAST) was used for the evaluation of homologies between selected bacterial proteins and the human proteome. This procedure is similar to a protein BLAST (BLASTp) at the NCBI (http://blast.ncbi.nlm.nih.gov/). The identification of identical 7-mers in the human proteome was done manually
Fig. 1. LC-MS survey scans of tryptic peptides were acquired as described in Materials and methods, and peptide ions were identified by MSMS fragmentation. Annotated are ions at m/z 916.44 for a light-Lys labelled peptide (residues 219–238), and m/z 920.20 for the heavy-Lys labelled peptide from flagellin. The ETEC specific peptide is labelled with heavy-Lys in the top and with light-Lys in the bottom of the figure. This figure shows that flagellin is up-regulated in the ETEC strain.
U. Sommer et al. / Journal of Microbiological Methods 83 (2010) 13–19
17
Fig. 2. LC-MS survey scans, the first and third panel with spectra with the ETEC strain labelled with heavy lysine, the second and fourth panel with the commensal strain labelled. They show, exemplified by the ions at m/z 685.3/689.3, and 543.7/547.7, that Outer Membrane Protein 1a is down-regulated in the ETEC strain.
with the in-house BLAST program. For PSORT, a computer program for the prediction of protein localization sites in cells, following website were used: http://psort.ims.u-tokyo.ac.jp/. 3. Results and discussion H10407 and BL21 were grown either in M9 minimal medium containing heavy lysine or a light lysine M9 media (Table 1). The use of M9 medium enables the SILAC technology for E. coli, because prototroph E. coli bacteria utilize the 13C15 6 N2 modified L-lysine for protein biosynthesis, if supplemented in M9 medium. However, the MC1061 strain could only be cultivated in Luria Bertani broth or on blood agar plates, as it is an auxotroph strain and does not grow in M9 minimal media without additions; therefore, cell labelling with SILAC of MC1061 was not performed. In contrast, we were able to apply SILAC to H10407 and Bl21, and to produce cultures with either lysine version. Equal amounts of “normal” and “heavy” isolated OM proteins were mixed for a 1-D or 2-D PAGE analysis. From all three strains, we identified more than 80 different surface proteins of E. coli (Table 2) and compared quantities of surface
proteins between the ETEC strain and the BL21 strain. Distinction of ETEC or BL21 specific peptides was uncomplicated using normal and heavy L-lysine for cell labelling. The SILAC method enables the determination of relative protein expression levels from two different strains in one experiment. Table 1 displays different SILAC experiments in detail. For relative quantification of ETEC and K12 protein concentration ratios ([ETEC]/[K12]) peptide signal intensities derived from the ETEC protein and the K12 protein were compared (Figs. 1 and 2). Differentially expressed ETEC proteins with a ratio of 2 or greater are shown in Table 3. Comparison of the ETEC strain with MC1061 gave a much larger amount of differentially expressed proteins, but this could well be due to the different growth conditions (minimal vs. rich medium; Table 3B). We identified seven proteins (Table 3) that are differentially expressed in the ETEC strain (ratio [ETEC}[:BL21] N [2]:[1]). Protein weights of these up-regulated proteins are between 25 kDa and 85 kDa. Interestingly, the number of identified proteins was higher with the 1D gel based strategy (GeLCMS/MS) than with the 2D gel based strategy (2D-gel MALDI-TOF/ TOF). We had obtained similar results in our previous proteomics approach with Streptococcus pneumomiae (Morsczeck et al., 2008).
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U. Sommer et al. / Journal of Microbiological Methods 83 (2010) 13–19
Table 3 Differentially expressed proteins. Anrp#
Protein
Number of peptides
A) Differentially expressed ETEC surface proteins (H10407/BL21). Identified proteins with a lower [ETEC]/[K12] ratio than 2 are not shown. AnrP17506 (NC_002655) pyridine nucleotide transhydrogenase, beta subunit 8 [Escherichia coli O157:H7 EDL933] (GI:15802016) AnrP4116560 Chain A, Outer Membrane Cobalamin Transporter (Btub) From E. coli (GI:145439) 11 AnrP553350 (AJ243795) flagellin [Escherichia coli] ALSO found in D63 (GI:6706172) 13 AnrP6997368 COG3203: Outer membrane protein (porin) [Escherichia coli F11] (GI:193070759) 4 AnrP7083944 COG4771: Outer membrane receptor for ferrienterochelin and colicins [Escherichia coli F11] 11 (GI:191172891) AnrP7963825 Putative inner membrane ABC-transporter [Escherichia coli UTI89] (GI:91072771) 6 AnrP96882 (Z38065) FyuA precursor [Escherichia coli] (GI:557754) 27 B) Differentially expressed proteins identified in H10407 (ETEC) and MC1061 (K12). AnrP204940 NC_002695) PTS system, glucose-specific IIBC component [Escherichia coli O157:H7] AnrP256328 (NC_002695) chaperonin GroEL [Escherichia coli O157:H7] AnrP324273 Putative ATPase subunit of translocase [Escherichia coli O157:H7 EDL933] AnrP3297020 Putative lipoprotein [Shigella flexneri 2a str. 301] AnrP417078 (NC_000913) L-lactate dehydrogenase [Escherichia coli K12] AnrP47616 (NC_002695) colicin I receptor precursor [Escherichia coli O157:H7] AnrP514889 (NC_000913) respiratory NADH dehydrogenase [Escherichia coli K12] AnrP619808 (NC_000913) outer membrane receptor for ferric enterobactin (enteroche AnrP679551 (NC_002655) putative transport protein [Escherichia coli O157:H7 EDL93 AnrP7083578 COG0541: Signal recognition particle GTPase [Escherichia coli F11] AnrP744366 AJ132668) lipoprotein inner membrane ABC-transporter, Irp6 [Yersinia pestis KIM] AnrP83888 (J04229) colicin I receptor [Escherichia coli] AnrP86905 (NC_000913) pyridine nucleotide transhydrogenase, alpha subunit [Escherichia coli K12] AnrP886047 (NC_002655) lipoamide dehydrogenase (NADH); component of 2-oxodehydrog AnrP939220 (NC_002655) putative function in exopolysaccharide production [Escheri AnrP954304 (NC_002655) yiaF gene product [Escherichia coli O157:H7 EDL933]
4 2 2 7 5 6 4 17 5 2 3 4 5 12 4 4
M-score
Ratio (ETEC:K12)
255
N2
134 648 120 284
2 N3 2.5 3–5
285 875
N2 2
109 124 96 240 223 240 155 727 230 71 155 131 230 462 117 124
3 N5 N2 N2 N2 N4 N2 N5 3 N5 3? N2 3 N3 N4 2
expression of the putative vaccine target, can the localization be verified by FACS or similar techniques. While all cell-surface preparation methods will have to deal with the possibility of leakage, it has on the other hand also been reported that real cell-surface proteins have been predicted not to be such (Tjalsma et al., 2006; Rodríguez-Ortega et al., 2008). Putative vaccine candidates should have low identities to human proteins (b30%) and should have no 7-mers consisting of seven consecutive amino acids identities with human proteins. Four out of seven (or 17 out of 23) proteins were excluded as a good protein vaccine candidate because of more than 30% identity to human proteins. After these experiments, only three of the identified proteins of Table 3 fit all restrictive selection criteria for a new protein vaccine candidate: BtuB, FyuA and flagellin. BtuB is achain A of the outer membrane cobalamin transporter; FyuA is a siderophore and probably localized on a pathogenicity island originally derived from the “High-Pathogenicity Island” (HPI) of Yersinia enterocolitica (Okeke et al., 2004; Schubert et al., 1998). Here, approx. 21% of ETEC strains harbour fyuA on their genome. However, 90% of enteroaggregative E. coli (EAEC) are HPI positive (Schubert et al., 1998). The protein flagellin is a novel interesting candidate for ETEC vaccines, even though it may cause an inflammatory response via the activation of IL8 production (Steiner et al., 2000). Recently, it was shown that vaccination against the flagellin proteins protects against colonization
Some identified surface proteins were not differentially expressed in all experiments or were not identified in all approaches. However, experiment #2006 (Table 1) demonstrates that labelling of the commensal bacterial strain instead of the ETEC strain with heavy lysine allows to reproduce the results; for the measurement of differentially expressed surface proteins it was secondary which strain was labelled (Figs. 1 and 2). Although there are visible differences between the ratios in the two parts of experiment 2006, most prominent visible in Fig. 1, where the signal for the commensal strain is barely visible when labelled with heavy lysine, but at about ¼ of the ETEC signal's height when labelled with light lysine, the by far stronger expression in the ETEC strain is obvious. This variance in results might be due to incomplete incorporation or other technical issues, and proves the worth of performing these complementary sets as well as the necessity of the (commonly used) 2-fold ratio cutoff. We localized differentially expressed proteins (including those from MC1061) with Psort and searched for homologue human proteins or peptides with a BLAST search (Table 4). Most proteins are localized on the OM, the periplasm and the cytoplasmic membrane. However, only 2 proteins are localized in the cytoplasm (and none of those from the BL21 comparisons), which verifies the reliability of cell envelope preparation strategy. Obviously prediction tools like Psort do not prove that a molecule is really presented on the bacterial surface. Only at a later stage, after
Table 4 Human BLAST and Psort analysis (ND: not determined). Proteins are in italic and were excluded as putative ETEC vaccine candidates after bioinformatics. Protein
% Identity (human BLAST)
AnrP17506 (NC_002695) pyridine nucleotide transhydrogenase beta AnrP4116560 Chain A, Outer Membrane Cobalamin Transporter AnrP553350 (AJ243796) flagellin [Escherichia coli] (AJ243795) AnrP6997368 COG3203: Outer membrane protein (porin) AnrP7083944 COG4771: Outer membrane receptor AnrP7963825 putative inner membrane ABC-transporter [Escherichia coli UTI89] AnrP96882 (Z38065) FyuA precursor [Escherichia coli
52 20 27 48 39 34 25
Comments
7-mers
Localization with Psort
(Short peptide) (Short peptide) (Short peptide)
ND No 7-mers No 7-mers ND ND ND No 7-mers
Cytoplasmic membrane Outer membrane Extracellular Outer membrane Unknown Cytoplasmic membrane Outer membrane
U. Sommer et al. / Journal of Microbiological Methods 83 (2010) 13–19
with ETEC (Roy, et al., 2009a, b). Moreover, this protein is also important for the chemotaxis of E. coli, which is an important characteristic of pathogenic bacteria. Interestingly the flagellin protein of Campylobacter jejuni was also discussed for a protective C. jejuni protein vaccine (Lee et al., 1999). 4. Conclusion To our knowledge, this study is the first to use SILAC for the identification of outer membrane proteins differentially expressed in enterotoxigenic E. coli. This technology should prove generally applicable to the detection of differentially expressed bacterial surface proteins, and therewith help to improve current technologies for the development of protein vaccines. In our example, SILAC enabled the detection of two OM proteins, which may be interesting candidates for the development of an ETEC vaccine. References Fountoulakis, M., Gasser, R., 2003. Proteomic analysis of the cell envelope fraction of Escherichia coli. Amino Acids 24, 19–41. Frech, S.A., Dupont, H.L., Bourgeois, A.L., McKenzie, R., Belkind-Gerson, J., Figueroa, J.F., Okhuysen, P.C., Guerrero, N.H., Martinez-Sandoval, F.G., Meléndez-Romero, J.H., Jiang, Z.D., Asturias, E.J., Halpern, J., Torres, O.R., Hoffman, A.S., Villar, C.P., Kassem, R.N., Flyer, D.C., Andersen, B.H., Kazempour, K., Breisch, S.A., Glenn, G.M., 2008. Use of a patch containing heat-labile toxin from Escherichia coli against travellers' diarrhoea: a phase II, randomised, double-blind, placebo-controlled field trial. Lancet 371, 2019–2025. Gaastra, W., Svennerholm, A.M., 1996. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 4, 444–452. Hyams, K.C., Bourgeois, A.L., Merrell, B.R., Rozmajzl, P., Escamilla, J., Thornton, S.A., Wasserman, G.M., Burke, A., Echeverria, P., Green, K.Y., 1991. Diarrheal disease during Operation Desert Shield. N Engl J. Med. 325, 1423–1428. Lee, L.H., Burg, E., Baqar, S., Bourgeois, A.L., Burr, D.H., Ewing, C.P., Trust, T.J., Guerry, P., 1999. Evaluation of a truncated recombinant flagellin subunit vaccine against Campylobacter jejuni. Infect. Immun. 67, 5799–5805.
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