Proteomic analysis of multidrug resistant Escherichia coli strains from scouring calves

Veterinary Microbiology 151 (2011) 363–371

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Proteomic analysis of multidrug resistant Escherichia coli strains from scouring calves Ablesh Gautam a,d, Heather M. Vinson a, Penelope S. Gibbs a,b, Susan Olet c, Robert Barigye a,b,* a

Department of Veterinary and Microbiological Sciences, North Dakota State University, 1523 Centennial Blvd, Fargo, ND 58108, USA Department of Veterinary Diagnostic Services, North Dakota State University, 1523 Centennial Blvd, Fargo, ND 58108, USA c Department of Statistics, North Dakota State University, P.O. Box 6060, Fargo, ND 58108, USA d Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546-0099, USA b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 November 2010 Received in revised form 7 March 2011 Accepted 28 March 2011

A number of researchers have used chemical inhibitors that target membrane efflux pumps as an experimental treatment strategy for multidrug resistant (MDR) bacterial infections. However, most of these compounds are toxic in vertebrate animals. The present research was therefore done to describe expression dynamics of drug resistanceassociated Escherichia coli proteins that could serve as novel drug targets. Proteomes of MDR and antimicrobial susceptible (AS) E. coli were studied in two dimensional (2-D) polyacrylamide gels and liquid chromatography–mass spectrometry (LC–MS) was performed on proteins of interest. The number of recovered peptides per protein was used to elucidate the amounts of target proteins expressed in MDR and AS E. coli strains. Eight proteins that may be potentially involved in mechanisms of drug resistance were analyzed and identified by LC–MS. These were grouped into membrane porins (TolC, OmpA, OmpC, Nmpc Precursor), proteins involved in microbial protein synthesis (EF-Ts, EF-Tu, RpsA) and Dps, a protein of unknown location and function. Experimental data demonstrated variability in the expression patterns and quantities of the four porins (TolC, OmpA, OmpC, Nmpc precursor), the three microbial protein synthesis associated proteins (EF-Ts, EF-Tu and RpsA), and Dps which has been previously associated with drug resistance. While variability was seen in quantities and expression patterns of some of the proteins of interest, the present data falls short of determining the suitability of these proteins as novel drug targets. Further studies are required to explore how these proteins could be targeted for drug development. Published by Elsevier B.V.

Keywords: LC–MS Multidrug resistance TolC Porins Dps

1. Introduction Multidrug resistant (MDR) pathogens are increasingly becoming an important animal health concern worldwide.

* Corresponding author at: Department of Veterinary and Microbiological Sciences, North Dakota State University, 1523 Centennial Blvd, 164 Van Es Hall, PO Box 6050, Fargo, ND 58108-6050, USA. Tel.: +1 701 231 5271. E-mail address: [email protected] (R. Barigye). 0378-1135/$ – see front matter . Published by Elsevier B.V. doi:10.1016/j.vetmic.2011.03.032

Over the years, both extremely-drug-resistant (XDR) and pan-drug-resistant (PDR) bacteria belonging to the Family Enterobacteriaciae have been reported (Bratu et al., 2007; Brink et al., 2008; Elliott et al., 2006). Many of these organisms have potential pathogenicity to both animals and humans. The majority of the MDR cases result from efflux of drug molecules by membrane-based protein transporters (Neyfakh et al., 1991; Tikhonova and Zgurskaya, 2004). In one study, platensimycin, a novel and promising antibiotic with potency against a wide range of MDR bacteria showed antibacterial activity against TolC-

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null but not against wild-type E. coli isolates (Wang et al., 2006). The AcrAB-TolC protein complex belongs to the resistance-nodulation-division (RND) family of bacterial membrane transporters (Ma et al., 1993, 1995; Tseng et al., 1999) that are responsible for most of the intrinsic drug resistance in Gram-negative bacteria (Nikaido, 1998; Nishino and Yamaguchi, 2001). Using quantitative real time polymerase chain reaction (qRT-PCR), we recently showed upregulated transcription of genes that encode TolC and the novel membrane porin YiaT in MDR E. coli strains cultured from scouring calves (Vinson et al., 2010). Targeting efflux pumps (EPs) with chemical inhibitors has become a popular research objective aimed at finding efficacious treatment for MDR infections (Kaatz, 2002; Lawrence and Barrett, 1998; Ryan et al., 2001). In one study (Neyfakh et al., 1991), investigators showed that effluxmediated multidrug resistance in Bacillus subtilis could be abrogated with the plant alkaloid reserpine. Rosenberg et al. were also able to show that, unlike wild strains, mutant E. coli isolates with a disrupted acrD gene became highly susceptible to a variety of aminoglycosides (Rosenberg et al., 2000). We have hypothesized that other multidrug-resistance-associated bacterial proteins might serve as novel drug targets including those with potential use in the design of immunogens that specifically target MDR/XDR/PDR bacteria. Because EP proteins have an elaborate tertiary structure that includes transmembrane and extracellular domains, we deduce their potential as suitable targets for combinatorial therapeutic agents that incorporate highly specific antibodies and conventional antibacterial agents. The present research was based on the premise that there ought to be detectable differences in the proteomic profiles of MDR and antimicrobial susceptible (AS) E. coli strains that may provide basis for identifying proteins that are crucial players in the mechanisms of multidrug resistance. The major objective of this research was therefore to identify and describe expression dynamics of potential MDR/XDR-associated proteins that could serve as novel drug targets. 2. Materials and methods 2.1. Study of E. coli strains All four multidrug resistant (MDR1, MDR2, MDR3 and MDR4), and one moderately antimicrobial agent-susceptible (AS2) E. coli strains used in this research were originally isolated from fecal samples and/or intestinal contents from scouring calves submitted for testing at the North Dakota State University Veterinary Diagnostic Laboratory (NDSU-VDL). The calves from which the study isolates were cultured were raised at various beef and dairy farms located within the State of North Dakota as well as western parts of Minnesota. The AS1 isolate (ATCC E. coli strain #25922) used as the experimental control was an American Type Culture Collection strain (Manassas, VA, U.S.A.) with known broad spectrum antimicrobial agent susceptibility. The antimicrobial susceptibility profiles were determined by the Sensititre method (TREK Diagnostic system, Cleveland, OH, U.S.A.). E. coli isolates that were resistant to 80% of the antimicrobial agents tested

were designated MDR and then selected for the study. The Sensititre panel included the following antibacterial agents: ampicillin, ceftiofur, chlortetracycline, clindamycin, danofloxacin, florfenicol, gentamicin, neomycin, oxytetracycline, penicillin, spectinomycin, sulfadimethoxime, tiamulin, tilmicosin, trimethoprim/sulfamethoxazole, tulathromycin, and tylosin–tartrate base. All the E. coli isolates studied are currently kept in cryopreservation at 80 8C in 20% glycerol and Luria–Bertani (LB) broth at Research Laboratory #154, Department of Veterinary & Microbiological Sciences, NDSU. The antimicrobial susceptibility profiles of the study E. coli isolates were determined by the bovine/porcine tulathromycin MIC sensititre microplate using the sensititre susceptibility system (Trek Diagnostic Systems, Ohio) according to manufacturers’ instructions. 2.2. Reactivation of E. coli Isolates Individual MDR E. coli strains were inoculated on MacConkey agar plates and incubated at 37 8C overnight. The following day, 3 colonies of each of the MDR E. coli isolates were inoculated in 3 mL of LB broth containing 100 mg/mL of ampicillin, and 50 mg/mL of tetracycline. The cultures were grown overnight at 37 8C with continuous shaking at 200 RPM. The AS E. coli strains were inoculated in 3 mL LB broth without antimicrobial agents and grown under conditions similar to those described for MDR strains above. 2.3. Extraction of E. coli proteins E. coli cells were harvested from cultures by centrifugation at 10,000  g for 10 min, 4 8C in sterile phosphate buffer saline, pH 7.2 (Sigma, Steinheim, Germany) containing 1 mM of the protease inhibitor phenylmethylsulphonyl fluoride (PMSF) (Amresco, Solon, OH, U.S.A.) or PBS/1 mM PMSF (Amresco). The supernatants were discarded, and the pellets subjected to two subsequent washes in PBS/1 mM PMSF (Amresco). For each of the washings, the pellet was resuspended in 2 mL of PBS/1 mM PMSF (Amresco), the mixture vortexed and centrifuged at 10,000  g for 20 min, 4 8C. After the second wash, the pellet was resuspended in 150 mL of SoluLyseTM Bacterial Protein Extraction Reagent (Genlantis, San Diego, CA, U.S.A.) followed by a 10 min. incubation on a rocking platform at room temperature (rT). This was followed by centrifugation at 14,000  g for 5 min, rT after which the supernatant containing the soluble protein fraction was transferred into a clean tube. The pellet constituting the insoluble protein fraction was resuspended in 300 mL SoluLyseTM reagent and added to the soluble protein fraction. Protein concentration was determined using the NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, U.S.A.). Outer membrane proteins (OMPs) were extracted from the E. coli strains according to the method described by Li et al. (2007), with a few modifications. Briefly, the E. coli isolates were cultured in 50 mL of LB broth overnight at 37 8C with continuous shaking at 200 RPM until an optical density reading of 1.0 at a wavelength of 600 nm was attained. The E. coli cells were harvested by centrifugation at 10,000  g

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for 10 min, 4 8C, the supernatants discarded, and the pellets subjected to two subsequent washes in PBS/1 mM PMSF. For each wash, the pellet was resuspended in 2 mL of PBS/ 1 mM PMSF, vortexed and then centrifuged at 10,000  g for 20 min at 4 8C. After the second wash, the pellet was resuspended in 10 mL of sonication buffer (50 mM Tris/ HCl, pH 7.4). While kept in ice, the bacterial cells disrupted by sonication at 1 min. intervals for a total 60 min. at an output of 6.0 by the Branson Sonifier 450 (Danbury, CT, U.S.A.) intervals. After sonication, samples were centrifuged for 20 min. at 5000  g, 4 8C to eliminate unbroken cells and cellular debris. The resultant turbid supernatant was fractionated for 1 h at 100,000  g, 4 8C in a Beckman ultracentrifuge (Beckman, Schaumburg, IL, U.S.A.) and the OMP-containing pellet was solubilized in 100 mL of sterile ddH2O. The protein concentration was determined using the NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, U.S.A.) according to the manufacturer’s instructions. 2.4. Sodium dodecyl polyacrylamide electrophoresis (SDSPAGE) Twenty mg of extracted bacterial total protein from each of the study E. coli strains were diluted 1:2 in sample buffer (625 mM Tris–HCl, pH 6.8; 10% glycerine; 5% SDS; 0.002% Bromophenol blue) plus 10% b-Mercaptoethanol. The samples were boiled for 4 min at 95 8C, cooled and then loaded onto a 12% Tris–HCl precast polyacrylamide gel (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). Five mL of Precision Plus Protein Standards, dual colored molecular weight marker (Bio-Rad Laboratories) were loaded in a separate lane to permit calculation of the apparent MW of interest proteins. SDS-PAGE was carried out in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 150 V for 2 h or until the bromophenol blue dye front reached the bottom of the separating gel. The separated proteins were visualized by staining the gels with Silver Stain Plus (BioRad Laboratories) as described by the manufacturer. After the desired staining of the gels, they were scanned (HP Scanjet G3010, CA, U.S.A.). 2.5. Isoelectric focusing and 2-D gel electrophoresis Three hundred mg of OMP from AS1 and MDR1 isolates was separately suspended in 0.625 mL of 0.5% ‘immobilized pH gradient (IPG) buffer’ (GE Healthcare, Wikstroms, Sweden) plus 111.9 mL of ‘destreak rehydration solution’ (GE Healthcare), and the volume brought to 125 mL. The mixtures were vortexed for 3 s and then loaded into the Ettan IPGphor ‘strip holder’ (GE Healthcare). Individual IPG strips (ImmobilineTM DryStrip; GE Healthcare) were placed into the ‘strip holder’ containing the sample mixture, with the positive end of the strip oriented towards the anode and plastic side facing upwards. Following a 5 min rT incubation, each IPG strip was flooded with 100 mL of ‘cover fluid’ (GE Healthcare), the ‘strip holders’ were then placed in the EttanTM IPGphorTM II IEF system (GE Healthcare) and isoelectric focusing carried out as per a user-defined program shown in Table 1. For the 2-DE, the IPG strips were washed twice at rT with gentle agitation in

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Table 1 Program used for isoelectric focusing of Escherichia coli proteins using an EttanTM IPGphorTM II IEF system (GE Healthcare). Steps

Voltage (V)

Timer/voltage hours (Vh)

1 2 3 4 5 6 7 8 9

100 200 500 1000 2000 3000 5000 5000 0

15:00 h:mm 200 Vh 300 Vh 600 Vh 1800 Vh 3000 Vh 7000 Vh 1250 Vh 00:00 h:mm

5 mL of ‘SDS equilibration buffer’ (6 M urea, 75 mM Tris– HCl, pH 8.8, 29.3% glycerol; 2% SDS; 0.002% bromophenol blue plus 1% dithiothreitol (DTT) for 15 min. The IPG strips were then loaded onto a 12% precast gel (Bio-Rad Laboratories) and overlaid with agarose sealing solution (25 mM Tris, 192 mM glycine, 0.1% SDS, 0.5% agarose, 0.002% bromophenol blue). After loading 5 mL of Precision Plus Protein Standards dual colored molecular weight markers (Bio-Rad Laboratories) in the small well, 2-DE was carried out in ‘running buffer’ (25 mM Tris, 192 mM glycine, and 0.1% SDS) at a constant voltage of 150 V for 2 h or until the bromophenol blue dye front reached the bottom of the separating gel. 2.6. Liquid chromatography mass spectrometry Following isoelectric focusing and 2-DE, the gels were stained by the silver-staining technique as per the silver kit manufacturer’s instructions (Bio-Rad Laboratories) and proteins of interest were selected on the basis of apparent molecular weight (MW) and isoelectric focusing point (pI). The gels were scanned, heat-sealed in plastic bags and sent to the Proteomics Laboratory, Lerner Research Institute, Cleveland OH for liquid chromatography–mass spectrometry (LC–MS) analysis. The protein identification by amino acid sequencing was done based on a standard LC– MS protocol routinely used at the institute. Briefly, the spots of interest were cut out, divided into smaller pieces, washed with water, and dehydrated in acetonitrile prior to trypsin digestion. Five mL equivalent to 20 ng/mL trypsin in 50 mM ammonium bicarbonate (Biorad) were added to the gel pieces and incubated overnight at rT to achieve complete digestion. The peptides that were formed were then extracted from the polyacrylamide in two aliquots of 30 mL of 50% acetonitrile with 5% formic acid. The extracts were combined and evaporated to <10 mL in a Speedvac (Thermo Scientific), and suspended in 1% acetic acid to make up a final volume of 30 mL. The resuspended peptides were analyzed by a Finnigan LTQ linear ion trap mass spectrometer system and a self-packed 9 cm  75 mm id Phenomenex Jupiter C18 reversed-phase capillary chromatography column (Phemomenex, Torrance, CA, U.S.A.). Ten mL volumes of the extract were injected and the peptides eluted from the column with an acetonitrile/0.1% formic acid gradient at a flow rate of 0.25 mL/min introduced into the source of the mass spectrometer on-line. The microelectrospray ion source

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was operated at 2.5 kV and the digests analyzed using the data dependent multitask capability of the instrument acquiring full scan mass spectra to determine peptide MWs. The product ion spectra were used to determine the amino acid sequence in successive instrument scans. The mode of analysis produced approximately 2500 Collisionally Induced Dissociation (CID) spectra of ions ranging in abundance over several orders of magnitude. The data were analyzed by using all CID spectra collected in the experiment to search the NCBI non-redundant database with the search program Mascot (Proteome Software, Inc., Portland, OR, U.S.A.) using a mammalian taxonomy filter. All matching spectra were verified by manual interpretation, and the process aided by additional searches using the programs Sequest (Sequest Technologies, Inc., Lisle, IL, U.S.A.) and Blast (Altschul et al., 1990) as needed.

19–100 kDa (Fig. 1). A number of differences were seen in the protein profiles of all the study E. coli strains and protein bands A–E were selected for LC–MS analysis (Fig. 1 and Table 2). Table 2 Summary of the Escherichia coli proteins that were identified by LC–MS in silver stained 1-D polyacrylamide gel. Band#

Protein

MW (kDa)

Identified peptides

A

ATP1 DLDH TF AspA RPOB GAD PDHE1

55 51 48 53 151 53 100

13 9 10 7 4 3 3

B

EF-Ts OmpA EF-Tu MDH NALase

31 37 43 32 33

15 10 6 5 6

C

MDH EF-Ts Gal-TP SCS-a Enoyl-ACP-R 50 S RPL2

32 31 36 30 28 30

10 11 7 6 4 5

D

DDPOA S-CoA SSa THP-2-CST PPH OmpA PPIase SCD OmpC PR

31 30 30 46 37 29 28 40 30

13 8 6 5 4 5 4 4 3

E

GAPDH TA-B OmpA OmpA S/P ABCTPSBP CysK AuxTP PGR NmpC Precusor L-Aspase PHADH

36 35 37 37 39 34 39 36 42 37 33

20 16 16 11 6 4 4 4 5 3 4

3. Results 3.1. SDS-PAGE Comparative protein profiles were obtained by SDSPAGE analysis of protein extracts from study E. coli strains. Silver-stained gels containing the fractionated proteins were visually evaluated for differences in the protein patterns of the different strains. All AS (AS1 and AS2) and MDR (MDR1, MDR2, MDR3 and MDR4) strains expressed numerous protein bands in the range of

Fig. 1. Silver stained 1-D polyacrylamide gel showing protein profiles of two antibiotic susceptible and four multidrug resistant Escherichia coli strains. Protein bands A–E were cut out of the gel and analyzed by LC–MS at a referral laboratory. MWM, molecular weight markers (Biorad Cat. #161-0374); AS, antibacterial agent susceptible E. coli strain; MDR, multidrug-resistant E. coli strain.

ATP1, F0F1 ATP synthase subunit alpha; DLDH, dihydrolipoamide dehydrogenase; TF, trigger factor; AspA, aspartate ammonia-lyase; RPOB, DNA-directed RNA polymerase subunit beta; GAD, glutamate decarboxylase isozyme; PDHE1, pyruvate dehydrogenase subunit E1; EF-Ts, elongation factor Ts; OmpA, outer membrane protein A; EF-Tu, elongation factor Tu; MDH, malate dehydrogenase; NALase, N-acetylneuraminate lyase; Gal-TP, galactose-binding transport protein; SCS-a, succinyl-CoA synthetase subunit alpha; Enoyl-ACP-R, enoyl-(acyl carrier protein) reductase; 50 S RPL2, 50 S ribosomal protein L2; DDPOA, 2dehydro-3-deoxyphosphooctonate aldolase; S-CoA SSa, succinyl-CoA synthetase subunit alpha; THP-2-CST, 2,3,4,5-tetrahydropyridine-2carboxylate N-succinyltransferase; PPH, phosphopyruvate hydratase; PPIase, FKBP-type peptidyl-prolyl cis-trans isomerase; SCD, short chain dehydrogenase; OmpC, outer membrane protein C; PR, putative regulator; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; TA-B, transaldolase B; S/P ABCTPSBP, sperimidine/putrescine ABC transporter periplasmic substrate binding protein; CysK, cysteine synthase A; AuxTP, auxiliary transport protein; PGR, putative global regulator; NmpC precursor, new membrane protein C; L-Aspase, L-asparaginase II; PHADH, putative 3-hydroxyacyl-CoA dehydrogenase. The proteins whose acronyms appear in bold font were assumed to be somewhat related to the mechanisms of drug resistance.

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Fig. 2. Silver stained 2-D polyacrylamide gel following isoelectric focusing and 2-D electrophoresis of protein extracts from strains of antibacterial-agent sensitive E. coli. Unlike the MDR strain, note the presence of protein spot #7 in the AS E. coli strain (A). AspA, aspartate ammonia-lyase; GadA, glutamate decarboxylase A, PLP-dependent; and TolC, component of the AcrAB-TolC drug efflux pump.

3.2. Isoelectric focusing and protein identification by LC–MS The data obtained from one dimensional SDS-PAGE (1DE) was validated by isoelectric focusing (IEF) of membrane protein extracts from MDR1 and AS1 strains (representative of MDR and AS strains, respectively). Two dimensional silver-stained gels for AS and MDR E. coli strains revealed variable expression patterns of protein which was consistent with 1-DE silver stained gels. Two sets of 2-D silver stained gels (Figs. 2A and 2B and 3A and 3B) were analyzed by LC–MS. In the first set of gels, a total of 8 protein spots were analyzed and protein identification was done by amino acid sequencing of gel-derived peptides in the AS1 strain (Fig. 2A, B and Table 3). Seven

protein spots were analyzed in the MDR1 strain gel (Fig. 3A, B and Table 3). In the second set of silver-stained gels, a total of 17 spots were selected for LC–MS analysis and definitive protein identification made for the AS1 and MDR1 strains (Figs. 2A and 2B and 3A and 3B and Table 3). For the 1-D silver gel (Fig. 1), a total of 32 proteins (Table 2) were identified by LC–MS in 5 bands of interest. Of the 32 proteins definitively identified in the 1-D gel, three were porins (OmpA, OmpC, and NmpC Precursor) (Fig. 1 and Table 2) with potential involvement in multidrug resitance while two were elongation factors (EF-Ts and EF-Ts) known to be targeted by antibiotics agents like tetracyclines. Overall analysis of gel-derived peptides by LC–MS led to the identification of multiple proteins in both the

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Fig. 3. Silver stained 2-D gel following isoelectric focusing and 2-D electrophoresis of protein extracts from a strains of multi-drug resistant E. coli. Unlike the AS strain, note the absence of protein spot #7 in the MDR E. coli strain (A). AspA, aspartate ammonia-lyase; GadA, glutamate decarboxylase A; PLPdependent TolC, component of the AcrAB-TolC drug efflux pump.

MDR1 and AS1 strains tested (Tables 2 and 3). Of the identified proteins, those deemed to be potentially involved in drug resistance mechanisms are indicated in gray-filled rows (Tables 2 and 3). Emphasis was particularly given to membrane porins and cytosolic proteins known to be sites of antibacterial agents and/or those reported to be involved in drug resistance mechanisms. The membrane channel protein TolC was definitively identified in 2-DE silver stained gel of fractionated MDR E. coli protein extracts by LC–MS analysis (Fig. 3A), but was undetectable in a silver gel of fractionated AS1 proteins (Fig. 2A). In total, four TolC peptides were detected in trypsin gel digests of the relevant MDR1 protein spot but

not in the corresponding gel area of the AS1 strain (Figs. 2A, 3A and 4; Table 2). The number of peptides detected in trypsin gel digests was taken as an indicator of the relative abundance of the protein in the MDR1 and AS1 strains tested (Tables 2 and 3 and Fig. 4). Even though thought to have no statistical significance, differences in the number of peptides recovered were apparent between the two E. coli strains for many of these proteins (Fig. 4). 4. Discussion A combination of proteomic techniques demonstrated some differences in protein expression profiles of study

A. Gautam et al. / Veterinary Microbiology 151 (2011) 363–371 Table 3 Summary of Escherichia coli proteins that were identified by LC–MS in silver stained 2-D polyacrylamide gels. Spot#

1 2 3 4 5 6 7 8

9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Protein

MW (kDa)

pI

Dps OmpA Dps Dps Dps Ahp PPase Ahp PPase Dps OmpA AspA GadA TolC DnaK RpsA HSP 90 FBP A EF-Tu FBP A TalB EF-Ts EF-Ts OmpA HYD 1 GAPDH OmpA OmpA PTS MsIIAB 2, 5DgrA PGM OmpA SDM Ahp PPase Ahp EF-Ts F0F1AS DLDH

19 38 19 19 19 21 20 21 20 19 38 53 53 53 69 61 72 40 43 40 35 31 31 37 41 36 37 37 35 38 29 37 21 21 20 21 31 55 51

5.7 6.0 5.7 5.7 5.7 5.0 – 5.0 – 5.7 6.0 5.2 5.2 5.5 4.8 4.9 – 5.5 5.3 5.5 5.1 5.2 5.2 6.0 6.7 6.3 6.0 6.0 5.7 6.0 5.9 6.0 5.6 5.0 5.0 5.0 5.2 5.8 –

Identified peptides AS

MDR

6 2 6 12 7 0 0 7 3 8 4 6 8 0 12 10 2 5 3 3 8 11 13 4 3 0 0 3 8 5 4 2 2 5 0 0 5 10 0

6 7 4 10 4 6 3 5 0 0 0 8 8 4 19 8 0 1 1 2 4 8 9 1 3 2 2 7 6 5 1 5 3 1 2 3 3 18 8

Dps, DNA starvation/stationary phase protection protein; OmpA, outer membrane protein A; TolC, TolC channel protein; Ahp, alkyl hydroperoxide reductase subunit C; PPase, inorganic pyrophosphatase; AspA, aspartate ammonia-lyase; GadA, glutamate decarboxylase A; PLP dependent; DnaK, molecular chaperone DnaK; RpsA, 30 S ribosomal protein S1; HSP90, heat shock protein 90; FBPA, fructose-biphosphate alodalse; EF-Tu, elongation factor-Tu; TalB, transaldolase B; EF-Ts, elongation factor-Ts; HYD1, hydrogenase 1; GAPDH, glyceraldehydes-3phosphate dehydrogenase A; PTS MsIIAB, PTS system, mannose-specific IIAB component; 2,5DgrA, 2,5-diketo-D-gluconate reductase A; PGM, phosphoglyceromutase; SDM, superoxide dismutase; Ahp, alkyl hydroperoxide reductase subunit C; PPase, inorganic pyrophosphatase; F0F1 AS, F0F1 ATP synthase subunit alpha; DLDH, dihydrolipoamide dehydrogenase. The proteins whose acronyms appear in bold font were assumed to be somewhat related to the mechanisms of drug resistance.

MDR and AS E. coli strains. Of the numerous proteins detected in 1-D and 2-D silver stained polyacrylamide gels and definitively identified by amino acid sequencing by LC–MS, 8 (TolC, OmpC, NmpC Precursor, OmpA, Dps, EF-Ts, EF-Tu and RspA) may potentially be involved in drug resistance mechanisms (Fralick, 1996; Goldman et al., 1983; Li et al., 2008; Nikaido, 1994; Paulsen et al., 1997; Pugsley and Schnaitman, 1978; Spahn et al., 2001; Sulavik

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et al., 2001; Wang, 2002; Zhai and Saier, 2002). In reference to SDS-PAGE data, the study E. coli strains showed variable protein expression patterns (Figs. 1–3). If taken as an indicator of the amount of protein expressed, the number of TolC peptides detected in the MDR1 strain (4 peptides) may suggest upregulation of the tolC gene product in the MDR1 but not the AS1 E. coli strain (0 peptides). This observation is further supported by qRT-PCR data that we recently reported (Vinson et al., 2010) and strongly suggests that the AS1 strain in question most likely expressed TolC at extremely low levels that were undetectable by LC–MS analysis. TolC has been widely reported for its drug efflux properties and plays a critical role in mediating clinically significant multidrug resistance (Fralick, 1996; Nikaido, 1994; Paulsen et al., 1997; Sulavik et al., 2001). The upregulation of TolC and other membrane proteins was also recently reported in E. coli strains with clinical resistance against chloramphenicol, streptomycin and nalidixic acid (Li et al., 2007; Li et al., 2008; Lin et al., 2008). Data from silver-stained gels showed a 42 kDa protein that was expressed in the 2 AS strains (AS1 and AS2) but absent or minimally expressed in four MDR isolates (MDR1, MDR2, MDR3, MDR4) (Fig. 1). Amino acid sequencing confirmed this protein to be NmpC precursor, an outer membrane porin (Zhai and Saier, 2002). Increased NmpC precursor expression has been associated with increased susceptibility to colicins and bacteriophages (Pugsley and Schnaitman, 1978). Supported by literature, results from the present study therefore strongly suggest that the apparent NmpC precursor downregulation in the study MDR E. coli strains (Fig. 1) is likely to be associated with multidrug resistance. LC–MS analysis identified 31/33 kDa proteins to be possible isoforms of elongation factor-Ts (EF-Ts). Despite the fact that EF-Ts is involved in bacterial systems affected by tetracyclines and other ribosome-targeting antimicrobial agents (Goldman et al., 1983), analysis of expression profiles of the protein in all the study E. coli strains did not show a pattern suggestive of the MDR phenomenon. Data from the present study also showed that EF-Tu was expressed in both AS1 and MDR1 strains. Three peptides were recovered from AS1 as compared to only one from the corresponding gel spot in the MDR1 strain. EF-Tu is involved in the elongation process of microbial protein biosynthesis. The present data does not suggest a relationship of this protein with drug resistance. The 2-D silver-stained gels also showed multiple protein spots in both MDR and AS strains that were confirmed by LC–MS analysis to be the outer membrane protein-A (OmpA). Depending on whether the protein sample is preheated ahead of electrophoresis or not, or due to the type of detergent used to solubilize the sample, OmpA may migrate at different locations in polyacrylamide gels thus giving spots of anomalous and variable apparent MW and pI (Kleinschmidt et al., 1999). The anomalous migration behavior displayed by OmpA in both the MDR1 and AS1 strains in this study is therefore suspected to be an artifact related to preanalysis sample treatment. In LC–MS analysis, 11 OmpA peptides with apparent MW of 19 kDa were recovered in the MDR1 strain

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Fig. 4. Bar graph showing distribution of counts of peptides recovered from silver stained 2-D polyacrylamide gels, definitively identified by liquid chromatography–mass spectrometry.

(Fig. 3A and B) compared to only 6 in the AS1 strain (Fig. 2A and B). In the second set of gels, 4 spots with variable pI and apparent MW of 36 kDa and 42 kDa were identified to be OmpA. The total OmpA peptide recovery was 15 in the AS1 strain but 22 in the MDR1 isolate (Fig. 4). OmpA is a major outer membrane porin (Wang, 2002) that allows transmembrane migration of multiple small solutes (Sugawara and Nikaido, 1992). It also functions to provide structural stabilization to the bacterium (Wang, 2002), acts as a colicin and phage receptor (Morona et al., 1984; Skurray et al., 1974); and is required in F-pilus conjugation (Schweizer and Henning, 1977). In addition, this protein imparts virulence to the bacterium, and mediates resistance against a number of substances (Wang, 2002). A study done by Wang (Wang, 2002) showed that OmpA null E. coli K-1 mutants were sensitive to SDS, cholate, acidic environment, high osmolarity, and serum when compared to the wild type strains. Supported by current literature (Wang, 2002), the difference in the number of peptides recovered from gels of fractionated AS1 and MDR1 protein extracts in the present study inconclusively suggests that a role in drug resistance may be possible. In 2-D silver stained gels, an entire protein spot confirmed to be a variant of the protein Dps was totally missing in MDR1 (Fig. 3A) but present in the AS1 strain (Spot #7; Fig. 2A). Furthermore, LC–MS analysis of 2-D gels revealed 31 Dps peptides in AS1 compared to 24 in the MDR1 strain (Table 3 and Fig. 4). Taken as a function of the amount of protein expressed, these findings, though not statistically significant, suggest the study AS1 strain most likely expressed more Dps than the MDR1 isolate. Previous work has shown downregulation of Dps in a streptomycin resistant E. coli strain (Li et al., 2008) but the exact role played by this protein in the mechanism of drug resistance remains uninvestigated. LC–MS analysis of 30 S ribosomal protein S1 (RpsA) spots detected 10 peptides in the AS1 strain, and only 8 in

the MDR1 strain (Table 3 and Fig. 4). The RpsA protein is a target site for antimicrobial agents in the tetracycline class (Goldman et al., 1983) as well as other antibacterial agents that inhibit the elongation process during bacterial protein synthesis (Agrawal et al., 2000; Spahn et al., 2001). However, data generated from this study does not seem to suggest any significant differences in the expression of this protein by the AS1 and MDR1 strains. 5. Conclusion The major goal of this research was to describe the expression dynamics of E. coli proteins with a potential role in multidrug resistance so they may serve as new drug targets in future. LC–MS data based on study MDR and AS E. coli strains confirmed variable expression patterns of TolC, three porins (OmpA, OmpC, and NmpC Precursor), three microbial protein synthesis-associated proteins (RspA, EFTs, and EF-Ts), plus Dps which might play a role in drug resistance mechanism. However, the present data falls short of determining the suitability of these proteins as drug targets. More research is needed to explore how these proteins could be targeted by novel drug agents. Funding This work was supported by funding provided through the United State Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS) Biosurveillance grant number FARG014465; and a North Dakota State University Development Foundation grant number FARG0013382. References Agrawal, R.K., Spahn, C.M., Penczek, P., Grassucci, R.A., Nierhaus, K.H., Frank, J., 2000. Visualization of tRNA movements on the Escherichia

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