Combining Mass Spectrometry, Surface Acoustic Wave Interaction

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Combining mass spectrometry, surface acoustic wave interaction analysis and cell viability assays for characterization of Shiga toxin subtypes of pathogenic Escherichia coli bacteria Daniel Steil, Gottfried Pohlentz, Nadine Legros, Michael Mormann, Alexander Mellmann, Helge Karch, and Johannes Müthing Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01189 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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Analytical Chemistry

Combining mass spectrometry, surface acoustic wave interaction analysis and cell viability assays for characterization of Shiga toxin subtypes of pathogenic Escherichia coli bacteria

Daniel Steil1, Gottfried Pohlentz1, Nadine Legros1, Michael Mormann1, Alexander Mellmann1,2, Helge Karch1,2, and Johannes Müthing1,2*

1

Institute for Hygiene, University of Münster, Robert-Koch-Strasse 41, D-48149 Münster, Germany

2

Interdisciplinary Center for Clinical Research (IZKF) Münster, Domagkstrasse 3, D-48149

Münster

*

To whom correspondence should be addressed. Phone: +49-(0)251-8355192. Fax: +49-

(0)251-8355341 E-mail: [email protected]

1 Environment ACS Paragon Plus

Analytical Chemistry 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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

Shiga toxin (Stx)-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) as a human-pathogenic subgroup of STEC are characterized by releasing Stx AB5-toxin as the major virulence factor. Worldwide disseminated EHEC strains cause sporadic infections and outbreaks in the human population and swine-pathogenic STEC strains represent greatly feared pathogens in pig breeding and fattening plants. Among the various Stx subtypes Stx1a and Stx2a are of eminent clinical importance in human infections being associated with lifethreatening hemorrhagic colitis and hemolytic uremic syndrome, whereas Stx2e subtype is associated with porcine edema disease with generalized fatal outcome for the animals. Binding towards the glycosphingolipid globotriaosylceramide (Gb3Cer) is a common feature of all Stx subtypes analyzed so far. Here we report on the development of a matched strategy combining (i) miniaturized one-step affinity purification of native Stx subtypes from culture supernatant of bacterial wild-type strains using Gb3-functionalized magnetic beads, (ii) structural analysis and identification of Stx holotoxins by electrospray ionization ion mobility mass spectrometry (ESI MS) (iii), functional Stx-receptor real-time interaction analysis employing the surface acoustic wave technology (SAW), and (iv) Vero cell culture assays for determining Stx-caused cytotoxic effects. Structural investigations revealed diagnostic tryptic peptide ions for purified Stx1a, Stx2a and Stx2e, respectively, and functional analysis resulted in characteristic binding kinetics of each Stx subtype. Cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e. Collectively, this matched procedure represents a promising clinical application for the characterization of lifeendangering Stx subtypes at the protein level.


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INTRODUCTION

Enterohemorrhagic Escherichia coli (EHEC) are the human pathogenic subgroup of Shiga toxin (Stx)-producing E. coli (STEC) that cause the life-threatening hemolytic uremic syndrome (HUS).1,2 STEC of human isolates produce mostly Stx1a and/or Stx2a (in previous publications imprecisely designated as Stx1 and Stx2; for revised nomenclature of Stx1 and Stx2 subtypes refer to Scheutz and collaborators.3 The large 2011 outbreak in Germany caused by an Stx2a-producing EHEC O104:H4 strain4,5 resulted in 855 HUS cases and 53 deaths.6 In swine, STEC cause the edema disease and the Stx2e subtype has been identified as the primary virulence factor involved in the pathogenesis of the infection.7 The presence of the stx2e gene in human STEC isolates may predict a milder disease with a minimal risk of HUS.8 All Stxs exhibit a common AB5 structure with a single A-subunit and five identical Bsubunits. The enzymatically active A-subunit halts protein biosynthesis at the ribosomal level.9 The pentameric B-subunit binds to glycosphingolipids (GSLs) of the globo-series,10 followed by internalization involving various endocytic mechanisms.11-13 Stx1a and Stx2a preferably bind to the receptor GSL globotriaosylceramide (Gb3Cer/CD77) with Galα4Galβ4Glcβ1Cer structure and to a lesser extent to globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1Cer),14-16 which are the major GSLs of human endothelial cells derived from various vascular beds.17-20 Stx2e binds with some preference to Gb4Cer,21 but recognizes Gb3Cer with the distal Galα4Galβ-disaccharide recognition motif as well.22-24 Magnetic

nanoparticles

functionalized

with

pigeon

ovalbumin,

which

contains

Galα4Galβ4GlcNAc termini, has been described as a useful tool to characterize Stx1 from complex samples.25 A procedure for affinity purification of Stx subtypes as native holotoxins from bacterial wild-type strains using Gb3-derivatized magnetic beads has so far not been described.



Identification of Stx can be achieved by peptide mass fingerprinting, a technology based on structural analysis of tryptic peptides obtained from in gel- or in solution-digested proteins utilising matrix-assisted laser desorption ionization (MALDI) or electrospray ionisation (ESI) mass spectrometry (MS),26-28 representing an indispensable tool and a still growing field in the post-genomic era working at the protein level.29 Real-time biomolecular interaction analysis should be the method of choice to elucidate the primary protein function, namely binding of the toxin towards its specific GSL receptor. For this purpose, surface acoustic wave (SAW) technology provides label-free sensing for determining binding kinetics using sensors that permit real-time monitoring.30 Sensor surfaces can be coated with model membranes that resemble the in vivo situation and can simulate biological processes.31,32 To date, the SAW technology has only been rarely employed for the investigation of protein-carbohydrate binding events, although this technique opens up promising perspectives for real-time and label-free interaction analysis employing biomimetic membranes.33 Here we report on the development of a matched strategy for the characterization of the clinically important Stx1a, Stx2a, and Stx2e subtypes. It includes affinity purification of Stx subtypes using Gb3-functionalized magnetic beads, structural analysis of Stxs by ESI MS, functional analysis of Stx-receptor binding using real-time interaction analysis employing an SAW biosensor, and biological analysis of the cytotoxic capacity of Stxs in cell cultures.


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EXPERIMENTAL SECTION

Stx1a, Stx2a and Stx2e subtypes.The Stx1a- and Stx2a-producing EHEC wild-type strains of serotype O145:H- (strain 2074/97)23 and O111:H- (strain 03-06016)34, respectively, and the Stx2e-releasing STEC wild-type strain of serotype ONT:H- (strain 2771/97)35 were from the collection of the Institute for Hygiene of the University of Münster (Münster, Germany). The strains are biosafety level (BSL) 2 pathogens according to international classifications36 and cultivated - according to national rules37 - in our BSL 3** laboratory. For production of Stxcontaining bacterial liquid culture supernatants, Luria Bertani (LB) broth growth medium, supplemented with 0.5 µg mL-1 mitomycin C (Sigma-Aldrich, Deisenhofen, Germany), was inoculated with the Stx-producing E. coli strains as previously described.23 Bacteria were removed by centrifugation followed by sterile filtration of the supernatant through membrane filters of 0.22 µm pore size (Schleicher and Schuell, Dassel, Germany). Sterility was determined by 48 h culture of the supernatant on blood agar plates (Columbia sheep blood agar, Oxoid GmbH, Wesel, Germany).

Preparation of Gb3-derivatized magnetic beads. Commercial Gb3-oligosaccharide (GLY120, Galα4Galβ4Glc, ELICITYL SA, Crolles, France) was coupled by reductive amination to the amino group of silanized magnetic iron oxide beads (MagnaBind™ Amine Derivatized Beads, size 1-4 µm, cat. 21352, ThermoFisher Scientific Inc., Rockford, IL, USA). For this purpose, beads corresponding to ~ 12 mM amine were taken up in 450 µL of water/methanol (1/1, v/v), followed by addition of 9 mg of Gb3, dissolved in 450 µL of water/methanol (1/1, v/v). The reaction mixture was incubated overnight at 50°C and 900 rpm. The reductive amination was performed by stepwise addition of 500 µL of a solution composed of 10 mg/mL of NaBH3CN in methanol with 1% acetic acid and subsequent



overnight incubation. The Gb3-loaded beads were rinsed three times with water/methanol (1/1, v/v) and three times with PBS and finally stored at 4°C in PBS until use.

Affinity purification of Stx subtypes. Stx-containing supernatants were reduced to a volume of 1 mL using centrifugal filters (Amicon® Ultra-Centrifugal Filters, Ultracel®-10K, Merck, Millipore Ltd., Cork, Ireland) and incubated with the Gb3-derivatized magnetic beads at 4°C and 1400 rpm overnight. Bound Stxs were eluted with 4.5 M MgCl2 in 7.5 x diluted PBS. Desalting of the samples was performed with centrifugal filters (Amicon® Ultra-Centrifugal Filters, Ultracel®-3K, Merck). The purified toxins were finally suspended in PBS with 5 mM MgCl2 and stored at -20°C until use. SDS PAGE was performed following standard procedures.38 Proteins were stained with Coomassie Brilliant Blue (Quick Coomassie Stain, Generon Ltd., Maidenhead, UK). The protein concentrations were determined using the bicinchoninic acid assay (PierceTM BCA Protein Assay Kit, #23225, ThermoFisher Scientific Inc.) following the supplier’s instructions.39 After desalting by use of centrifugal filters (Amicon® Ultra-Centrifugal Filters, Ultracel®-3K) solutions with approximate protein concentrations of 2 µg µL-1 in 40% methanol/2% formic acid were prepared for MS analysis.

Vero cell cytotoxicity assay. The cytotoxic effects of Stx1a, Stx2a and Stx2e were determined using the crystal violet assay as previously described.40,41 Briefly, Vero-B4 cells were treated with each Stx subtypes for 1 h, followed by 48 h incubation with cell culture medium lacking Stx. After suction of spent medium, survived cells were stained with crystal violet and cell viability was determined photometrically as previously described.40,41

Thin-layer chromatography (TLC) overlay assay. The binding specificity of affinitypurified Stx1a, Stx2a and Stx2e was evaluated using a neutral GSL preparation from Vero cells harboring globopentaosylceramide (Gb5Cer, Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer) in


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addition to Gb3Cer and Gb4Cer. The TLC binding procedure has been recently described in detail for Stx-containing bacterial liquid culture supernatants.24,40

Proteolytic digestion of Stx subtypes. Trypsin and chymotrypsin of sequencing grade were from Roche Diagnostics (Mannheim, Germany; no. 11418475001 and 11418467001, respectively). For tryptic in-solution digestion of Stx1a, the toxin was denatured with 6 M guanidine hydrochloride in 100 mM Tris buffer (pH 7.5) for 2 h at 56°C and subsequently desalted by size exclusion chromatography (Micro BioSpinTM P-6 Gel Columns, #7326221, Bio-Rad Laboratories). An aliquot of ~ 5 µg of desalted Stx1a was incubated with 80 µL of a mix of 20 µL of trypsin solution in 1 mM HCl (0.1 µg µL-1) and 60 µL of 25 mM NH4HCO3 overnight at 37°C. Proteolytic in-gel digest of SDS PAGE-separated B-subunits of Stx2a and Stx2e (~ 5 µg each) was performed following standard protocols26 with some modifications. Excised gel particles were dried and then soaked with 80 µL of trypsin, consisting of 20 µL of protease solution in 1 mM HCl (0.1 µg µL-1) and 60 µL of 25 mM NH4HCO3. Proteolytic peptides were successively extracted with 200 µL each of 25 mM NH4HCO3, 3 x 50% acetonitrile/2.5% formic acid, 80% acetonitrile/2.5% formic acid and 100% acetonitrile. For desalting by use of C18 ZipTips (Merck Millipore Corporation #Z720070-96EA) the dried samples were dissolved in 10 µL of 0.5% trifluoroacetic acid (TFA) and loaded to ZipTips equilibrated with 0.1% TFA. Peptides were eluted with 50 µL each of 50% acetonitrile/0.1% TFA and 80% acetonitrile/0.1% TFA and, finally, with 30 µL of 100% acetonitrile. For MS analysis the dried residues were dissolved in 40% methanol/0.5% formic acid.

Electrospray ionization mass spectrometry (ESI MS). NanoESI MS analyses of affinitypurified Stx holotoxins and proteolytic peptides were performed by use of a SYNAPT G2-S mass spectrometer (Waters, Manchester, UK) in the positive ion sensitivity mode. The source settings were: source temperature 80°C, capillary voltage 0.8 kV, sampling cone voltage 20



V, and offset voltage 50 V. For low energy collision-induced dissociation (CID) experiments (MS2), peptide precursor ions were selected with the quadrupole analyser, subjected to ion mobility separation with the following settings: wave velocity 800 - 1200 m s-1, wave height 40 V, nitrogen gas flow rate 90 mL min-1, and helium gas flow rate 180 mL min-1, and fragmented in the transfer cell using a collision gas (Ar) flow rate of 2.0 mL min-1 and collision energies up to 100 eV (Elab). Lipids required for the manufacturing of vesicles were dissolved in chloroform/methanol (1/4, v/v) and subjected to ESI MS as described in previous publications.19,20,40-42

Fabrication of vesicles. Commercial lipids employed for the preparation of large multilamellar vesicles (LMVs) and small unilamellar vesicles (SUVs) were 1,2-dioleoyl-snglycero-3-phosphatidylcholine (DOPC; Sigma-Aldrich, Steinheim, Germany; P6354), 1,2diacyl-sn-glycero-3-phosphatidyl-L-serine (DAPS; Sigma-Aldrich; P7769), and cholesterol (Sigma-Aldrich; C8667). Sphingomyelin (SM) was from calf brain and purified Gb3Cer has been prepared from the heart muscle of a Morbus Fabry patient in the late 1980s following standard procedures.43 Three different types of vesicles were produced for immobilization on the surface of the biosensor. Type 1 vesicles (without DAPS and without Gb3Cer) were composed of matrix lipids DOPC, SM and cholesterol in a 7:1:2 ratio (each by weight); type 2 vesicles (with DAPS and without Gb3Cer) contained DOPC, SM, cholesterol and DAPS in a 5:1:2:2 ratio (each by weight); type 3 vesicles (with DAPS and with Gb3Cer) harbored DOPC, SM, cholesterol, DAPS and Gb3Cer in a 4.5:1:2:2:0.5 ratio (each by weight). For preparing LMVs, lipids were dissolved in PBS or in PBS with 5 mM MgCl2 and incubated 2 x for 10 min at 45°C in a sonication water bath (Sonorex, Bandelin, Berlin, Germany). SUVs were prepared with a mini-extruder (Avanti Polar Lipids Inc., Alabaster, AL, USA) by extrusion of the LMVs through a polycarbonate membrane with 100 nm pore size (Whatman® NucleoporeTM Track-Etched Membranes, GE Healthcare, Maidstone, UK) at 45°C44 and


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stored at 4°C no longer than 24 h until use. The particle size distribution of the resulting SUVs was determined by dynamic light scattering (DLS) measurements with a Zetasizer Nano SZ instrument (Malvern instruments, Malvern, UK).

Biomolecular real-time interaction analysis using surface acoustic wave. The surface acoustic wave (SAW) instrument SAM®5 blue (SAW Instruments GmbH, Bonn, Germany) with 5-channel configuration was employed for advanced real-time, label-free measurement of biomolecular interaction. The gold surface of the ST-cut microfluidic SiO2 biosensor chips was functionalzed by a self-assembled monolayer of 5 mM 11-mercaptoundecanoic acid (98%; Sigma-Aldrich) in pure ethanol (≥ 99.5%; Carl Roth GmbH, Karlsruhe, Germany) overnight. After washing with Millipore® water (Synergy® UV water purification system, Millipore GmbH, Schwalbach, Germany) and pure ethanol, the chip was transferred into the chip reader unit of the SAW instrument and the frequency optimum was determined. Measurements were performed at 25°C and the flow rate was adjusted to 20 µL min-1 for the injection of 400 µL of SUVs (1 mg mL-1). After 20 min of loading, the flow rate was increased to 40 µL min-1 for the ensuing 60 min of washing with PBS. Unspecifically adsorbed SUVs were removed with 400 µL of 10 mM aqueous NaOH for 10 min. After flushing with PBS, the chip was ready for Stx interaction analysis. To this end, 400 µL of bovine serum albumin (fraction V, cat. 11930.04, Serva Electrophoresis GmbH, Heidelberg, Germany) in PBS with 5 mM MgCl2 (1 mg mL-1) were injected with a flow rate of 40 µL min-1 for 10 min to block unspecific binding of the Stxs. Volumes of 150 µL of each Stx subtype were applied with increasing concentrations of 20, 50, 80, 120, 180, and 250 nM in PBS with 5 mM MgCl2 using a flow rate of 20 µL min-1. After a dissociation phase of 30 min remaining Stx was removed with 0.1 M Gb3 in PBS with 5 mM MgCl2 with a flow rate of 40 µL min-1. Changes in the phase shift φ were registered online. The response curves obtained with increasing Stx concentrations were fitted to an 1:1 binding model. From the calculated



values of kobs (“obs” stands for “observed”) and the concentration c of the ligand (videlicet Stx) kass can be mathematically defined from the slope of the linear regression function according to the equation kobs = kass · c + kdiss. The equilibrium dissociation constant KD was calculated from the quotient KD = kdiss / kass.


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RESULTS

We investigated the three clinically important Stx1a, Stx2a and Stx2e subtypes at the protein level for their structural features, receptor binding characteristics, and cytotoxic activity aimed at detection of differences, which might be applicable for their diagnostic distinction. The matched strategy is illustrated in Scheme 1 showing the single steps of the procedure.

Production of purified Stx1a, Stx2a, and Stx2e subtypes using Gb3-functionalized magnetic beads. The sterile-filtered Stx-containing supernatants of the bacterial liquid cultures were approximately 50-fold concentrated to a volume of 1 mL for each Stx subtype (Scheme 1, step 1). The concentrate was incubated with Gb3-functionalized magnetic beads (Scheme 1, step 2) and bound Stxs were eluted from the beads, followed by desalting and concentration to reduced volume (Scheme 1, step 3). The affinity purification yielded on average approximately 1.5 µg of Stx1a, 5.7 µg of Stx2a and 2.3 µg of Stx2e per mL of supernatant.

MS1 and MS2 analysis of Stx holotoxins. First, we determined the exact molecular weights of the respective A- and B-subunits of the three Stx subtypes by MS1 analysis (Scheme 1, step 4). The positive ion overview ESI mass spectra of Stx1a, Stx2a and Stx2e are shown in Figure 1A, 1B and 1C, respectively, and the SDS PAGE of the purified Stx subtypes (Scheme 1, step 5) is shown in Figure 1D.

Stx1a In Figure 1A the m/z-values between 921.75+35 and 1791.25+18 correspond to a series of positively charged [M+35H]35+ to [M+18H]18+ ions of the A-subunit of Stx1a, which are marked in red in the spectrum. The average molecular weight of 32,225.51±0.52 Da coincides



very well with the calculated molecular weight of 32,225.15 Da (UniprotKB, accession number Q8X696) for the A-subunit. The ions at m/z 961.48+8, 1098.69+7, 1281.64+6, and 1537.77+5 represent a series of discrete positively charged monoisotopic ions of the B-subunit and are colored in blue (Figure 1A). The monoisotopic molecular weight of 7,683.79±0.01 Da is virtually identical to 7,683.78 Da calculated from the amino acid sequence of the B-subunit of Stx1a (UniprotKB, accession number K4VUY1). ESI CID MS analysis of Stx1a-derived tryptic peptides revealed a number of sequenced peptides of the A-subunit (listed in Table S-1 and Scheme S-1) and the B-subunit (listed in Table S-2 and Scheme S-2).

Stx2a The evaluation of the MS1 spectrum of Stx2a in Figure 1B shows a series of discrete positively charged ions of the A-subunit from [M+27H]27+ at m/z 1219.76 to [M+16H]16+ at m/z 2057.71 with an average molecular weight of 32,906.78±1.25 Da. This value is in agreement with the calculated molecular weight of 32,907.24 Da for the A-subunit of Stx2a (UniprotKB, accession number Q8XBV2), corresponding to the mature protein lacking the first two N-terminal amino acids and containing one disulfide bond (C263-C282). The discrete positively charged monoisotopic ions of the B-subunit at m/z 1953.75+4, m/z 1563.14+5 and m/z 1302.79+6, respectively, resulted in determining a monoisotopic molecular weight of 7,810.66±0.17 Da (Figure 1B) being almost identical to calculated 7,810.68 Da (UniprotKB, accession number Q7DJJ2). ESI CID MS analysis of Stx2a-derived tryptic peptides provided a number of sequenced peptides of the A-subunit (listed in Table S-3 and Scheme S-3) and the B-subunit (listed in Table S-4 and Scheme S-4).

Stx2e From the series of [M+33H]33+ to [M+19H]19+ ions with m/z values of 1004.18 to 1743.36 in the MS1 spectrum shown in Figure 1C an average molecular weight of 33,104.49±0.31 Da


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could be deduced for Stx2e. This nicely fits with the calculated molecular weight of 33,105.57 Da (UniprotKB, accession number C7TQM6) for the A-subunit of Stx2e. From the heavily abundant monoisotopic positively charged [M+6H]6+ ions at m/z 1263.79, accompanied by [M+5H]5+ ions at m/z 1516.35 and [M+7H]7+ ions at m/z 1083.39, a monoisotopic molecular weight of 7,576.69±0.01 Da could be deduced for the B-subunit of Stx2e. This value is identical to the calculated molecular weight of 7,576.69 (UniprotKB, accession number G0FDA1). ESI CID MS analysis of Stx2e-derived tryptic peptides yielded a number of sequenced peptides of the A-subunit (listed in Table S-5 and Scheme S-5) and the B-subunit (listed in Table S-6 and Scheme S-6).

Diagnostic ions derived from Stx B-subunits. We could detect and sequence highly abundant and at the same time Stx subtype-specific diagnostic peptide ions in the m/z range between 608 and 618 as shown in Figure 2A. The peptide positions and their amino acid sequences within the B-subunit are highlighted in yellow in Figure 2B. Doubly charged ions derived from the tryptic decapeptides 32YNEDDTFTVK41

34YNDDDTFTVK43

of Stx2a at m/z 616.29 and

of Stx1a at m/z 609.27,

32YNEDNTFTVK41

of Stx2e at m/z 615.79

represent diagnostic ions for the respective Stx subtype and are suitable for a fast and facile MS1-based identification of Stx subtypes.

Structures of lipids employed for vesicle preparation and vesicle size distribution. Lipid structures were verified by MS1 and MS2 analysis, giving evidence for pure DOPC (C18:1, C18:1) as shown in Figure S-1 and prevalent DAPS (C18:1, C18:0), accompanied by traces of DAPS variants with C18:0/C18:1-OH and C20:1 fatty acid, in the DAPS sample (see Figure S-2). The SM preparation harbored predominant SM (d18:1, C18:0) and SM (d18:1, C24:1) species as documented in Figure S-3. The purity of cholesterol is shown in Figure S-4 and the analysis of Gb3Cer revealed Gb3Cer species with Cer (d18:1, C18:0), Cer (d18:1,



C22:1/C22:0) and Cer (d18:1, C24:1/C24:0) lipid anchors as the dominant variants (see Figure S-5). The size distribution of SUVs, produced by extrusion through 100 nm pores and determined by means of dynamic light scattering (DLS), is documented for SUVs of type 1 (DOPC, SM, and cholesterol), type 2 (DOPC, SM, cholesterol, and DAPS) and type 3 (DOPC, SM, cholesterol, DAPS, and Gb3Cer) in Figure S-6A, S-6B and S-6C, respectively.

Construction of stable lipid bilayer membranes. As a proof of principle of SUV adsorption to the negatively charged biosensor surface, which was achieved by covering the gold layer with 11-mercaptoundecanoic acid, Figure S-7 demonstrates the requirement of the linker lipid DAPS and divalent Mg2+ bridging cations for generating a stable lipid bilayer. This is exemplarily shown by a phase shift of 50° accomplished upon exposure of the biosensor surface to type 2 SUVs (containing DAPS) (Figure S-7B) in contrast to type 1 SUVs (lacking DAPS) (Figure S-7A). The accessibility of the Stx GSL receptor Gb3Cer was evaluated using biosensors coated with SUVs of type 2 (without Gb3Cer) and type 3 (with Gb3Cer) by measuring the phase shift ∆φ obtained after incubation with the 3 Stx subtypes (Figure S8). While none of the Stxs adhered to type 2 SUVs (without Gb3Cer) (Figure S-8A), Stxs specifically bound to SUVs of type 3 (with Gb3Cer) evidenced by the phase shifts attained with Stx1a, Stx2a and Stx2e (Figure S-8B).

SAW binding kinetics of Stx subtypes employing model membranes. After the preparatory experiments we created Stx real-time binding kinetics (Scheme 1, step 6) employing biosensor surfaces coated with type 3 SUVs forming a stable Gb3Cer-spiked model membrane. Affinity-purified Stx1a, Stx2a and Stx2e were applied with increasing concentrations allowing for determination of association and dissociation rate constants as shown in Figure 3. The time course of SAW sensorgrams obtained with Gb3Cer-spiked type 3 SUVs is exemplarily shown for Stx2a in Figure S-9, which elaborates the consecutive


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application of increasing Stx concentrations and intermediate elution of bound Stx from the membrane using Gb3 oligosaccharide. The dissociation constant KD, which describes the strength of binding between the Gb3Cer-doped membrane and the respective Stx, was calculated from the binding kinetics using 20, 50, 80, 120, 180 and 250 nM concentrations of Stx1a, Stx2a and Stx2e as shown in Figure 3A, 3B and 3C, respectively. KD-values obtained from binding kinetics were 103.88±17.31 nM for Stx1a, 124.38±6.69 nM for Stx2a and 1481.41±660.21 nM for Stx2e as depicted in Figure 3D, clarifying visually distinct differences in the association and dissociation curves regarding distinct receptor binding strength of the 3 Stx subtypes.

Cytotoxic activity of affinity-purified Stx subtypes. Vero cells were challenged with increasing concentrations of purified Stxs (Scheme 1, step 7) ranging from 1 pg mL-1 (10-3 ng mL-1) up to 1 µg mL-1 (103 ng mL-1) as shown in Figure 4A. All Stx subtypes exerted a concentration-dependent reduction in cell viability, but to a different extent. The strongest cytotoxic effect was detected for Stx1a, which caused a decline in cell viability to 14.4±3.3% at the highest concentration of 1 µg mL-1 of Stx1a, followed by Stx2a-mediated decrease in cell survival to 38.6±3.6% after treatment with the uppermost toxin concentration. Vero cells exhibited only a weak response upon exposure to 1 µg mL-1 of Stx2e resulting in cell viability of 64.7±2.8%. Thus, Vero cells were sensitive towards the affinity-purified Stxs ranked in descending order Stx1a > Stx2a > Stx2e. Stx TLC overlay assays revealed binding towards Gb3Cer as a common feature of the 3 subtypes as shown in Figure 4B. In contrast to Stx1a and Stx2a, Stx2e recognized Gb4Cer and Gb5Cer in addition to Gb3Cer. This binding profile acts as a distinguishing mark for Stx2e. Interestingly, despite binding to more receptor GSLs, Stx2e exhibited the lowest cytotoxic effect on Vero cells when compared to Stx1a and Stx2a (Figure 4A). On the other hand, poor cytotoxicity of Stx2e coincides with low binding strength determined by SAW measurements (see Figure 3).



DISCUSSION

We investigated in this study three clinically important affinity-purified Stx subtypes for their structural and functional differences based on ESI MS and SAW real-time interaction analysis, respectively, as well as TLC overlay and cell culture cytotoxicity assays as an improved combined methodological approach aimed at distinguishing Stx1a, Stx2a and Stx2e subtype at the protein level. It is known that STEC (by definition Stx-producing E. coli) also produce non-Stx molecules, such as cytolethal distending toxin, which can contribute to the endothelial or vascular injury.1 Thus, the application of purified Stxs is of outstanding importance for defining specific receptor binding and determining Stx-mediated cytotoxic activity on target cells. Using the purified Stx subtypes we can exclude unspecific binding and any cytotoxic effect of other virulence factors released by STEC. Isolation of Stxs based on affinity binding offers the development of efficient isolation procedures. An example is the use of an affinity column conjugated with a monoclonal antibody against the Stx2 A-subunit for purification of various Stx2 subtypes.45 Alternatively, Stxs can be purified via the carbohydrate-binding B-subunit that avoids tedious column chromatographic purification steps. Such improved one step affinity purification of Stx variants has been reported using P1 glycoprotein from hydatid cyst material,46,47 which carries glycans with terminal Galα4Galβ4GlcNAc-sequence. Reported procedures started from 20 liter batches of Stx-containing liquid culture broths produced in 9.9 liter fermenter cultures46,47 with yields of 5 to 10 mg pure toxin. Further purification approaches have been published using the native Stx receptor Gb3Cer adsorbed onto silica gel columns as affinity matrix48 or conjugated to Octyl Sepharose CL-4B as a carrier.49 Furthermore, Gb3 covalently linked to Fractogel50 or galabiose (Galα4Gal) coupled to agarose resin51 have been employed for Stx and Stx B-subunit purification, respectively. The same holds true for a trisaccharide-based affinity matrix using synthetic Galα4Galβ4GlcNAc


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glycan, which was covalently attached to hydrazine group-containing agarose gel by reductive amination.52 Here we performed reductive amination for coupling the proximal Glc aldehyde of Gb3 to amino groups on magnetic beads and report for the first time on the advantages of single-step purification of 3 different Stx subtypes using Gb3-functionalized magnetic beads. The novelty of our procedure lies in the fast and facile purification of Gb3-binding Stx subtypes on microgram scale, handling small volumes on milliliter scale, and avoiding extended column chromatography separation. Vero cell culture cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e, which has been shown in previous studies using Stx1a and Stx2a from otherwise purified Stx batches53,54 or employing Stx-containing bacterial supernatants including the Stx2e subtype.40 The recognition of Gb5Cer by Stx2e distinguishes this subtype from Stx1a and Stx2a. Importantly, the affinity-purified Stx subtypes exhibited the same binding pattern towards GSLs from Vero cells when compared to Stxs in crude STEC supernatants40 indicating that the Stx binding properties were retained unchanged throughout affinity purification. Sophisticated ESI MS technologies have been described allowing for determination of the stoichiometry of multivalent complexes of Stx with Gb3 and the affinities of Stx1 and Stx2 to Gb3 analogs,55,56 localization of the carbohydrate binding sites of the B-subunit polymer of Stx1a,57 detection of specific interactions between Gb3Cer and Stx,58 and investigations on the assembly and stability of Stx.59 Safe and effective means of detecting and quantitating various Stx subtypes have been recently reported by Silva and co-workers.28 They were able to distinguish among most of the known subtypes including Stx1a, Stx2a and Stx2e. Identified diagnostic B-subunit-derived decapeptides were 32YNEDDTFTVK41

34YNDDDTFTVK43

for Stx1a,

for Stx2a and 32YNEDNTFTVK41 for Stx2e.28,60 We identified the same

diagnostic ions being in perfect agreement with those scrutinized by Silva and collaborators. Similar to their approach, an optimized MS method for Stx distinction has been recently published employing LC-MS/MS detection of tryptic digests of Stx preparations enriched by



Synsorb-digalactoside affinity binding utilizing the distal Galα4Galβ-binding motif of Gb3Cer.61,62 Another approach applying top-down proteomic identification of Stx2 subtypes by MALDI tandem MS has been reported, which is based on the mass determination of the A2 fragment and the B-subunit as well as from their sequence-specific fragment ions.63-65 The Stx2 subtypes a, c, d, f, and g were identified classifying top-down proteomic identification as a rapid, highly specific technique for distinguishing Stx2 subytpes.65 Thus, protein sequencing by mass spectrometry is an important tool in the context of verifying the biological activity of a protein upon purification and to discern structural differences of closely related proteins such as the various Stx subtypes as described in this report. Surface plasmon resonance (SPR), quartz crystal microbalance (QCM), and surface acoustic wave (SAW) technologies enable label-free real-time monitoring of biomolecular interactions.66-68 For protein-glycolipid interactions, SPR represents, thus far, the most frequently used biosensor technology.69-73 Initial SPR experiments creating differential binding kinetics for Stx1a and Stx2a and membrane-embedded glycolipid ligands have been conducted by Nakajima and co-workers.14 For instance, Stx2a bound more slowly to Gb3Cer than Stx1a but, once bound, was difficult to dissociate. A similar trend has been detected by us using SAW, whereby Stx2e exhibited the most pronounced differences due to its extremely slow binding and poor dissociation. Interestingly, weak binding towards Gb3Cer-doped model membranes paralleled low cytotoxic activity of Stx2e. When combined with surfaceimmobilized model membranes, the analytical value of SAW biosensors has strongly been increased and extended to various topics of biomedical investigations.31,32 Our study is the first that evidenced the general applicability of SAW for determining the binding strength of Stxs towards Gb3Cer-doped model membranes and detection of differences between Stx1a, Stx2a and Stx2e subtype. The application of SAW in conjunction with immobilized SUVs using one step-purified Stxs for biomolecular real-time interaction analysis is an essential proof for the reliability of the purification procedure. This is not trivial since extended


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column-based purification strategies may result in loss of biological activity of Stx. Based on our experience, the SAW technology opens up promising perspectives for real-time and labelfree interaction analysis avoiding derivatization procedures of the analytes.33 Specific detection of the presence of stx genes coding for the various Stx1- and Stx2-subtypes of STEC is usually performed by molecular typing using conventional polymerase chain reaction (PCR) as well as real-time or multiplex PCR analysis.74-76 This includes the specific detection of the stx1a and stx2a genes of the HUS-relevant Stx1a- and Stx2a-subtypes as well as the stx2e gene coding for pig edema disease-associated Stx2e. However, the presence of a given stx gene does necessarily imply the expression of the corresponding Stx protein. The production of Stxs can be indirectly determined by antibody-based Stx1 and Stx2 serotyping.77-79 However, discrepancies between commercial PCR and serological assays have been observed, which may be due to differences in the specificities of stx PCR primers or anti-Stx antibodies for differentiation between subtypes of Stx of type 1 and type 2. Thus, results, especially those of serological assays, may need to be interpreted with caution80 and the inability of several serological assays to differentiate between the various Stx1 and Stx2 subtypes and in particular the failure to detect the Stx2e subtype may be of concern.80,81 These shortcomings of PCR analysis and serotyping point to the advantage of our combined strategy working with purified Stx subtypes of STEC wild-type strains and suggest future clinical applications such as the clinical diagnosis based on STEC-released Stx subtypes at the protein level.



AUTHOR INFORMATION

Corresponding author *Fax: +49-(0)251-83 55341. E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT

This work was supported by grants from the German Federal Ministry of Education and Research (BMBF), InfectControl 2020 IRMRESS (ref. no. 03ZZ0805B) and InfectControl 2020 (TFP-TV8-AS12, ref. no. 03ZZ0802H), with assistance of the German Center for Infection Research (DZIF, TTU 06.801), the Interdisciplinary Center for Clinical Research (IZKF) Münster project no. Müth2/021/15 (J.M.) and Me2/010/16 (A.M. and H.K.) and the German Research Foundation (DFG) Collaborative Research Centre (SFB) 1009 (B04 A.M.). We thank Dagmar Mense, Nikola Skutta, and Ralph Fischer for excellent technical assistance. We apologize to investigators whose work was not cited due to limited space.

SUPPORTING INFORMATION AVAILABLE

Additional experimental details are provided as tables, schemes and figures (PDF) as noted in the text. This material is available free of charge on the ACS Publications website via the Internet at http://pubs.acs.org.


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Scheme 1. Flowsheet of the matched strategy including mass spectrometry, surface acoustic wave interaction analysis and cell viability assays for distinction of Stx subtypes prepared by affinity-purification. Sterile filtered supernatants from bacterial liquid cultures of Stxproducing E. coli were concentrated by spin ultrafiltration (1) followed by affinity purification of Stxs using Gb3-functionalized magnetic beads (2-3). Purified holotoxins were probed by ESI MS1 analysis, followed by detection of Stx subtype-specific diagnostic ions derived from tryptic peptides using ion mobility MS2 analysis (4). Stxs were further verified by SDS PAGE (5), functionally characterized by SAW interaction analysis using Gb3Cer-spiked model membranes (6) and biologically characterized by their cytotoxicity exerted in Vero cell cultures (7).



Figure 1. ESI MS1 spectra of purified Stx1a (A), Stx2a (B) and Stx2e (C), and SDS PAGE of purified Stxs (D). (A-C) The averaged m/z values of the A [M+nH]n+ ions of the A-subunit are depicted in red and the monoisotopic [M+nH]n+ ions of the B-subunit are portrayed in blue. The high resolution of the spectra is illustrated by zoomed inserts showing isotope distribution of selected multiply charged monoisotopic [M+nH]n+ ions: [M+6H]6+ ions of the B-subunit of Stx1a at m/z 1281.64 (A), [M+5H]5+ ions of the B-subunit of Stx2a with increasing exchange of H+ against Na+ ions in the range from m/z 1560 to m/z 1600 (B), and [M+6H]6+ ions of the B-subunit of Stx2e at m/z 1263.76 (C). (D) Purified Stxs applied to SDS PAGE correspond to 10 µg of each Stx subtype. A-SU, A-subunit; B-SU, B-subunit.


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A

609.27+2

Stx1a 34YNDDDTFTVK43

605

610

615

620

m/z

620

m/z

620

m/z

616.29+2

Stx2a 32YNEDDTFTVK41

605

610

615

615.79+2

Stx2e 32YNEDNTFTVK41

605

B

…28KVE …26KIE …26KIE

610

615

YTKYNDDDTF TVKVGD46…

Stx1a

FSKYNEDDTF TVKVDG44…

Stx2a

FSKYNEDNTF TVKVSG44…

Stx2e

Figure 2. Diagnostic ions derived from the tryptic peptides of the B-subunits of Stx1a, Stx2a, and Stx2e (A) and localization of the corresponding decapeptide amino acid sequences within the B-subunits highlighted in yellow (B).



Figure 3. SAW real-time interaction of Stx1a (A), Stx2a (B) and Stx2e (C) with Gb3Cerspiked model membranes and function graph of calculated dissociation constants KD of the 3 Stx subtypes (D). Interactions between Stxs and Gb3Cer are portrayed as association curves (ascending bold lines in the left hand panels) and dissociation curves (descending bold lines in the right hand panels). Kinetics were fitted to the association and dissociation curves using increasing toxin concentrations as indicated. a, start of Stx injection; b, end of Stx injection.


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Figure 4. Susceptibility of Vero cells towards affinity-purified Stxs (A) and Stx TLC overlay assay detection of Stx receptors in the neutral GSL fraction of Vero cells (B). (A) Epithelial cells were exposed to Stx1a, Stx2a or Stx2e with increasing toxin concentrations ranging from lowest concentration of 10-3 ng mL-1 to highest concentration of 103 ng mL-1. Viability measurements of six biological replicates are depicted as mean percentage values ±SD related to two untreated controls. (B) Applied GSL amounts correspond to 1 x 106 cells for the orcinol stain and 0.5 x 106 cells for the Stx TLC overlay assays, respectively.



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ABSTRACT GRAPHIC (for TOC only)


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Combining Mass Spectrometry, Surface Acoustic Wave Interaction

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