Supporting Information

Transkript

1 Supporting Information 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 1, Gottfried Pohlentz 1, Nadine Legros 1, Michael Mormann 1, Alexander Mellmann 1,2, Helge Karch 1,2, and Johannes Müthing 1,2* 1 Institute for Hygiene, University of Münster, Robert-Koch-Strasse 41, D Münster, Germany 2 Interdisciplinary Center for Clinical Research (IZKF) Münster, Domagkstrasse 3, D Münster * To whom correspondence should be addressed. Phone: +49-(0) Fax: +49-(0) jm@uni-muenster.de S-1

2 TABLE OF CONTENTS Supporting Tables Table S-1. Experimental values of sequenced tryptic peptides from the A-subunit of Stx1a Table S-2. Experimental values of sequenced tryptic peptides from the B-subunit of Stx1a Table S-3. Experimental values of sequenced tryptic peptides from the A-subunit of Stx2a Table S-4. Experimental values of sequenced tryptic peptides from the B-subunit of Stx2a Table S-5. Experimental values of sequenced tryptic peptides from the A-subunit of Stx2e Table S-6. Experimental values of sequenced tryptic peptides from the B-subunit of Stx2e Supporting Schemes Scheme S-1. Amino acid sequence deduced from tryptic peptides of the A-subunit of Stx1a. Scheme S-2. Amino acid sequence deduced from tryptic peptides of the B-subunit of Stx1a. Scheme S-3. Amino acid sequence deduced from tryptic peptides of the A-subunit of Stx2a. Scheme S-4. Amino acid sequence deduced from tryptic peptides of the B-subunit of Stx2a. Scheme S-5. Amino acid sequence deduced from tryptic peptides of the A-subunit of Stx2e. Scheme S-6. Amino acid sequence deduced from tryptic peptides of the B-subunit of Stx2e. Supporting Figures Figure S-1. ESI MS 1 spectrum of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Figure S-2. ESI MS 1 spectrum of 1,2-diacyl-sn-glycero-3-phosphatidyl-L-serine (DAPS). Figure S-3. ESI MS 1 spectrum of sphingomyelin (SM). Figure S-4. ESI MS 1 spectrum of cholesterol (Chol). Figure S-5. ESI MS 1 spectrum of Gb3Cer. Figure S-6. Size distribution of small unilamellar vesicles (SUVs) of type 1 (A), type 2 (B), and type 3 (C). Figure S-7. Lipid bilayer formation by SUVs of type 1 (A) and type 2 (B). Figure S-8. Binding of Stx1a, Stx2a and Stx2e subtypes to SUVs of type 2 (A) and type 3 (B). Figure S-9. SAW sensorgram of real-time interaction analysis of Stx2a with Gb3Cer-spiked SUVs of type 3. S-2

3 Supporting Tables and Schemes Table S-1. Experimental values of sequenced tryptic peptides from the A-subunit of Stx1a Peptide FADFSHV TFPGTTAVTL SGDSSYTTLQ R TTLDDLSGR FVTVTAEA LR LSS VLPDYHGQDS VR GID PEEGRFNNLR TGM QINR SYVMTAEDVD LTLNWGR MKIIIFRVLT FFFVIFSVNV VAKEFTLDFS TAKTYVDSLN VIRSAIGTPL QTISSGGTSL 60 GID PEEGRFNNLR FADFSHV 61 LMIDSGTGDN LFAVDVRGID PEEGRFNNLR LIVERNNLYV TGFVNRTNNV FYRFADFSHV 120 TFPGTTAVTL SGDSSYTTLQ R TGM QINR 121 TFPGTTAVTL SGDSSYTTLQ RVAGISRTGM QINRHSLTTS YLDLMSHSGT SLTQSVARAM 180 SYVMTAEDVD LTLNWGR FVTVTAEA LR TTLDDLSGR LSS VLPDYHGQDS 181 LRFVTVTAEA LRFRQIQRGF RTTLDDLSGR SYVMTAEDVD LTLNWGRLSS VLPDYHGQDS 240 VR 241 VRVGRISFGS INAILGSVAL ILNCHHHASR VARMASDEFP SMCPADGRVR GITHNKILWD SSTLGAILMR RTISS 315 (Q8X696) Scheme S-1. Amino acid sequence deduced from fragment ion spectra of tryptic peptide ions derived from the A-subunit of Stx1a. Sequenced peptides (colored in green), obtained by in solution digest of Stx1a, were aligned with the amino acid sequence taken from the UniprotKB database (Q8X696, displayed in black). The sequenced peptides account for an amino acid coverage of 63.8 % (187 of 293 amino acids). S-3

4 Table S-2. Experimental values of sequenced tryptic peptides from the B-subunit of Stx1a Peptide WNLQSLL LSAQITGMTV TIK YNDDDTF TVK VGDK VE YTK ELF TNR TPDCVTGK TNACHNG GGFSEVIFR TPDCVTGKVE YTK TNACHNG GGFSEVIFR TPDCVTGKVE YTKYNDDDTF TVK TNACHNG GGFSEVIFR VGDKELF TNR VE YTKYNDDDTF TVK VE YTKYNDDDTF TVK VGDKELF TNR TPDCVTGKVE YTKYNDDDTF TVK TPDCVTGKVE YTK ELF TNR VE YTK VGDK TPDCVTGK YNDDDTF TVK WNLQSLL 1 MKKTLLIAAS LSFFSASALA TPDCVTGKVE YTKYNDDDTF TVKVGDKELF TNRWNLQSLL 60 TNACHNG GGFSEVIFR LSAQITGMTV TIK 61 LSAQITGMTV TIKTNACHNG GGFSEVIFR 89 (K4VUY1) Scheme S-2. Amino acid sequence deduced from fragment ion spectra of tryptic peptide ions derived from the B-subunit of Stx1a. Sequenced peptides (colored in green), obtained by in solution digest of Stx1a, were aligned with the amino acid sequence taken from the UniprotKB database (K4VUY1, displayed in black). The cysteins Cys 24 and Cys 77, involved in the formation of the disulfide bridge, are highlighted in red. The N-terminal signal sequence composed of 20 amino acids is displayed in grey. The experimentally determined sequence coverage was 100 % (69 of 69 amino acids). S-4

5 Table S-3. Experimental values of sequenced tryptic peptides from the A-subunit of Stx2a Peptide GLD VYQAR FSDFTHI SVPGVTTVSM TTDSSYTTLQ R TTDSSYTTLQ R VAALER SGM QISR FVTVTAEA LR QIQR QALSETAPV YTMTPGDVDL TLNWGR qalsetapv YTMTPGDVDL TLNWGR ISNV LPEYR ISNV LPEYRGEDGV R GEDGV R INNTLWES NTAAAFLNR INNTLWES NTAAAFLNRK SQFLYTTGK MKCILFKWVL CLLLGFSSVS YSREFTIDFS TQQSYVSSLN SIRTEISTPL EHISQGTTSV 60 GLD VYQAR FSDFTHI 61 SVINHTPPGS YFAVDIRGLD VYQARFDHLR LIIEQNNLYV AGFVNTATNT FYRFSDFTHI 120 VAALER TTDSSYTTLQ R SGM QISR SVPGVTTVSM TTDSSYTTLQ R 121 SVPGVTTVSM TTDSSYTTLQ RVAALERSGM QISRHSLVSS YLALMEFSGN TMTRDASRAV 180 QALSETAPV YTMTPGDVDL TLNWGR qalsetapv YTMTPGDVDL TLNWGR GEDGV FVTVTAEA LR ISNV LPEYR QIQR ISNV LPEYRGEDGV 181 LRFVTVTAEA LRFRQIQREF RQALSETAPV YTMTPGDVDL TLNWGRISNV LPEYRGEDGV 240 R INNTLWES R INNTLWES 241 RVGRISFNNI SAILGTVAVI LNCHHQGARS VRAVNEESQP ECQITGDRPV IKINNTLWES 300 NTAAAFLNRK NTAAAFLNR SQFLYTTGK 301 NTAAAFLNRK SQFLYTTGK 319 (Q8XBV2) Scheme S-3. Scheme S3. Amino acid sequence deduced from fragment ion spectra of tryptic peptide ions derived from the A-subunit of Stx2a. Sequenced peptides (colored in green), obtained by in solution digest of Stx2a, were aligned with the amino acid sequence taken from the UniprotKB database (Q8XBV2, displayed in black). Red marked Met 150, Met 165, Met 172 and Met 213 were present in an oxidized state; red marked Gln 202 represents its deaminated pendant (q). The N-terminal signal sequence composed of 22 amino acids is displayed in grey. The sequenced peptides account for an amino acid coverage of 41.4 % (123 of 297 amino acids). S-5

6 Table S-4. Experimental values of sequenced tryptic peptides from the B-subunit of Stx2a Peptide A DCAK SSTCESGSG FAEVQFNND YNEDDTFTV K IEFS K LQS AQLTGMTVTI K YNEDDTFTV KVDGK EYWTS R GKIEFS KYNEDDTFTV K WNLQPLLQS AQLTGMTVTI K GKIEFS KYNEDDTFTV K YNEDDTFTV KVDGK IEFS K WNLQPLLQS A DCAK YNEDDTFTV K EYWTS R LQS 1 MKKMFMAVLF ALVSVNAMAA DCAKGKIEFS KYNEDDTFTV KVDGKEYWTS RWNLQPLLQS 60 AQLTGMTVTI K AQLTGMTVTI K SSTCESGSG FAEVQFNND 61 AQLTGMTVTI KSSTCESGSG FAEVQFNND 89 (Q7DJJ2) Scheme S-4. Amino acid sequence deduced from fragment ion spectra of tryptic peptide ions derived from the B-subunit of Stx2a. Sequenced peptides (colored in green), obtained by in gel digest of Stx2a, were aligned with the amino acid sequence taken from the UniprotKB database (Q7DJJ2, displayed in black). Red marked Met 66 was present in an oxidized state. Cys 22 and Cys 75, involved in the formation of the disulfide bridge, are highlighted in red. The N-terminal signal sequence composed of 19 amino acids is displayed in grey. The experimentally determined sequence coverage was 100 % (70 of 70 amino acids). S-6

7 Table S-5. Experimental values of sequenced tryptic peptides from the A-subunit of Stx2e Peptide FSDFAHI SLPGVTTISM TTDSSYTTLQ R NNLYV AGFVNTTTNT FYR AVNEESQP ECQITGDRPV IK HSLVSS YLALMEFSGN TMTR qeftidfs TQQSYVSSLN SIR QEFTIDFS TQQSYVSSLN SIR GLD VYQER ISNV LPEYR ISNV LPEYRGEAGV R FVTVTAEA LR K SQPLYTTGE LIIER INNTLWES NTAAAFLNR VAALER TAISTPL EHISQGATSV SVINHTPPGS YISVGIR LALSETAPV YTMTPEDVDL TLNWGR LALSETAPV YTMTPEDVDL TLNWGR TAISTPL EHISQGATSV QEFTIDFS TQQSYVSSLN SIR qeftidfs TQQSYVSSLN SIR 1 MKCILLKWIL CLLLGFSSVS YSQEFTIDFS TQQSYVSSLN SIRTAISTPL EHISQGATSV 60 SVINHTPPGS YISVGIR NNLYV AGFVNTTTNT FYR GLD VYQER LIIER FSDFAHI 61 SVINHTPPGS YISVGIRGLD VYQERFDHLR LIIERNNLYV AGFVNTTTNT FYRFSDFAHI 120 VAALER SLPGVTTISM TTDSSYTTLQ R HSLVSS YLALMEFSGN TMTR 121 SLPGVTTISM TTDSSYTTLQ RVAALERSGM QISRHSLVSS YLALMEFSGN TMTRDASRAV 180 LALSETAPV YTMTPEDVDL TLNWGR LALSETAPV YTMTPEDVDL TLNWGR ISNV LPEYRGEAGV FVTVTAEA LR ISNV LPEYR 181 LRFVTVTAEA LRFRQIQREF RLALSETAPV YTMTPEDVDL TLNWGRISNV LPEYRGEAGV 240 INNTLWES R AVNEESQP ECQITGDRPV IK 241 RVGRISFNNI SAILGTVAVI LNCHHQGARS VRAVNEESQP ECQITGDRPV IKINNTLWES 300 NTAAAFLNR K SQPLYTTGE 301 NTAAAFLNRK SQPLYTTGE 319(C7TQM6) Scheme S-5. Amino acid sequence deduced from fragment ion spectra of tryptic peptide ions derived from the A-subunit of Stx2e. Sequenced peptides (colored in green), obtained by in solution digest of Stx2e, were aligned with the amino acid sequence taken from the UniprotKB database (C7TQM6, displayed in black). The N-terminal signal sequence composed of 22 amino acids is displayed in grey. Red marked Met 130, Met 165, Met 172, Met 213 and Trp 224 were present in an oxidized state. The sequenced peptides account for an amino acid coverage of 79.1 % (235 of 297 amino acids). S-7

8 Table S-6. Experimental values of sequenced tryptic peptides from the B-subunit of Stx2e Peptide GKIEFS K YNEDNTFTV K IEFS KYNEDNTFTV K GKIEFS KYNEDNTFTV K FS KYNEDNTFTV K A DCAK TVTI ISNTCSSGSG FAQVK YNEDNTFTV KVSGR EFS KYNEDNTFTV K EYWTN R WNLQPLLQSAQLTGM YNEDNTFTV KVSGR FS KYNEDNTFTV K IEFS KYNEDNTFTV K A DCAK YNEDNTFTV K GKIEFS K GKIEFS KYNEDNTFTV K EYWTN R WNLQPLLQS 1 MKKMFIAVLF ALVSVNAMAA DCAKGKIEFS KYNEDNTFTV KVSGREYWTN RWNLQPLLQS 60 TVTI ISNTCSSGSG FAQVK AQLTGM 61 AQLTGMTVTI ISNTCSSGSG FAQVKFN 87 (G0FDA1) Scheme S-6. Amino acid sequence deduced from fragment ion spectra of tryptic peptide ions derived from the B-subunit of Stx2e. Sequenced peptides (colored in green), obtained by in gel digest of Stx2e, were aligned with the amino acid sequence taken from the UniprotKB database (G0FDA1, displayed in black). Cys 22 and Cys 75, involved in the formation of the disulfide bridge, are highlighted in red. Red marked Met 66 was present in an oxidized state. The N-terminal signal sequence composed of 19 amino acids is displayed in grey. The sequenced peptides account for an amino acid coverage of 97.1 % (66 of 68 amino acids). S-8

9 Supporting Figures DOPC C18:1, C18: [M+H] + C18:1, C18: Figure S-1. ESI MS 1 spectrum of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). [M+H] + ions at and ions at detected in the positive ion mode indicate the proposed DOPC (C18:1, C18:1) structure. S-9

10 [M H] C18:1, C18: DAPS [M H] C18:1, C18:1-OH [M H] C18:1, C18:0-OH [M H] C18:1, C18: [M H] C18:0, C20:1 [M H] C18:1, C20: Figure S-2. ESI MS 1 spectrum of 1,2-diacyl-sn-glycero-3-phosphatidyl-L-serine (DAPS). Major deprotonated [M-H] - species at detected in the negative ion mode indicate the prevalence of DAPS (C18:1, C18:0). S-10

11 SM C18: C24: [M+H] + C18: [M+H] + C18: C22: [M+H] + C24: [M+H] + C16: C16: C18: C20: C22: Figure S-3. ESI MS 1 spectrum of sphingomyelin (SM). Major [M+H] + ions at and as well as ions at and detected in the positive ion mode indicate the prevalence of SM (d18:1, C18:0) and SM (d18:1, C24:1) lipoforms, respectively. All SM species harbor d18:1 sphingosine (2-amino-4-octadecene-1,3-diol) as the long-chain amino alcohol. S-11

12 x10 x10 x NaOH [M 1 +Na] Chol [M 2 +Na] [M 3 +H] [M 3 +Na] Figure S-4. ESI MS 1 spectrum of cholesterol (Chol). [M 1-3 +Na] + ions at , and , respectively, as well as [M 3 +H] + ions at detected in the positive ion mode are evident for cholesterol. S-12

13 Gb3Cer [M+H] + C18: C16: C18: C18:0-OH C22: C20: C22: C24: C24: C18: [M+H] + C20: C18:1-OH C20: [M+H] + C22: C20:1-OH C20:0-OH [M+H] + C24: C22:2-OH C22:1-OH C24: C24:2-OH C24:1-OH C24:0-OH Figure S-5. ESI MS 1 spectrum of Gb3Cer. Major ions at , / and / detected in the positive ion mode indicate the prevalence of Gb3Cer (d18:1, C18:0), Gb3Cer (d18:1, C22:1/C22:0) and Gb3Cer (d18:1, C24:1/C24:0) lipoforms, respectively. S-13

14 Figure S-6. Size distribution of small unilamellar vesicles (SUVs) of type 1 (A), type 2 (B), and type 3 (C). Type 1 vesicles (DOPC, SM and cholesterol in a 7:1:2 ratio), type 2 vesicles (DOPC, SM, cholesterol and DAPS in a 5:1:2:2 ratio) and type 3 vesicles (DOPC, SM, cholesterol, DAPS and Gb3Cer in a 4.5:1:2:2:0.5 ratio) were extruded through a polycarbonate membrane with 100 nm pore size. SUV diameters were determined in triplicate by means of dynamic light scattering (DLS) shown by the different colored lines. S-14

15 Figure S-7. Lipid bilayer formation by SUVs of type 1 (A) and type 2 (B). SUVs of type 2 containing negatively charged DAPS formed a stable lipid bilayer on the negatively charged 11- mercaptoundecanoic acid (MUA)-coated biosensor gold surface mediated by positively charged Mg 2+ bridging ions (B), whereas SUVs of type 1 without DAPS failed in formation of a stable lipid bilayer (A). For lipid composition of type 1 and type 2 vesicles refer to Supporting Figure S6. S-15

16 Figure S-8. Binding of Stx1a, Stx2a and Stx2e subtypes to SUVs of type 2 (A) and type 3 (B). Stx1a (black bars), Stx2a (grey bars) and Stx2e (white bars) were applied to the MUA-coated biosensor surface using 250 nm toxin concentrations, respectively. Changes in the phase shift φ were measured 750 s after Stx injection. Stxs specifically bound to SUVs of type 3 (with Gb3Cer), whereas SUVs of type 2 (without Gb3Cer) failed in Stx binding. Experiments were performed in triplicate. S-16

17 Figure S-9. SAW sensorgram of real-time interaction analysis of Stx2a with Gb3Cer-spiked SUVs of type 3. After coating of the sensor surface with SUVs, the stability of the lipid bilayer was checked by rinsing with 10 mm NaOH, followed by blocking the model membrane with BSA to prevent unspecific toxin binding. Stx2a was injected and after the association and dissociation period remaining bound toxin was released using 0.1 M Gb3 oligosaccharide solution. This procedure was consecutively performed with increasing concentrations of 50, 80, 120, 180 and 250 nm Stx2a as indicated. S-17