Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis

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1 Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis Claudio Peri 1, Alessandro Gori 1, Paola Gagni 1, Laura Sola 1, Daniela Girelli 2, Samantha Sottotetti 2, Lisa Cariani 2, Marcella Chiari 1, Marina Cretich 1*, and Giorgio Colombo 1 * 1 Istituto di Chimica del Riconoscimento Molecolare, ICRM, CNR. Via Mario Bianco 9, 2131, Milano (Italy) 2 Cystic Fibrosis Microbiology Laboratory, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milano (Italy) SUPPLEMENTARY INFORMATION

2 ANTIGEN ORGANISM #num E-value al. Length IDENTITY (%) GENOMIC LOCATION GKEKPVALGHDEASWAQNRRADLVYQ GVGDAQMEAV-YLKSHPQRHIL SYSVQDQYQALLQQHAQYLK BPSL2765 B. thailandeis 1 2.4E :21718 to (+) GKEKPVALGHDEASWAQNRRADLVYQ GVGDSQMEAV-YLKSHPQRHIL SYSVQDQYQPLLQQHAQYLK (17 res) B. mallei 1 4.3E : to (-) GKEKPVALGHDEASWAQNRRADLVYQ GVGDAQMEAV-YLKSHPQRHIL SYSVQDQYQALLQQHAQYLK B. cepacia 1 1.8E : to (+) GKEKPQATGHDEASWAQNRRADLVYQ GVNDSQMEAV-YLKSHPQRHVL SYSVKDEYQPLMQQHAQYLK 2 7.8E :29687 to 376 (-) GKEKPVALGHDEEAWAQNRRADLVYR GVPATQLEAV-YLRSHPARHVL QYSVKPEFQSLLQAHADYLR B. multivora 1 2.7E BMD22_12: to (-) GKEKPQATGHDEASWAQNRRADLVYQ GVADSQMEAV-YLKSHPQRHVL SYSVKDEYQPLLQQHAQYLK 2 8.5E BMD22_4: to (-) GKEKPVALGHDEAAWAQNRRADLVYR GVSASQLEAV-YLRSHPSRHVL SYTVKPEDQSLLQAHANYLR B. cenocepacia 1 5.1E : to (-) GKEKPQATGHDEASWAQNRRADLVYQ GVNDSQMEAV-YLKSHPQRHVL SYSVKDEYQPLLQQHAQYLK 2 8.7E : to 1776 (+) GKEKPVALGHDEESWSQNRRADLVYR GVQASQLEAV-YLRSHPSRHVL QYSVKPEYQSLLQAHADYLR B. vietnamieis 1 3.8E : to (-) GKEKPQATGHDEASWAQNRRADLVYQ GVNDSQMEAV-YLKSHPQRHVL SYSVKDEYQPLLQQHAQYLK B. dolosa 1 3.5E CH48238: to (+) GKEKPLATGHDEASWAQNRRADLVYQ GVADSQMEAV-YLKSHPQRHVL SYSVKDEYQPLLQQHAQYLK B. ambifaria 1 3.1E : to (+) GKEKPQASGHDEASWAQNRRADLVYQ GVNDSQMEAV-YLKSHPQRHVL SYSVKDEYQPLMQQHAQYLK 2 2.3E : to (+) GKEKPVALGHDEDSWAQNRRADLVYR GVQATQLEAV-YLRNHPSRRVL QYSVKPEFQSLLQSHADYLR P. aeruginosa 1 8.7E contig1: 7116 to 7328 (-) GKERPVATGHDEQSWAQNRRVEL--- GVSPAQLELV QRVV n.a. A. xylosoxida 1 1.7E quiver: to (-) GKEKPKATGTSEADFAENRRADIVYR GVSDNQIETI----SHQQQTIK n.a. S. maltophilia 1 6.8E CP8838:2747 to 2788 (+) GEERPVCTESNESCWSQNRRVEIVY- GGSASQLTVV-YLRDRPSSRIT KEDVKPEFQAIMACHAKYLR S. aureus 1 1.1E CP8838:27734 to 2788 (+) n.a. n.a. n.a. C. albica 1 8.4E cont1.27: to (+) n.a. n.a. n.a. ANTIGEN ORGANISM #num E-value al. Length IDENTITY (%) GENOMIC LOCATION KAPDT-KTEVPVSY GVKGVQRPFTPDWA AEANQKLDDGARAALLTQAHDLA WSNGQPVTAADFVYAWQR ELRPGLQLATYYYYLK BPSS2141 B. thailandeis 1.E : to (-) KTPDT-TTEVPVSY GVKGVQRPFTPDWA AEANQKLDDGARSALLTQAHDLA WSNGQPVTAADFVYSWQR ELRPGLQLATYYYYLK (554 res) B. mallei 1.E DM79.Contig17: to (+) KAPDT-KTEVPVSY GVKGVQRPFTPDWA AEANQKLDDGARAALLTQAHDLA WSNGQPVTAADFVYAWQR ELRPGLQLATYYYYLK B. cepacia 1.E : to (+) KTPQT-TTDVPVAF GTKGVQQPFTPEWA DEGNQKLDDQARTTLLTQAHDMA WSNGQPVTAADFVYSWQR ELRPGLQLATYYYYLN 1 5.2E : to (+) B. multivora 1.E BMD22_9: to (-) KTPQT-TTDVPVAF GTKGVQQPFTPEWA DEGNQKLDDKARAALLTQAHDTA WSNGQPVTAADFVYSWQR ELRPGLQLATYYYYLN 1 2.7E BMD22_9: to (-) B. cenocepacia 1.E : to (-) KTPQT-TTDVPVAF GTKGVQQPFTPEWA EEGNQKLDDKARTALLTQAHDMA WSNGQPVTAADFVYSWQR ELRPGLQLATYYYYLN 1 1.4E : to 3669 (-) B. vietnamieis 1.E : to (-) KTPQT-TTDVPVAF GTKGVRRPYTPDWA DEGNQKLDDAARTALLTQAHDLA WSNGQPVTAADFVYSWQR ELRPGLQLATYYYYLN 1 3.2E : to (-) B. dolosa 1.E CH482381: to (-) KTPQT-TTDVPVAF GTKGVQQPFTPEWA DDGNQKLDDSARAALLTQAHDAA WSNGQPVTAADFVYSWQR ELRPGLQLATYYYYLN 1 8.4E CH482381: to (-) B. ambifaria 1.E : to (-) KTPQT-TTDVPVAF GTKGAQQPFTPDWA DEGNQKLDDKARTALLTQAHDTA WSNGQPVTAADFVYSWQR ELRPGLQLATYYYYLN 1 5.2E : to (-) P. aeruginosa 1 1.9E contig_61: 692 to 799 (-) n.a. n.a. n.a. n.a. n.a. A. xylosoxida 1 2.2E quiver: to (+) n.a. n.a. n.a. FHDGSPFTADDVIFSWKR n.a. 2 1.E quiver: to (+) n.a. n.a. n.a. FSNGQPFTAQDVLFTFCR n.a. S. maltophilia 1 2.E contigid18:2689 to (+) n.a. n.a. n.a. n.a. n.a. S. aureus 1 7.5E CPJO11:88198 to (+) n.a. n.a. KVARTKL WSNGDKVTAQDFVYAWRK n.a E CPJO11:89134 to 89346(+) 1 7.7E CPJO11:8851 to (+) C. albica n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. ANTIGEN ORGANISM #num E-value al. Length IDENTITY (%) GENOMIC LOCATION VGYGGHGHPTQVRIVAPHAEHVRGYAH DGAARFERYLAALPRKLAAWENARGVDFGSRTQADAL BPSL15 B. thailandeis 1 9.3E : to (-) VGYGGHGHPTQVRIAAPPAEHVRGYAH DGAARFERYLAALPRKLPAWENARGIDFGSRTQTDAL (126 res) B. mallei 1 2.3E DM79.Contig17: to (+) VGYGGHGHPTQVRIVAPHAEHVRGYAH DGAARFERYLAALPRKLAAWENARGVDFGSRTQADAL B. cepacia 1 8.1E : to (-) VPYGGHGHPTRVQIRSAPHEHVSGFVH DGPQRFEHYLAALPRKLGAWQGARDIDLASRTQADPL B. multivora 1 3.7E BMD22_6: to (+) VPYGGHGHPTRVRIRSAPHEHVSGFAH DGAQRFEHYLAALPKKLNAWQGARDIDLASRTQADPL B. cenocepacia 1 1.4E : to 2911 (+) VPYGGHGHPTCVQIRSAPHEHVSGFAH DGPARFEHYLSALPRKLDAWQGARDIDLASRTQADPL B. vietnamieis 1 5.7E : to (+) VPYGGHGQPTRVHISSAPHEHVSGFAY DGAARFEHYLSALPKKLNAWESARDIDLASRTQEEPL B. dolosa 1 1.6E CH48238: to (-) VPYGGHGHPTCVRVRSAPHEHVSGFAH DGRARFDRYLAALPRKLAAWQDARDIDLPSRTQAEPI B. ambifaria 1 9.8E : to (-) APYGGHGQPTRVRIRSAPHEHVSGFVH DGPQRFEHYLAALPKKLGAWQGARDIDLASRTQADPL P. aeruginosa 1 4.4E contig_1: 2882 to (-) ----GYFRTFEQKIATPHGEEVRGYHK n.a. A. xylosoxida n.a. n.a. n.a. n.a. n.a. n.a. n.a. S. maltophilia n.a. n.a. n.a. n.a. n.a. n.a. n.a. S. aureus n.a. n.a. n.a. n.a. n.a. n.a. n.a. C. albica n.a. n.a. n.a. n.a. n.a. n.a. n.a. ANTIGEN ORGANISM #num E-value al. Length IDENTITY (%) GENOMIC LOCATION ALEGIEENVSFPLPRGL BPSL919 B. thailandeis 1.E : to (+) ALEGIEENVSFPLPRGL (326 res) 2 3.7E : to (+) TMSGREEKVEFKLPAKL B. mallei 1.E DM79.Contig17: to (-) ALEGIEENVSFPLPRGL 2 3.2E DM79.Contig17: to (+) TMSGREEKVEFKLPAKL B. cepacia 1.E : to (+) ALDGIEENVSFPLPRGL 2 8.7E : to (-) TMAGREEKVEFKLPAKL B. multivora 1.E BMD22_6: to (-) ALEGIEENVSFPLPRGL 2 9.4E BMD22_9: to 2626 (+) TMAGREEKVEFKLPAKL B. cenocepacia 1.E : to (-) ALEGIEENVSFPLPRGL 2 5.1E : to (+) TMAGREEKVEFKLPAKL B. vietnamieis 1.E : to (-) ALEGIEENVAFPLPRGL 2 6.4E : to (+) TMAGREEKVEFKLPAKL B. dolosa 1.E CH48238: to (+) ALEGIEENVSFPLPRGL 2 4.4E CH482381: to (+) TMAGREEKVEFKLPAKL B. ambifaria 1.E : to (+) ALDGIEENVSFPLPRGL 2 4.6E : to (+) TMAGREEKVEFKLPAKL P. aeruginosa 1 4.1E contig_5: to (+) ELEGREENITFSMPKEL 2 6.1E contig_5: to (+) A. xylosoxida 1 4.5E quiver: to (+) TMPGLEENVAFPLPKGL S. maltophilia n.a. n.a. n.a. n.a. n.a. n.a. S. aureus n.a. n.a. n.a. n.a. n.a. n.a. C. albica n.a. n.a. n.a. n.a. n.a. n.a.

3 Figure S1. Epitope coervation among Burkholderia species and common FC superinfection agents: the table is subdivided in four sectio, one for each antigen. The coervation has been evaluated via BLASTp agait the proteome of 11 bacteria and 1 fungus using the EemblGenomes database ( For each query, only clear homologues have been reported in case of strong similarities, and only the best hit in case of poor similarities. A color code is adopted to underline the similarity level: exact or almost exact matches are shown in green background. Blue background is for homologous protei, yellow stands for poor similarity and red is for no similarity. For each genome, the table shows the number of similar protei coidered, the length of the aligned region, the percentage of identity between the aligned sequences, the genomic location on the reference genome and its orientation on the DNA strand (forward = +, reverse = -) and the epitope sequences (mutatio highlighted in red). One locus has been split by EemblGenomes during alignment. Multiple results here are actually a single one. Infectio N N BCC positive CF patients Genomovar I 2 14 (B+) Genomovar II 2 Genomovar III 6 Genomovar IV 2 Genomovar V 1 UNK 1 BCC negative CF patients P. aeruginosa 6 11 (I) (B-) A. xylosoxida 4 S. maltophilia 1 Healthy controls (H) Table S1. Details on the tested serum samples. The screening method was tested agait sera collected from 39 donors, subdivided in three main categories: 14 cystic fibrosis patients (CF)

4 positive to Burkholderia cepacia complex (BCC) infectio (B+); 25 individuals negative to BCC (B-), subdivided in turn by 11 cystic fibrosis patients infected by other pathoge (I) and 14 healthy controls (H). Colum Infectio and N subdivide the CF patients based on the specific BCC genomovar, if known, or other pathogenic microbes. Genomovar I-V correspond in turn to species B. cepacia, B. multivora, B. cenocepacia, B. stabilis, B. vietnamieis. UNK indicates the BCC genotype for the Burkholderia infected patient is unknown. Probe AUC: Area Under the ROC Curve and 95% Confidence Interval (95% CI) (B+): BCC positive patients, 14 samples (H): healthy controls, 14 samples (I): Infected individuals (Negative for Burkholderia), 11 samples AUC B+ vs H AUC B+ vs I PAL to to 1. PAL to to 1. PAL to to 1. PAL3H to to 1. OPPA to to.7483 OPPA to to 1. OPPA to to 1. OPPA to to 1. OPPA to to 1. 2MPE to to 1. 2MPE to 1. ISPH to to to.8213 Table S2: AUC and 95% Confidence Interval ROC Curve

5 PAL1 PAL2 PAL * * PAL3H 2MPE1 2MPE * Figure S2: Antibody recognition signals subdivided by probe and patient category (part I). IgG levels specific for peptides PAL1, PAL2, PAL3, PAL3H, 2MPE1 and 2MPE2 in healthy controls (H), CF patients negative for BCC and positive for other respiratory infectio (I) and BCC positive patients (B+). The mean relative fluorescence inteity (), detected at 7% laser power and photomultiplier and P-values of test significance are reported for each peptide.

6 : not significative. Significative: p<.5; * = p<.5; = p<.1; * = p<.1; = p<.1. OPPA1 OPPA2 OPPA * * OPPA4 OPPA5 ISPH Figure S3: Antibody recognition signals subdivided by probe and patient category (part II) : IgG levels specific for peptides OPPA1, OPPA2, OPPA3, OPPA4, OPPA5 and ISPH1 in healthy controls (H), CF patients negative for BCC and positive for other respiratory infectio (I) and BCC positive patients (B+). The mean relative fluorescence inteity (), detected at 7% laser power and photomultiplier and P-values of test significance are reported for each peptide. : not significative. Significative: p<.5; * = p<.5; = p<.1; * = p<.1; = p<.1.

Cystic Fibrosis. Cystic Fibrosis. Cystic Fibrosis 5/01/2011 CYSTIC FIBROSIS OF THE PANCREAS AND ITS RELATION TO CELIAC DISEASE. D ANDERSEN.

Cystic Fibrosis. Cystic Fibrosis. Cystic Fibrosis 5/01/2011 CYSTIC FIBROSIS OF THE PANCREAS AND ITS RELATION TO CELIAC DISEASE. D ANDERSEN. 1938 OF THE PANCREAS AND ITS RELATION TO CELIAC DISEASE. D ANDERSEN. American Journal Diseases Children. : The beginning May 1938: 49 cases 25 20 15 Nos of cases 10 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Age