A proteogenomics approach to investigating snail-schistosome interactions

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1 A proteogenomics approach to investigating snail-schistosome interactions Timothy P. Yoshino, Ph.D. Dept. of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison

2 * WI University of Wisconsin-Madison

3 Schistosoma mansoni intestinal schistosomiasis Sexual phase Miracidium Cercaria Asexual phase 2 o /daughter sporocyst 1 o /mother sporocyst

4 Model host-parasite system Schistosoma mansoni Biomphalaria glabrata Susceptible: NMRI Compatible NMRI strain Hemocytes? Plasma Normal development Resistant: BS-90 Incompatible Encapsulated Parasite death

5 HOST-PARASITE INTERFACE Hemocytes Plasma Pathogen Recognition Receptors (PRR) Hemocyte/plasma Fibrinogen-related proteins (FREPs) C- and S-type lectins Toll-like receptors Integrin-like receptors Peptidoglycan receptors Path.-Associated Mol. Patterns (PAMP) Larval surface/larval transformation proteins (=LTP) polymorphic mucins O/N-glycans glycoprotein N-glycans Sporocyst How can these very complex, multi-receptor, multi-ligand molecular interactions be investigated?

6 In vitro snail-larval schistosome system In vitro Larval transformation Snail hemolymph 0 hr 24 hr Miracidium Sporocyst Plasma ** ** Larval Transformation Proteins (LTP): 24-hr culture supernatant Hemocytes

7 In vitro snail-larval schistosome system In vitro Larval transformation Snail hemolymph 0 hr 24 hr Miracidium Sporocyst ** ** ** Hemocytes

8 In vitro snail-larval schistosome system In vitro Larval transformation Snail hemolymph 0 hr 24 hr Miracidium Sporocyst Plasma Larval Transformation Proteins (LTP): 24-hr culture supernatant Hemocytes

9 Proteogenomics: Genome annotation approach that combines proteomics and genomics Proteins of interest are subjected to proteomic analysis: nanolc-ms/ms Peptide sequences are searched against protein databases in nrncbi protein db and a 6-frame translation of the B. glabrata genome Sequence and location of genes encoding identified proteins are annotate within the genome Database Search Genome Annotation peptides LC separation Fragmentation MS/MS scan Peptide sequences 6-frame translated snail genome Protein ID Protein db (NCBI)

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11 Proteogenomics: Genome annotation approach that combines proteomics and genomics Genome annotation Miracidium Larval Transformation Proteins (LTP): 24-hr culture supernatant Sporocyst?? Plasma Protein ID Other applications of proteogenomic data Proteins as molecular phenotypes Identification of expressed proteins provide insights into their function Analysis of systems with complex molecular interactions (parasite-host)

12 Larval target tissue preparation: Affinity matrices Using biotinylated sporocyst proteins as Baits or Capture proteins b b b Subcellular fractionation of sporocyst proteins: (ProteoExtract Proteome Extraction) b b F1: Cytosol F2: Memb F3: Nucl F4: Cytoskel b b b b b Harvest larval transformation proteins (LTP) from culture supernatant -> biotinylate LTP F2 [ Membrane-enriched Fraction ]

13 Affinity isolation of larval-reactive snail plasma proteins: NMRI vs. BS-90 B. glabrata comparison Strepavidinbeads Biotinyl-F2 or -LTP NMRI Plasma BS-90 Streptavidinagarose column Streptavidin + biotinyl-proteins Plasma protein capture Plasma protein elution Peptides In-liquid digestion of eluted proteins Mass Spectrometry (LC-MS/MS) Proteomic Analysis* *B. glabrata genome using an in-house Mascot search engine

14 Summary of plasma proteins recovered from larval affinity columns F2 (Sporocyst membrane matrix) NMRI BS-90 # unique peptides # proteins ID # unidentified proteins 4 3 LTP (Larval Transform. Protein matrix) # unique peptides # proteins ID # unidentified proteins 3 4

15 Immune-related plasma proteins recovered from larval affinity columns Fibrinogen-related proteins (FREPS) Protein ID Membrane (F2)-reactive LTP-reactive NMRI BS-90 NMRI BS-90 FREP (4) FERP (2) 1 (2) FERP (6) 2 (5) FERP 5 4 (8) 3 (9) 13 (53) 11 (56) FERP 7 2 (4) 3 (5) 1 (1) 3 (9) FERP 12 7 (24) 9 (25) 13 (41) 11 (39) *FERP 12.1-pre [BGLB000116] (22) 0 FREP (2) 2 (3)

16 Immune-related plasma proteins recovered from larval affinity columns Other lectins/adhesion proteins Protein ID Membrane (F2)-reactive LTP-reactive NMRI BS-90 NMRI BS-90 *GREP 0 5 (13) 0 9 (40) (Galectin-relate protein) CREP 8 (27) 8 (21) 7 (23) 3 (3) (C-type lectin-related protein) Hemagglutinin/aggregation 2 (3) 1 (2) 10 (25) 1 (2) factor [Aplysia] Agglutinin-HPA type [Helix] (23) 5 (18) Dermatopontin 2-like (4) 3 (3) TEP (3) 2 (2) (Thioester-containing protein)

17 FREPs, GREPs and CREPs New families of immune lectins Signal peptide IgSF 1 Ig superfamily domain IgSF 2 Lectin domain FREP Fibrinogen-binding domain GREP Galectin-CRD CREP C-type lectin-crd

18 Proportion (%) of unique Frep vs. non-frep lectin peptides compared to total identified pool of unique peptides identified by tandem MS analyses Membrane LTP NMRI BS-90 NMRI BS-90 FREPs 15/74 (20%) 14/54 (26%) 35/74 (47%) 30/54 (56%) GREPs 0/74 (0%) 5/54 ( 9%) 0/74 (0%) 9/54 (17%) CREPs 8/74 (11%) 8/54 (15 %) 7/74 (10%) 3/54 (6%)

19 Lectin-like proteins differentially expressed by S. mansonisusceptible (NMRI) and resistant (BS-90) snails Protein ID Membrane (F2)-reactive LTP-reactive 90 NMRI BS-90 NMRI BS- GREP 0 5 (13) 0 9 (40) (Galectin-relate protein) FERP 12.1-precursor (22) 0 (BGLB000116)

20 Differential expression of transcripts encoding GREP-like sequences between resistant BS-90 and susceptible NMRI strains of B. glabrata GREP ~8 mm X 10 a-actinin RNA isolation cdna syn GREP a-actinin PCR agarose gel

21 Differential expression of transcripts encoding FREP 12.1 [Scaffold 28775] sequences between BS-90 and NMRI strains of B. glabrata. 10 NMRI snails 10 BS-90 snails FREP 12.1 pre a-actinin

22 Summary and Conclusions Proteogenomics is an effective approach to genome annotation and identifying proteins involved in complex host-parasite interactions Using a affinity chromatography and proteomic analyses novel plasma FREP variants have been identified, plus other immune-related molecules GREP, CREP Differential expression and binding of selected GREP and FREPs to larval proteins suggest a functional role in determining host compatibility and infection outcomes Currently we are applying this comparative proteomics approach to S and R strain hemocytes during in vitro encapsulation using Laser-capture microdissection

23 Acknowledgements X. J. Wu N. Dinguirard U. Bickham-Wright L. A. Gonzalez J. Chung G. Sabat - LC-MS/MS Funding support: NIH, NIAID Snail and parasites provided by the Schistosome Research Reagent Resource Center (BRI/BEI Resources, NIAID) Prof. Paron Dekumyoy and Dept of Helminthology Mahidol University Royal Thai Government

24 Acknowledgements X. J. Wu N. Dinguirard U. Bickham-Wright L. A. Gonzalez J. Chung G. Sabat - Mass Spectroscopy and Proteomic Facility UW-Madison Biotechnology Center Funding support: NIH, NIAID Snail and parasites provided by the Schistosome Research Reagent Resource Center (BEI Resources, NIAID) Prof. Paron Dekumyoy and Dept of Helminthology Mahidol University Royal Thai Government