Carlos Chagas CHAGAS DISEASE. Walter Colli Institute of Chemistry University of São Paulo

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1 Carlos Chagas CHAGAS DISEASE Walter Colli Institute of Chemistry University of São Paulo

2 Chagas disease in the U.S. Chagas disease is an emerging disease in the U.S. (and Europe as well): migration of chronically infected individuals from endemic countries no regular screening in blood banks and hospitals (few exceptions) November 2008 November 2009 Confirmed cases: (54% increase!!) Continental U.S. Map: RIPA Positives (January 2007 to present) AABB Chagas' Biovigilance Network:

3 ASSASSIN BUGS Panstrongilus megistus Triatoma infestans Triatoma maculata Panstrongilus geniculatus Triatoma brasiliensis Triatoma sordida Rhodnius prolixus

4 CHAGOMA

5 Leslie, M (2011) Drug developers finally take aim at a Neglected Disease. Science 333,

6 BENZNIDAZOLE N-benzyl-2-(2-nitro-1H-imidazol-1-yl)acetamide NIFURTIMOX (RS)-3-methyl-N-[(1E)-(5-nitro-2-furyl)methylene] thiomorpholin-4-amine 1,1-dioxide

7 THE PARASITE TRYPANOSOMA CRUZI

8 TRYPANOSOMA CRUZI CYCLE

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10 Molecules involved in invasion by T. cruzi Adhesion Invasion Lysosome-dependent Lysosome-independent Survival

11 T. cruzi Trypanosoma cruzi surface is covered by glycoconjugates Glycoconjugates Plasma membrane 60-80% (Glycoinositolphospholipid)

12 Trypanosoma cruzi GIPL 1

13 Major Surface Molecules of Trypanosoma cruzi Mucin Sialic acid galactose trans-sialidase/ gp85 glycoprotein Glycoinositolphospholipid (GIPL) Plasma Membrane Cytoplasm

14 Two-dimensional electrophoresis of 14 C-mannose labeled Tc-85 Couto, Katzin, Colli, Lederkremer (1987) Mol. Biochem. Parasitol. 26:145

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16 Overall Primary Structure of gp85/ts Family ~1,400 genes Frasch (2000) Parasitol. Today 16:282

17 Large gene families in T. cruzi Gene product Members (pseudogenes) Gp85/Trans-sialidase 1430 (693) MASP 1377 (433) Mucin 863 (201) Retrotransposon (RHS) 752 (557) Surface protease 425 (251) Mucin-like members encoding active TS 70 members encoding inactive TS TS-like/gp85 (subset Tc85) 86 phosphatases 190 protein kinases El Sayed et al. (2005) Science 309:409

18 Binding of laminin to purified Tc85-11 fusion protein Giordano et al. (1999) JBC. 274:3461

19 Identification of Host-Cell Binding Sites

20 C-terminal domain FLY binds to the surface of LLC-MK 2 in a specific and saturable manner (1.66 x 10 6 binding sites) Fly binds to tumor cells (B16F10), human umbilical cord endothelial cells (ECV), macrophagelike cell lines (J774), mouse fibroblast cells (3T3) Magdesian et al. (2001) JBC 276:19382

21 Alanine scanning VT-VFLY-R

22 FLY-beads bind to epithelial cells Magdesian et al. (2007) Exp Cell Res 313:210

23 Infected cells (%) FLY enhances infection of epithelial cells by T. cruzi Control FLY Infected cells (%) Control A 1xJ 2xJ Tc85-11 IgG Anti- J+Anti- CK18 CK18 Additions Magdesian et al. (2001) JBC 276:19382

24 FLY increases parasitemia and mortality FLY ~ 2

25 FLY increases tissue infection 3.0 FOLD 7.6 FOLD 3.6 FOLD

26 BINDING OF FLY TO THE VASCULATURE IN VIVO FLY phage (phage displaying the FLY peptide sequence) FAY-phage (phage displaying the FAY peptide sequence) fd-tet phage (control phage without the inserts)

27 FLY-phage binding to organ-derived endothelial cells Tonelli et al., Plosntds 4 (11) e864, Nov 2010 Tissue specific endothelial cells from H-2K(b)-tsA58 mouse were kindly provided by Isaiah J. Fidler

28 FLY-phage binding in vivo 0 30 min FLY- FAY- fd-tet phage (10 10 ) Perfusion procedure DNA extraction Quantification of phages

29 FLY-phage binding to the vasculature of the mouse Tonelli et al., Plosntds 4 (11) e864, Nov 2010

30 Conclusions 1-Members of the gp85/ts protein family bind to extracellular matrix elements 2-The C-terminal invariant sequence VTVXNVFLYNR binds to the surface membrane of host cells 3-The FLY sequence is a virulence factor to the host facilitating binding and internalization

31 Acknowledgements First phase ( ) Alejandro M. Katzin Bianca Zingales Norma W. Andrews Second phase ( ) Grace Abuin Ricardo Giordano Vera Kuwajima Third phase ( ) Ana Claudia Torrecilhas Ariel Silber Henning Ulrich Marinez Almeida-de-Faria Margaret Magdesian Melissa R. Fessel Miryam O. Marroquin-Quelopana Renata R. Tonelli Ricardo Giordano Collaborators A.Couto, UBA J. Manning, UCI E. Freymüller, UNIFESP I. Abrahamsohn, ICBUSP J. Santana da Silva, FMRP L. Juliano, UNIFESP M. A. Juliano, UNIFESP Mariana S. Silveira, UFRJ N. Cunha-e-Silva, UFRJ R. I. Schumacher, USP R. Chammas, USP R. Linden, UFRJ R. M. de Lederkremer, UBA S. Schenkman, UNIFESP S. S. Veiga, UFPr T. Pertinhez, LNLS Maria Júlia Manso Alves Technical support Marinei Ferreira Gonçalves Roberto Zangrandi Célia Ludio Braga

32 Thank you

33 Host cell invasion by T. cruzi may be the result of multiple molecular interactions with the ensuing activation of a number of signaling pathways De Souza, Carvalho & Barrias (2010) Int. J. Cell Biol. 2010, 1-18

34 Triazenos

35 FLY-phage interaction with intermediate filament proteins Tonelli et al., Plosntds 4 (11) e864, Nov 2010

36 Clones Consensus Displaced by Kd (nm) 8/24 12/24 13/28 8/20 GAUCCCC CGGACCAC(A/U)GAUGCCA UUGGGC AAGCGXGCXCC Laminin Fibronectin Heparan sulfate Thrombospondin

37 Elimination of Triatoma infestans Ministry of Health in Coura JC & Dias JCP (2009) Mem. Inst. Oswaldo Cruz 104, 31-40

38 Interruption of transmission of Chagas disease Southern cone countries Moncayo, A. & Silveira, A.C. (2009) Mem. Inst. Oswaldo Cruz, 104, 17-30

39 Trypomastigote vesicles Torrecilhas et al (2009) Microbes Infect 11, 29

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