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Host-Parasite Interactions of Cryptosporidium of Cryptosporidium Molecular Basis of Attachment and Invasion.

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Presentation on theme: "Host-Parasite Interactions of Cryptosporidium of Cryptosporidium Molecular Basis of Attachment and Invasion."— Presentation transcript:

1 Host-Parasite Interactions of Cryptosporidium of Cryptosporidium Molecular Basis of Attachment and Invasion

2 Ultrastructural Aspects of Cryptosporidium Attachment and Invasion Marcial and Madara, 1986; Lumb et al, 1988; Tzipori, 1988; Fayer et al, 1990, Yoshikawa and Iseki, 1992; Fayer et al, 1997; Griffiths and Tzipori, 1998, Zoites attach to host cells by their anterior pole Zoites attach to host cells by their anterior pole Rhoptries and micronemes discharge their contents Rhoptries and micronemes discharge their contents Electron-dense bands form in host cell cytoplasm Electron-dense bands form in host cell cytoplasm Zoites invaginate the host cell plasma membrane which eventually engulfs the parasite within the parasitophorus vacuole Zoites invaginate the host cell plasma membrane which eventually engulfs the parasite within the parasitophorus vacuole Parasite remains in parasitophorus vacuole in unique intracellular but extracytoplasmic location Parasite remains in parasitophorus vacuole in unique intracellular but extracytoplasmic location Unique feeder organelle membrane forms at the site of attachment Unique feeder organelle membrane forms at the site of attachment

3 Electron Micrograph of Cryptosporidium Sporozoite Attaching to Intestinal Microvillus Membrane Tzipori, 1988

4 Electron Micrograph of Cryptosporidium Merozoite Invading Intestinal Epithelial Cell Membrane Tzipori, 1988

5 Factors affecting Cryptosporidium sporozoite attachment in vitro Hamer et al, 1994; Joe et al, 1998; Chen et al 1998; Chen et al 2000 Time Time Number of sporozoites Number of sporozoites Temperature Temperature Divalent cations Divalent cations pH pH Host cell type Host cell type Differentiation status of host cells Differentiation status of host cells Host plasma membrane domain Host plasma membrane domain

6 Role of Parasite and Host Cytoskeletal Elements in Cryptosporidium Motility, Attachment and Invasion in vitro Forney et al, 1998, Forney et al, 1999, Yu and Lee, 1996; Bonin et al, 1999, Chen et al 2000, Elliot and Clark, 2000 Sporozoite motility is powered by actin- myosin motor system Sporozoite motility is powered by actin- myosin motor system Host cell actin is recruited to the host-parasite interface during invasion. Host cell actin is recruited to the host-parasite interface during invasion. Filamentous actin is assembled into a plaque-like structure Filamentous actin is assembled into a plaque-like structure Host cytoskeletal molecules may be involved in parasitophorus vacuole formation Host cytoskeletal molecules may be involved in parasitophorus vacuole formation

7 Surface/Apical Proteins of Cryptosporidium >20 sporozoite surface proteins 11-~1300 kDa identified >20 sporozoite surface proteins 11-~1300 kDa identified Surface/apical proteins implicated in attachment and/or invasion Surface/apical proteins implicated in attachment and/or invasion many identified by antibodies which inhibit infection in vitro and/or in vivo in animal models many identified by antibodies which inhibit infection in vitro and/or in vivo in animal models many proteins glycosylated many proteins glycosylated many proteins shed in trails during gliding motility many proteins shed in trails during gliding motility

8 Surface/Apical Proteins of Cryptosporidium >200 kDa-~1300 kDa >200 kDa-~1300 kDa Petersen et al 1992; Doyle et al, 1993; Barnes et al, 1998; Langer and Riggs, 1996; Riggs, 1997; Riggs et al, 1997; Langer et al 1999; McDonald et al, 1995, Robert et al, 1994) kDa kDa Nesterenko et al, 1999; Cevallos et al, 2000; Strong et al, kDa kDa Ungar and Nash, 1986; Mead et al, 1988, Arrowood et al, 1989; Arrowood et al, 1991; Perryman et al, 1996; Perryman et al, 1999; Enriquez and Riggs, 1998; Lumb et al, 1989; Tilley et al, 1993; Tilley and Upton, kDa kDa Tilley et al, 1991; Tilley et al, 1993,; Tilley and Upton, 1994; Jenkins et al 1993; Jenkins and Fayer, 1995; Khramtsov et al, 1993; Sagodira, 1999; Gut and Nelson, 1994; Strong et al, 2000; Cevallos et al, 2000; El Shewy et al, 1994; Mead et al, 1988; Moss et al 1994, 1998; Reperant et al 1992, 1994; Peeters et al, 1992; Ortega- Mora et al 1994; Priest et al, 1999; Priest et al, 2000 TRAP C1 (Spano et al, 1998) TRAP C1 (Spano et al, 1998) Gal/GalNAc-specific lectin/s (Joe et al. 1994; Joe et al, 1998; Chen et al, 2000) Gal/GalNAc-specific lectin/s (Joe et al. 1994; Joe et al, 1998; Chen et al, 2000)

9 Effect of MAb 4E9 IgM on C. parvum infection of Caco-2A cells Infection (A405nm) Cevallos et al, 2000 IgM µg/ml 4E9 B9A4

10 Effect of MAb 4E9 IgM on C. parvum infection of neonatal Balb/c mice No. of oocysts/5µl Hamer, Ward and Tzipori,

11 Reactivity of MAb 4E9 with C. parvum developmental stages by immunofluorescence Cevallos et al, 2000

12 Reactivity of MAb 4E9 with C. parvum developmental stages by immunoelectron microscopy Cevallos et al, 2000

13 12kDa Immunoblot analysis of C. parvum antigens recognized by MAb 4E9 1, Oocysts 2, Sporozoites Cevallos et al, 2000

14 GP900 >900kDa glycoprotein present in sporozoites and merozoites; shed from surface of sporozoites during gliding motility >900kDa glycoprotein present in sporozoites and merozoites; shed from surface of sporozoites during gliding motility localized to micronemes of invasive stages by IEM localized to micronemes of invasive stages by IEM encoded by 7.5kb gene locus, 5.5kb ORF, corresponding to predicted 1832 amino acid proteinencoded by 7.5kb gene locus, 5.5kb ORF, corresponding to predicted 1832 amino acid protein deduced amino acid sequence shows a mucin-like protein containing cysteine-rich and polythreonine domains deduced amino acid sequence shows a mucin-like protein containing cysteine-rich and polythreonine domains native GP900 binds to intestinal epithelial cells and competitively inhibits infection in vitro native GP900 binds to intestinal epithelial cells and competitively inhibits infection in vitro cysteine-rich domain of recombinant GP900 as well as antibodies to it inhibit infection in vitro cysteine-rich domain of recombinant GP900 as well as antibodies to it inhibit infection in vitro Petersen et al 1992, Barnes et al, 1998, Ward and Cevallos, 1998 Petersen et al 1992, Barnes et al, 1998, Ward and Cevallos, 1998

15 CSL (circumsporozoite precipitate-like) glycoprotein identified by surface and apical-reactive MAbs C4A1, 3E2 identified by surface and apical-reactive MAbs C4A1, 3E2 localized to surface and apical region (micronemes and dense bodies) of sporozoites and merozoites localized to surface and apical region (micronemes and dense bodies) of sporozoites and merozoites MAb 3E2 elicits CSP-like reaction (formation, posterior movement and release of membraneous Ag-MAb precipitates MAb 3E2 elicits CSP-like reaction (formation, posterior movement and release of membraneous Ag-MAb precipitates MAb 3E2 neutralizes sporozoite infectivity and prevents infection in neonatal Balb/c mice MAb 3E2 neutralizes sporozoite infectivity and prevents infection in neonatal Balb/c mice soluble glycoprotein exoantigen comprised of multiple ~1300 kDa molecular species with differing pI’s soluble glycoprotein exoantigen comprised of multiple ~1300 kDa molecular species with differing pI’s isolated native CSL binds to intestinal epithelial cells and inhibits sporozoite attachment to and invasion of these cellsisolated native CSL binds to intestinal epithelial cells and inhibits sporozoite attachment to and invasion of these cells Langer and Riggs, 1996, Riggs, 1997, Riggs et al, 1997, Langer et al 1999, Langer and Riggs, 1996, Riggs, 1997, Riggs et al, 1997, Langer et al 1999,

16 kDa kDa14.3 Silver stain 4E9 Immunoblot 1, total proteins 2, shed proteins Reactivity of MAb 4E9 with C. parvum “shed” proteins Cevallos et al, 2000

17 GP900 is not related to gp40 MAb4E9anti-gp GP900 gp kDakDa Silver stain of HPA-affinity purified glycoproteins Immunoblot of GalNAc eluate lysateeffluentGalNAceluate Cevallos et al, 2000

18 gp40-specific antisera inhibit C. parvum infection of intestinal epithelial Caco 2A cells Infection (A405nm) Cevallos et al, 2000

19  -galactosidase HPA-glycoproteins Shed proteins Binding (A405nm) gp40 binds to intestinal epithelial Caco 2A cells Cevallos et al, 2000

20 Analysis of Cpgp40/15 deduced amino acid sequence Signal peptide 326 aa /33.6 kDa protein O-glycosylation site GPI anchor site Polyserine region N-glycosylation site 981 bp Cevallos et al, 2000

21 Analysis of Cpgp40/15 deduced amino acid sequence gp40 N-terminus gp15 N-terminus Cpgp40/15 MRLSLIIVLLSVIVSAVFSAPAVPLRGTLKDVPVEGSSSSSSSSSSSSSSSSSSSTSTVAPA NKARTGEDAEGSQDSSGTEASGSQGSEEEGSEDDGQTSAASQPTTPAQSEGATTETI EATPKEECGTSFVMWFGEGTPAATLKCGAYTIVYAPIKDQTDPAPRYISGEVTSVTF EKSDNTVKIKVNGQDFSTLSANSSSPTENGGSAGQASSRSRRSLSEETSEAAATVDLF AFTLDGGKRIEVAVPNVEDASKRDKYSLVADDKPFYTGANSGTTNGVYRLNENGDL VDKDNTVLLKDAGSSAFGLRYIVPSVFAIFAALFVL gp40 N-terminus gp15 N-terminus

22 gp15/17 kDa immunodominant antigen 15 kDa protein localized to surface of sporozoites and merozoites and shed in “trails” during gliding motility 15 kDa protein localized to surface of sporozoites and merozoites and shed in “trails” during gliding motility contains  GalNAc residuescontains  GalNAc residues Gut and Nelson, 1994; Strong et al kDa protein localized to surface of sporozoites and merozoites 15 kDa protein localized to surface of sporozoites and merozoites recognized by IgA MAbs CrA1 and CrA2 which are partially protective against C. parvum infection in scid mouse backpack tumor modelrecognized by IgA MAbs CrA1 and CrA2 which are partially protective against C. parvum infection in scid mouse backpack tumor model Zhou et al, Cevallos et al kDa immunodominant antigen recognized by serum antibodies from infected humans 17 kDa immunodominant antigen recognized by serum antibodies from infected humans present in TX-114 extracts of sonicated oocysts present in TX-114 extracts of sonicated oocysts Priest et al, 1999, Priest et al, 2000

23 123kDa kDa CrA1anti-gp15 anti-gp40 gp40 and gp15 are antigenically distinct proteins 1, oocysts 2, sporozoites 3, shed proteins Cevallos et al, 2000

24 CrA1anti-gp15 kDakDa anti-gp40 Antibodies to native gp40 and gp15 recognize the corresponding recombinant fusion proteins 1, control 2, r40/15 3, r15 4, r40 Cevallos et al, 2000

25 SporozoitesMerozoites Reactivity of anti-gp40 antisera with C. parvum sporozoites and merozoites by immunofluorescence Cevallos et al, Infect. Immun 68: , 2000

26 SporozoitesMerozoites Reactivity of MAb CrA1 (anti-gp15) with C. parvum sporozoites and merozoites by immunofluorescence Cevallos et al, 2000

27 kDa kDa anti-gp40 anti-gp15anti-r40anti-r15 gp40 and gp15 are products of proteolytic cleavage of a 49kDA precursor protein Cevallos et al, 2000

28 Polymorphisms at gp15/45/60 locus sequence analysis of PCR amplified gp15/45/60 ORF from 29 diverse C. parvum isolates magnitude of sequence polymorphism identified at this locus is far greater than that detected at any other C. parvum locus identified to date magnitude of sequence polymorphism identified at this locus is far greater than that detected at any other C. parvum locus identified to date 77-88% nucleotide sequence identity 77-88% nucleotide sequence identity 67 to 80% amino acid sequence identity 67 to 80% amino acid sequence identity numerous SNPs and SAAPs in these sequences defined at least 5 distinct allelic groupings numerous SNPs and SAAPs in these sequences defined at least 5 distinct allelic groupings Ia, Ib, Ic, Id, (human/genotype I) Ia, Ib, Ic, Id, (human/genotype I) II (calf/genotype II) II (calf/genotype II) conserved regions conserved regions putative signal peptide putative signal peptide putative GPI anchor site putative GPI anchor site putative proteolytic processing site putative proteolytic processing site Strong et al, 2000

29 Comparison of Type II (calf) and Type I (human) Cpgp40/15 deduced AA sequences 69% identity at amino acid level, 84% identity at nucleotide level

30 Southern blot analysis of Cpgp40/15 EcoRI HindIII PstI SspI EcoRI/HindII 23, EcoRI HindIII PstI SspI EcoRI/HindII 23, Type II GCH1 Type I UG502

31 Reactivity of anti-gp40 antibodies with Type I (GCH1) and Type II (UG502) isolates kDa I II I II I II

32 Genotyping of C. parvum isolates from HIV-Infected Children with Persistent Diarrhea in South Africa bp bp HHHCH HH HCH?HHCC C. parvum DNA PCR amplified from 21/24 stool samples C. parvum DNA PCR amplified from 21/24 stool samples Genotype of isolates determined by PCR-RFLP at TRAP C1 and COWP loci Genotype of isolates determined by PCR-RFLP at TRAP C1 and COWP loci 16/21 (76%) of isolates were of the human genotype at both loci 16/21 (76%) of isolates were of the human genotype at both loci PCR amplification of Cpgp40/15 locus PCR amplification of Cpgp40/15 locus

33 gp40  gp40 is a mucin-like glycoprotein containing terminal  GalNAc residues F gp40-specific antibodies neutralize infection in vitro and gp40 binds to intestinal epithelial cells. F The gene encoding gp40 also encodes gp15, an antigenically distinct protein. F gp40 is localized to the surface and apical region of invasive stages. F gp40 and gp15, are products of proteolytic cleavage of a 49 kDa precursor protein expressed in intracellular stages. F The Cpgp40/15 locus is highly polymorphic

34 New England Medical Center Tufts University, Boston Ana Maria Cevallos Najma Bhat Smitha Jaison Brett Leav Roberta O’Connor Renaud Verdon David Hamer Xiaoping Zhang Matt Waldor Gerald Keusch Miercio Pereira Children’s Hospital, Harvard University, Boston Marian Neutra Xiaoyin Zhou University of California San Francisco Carolyn Petersen Bill Strong Richard Nelson University of Texas, Houston Sara Dann Cynthia Chappell University of Natal, Durban, Africa Centre for Health and Population Research, Mtubatuba Michael Bennish Nigel Rollins CDC, Atlanta Jeff Priest Tufts University School of Veterinary Medicine, Grafton Barry Stein Giovanni Widmer Donna Akiyoshi Inderpal Singh Cindy Theodos Saul Tzipori ACKNOWLEDGEMENTS


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