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Host-Parasite Interactions of Cryptosporidium Molecular Basis of Attachment and Invasion Ultrastructural Aspects of Cryptosporidium Attachment and Invasion • • Zoites attach to host cells by their anterior pole Rhoptries and micronemes discharge their contents • • Electron-dense bands form in host cell cytoplasm 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 Unique feeder organelle membrane forms at the site of attachment • 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, Electron Micrograph of Cryptosporidium Sporozoite Attaching to Intestinal Microvillus Membrane Tzipori, 1988 Electron Micrograph of Cryptosporidium Merozoite Invading Intestinal Epithelial Cell Membrane Tzipori, 1988 Factors affecting Cryptosporidium sporozoite attachment in vitro • Time • Number of sporozoites • Temperature • Divalent cations • pH • Host cell type • Differentiation status of host cells • Host plasma membrane domain Hamer et al, 1994; Joe et al, 1998; Chen et al 1998; Chen et al 2000 Role of Parasite and Host Cytoskeletal Elements in Cryptosporidium Motility, Attachment and Invasion in vitro • Sporozoite motility is powered by actinmyosin motor system • Host cell actin is recruited to the host-parasite interface during invasion. • Filamentous actin is assembled into a plaque-like structure • Host cytoskeletal molecules may be involved in parasitophorus vacuole formation Forney et al, 1998, Forney et al, 1999, Yu and Lee, 1996; Bonin et al, 1999, Chen et al 2000, Elliot and Clark, 2000 Surface/Apical Proteins of Cryptosporidium • >20 sporozoite surface proteins 11-~1300 kDa identified • 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 proteins glycosylated • many proteins shed in trails during gliding motility Surface/Apical Proteins of Cryptosporidium • >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) • 40-47 kDa Nesterenko et al, 1999; Cevallos et al, 2000; Strong et al, 2000 • 20-27 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, 1994 • 15-17 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; OrtegaMora et al 1994; Priest et al, 1999; Priest et al, 2000 • TRAP C1 (Spano et al, 1998) • Gal/GalNAc-specific lectin/s (Joe et al. 1994; Joe et al, 1998; Chen et al, 2000) Effect of MAb 4E9 IgM on C. parvum infection of Caco-2A cells Infection (A405nm) 0.5 4E9 B9A4 0.4 0.3 0.2 0.1 0.0 100 100 50 10 IgM µg/ml Cevallos et al, 2000 5 1 Effect of MAb 4E9 IgM on C. parvum infection of neonatal Balb/c mice No. of oocysts/5µl 30 25 20 15 10 5 0 Control Hamer, Ward and Tzipori, MAb 4E9 Reactivity of MAb 4E9 with C. parvum developmental stages by immunofluorescence Cevallos et al, 2000 Reactivity of MAb 4E9 with C. parvum developmental stages by immunoelectron microscopy Cevallos et al, 2000 Immunoblot analysis of C. parvum antigens recognized by MAb 4E9 kDa 200 116.3 97.4 66.2 45 31 1, Oocysts 2, Sporozoites Cevallos et al, 2000 21.5 1 2 GP900 • >900kDa glycoprotein present in sporozoites and merozoites; shed from surface of sporozoites during gliding motility • localized to micronemes of invasive stages by IEM • encoded 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 • 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 Petersen et al 1992, Barnes et al, 1998, Ward and Cevallos, 1998 CSL (circumsporozoite precipitate-like) glycoprotein • identified by surface and apical-reactive MAbs C4A1, 3E2 • 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 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 • isolated 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, Reactivity of MAb 4E9 with C. parvum “shed” proteins kDa 1 2 200 2 97.4 69 46 46 30 21.5 14.3 30 21.5 14.3 Silver stain Cevallos et al, 2000 1 200 97.4 69 1, total proteins 2, shed proteins kDa 4E9 Immunoblot GP900 is not related to gp40 kDa kDa GP900 225 225 150 150 100 75 100 50 50 75 gp40 35 35 25 25 15 15 lysate effluent GalNAc eluate Silver stain of HPA-affinity purified glycoproteins Cevallos et al, 2000 MAb anti4E9 gp40 Immunoblot of GalNAc eluate gp40-specific antisera inhibit C. parvum infection of intestinal epithelial Caco 2A cells 0.25 Infection (A405nm) 0.20 0.15 0.10 0.05 0.00 preimmune-1 Cevallos et al, 2000 anti-gp40 1 preimmune-2 anti-gp40 2 gp40 binds to intestinal epithelial Caco 2A cells Binding (A405nm) 1.60 Shed proteins 1.20 HPA-glycoproteins 0.80 0.40 0.00 0.00 b-galactosidase 0.50 1.00 1.50 gp40 (µg/ml) Cevallos et al, 2000 2.00 Analysis of Cpgp40/15 deduced amino acid sequence 981 bp 326 aa /33.6 kDa protein Signal Polyserine peptide region GPI anchor site N-glycosylation site O-glycosylation site Cevallos et al, 2000 Analysis of Cpgp40/15 deduced amino acid sequence gp40 N-terminus MRLSLIIVLLSVIVSAVFSAPAVPLRGTLKDVPVEGSSSSSSSSSSSSSSSSSSSTSTVAPA NKARTGEDAEGSQDSSGTEASGSQGSEEEGSEDDGQTSAASQPTTPAQSEGATTETI EATPKEECGTSFVMWFGEGTPAATLKCGAYTIVYAPIKDQTDPAPRYISGEVTSVTF gp15 N-terminus EKSDNTVKIKVNGQDFSTLSANSSSPTENGGSAGQASSRSRRSLSEETSEAAATVDLF AFTLDGGKRIEVAVPNVEDASKRDKYSLVADDKPFYTGANSGTTNGVYRLNENGDL VDKDNTVLLKDAGSSAFGLRYIVPSVFAIFAALFVL Cpgp40/15 gp40 N-terminus gp15 N-terminus gp15/17 kDa immunodominant antigen Gut and Nelson, 1994; Strong et al 2000 • 15 kDa protein localized to surface of sporozoites and merozoites and shed in “trails” during gliding motility • contains aGalNAc residues Zhou et al, Cevallos et al 2000 • • 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 model Priest et al, 1999, Priest et al, 2000 • • 17 kDa immunodominant antigen recognized by serum antibodies from infected humans present in TX-114 extracts of sonicated oocysts gp40 and gp15 are antigenically distinct proteins kDa 225 150 1 2 3 kDa 1 2 3 225 150 100 75 50 100 75 50 35 35 25 25 15 15 anti-gp40 1, oocysts 2, sporozoites 3, shed proteins CrA1 anti-gp15 Cevallos et al, 2000 Antibodies to native gp40 and gp15 recognize the corresponding recombinant fusion proteins kDa 1 2 3 4 kDa 150 100 75 50 150 35 35 1 2 3 4 100 75 50 25 25 15 15 1, control 2, r40/15 3, r15 4, r40 CrA1 anti-gp15 anti-gp40 Cevallos et al, 2000 Reactivity of anti-gp40 antisera with C. parvum sporozoites and merozoites by immunofluorescence Sporozoites Cevallos et al, Infect. Immun 68: 4108-4116, 2000 Merozoites Reactivity of MAb CrA1 (anti-gp15) with C. parvum sporozoites and merozoites by immunofluorescence Sporozoites Cevallos et al, 2000 Merozoites gp40 and gp15 are products of proteolytic cleavage of a 49kDA precursor protein kDa 1 2 1 2 225 150 100 75 50 35 25 225 150 100 75 50 35 25 15 15 anti- antigp40 gp15 Cevallos et al, 2000 kDa 1 2 antir40 1 2 antir15 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 • 77-88% nucleotide sequence identity • 67 to 80% amino acid sequence identity • numerous SNPs and SAAPs in these sequences defined at least 5 distinct allelic groupings • Ia, Ib, Ic, Id, (human/genotype I) • II (calf/genotype II) • conserved regions • putative signal peptide • putative GPI anchor site • putative proteolytic processing site Strong et al, 2000 Comparison of Type II (calf) and Type I (human) Cpgp40/15 deduced AA sequences human gp 40/15 calf gp40/15 1 DVPVEGSSSSSSSSSSSSSSSSSSSSSSSSSTSTVAPAPK 1 DVPVEGSSSSSSSSSSSSSSSS------SSSTSTVAPAN********************** ********** 40 33 human gp 40/15 calf gp40/15 41 KERTVEGGTEGKNEESSPGSEEQDGGKEDGGKENGEGDTV 34 KARTGEDAE-GSQDSSGTEASGSQGSEEEGSEDDG----Q * ** * * .. * . * *.* . * 80 68 human gp 40/15 calf gp40/15 81 DGEQTGSGSQVTPSGSAGTATESTATTTPKEECGTSFVMW 120 69 ----TSAASQPTTPAQSEGATTETIEATPKEECGTSFVMW 104 * . ** * . ** * .************* human gp 40/15 calf gp40/15 121 FEKGTPVATLKCGDYTIVYAPIKDQTDPAPRYISGEVTSV 160 105 FGEGTPAATLKCGAYTIVYAPIKDQTDPAPRYISGEVTSV 144 * *** ****** ************************** human gp 40/15 calf gp40/15 161 SFEKSESTVTIKVNGKEFSTLSANSSSPTKDNGESSDSQV 200 145 TFEKSDNTVKIKVNGQDFSTLSANSSSPTENGG--SAGQA 182 .****. ** *****..************ .* * * human gp 40/15 calf gp40/15 201 QSRSRRSLAEENGETVATVDLFAFTLDGGRRIEVAVPKDE 240 183 SSRSRRSLSEETSEAAATVDLFAFTLDGGKRIEVAVPNVE 222 *******.**. *. *************.******* * human gp 40/15 calf gp40/15 241 NADKRSEYSLVADDKPFYTGANSGITNGVYKLDENGNLVD 280 223 DASKRDKYSLVADDKPFYTGANSGTTNGVYRLNENGDLVD 262 * ** ***************** *****.* *** *** human gp 40/15 calf gp40/15 281 KDNKVLLKDAGSSAFGFRYIVPSVFAIFAALFVL 314 263 KDNTVLLKDAGSSAFGLRYIVPSVFAIFAALFVL 296 *** ************ ***************** 69% identity at amino acid level, 84% identity at nucleotide level SspI EcoRI/HindII HindIII PstI EcoRI SspI EcoRI/HindII HindIII PstI EcoRI Southern blot analysis of Cpgp40/15 23,130 23,130 9416 6557 4361 9416 6557 4361 2322 2027 2322 2027 564 Type II GCH1 Type I UG502 Reactivity of anti-gp40 antibodies with Type I (GCH1) and Type II (UG502) isolates I kDa 225 150 100 75 50 35 25 15 II I II I II Genotyping of C. parvum isolates from HIV-Infected Children with Persistent Diarrhea in South Africa • C. parvum DNA PCR amplified from 21/24 stool samples • 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 • PCR amplification of Cpgp40/15 locus bp 22 23 24 25 26 27 28 bp 14 15 16 17 18 19 20 21 2.0 1.5 1.0 0.6 2.0 1.5 1.0 0.6 H H H C H H H C H C H ? H H C gp40 gp40 is a mucin-like glycoprotein containing terminal aGalNAc residues gp40-specific antibodies neutralize infection in vitro and gp40 binds to intestinal epithelial cells. The gene encoding gp40 also encodes gp15, an antigenically distinct protein. gp40 is localized to the surface and apical region of invasive stages. gp40 and gp15, are products of proteolytic cleavage of a 49 kDa precursor protein expressed in intracellular stages. The Cpgp40/15 locus is highly polymorphic ACKNOWLEDGEMENTS New England Medical Center Tufts University, Boston Ana Maria Cevallos Najma Bhat Smitha Jaison Brett Leav Roberta O’Connor Renaud Verdon David Hamer Children’s Hospital, Harvard University, Boston Marian Neutra Xiaoyin Zhou University of California San Francisco Carolyn Petersen Xiaoping Zhang Matt Waldor Bill Strong Richard Nelson Gerald Keusch Miercio Pereira University of Natal, Durban, Africa Centre for Health and Population Research, Mtubatuba Tufts University School of Veterinary Medicine, Grafton Barry Stein Giovanni Widmer Donna Akiyoshi Inderpal Singh Cindy Theodos Saul Tzipori Michael Bennish Nigel Rollins University of Texas, Houston Sara Dann Cynthia Chappell CDC, Atlanta Jeff Priest