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

Host-Parasite Interactions of Cryptosporidium

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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 - PowerPoint PPT Presentation

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Page 1: Host-Parasite Interactions of  Cryptosporidium

Host-Parasite InteractionsHost-Parasite Interactions ofof Cryptosporidium Cryptosporidium

Molecular Basis of Attachment Molecular Basis of Attachment and Invasionand Invasion

Page 2: Host-Parasite Interactions of  Cryptosporidium

Ultrastructural Aspects of Ultrastructural Aspects of Cryptosporidium Cryptosporidium Attachment and Invasion Attachment and Invasion

Marcial and Madara, 1986; Lumb et al, 1988; Tzipori, 1988; Fayer et al, 1990, Yoshikawa and 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, Iseki, 1992; Fayer et al, 1997; Griffiths and Tzipori, 1998,

• Zoites attach to host cells by their anterior poleZoites attach to host cells by their anterior pole

• Rhoptries and micronemes discharge their Rhoptries and micronemes discharge their contents contents

• Electron-dense bands form in host cell cytoplasmElectron-dense bands form in host cell cytoplasm

• Zoites invaginate the host cell plasma membrane Zoites invaginate the host cell plasma membrane which eventually engulfs the parasite within the which eventually engulfs the parasite within the parasitophorus vacuoleparasitophorus vacuole

• Parasite remains in parasitophorus vacuole in Parasite remains in parasitophorus vacuole in unique intracellular but extracytoplasmic unique intracellular but extracytoplasmic locationlocation

• Unique feeder organelle membrane forms at the site Unique feeder organelle membrane forms at the site of attachmentof attachment

Page 3: Host-Parasite Interactions of  Cryptosporidium

Electron Micrograph of Electron Micrograph of Cryptosporidium Cryptosporidium Sporozoite Sporozoite Attaching to Intestinal Microvillus MembraneAttaching to Intestinal Microvillus Membrane

Tzipori, 1988Tzipori, 1988

Page 4: Host-Parasite Interactions of  Cryptosporidium

Electron Micrograph of Electron Micrograph of Cryptosporidium Cryptosporidium MerozoiteMerozoite Invading Intestinal Epithelial Cell MembraneInvading Intestinal Epithelial Cell Membrane

Tzipori, 1988Tzipori, 1988

Page 5: Host-Parasite Interactions of  Cryptosporidium

Factors affecting Factors affecting Cryptosporidium Cryptosporidium sporozoite attachment in vitro sporozoite attachment in vitro

Hamer et al, 1994; Joe et al, 1998; Chen et al 1998; Chen et al 2000Hamer et al, 1994; Joe et al, 1998; Chen et al 1998; Chen et al 2000

• TimeTime

• Number of sporozoitesNumber of sporozoites

• TemperatureTemperature

• Divalent cations Divalent cations

• pHpH

• Host cell typeHost cell type

• Differentiation status of host Differentiation status of host cellscells

• Host plasma membrane domainHost plasma membrane domain

Page 6: Host-Parasite Interactions of  Cryptosporidium

Role of Parasite and Host Cytoskeletal Elements Role of Parasite and Host Cytoskeletal Elements in in Cryptosporidium Cryptosporidium Motility, Attachment and Motility, Attachment and

Invasion in vitro Invasion in vitro

Forney et al, 1998, Forney et al, 1999, Yu and Lee, 1996; Bonin et al, 1999, Chen et al Forney et al, 1998, Forney et al, 1999, Yu and Lee, 1996; Bonin et al, 1999, Chen et al 2000, Elliot and Clark, 2000 2000, Elliot and Clark, 2000

• Sporozoite motility is powered by actin- Sporozoite motility is powered by actin- myosin motor systemmyosin motor system

• Host cell actin is recruited to the host-parasite Host cell actin is recruited to the host-parasite interface during invasion. interface during invasion.

• Filamentous actin is assembled into a plaque-like Filamentous actin is assembled into a plaque-like structure structure

• Host cytoskeletal molecules may be involved in Host cytoskeletal molecules may be involved in parasitophorus vacuole formationparasitophorus vacuole formation

Page 7: Host-Parasite Interactions of  Cryptosporidium

Surface/Apical Proteins of Surface/Apical Proteins of CryptosporidiumCryptosporidium

• >20 sporozoite surface proteins 11-~1300 kDa >20 sporozoite surface proteins 11-~1300 kDa identifiedidentified

• Surface/apical proteins implicated in attachment Surface/apical proteins implicated in attachment and/or invasionand/or invasion

• many identified by antibodies which inhibit many identified by antibodies which inhibit infection in vitro and/or in vivo in infection in vitro and/or in vivo in

animal animal modelsmodels

• many proteins glycosylated many proteins glycosylated

• many proteins shed in trails during gliding many proteins shed in trails during gliding motilitymotility

Page 8: Host-Parasite Interactions of  Cryptosporidium

Surface/Apical Proteins of Surface/Apical Proteins of CryptosporidiumCryptosporidium• >200 kDa-~1300 kDa>200 kDa-~1300 kDa

Petersen et al 1992; Doyle et al, 1993; Barnes et al, 1998Petersen et al 1992; Doyle et al, 1993; Barnes et al, 1998; ; Langer and Riggs, Langer and Riggs, 1996; 1996; Riggs, 1997; Riggs et al, 1997; Langer et al 1999; McDonald et al, 1995, Riggs, 1997; Riggs et al, 1997; Langer et al 1999; McDonald et al, 1995, Robert et Robert et al, 1994)al, 1994)

• 40-47 kDa40-47 kDa

Nesterenko et al, 1999; Cevallos et al, 2000; Strong et al, 2000Nesterenko et al, 1999; Cevallos et al, 2000; Strong et al, 2000

• 20-27 kDa20-27 kDa

Ungar and Nash, 1986; Mead et al, 1988, Arrowood et al, 1989; Arrowood et al, 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 1991; Perryman et al, 1996; Perryman et al, 1999; Enriquez and Riggs, 1998; Lumb et al, 1989; Tilley et al, 1993; Tilley and Upton, 1994et al, 1989; Tilley et al, 1993; Tilley and Upton, 1994

• 15-17 kDa15-17 kDa

Tilley et al, 1991; Tilley et al, 1993,; Tilley and Upton, 1994; Jenkins et al 1993; 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, 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, 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-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, 2000Mora et al 1994; Priest et al, 1999; Priest et al, 2000

• TRAP C1TRAP C1 (Spano et al, 1998)(Spano et al, 1998)

• Gal/GalNAc-specific lectin/sGal/GalNAc-specific lectin/s (Joe et al. 1994; Joe et al, 1998; Chen et al, 2000)(Joe et al. 1994; Joe et al, 1998; Chen et al, 2000)

Page 9: Host-Parasite Interactions of  Cryptosporidium

Effect of MAb 4E9 IgM on Effect of MAb 4E9 IgM on C. parvumC. parvum infection of Caco-2A cells infection of Caco-2A cells

Infe

ctio

n (

A40

5nm

)

Cevallos et al, 2000 Cevallos et al, 2000

IgM µg/ml

4E9 B9A4

0.0

0.1

0.2

0.3

0.4

0.5

100 100 50 10 5 1

Page 10: Host-Parasite Interactions of  Cryptosporidium

Effect of MAb 4E9 IgM on Effect of MAb 4E9 IgM on C. parvumC. parvum infection of neonatal Balb/c miceinfection of neonatal Balb/c mice

No.

of

oocy

sts/

5µl

Hamer, Ward and Tzipori, Hamer, Ward and Tzipori,

0

5

10

15

20

25

30

Control MAb 4E9

Page 11: Host-Parasite Interactions of  Cryptosporidium

11

Reactivity of MAb 4E9 with Reactivity of MAb 4E9 with C. parvumC. parvum developmental stages by immunofluorescencedevelopmental stages by immunofluorescence

Cevallos et al, 2000 Cevallos et al, 2000

Page 12: Host-Parasite Interactions of  Cryptosporidium

Reactivity of MAb 4E9 with Reactivity of MAb 4E9 with C. parvumC. parvum developmental stages by immunoelectron microscopydevelopmental stages by immunoelectron microscopy

Cevallos et al, 2000 Cevallos et al, 2000

Page 13: Host-Parasite Interactions of  Cryptosporidium

11 22kDakDa

200200

97.497.4

66.266.2

4545

3131

21.521.5

116.3116.3

Immunoblot analysis of Immunoblot analysis of C. parvumC. parvum antigens antigens recognized by MAb 4E9recognized by MAb 4E9

1, Oocysts1, Oocysts2, Sporozoites2, Sporozoites

Cevallos et al, 2000 Cevallos et al, 2000

Page 14: Host-Parasite Interactions of  Cryptosporidium

14

GP900GP900• >900kDa glycoprotein present in sporozoites and merozoites; >900kDa glycoprotein present in sporozoites and merozoites;

shed from surface of sporozoites during gliding motility shed from surface of sporozoites during gliding motility

• localized to micronemes of invasive stages by IEMlocalized to micronemes of invasive stages by IEM

• encoded by 7.5kb gene locus, 5.5kb ORF, corresponding to encoded by 7.5kb gene locus, 5.5kb ORF, corresponding to predicted 1832 amino acid protein predicted 1832 amino acid protein

• deduced amino acid sequence shows a mucin-like protein deduced amino acid sequence shows a mucin-like protein containing cysteine-rich and polythreonine domains containing cysteine-rich and polythreonine domains

• native GP900 binds to intestinal epithelial cells and native GP900 binds to intestinal epithelial cells and competitively competitively inhibits infection in vitroinhibits infection in vitro

• cysteine-rich domain of recombinant GP900 as well as cysteine-rich domain of recombinant GP900 as well as antibodies to it inhibit infection in vitroantibodies to it inhibit infection in vitro

Petersen et al 1992, Barnes et al, 1998, Ward and Cevallos, 1998Petersen et al 1992, Barnes et al, 1998, Ward and Cevallos, 1998

Page 15: Host-Parasite Interactions of  Cryptosporidium

15

CSL (circumsporozoite precipitate-like) glycoproteinCSL (circumsporozoite precipitate-like) glycoprotein

• identified by surface and apical-reactive MAbs C4A1, 3E2identified by surface and apical-reactive MAbs C4A1, 3E2

• localized to surface and apical region (micronemes and dense localized to surface and apical region (micronemes and dense bodies) of sporozoites and merozoitesbodies) of sporozoites and merozoites

• MAb 3E2 elicits CSP-like reaction (formation, posterior MAb 3E2 elicits CSP-like reaction (formation, posterior movement and release of membraneous Ag-MAb precipitatesmovement and release of membraneous Ag-MAb precipitates

• MAb 3E2 neutralizes sporozoite infectivity and prevents MAb 3E2 neutralizes sporozoite infectivity and prevents infection in neonatal Balb/c miceinfection in neonatal Balb/c mice

• soluble glycoprotein exoantigen comprised of multiple ~1300 soluble glycoprotein exoantigen comprised of multiple ~1300 kDa molecular species with differing pI’s kDa molecular species with differing pI’s

• isolated native CSL binds to intestinal epithelial cells and inhibits isolated native CSL binds to intestinal epithelial cells and inhibits sporozoite attachment to and invasion of these cellssporozoite 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,

Page 16: Host-Parasite Interactions of  Cryptosporidium

16

200200

6969

4646

3030

21.521.5

kDakDa

97.497.4

11 22 11 22

200200

97.497.46969

4646

3030

14.314.321.521.5

kDakDa

14.314.3Silver stainSilver stain 4E9 Immunoblot4E9 Immunoblot

1, total proteins1, total proteins2, shed proteins2, shed proteins

Reactivity of MAb 4E9 with Reactivity of MAb 4E9 with C. parvumC. parvum “shed” proteins “shed” proteins

Cevallos et al, 2000Cevallos et al, 2000

Page 17: Host-Parasite Interactions of  Cryptosporidium

GP900 is not related to gp40GP900 is not related to gp40

MAbMAb4E94E9

anti-anti-gp40gp40

1515

2525

3535

5050

100100

150150

7575

225225

GP900GP900

gp40gp40

1515

2525

3535

5050

100100

150150

7575

225225

kDakDa kDakDa

Silver stain of HPA-affinity Silver stain of HPA-affinity purified glycoproteinspurified glycoproteins

Immunoblot of Immunoblot of GalNAc eluateGalNAc eluate

lysatelysate effluenteffluent GalNAcGalNAceluateeluate

Cevallos et al, 2000Cevallos et al, 2000

Page 18: Host-Parasite Interactions of  Cryptosporidium

gp40-specific antisera inhibit gp40-specific antisera inhibit C. parvumC. parvum infection of infection of intestinal epithelial Caco 2A cellsintestinal epithelial Caco 2A cells

Infe

ctio

n (A

405n

m)

0.00

0.05

0.10

0.15

0.20

0.25

preimmune-1 anti-gp40 1 preimmune-2 anti-gp40 2

Cevallos et al, 2000 Cevallos et al, 2000

Page 19: Host-Parasite Interactions of  Cryptosporidium

0.00

0.40

0.80

1.20

1.60

0.00 0.50 1.00 1.50 2.00

gp40 (µg/ml)

-galactosidase-galactosidase

HPA-glycoproteinsHPA-glycoproteins

Shed proteinsShed proteins

Bin

ding

(A

405n

m)

gp40 binds to intestinal epithelial Caco 2A cellsgp40 binds to intestinal epithelial Caco 2A cells

Cevallos et al, 2000 Cevallos et al, 2000

Page 20: Host-Parasite Interactions of  Cryptosporidium

20

Analysis ofAnalysis of Cpgp40/15 Cpgp40/15 deduced amino acid sequence 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 Cevallos et al, 2000

Page 21: Host-Parasite Interactions of  Cryptosporidium

21

Analysis ofAnalysis of Cpgp40/15 Cpgp40/15 deduced amino acid sequence deduced amino acid sequence

gp40 N-terminusgp40 N-terminus gp15 N-terminusgp15 N-terminus

Cpgp40/15Cpgp40/15

MRLSLIIVLLSVIVSAVFSAPAVPLRGTLKMRLSLIIVLLSVIVSAVFSAPAVPLRGTLKDVPVEGSSSSSSSSSSSSSSSSSSSTSTVAPDVPVEGSSSSSSSSSSSSSSSSSSSTSTVAP

ANKARTGEDAEGSQDSSGTEASGSQGSEEEGSEDDGQTSAASQPTTPAQSEGATTEANKARTGEDAEGSQDSSGTEASGSQGSEEEGSEDDGQTSAASQPTTPAQSEGATTE

TIEATPKEECGTSFVMWFGEGTPAATLKCGAYTIVYAPIKDQTDPAPRYISGEVTSVTIEATPKEECGTSFVMWFGEGTPAATLKCGAYTIVYAPIKDQTDPAPRYISGEVTSV

TFEKSDNTVKIKVNGQDFSTLSANSSSPTENGGSAGQASSRSRRSLSETFEKSDNTVKIKVNGQDFSTLSANSSSPTENGGSAGQASSRSRRSLSEETSEAAATVDETSEAAATVD

LFAFTLDGGKRIEVAVPNVEDASKRDKYSLVADDKPFYTGANSGTTNGVYRLNENLFAFTLDGGKRIEVAVPNVEDASKRDKYSLVADDKPFYTGANSGTTNGVYRLNEN

GDLVDKDNTVLLKDAGSSAFGLRYIVPSVFAIFAALFVLGDLVDKDNTVLLKDAGSSAFGLRYIVPSVFAIFAALFVL

gp40 N-terminusgp40 N-terminus

gp15 N-terminusgp15 N-terminus

Page 22: Host-Parasite Interactions of  Cryptosporidium

22

gp15/17 kDa immunodominant antigengp15/17 kDa immunodominant antigen

• 15 kDa protein localized to surface of sporozoites and merozoites and shed in 15 kDa protein localized to surface of sporozoites and merozoites and shed in “trails” during gliding motility“trails” during gliding motility

• contains contains GalNAc residuesGalNAc residues

Gut and Nelson, 1994; Strong et al 2000 Gut and Nelson, 1994; Strong et al 2000

• 15 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 recognized by IgA MAbs CrA1 and CrA2 which are partially protective partially protective against against C. parvumC. parvum infection in infection in scidscid mouse backpack tumor model mouse backpack tumor model

Zhou et al, Cevallos et al 2000Zhou et al, Cevallos et al 2000

• 17 kDa immunodominant antigen recognized by serum antibodies from 17 kDa immunodominant antigen recognized by serum antibodies from infected humansinfected humans

• present in TX-114 extracts of sonicated oocystspresent in TX-114 extracts of sonicated oocysts

Priest et al, 1999, Priest et al, 2000Priest et al, 1999, Priest et al, 2000

Page 23: Host-Parasite Interactions of  Cryptosporidium

11 22 33kDakDa

3535

5050

7575100100

150150

2525

1515

22522511 22 33kDakDa

3535

50507575

100100150150

2525

1515

225225

CrA1CrA1anti-gp15anti-gp15anti-gp40anti-gp40

gp40 and gp15 are antigenically distinct proteinsgp40 and gp15 are antigenically distinct proteins

1, oocysts1, oocysts2, sporozoites2, sporozoites3, shed proteins3, shed proteins Cevallos et al, 2000Cevallos et al, 2000

Page 24: Host-Parasite Interactions of  Cryptosporidium

11 22 33 44

1515

2525

3535

5050

100100150150

7575

11 22 33 44

1515

2525

3535

5050

100100150150

7575

CrA1CrA1anti-gp15anti-gp15

kDakDa kDakDa

anti-gp40anti-gp40

Antibodies to native gp40 and gp15 recognize the Antibodies to native gp40 and gp15 recognize the corresponding recombinant fusion proteinscorresponding recombinant fusion proteins

1, control1, control2, r40/152, r40/153, r153, r154, r404, r40 Cevallos et al, 2000Cevallos et al, 2000

Page 25: Host-Parasite Interactions of  Cryptosporidium

25

SporozoitesSporozoites MerozoitesMerozoites

Reactivity of anti-gp40 antisera with Reactivity of anti-gp40 antisera with C. parvumC. parvum sporozoites and merozoites by immunofluorescencesporozoites and merozoites by immunofluorescence

Cevallos et al, Infect. Immun 68: 4108-4116, 2000Cevallos et al, Infect. Immun 68: 4108-4116, 2000

Page 26: Host-Parasite Interactions of  Cryptosporidium

26

SporozoitesSporozoites MerozoitesMerozoites

Reactivity of MAb CrA1 (anti-gp15) with Reactivity of MAb CrA1 (anti-gp15) with C. parvumC. parvum sporozoites and merozoites by immunofluorescencesporozoites and merozoites by immunofluorescence

Cevallos et al, 2000Cevallos et al, 2000

Page 27: Host-Parasite Interactions of  Cryptosporidium

75

15

253550

100150

kDa

225

75

15

253550

100150

kDa

225

1 2 1 2 1 2 1 2

anti-anti-gp40gp40

anti-anti-gp15gp15

anti-anti-r40r40

anti-anti-r15r15

gp40 and gp15 are products of proteolytic gp40 and gp15 are products of proteolytic cleavage of a 49kDA precursor proteincleavage of a 49kDA precursor protein

Cevallos et al, 2000Cevallos et al, 2000

Page 28: Host-Parasite Interactions of  Cryptosporidium

Polymorphisms at Polymorphisms at gp15/45/60gp15/45/60 locus locus• sequence analysis ofsequence analysis of PCR amplifiedPCR amplified gp15/45/60 gp15/45/60 ORF from 29 ORF from 29 diverse diverse C. parvumC. parvum isolates isolates

• magnitude of sequence polymorphism identified at this locus is far magnitude of sequence polymorphism identified at this locus is far greater than that detected at any other greater than that detected at any other C. parvumC. parvum locus identified to locus identified to datedate

• 77-88% nucleotide sequence identity77-88% nucleotide sequence identity• 67 to 80% amino acid sequence identity67 to 80% amino acid sequence identity

• numerous SNPs and SAAPs in these sequences defined at least 5 numerous SNPs and SAAPs in these sequences defined at least 5 distinct allelic groupingsdistinct 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 regionsconserved regions• putative signal peptideputative signal peptide• putative GPI anchor siteputative GPI anchor site• putative proteolytic processing siteputative proteolytic processing site

Strong et al, 2000Strong et al, 2000

Page 29: Host-Parasite Interactions of  Cryptosporidium

human gp 40/15 1 DVPVEGSSSSSSSSSSSSSSSSSSSSSSSSSTSTVAPAPK 40calf gp40/15 1 DVPVEGSSSSSSSSSSSSSSSS------SSSTSTVAPAN- 33 ********************** **********

human gp 40/15 41 KERTVEGGTEGKNEESSPGSEEQDGGKEDGGKENGEGDTV 80calf gp40/15 34 KARTGEDAE-GSQDSSGTEASGSQGSEEEGSEDDG----Q 68 * ** * * .. * . * *.* . *

human gp 40/15 81 DGEQTGSGSQVTPSGSAGTATESTATTTPKEECGTSFVMW 120calf gp40/15 69 ----TSAASQPTTPAQSEGATTETIEATPKEECGTSFVMW 104 * . ** * . ** * .*************

human gp 40/15 121 FEKGTPVATLKCGDYTIVYAPIKDQTDPAPRYISGEVTSV 160calf gp40/15 105 FGEGTPAATLKCGAYTIVYAPIKDQTDPAPRYISGEVTSV 144 * *** ****** **************************

human gp 40/15 161 SFEKSESTVTIKVNGKEFSTLSANSSSPTKDNGESSDSQV 200calf gp40/15 145 TFEKSDNTVKIKVNGQDFSTLSANSSSPTENGG--SAGQA 182 .****. ** *****..************ .* * *

human gp 40/15 201 QSRSRRSLAEENGETVATVDLFAFTLDGGRRIEVAVPKDE 240calf gp40/15 183 SSRSRRSLSEETSEAAATVDLFAFTLDGGKRIEVAVPNVE 222 *******.**. *. *************.******* *

human gp 40/15 241 NADKRSEYSLVADDKPFYTGANSGITNGVYKLDENGNLVD 280calf gp40/15 223 DASKRDKYSLVADDKPFYTGANSGTTNGVYRLNENGDLVD 262 * ** ***************** *****.* *** ***

human gp 40/15 281 KDNKVLLKDAGSSAFGFRYIVPSVFAIFAALFVL 314calf gp40/15 263 KDNTVLLKDAGSSAFGLRYIVPSVFAIFAALFVL 296 *** ************ *****************

Comparison of Type II (calf) and Type I (human) Comparison of Type II (calf) and Type I (human) Cpgp40/15Cpgp40/15 deduced AA sequences deduced AA sequences

69% identity at amino acid level, 84% identity at nucleotide level69% identity at amino acid level, 84% identity at nucleotide level

Page 30: Host-Parasite Interactions of  Cryptosporidium

Southern blot analysis of Southern blot analysis of Cpgp40/15Cpgp40/15

Eco

Eco

RI

RI

Hin

Hin

dIII

dIII

Pst

Pst

II

Ssp

Ssp II

Eco

Eco

RI/

RI/

Hin

Hin

dII

dII

23,13023,130

9416941665576557

43614361

2322232220272027

564564

Eco

Eco

RI

RI

Hin

Hin

dIII

dIII

Pst

Pst

II

Ssp

Ssp II

Eco

Eco

RI/

RI/

Hin

Hin

dII

dII

23,13023,130

9416941665576557

43614361

2322232220272027

Type II GCH1Type II GCH1 Type I UG502Type I UG502

Page 31: Host-Parasite Interactions of  Cryptosporidium

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

1515

2525

3535

5050

100100

150150

7575

225225

kDakDa

I II I II I II

Page 32: Host-Parasite Interactions of  Cryptosporidium

Genotyping of Genotyping of C. parvumC. parvum isolates from HIV-Infected isolates from HIV-Infected Children with Persistent Diarrhea in South AfricaChildren with Persistent Diarrhea in South Africa

bpbp

0.60.6

1.01.0

1.51.52.02.0

0.60.61.01.01.51.52.02.0

bpbp 1414 1515 1616 1717 1818 1919 2020 21212222 2323 2424 2525 2626 2727 2828

HH HH HH CC HH HH HH HH CC HH ?? HH HH CCCC

• C. parvumC. parvum DNA PCR amplified from 21/24 stool samples DNA PCR amplified from 21/24 stool samples

• Genotype of isolates determined by PCR-RFLP at TRAP C1 Genotype of isolates determined by PCR-RFLP at TRAP C1 and COWP lociand COWP loci

• 16/21 (76%) of isolates were of the human genotype at both 16/21 (76%) of isolates were of the human genotype at both lociloci

• PCR amplification of PCR amplification of Cpgp40/15Cpgp40/15 locus locus

Page 33: Host-Parasite Interactions of  Cryptosporidium

gp40gp40

gp40 is a mucin-like glycoprotein containing terminal gp40 is a mucin-like glycoprotein containing terminal GalNAc GalNAc residuesresidues

gp40-specific antibodies neutralize infection gp40-specific antibodies neutralize infection in vitroin vitro and gp40 and gp40 binds to intestinal epithelial cells.binds to intestinal epithelial cells.

The gene encoding gp40 also encodes gp15, an antigenically The gene encoding gp40 also encodes gp15, an antigenically distinct protein. distinct protein.

gp40 is localized to the surface and apical region of invasive gp40 is localized to the surface and apical region of invasive stages. stages.

gp40 and gp15, are products of proteolytic cleavage of a 49 kDa gp40 and gp15, are products of proteolytic cleavage of a 49 kDa precursor protein expressed in intracellular stages. precursor protein expressed in intracellular stages.

The The Cpgp40/15Cpgp40/15 locus is highly polymorphic locus is highly polymorphic

Page 34: Host-Parasite Interactions of  Cryptosporidium

New England Medical CenterNew England Medical CenterTufts University, BostonTufts University, BostonAna Maria CevallosAna Maria CevallosNajma BhatNajma BhatSmitha JaisonSmitha Jaison Brett LeavBrett Leav Roberta O’ConnorRoberta O’Connor

Renaud VerdonRenaud VerdonDavid HamerDavid Hamer

Xiaoping ZhangXiaoping ZhangMatt WaldorMatt Waldor

Gerald KeuschGerald KeuschMiercio PereiraMiercio Pereira

Children’s Hospital,Children’s Hospital,Harvard University, BostonHarvard University, BostonMarian NeutraMarian NeutraXiaoyin ZhouXiaoyin Zhou

University of CaliforniaUniversity of CaliforniaSan FranciscoSan FranciscoCarolyn PetersenCarolyn Petersen

Bill StrongBill StrongRichard NelsonRichard Nelson

University of Texas, HoustonUniversity of Texas, HoustonSara DannSara DannCynthia ChappellCynthia Chappell

University of Natal, Durban, University of Natal, Durban, Africa Centre for Health and Africa Centre for Health and Population Research, MtubatubaPopulation Research, Mtubatuba Michael BennishMichael Bennish Nigel RollinsNigel Rollins

CDC, AtlantaCDC, AtlantaJeff PriestJeff Priest

Tufts University School of Tufts University School of Veterinary Medicine, GraftonVeterinary Medicine, GraftonBarry SteinBarry SteinGiovanni WidmerGiovanni WidmerDonna AkiyoshiDonna AkiyoshiInderpal SinghInderpal SinghCindy TheodosCindy TheodosSaul TziporiSaul Tzipori

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS