host-parasite interactions of cryptosporidium
DESCRIPTION
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 PresentationTRANSCRIPT
Host-Parasite InteractionsHost-Parasite Interactions ofof Cryptosporidium Cryptosporidium
Molecular Basis of Attachment Molecular Basis of Attachment and Invasionand Invasion
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
Electron Micrograph of Electron Micrograph of Cryptosporidium Cryptosporidium Sporozoite Sporozoite Attaching to Intestinal Microvillus MembraneAttaching to Intestinal Microvillus Membrane
Tzipori, 1988Tzipori, 1988
Electron Micrograph of Electron Micrograph of Cryptosporidium Cryptosporidium MerozoiteMerozoite Invading Intestinal Epithelial Cell MembraneInvading Intestinal Epithelial Cell Membrane
Tzipori, 1988Tzipori, 1988
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
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
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
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)
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
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
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
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
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
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
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,
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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