Transcript No Slide Title

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
h u ma n g p 4 0 / 1 5
c a lf g p 4 0/ 1 5
1
1
D V P V EG S S S SS S S S SS S S S S SS S S S SS S S S ST S T V A
D V P V EG S S S SS S S S SS S S S S SS - - - -- - S S ST S T V A
* * * * ** * * * ** * * * ** * * * * **
* * ** * * * *
h u ma n g p 4 0 / 1 5
c a lf g p 4 0/ 1 5
41
34
K E R T VE G G T EG K N E ES S P G S EE Q D G GK E D G GK E N G E
K A R T GE D A E -G S Q D SS G T E A SG S Q G SE E E G SE D D G * ** *
* .. *
.
*
*.*
. *
h u ma n g p 4 0 / 1 5
c a lf g p 4 0/ 1 5
81
69
D G E Q TG S G S QV T P S GS A G T A TE S T A TT T P K EE C G T S
- - - - TS A A S QP T T P AQ S E G A TT E T I EA T P K EE C G T S
* . ** *
.
**
*
. * * * ** * * * *
h u ma n g p 4 0 / 1 5
c a lf g p 4 0/ 1 5
121
105
F E K G TP V A T LK C G D YT I V Y A PI K D Q TD P A P RY I S G E
F G E G TP A A T LK C G A YT I V Y A PI K D Q TD P A P RY I S G E
*
* ** * * ** * * ** * * * * ** * * * ** * * * ** * * * *
h u ma n g p 4 0 / 1 5
c a lf g p 4 0/ 1 5
161
145
S F E K SE S T V TI K V N GK E F S T LS A N S SS P T K DN G E S S
T F E K SD N T V KI K V N GQ D F S T LS A N S SS P T E NG G - - S
. * * * *. * * * * * * *. . * * * ** * * * ** * *
.*
*
h u ma n g p 4 0 / 1 5
c a lf g p 4 0/ 1 5
201
183
Q S R S RR S L A EE N G E TV A T V D LF A F T LD G G R RI E V A V
S S R S RR S L S EE T S E AA A T V D LF A F T LD G G K RI E V A V
* * * ** * * . ** . * . * * * * ** * * * ** * * . ** * * * *
h u ma n g p 4 0 / 1 5
c a lf g p 4 0/ 1 5
241
223
N A D K RS E Y S LV A D D KP F Y T G AN S G I TN G V Y KL D E N G
D A S K RD K Y S LV A D D KP F Y T G AN S G T TN G V Y RL N E N G
* **
* * ** * * * ** * * * * ** * * ** * * * .* * * *
h u ma n g p 4 0 / 1 5
c a lf g p 4 0/ 1 5
281
263
K D N K VL L K D AG S S A FG F R Y I VP S V F AI F A A LF V L
K D N T VL L K D AG S S A FG L R Y I VP S V F AI F A A LF V L
* * * ** * * * ** * * * ** * * * ** * * * ** * * * ** * *
69% identity at amino acid level, 84% identity at nucleotide level
3
2
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