HostParasite Interactions

1
Host-Parasite Interactions of Cryptosporidium
Molecular Basis of Attachment and Invasion
2
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,
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

• 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
6
Role of Parasite and Host Cytoskeletal Elements
in Cryptosporidium Motility, Attachment and
Invasion in vitro

• Sporozoite motility is powered by actin-
  myosin 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
7
Surface/Apical Proteins of Cryptosporidium

• gt20 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

8
Surface/Apical Proteins of Cryptosporidium

• gt200 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 Ortega-Mora
  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)

9
Effect of MAb 4E9 IgM on C. parvum infection of
Caco-2A cells
4E9
B9A4
Infection (A405nm)
IgM µg/ml
Cevallos et al, 2000
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
Immunoblot analysis of C. parvum antigens
recognized by MAb 4E9
1
2
kDa
200
116.3
97.4
66.2
45
31
1, Oocysts 2, Sporozoites
21.5
Cevallos et al, 2000
14
GP900

• gt900kDa 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
15
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 pIs
• 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,
16
Reactivity of MAb 4E9 with C. parvum shed
proteins
1, total proteins 2, shed proteins
Cevallos et al, 2000
17
GP900 is not related to gp40
MAb 4E9
anti- gp40
lysate
effluent
GalNAc eluate
Silver stain of HPA-affinity purified
glycoproteins
Immunoblot of GalNAc eluate
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
gp40 binds to intestinal epithelial Caco 2A cells
Shed proteins
HPA-glycoproteins
Binding (A405nm)
b-galactosidase
Cevallos et al, 2000
20
Analysis of Cpgp40/15 deduced amino acid sequence
981 bp
326 aa /33.6 kDa protein
Signal peptide
GPI anchor site
Polyserine region
O-glycosylation site
Cevallos et al, 2000
21
Analysis of Cpgp40/15 deduced amino acid sequence
gp40 N-terminus
MRLSLIIVLLSVIVSAVFSAPAVPLRGTLKDVPVEGSSSSSSSSSSSSSS
SSSSSTSTVAPANKARTGEDAEGSQDSSGTEASGSQGSEEEGSEDDGQTS
AASQPTTPAQSEGATTETIEATPKEECGTSFVMWFGEGTPAATLKCGAYT
IVYAPIKDQTDPAPRYISGEVTSVTFEKSDNTVKIKVNGQDFSTLSANSS
SPTENGGSAGQASSRSRRSLSEETSEAAATVDLFAFTLDGGKRIEVAVPN
VEDASKRDKYSLVADDKPFYTGANSGTTNGVYRLNENGDLVDKDNTVLLK
DAGSSAFGLRYIVPSVFAIFAALFVL
gp15 N-terminus
Cpgp40/15
gp40 N-terminus
gp15 N-terminus
22
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

23
gp40 and gp15 are antigenically distinct proteins
1
2
3
kDa
1
2
3
kDa
225
225
150
150
100
100
75
75
50
50
35
35
25
25
15
15
anti-gp40
CrA1 anti-gp15
1, oocysts 2, sporozoites 3, shed proteins
Cevallos et al, 2000
24
Antibodies to native gp40 and gp15 recognize the
corresponding recombinant fusion proteins
kDa
kDa
150
150
100
100
75
75
50
50
35
35
25
25
15
15
anti-gp40
CrA1 anti-gp15
1, control 2, r40/15 3, r15 4, r40
Cevallos et al, 2000
25
Reactivity of anti-gp40 antisera with C. parvum
sporozoites and merozoites by immunofluorescence
Sporozoites
Merozoites
Cevallos et al, Infect. Immun 68 4108-4116, 2000
26
Reactivity of MAb CrA1 (anti-gp15) with C. parvum
sporozoites and merozoites by immunofluorescence
Sporozoites
Merozoites
Cevallos et al, 2000
27
gp40 and gp15 are products of proteolytic
cleavage of a 49kDA precursor protein
kDa
kDa
1
2
1
2
1
2
1
2
225
225
150
150
100
100
75
75
50
50
35
35
25
25
15
15
anti- gp15
anti- r40
anti- r15
anti- gp40
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
• 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
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
Type II GCH1
Type I UG502
31
Reactivity of anti-gp40 antibodies with Type I
(GCH1) and Type II (UG502) isolates
I II I II I II
kDa
225
150
100
75
50
35
25
15
32
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

33
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

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