Transcript Reorientation (AMA-1)

Cell Biology of Plasmodium
Mark F. Wiser
http://www.tulane.edu/~wiser/malaria/
• Merozoite invasion
involves specific
interactions with the
host erythrocyte.
• The actively growing
parasite places metabolic
and other demands on
the host cell.
• Ultrastructural modifications are evident in the
infected erythrocyte.
Plasmodium Invasive Stages
ookinete (motile)
• mosquito gut
epithelial cells
sporozoite (motile)
• mosquito salivary
glands
• hepatocytes
merozoite (non-motile)
• erythrocytes
Apical Organelles
• found in invasive stages
of apicomplexans
• participate in invasion
process
Steps in Merozoite Invasion
Reorientation
• accompanied by
erythrocyte deformation
• AMA-1 implicated*
• apical membrane antigen-1
• binds erythrocytes
• antibodies inhibit invasion
and reorientation
• antibodies do not inhibit
initial attachment
*Apical Membrane Antigen-1
Mitchell et al (2004) Inf. Imm. 72, 154.
Secretory (Apical) Organelles
Organelle
Shape
Rhoptry
Microneme
Dense Granule
Teardrop
Ellipsoidal
Spherical
Size (nm)
300 x 600
40 x 100
120 - 140
Adhesins Localized to Micronemes
•Merozoite proteins:
• EBA-175 (sialic binding protein of P. falciparum)
• Duffy-binding protein (P. vivax and P. knowlesi)
•TRAP family*:
• SSP2 (sporozoite surface protein-2)  TRAP
(thrombospondin-related adhesive protein)
• Toxoplasma, Eimeria and Cryptosporidium
proteins with homology to SSP2/TRAP
• CTRP, circumsporozoite- and TRAP-related
protein (Plasmodium ookinete stage)
*Thrombospondin family characterized by von Willebrand factor
type A domain. Functions in cell-cell and cell-matrix interactions.
Invasion Receptors/Ligands
Species
P. falciparum
P. vivax,
P. knowlesi
Host
Receptor
Merozoite
Ligand
glycophorins
(sialic acid)
EBA-175
Duffy Ag
DBP
Tham et al (2012) Tr. Parasitol. 28:23
• microneme secretion
• receptor-ligand
interactions
• junction formation
Electron micrograph from Aikawa et al
(1978) J. Cell Biol. 77:72
Events correlated with entry
• clearance of erythrocyte
membrane proteins
• host membrane
invagination
• parasitophorous vacuolar
membrane (PVM) formation
• discharge of rhoptries
• junction becomes
an annulus (ring)
Rhoptries also participate
in junction formation
• Rhoptry neck proteins
(RONs) inserted into host
membrane
• RON2 interacts with
AMA-1
• Forms part of the moving
junction
• Highly conserved in
Apicomplexa
Tonkin et al 2011, Science 233,463
Rhoptries are likely involved in PVM formation
• Junction Formation
– microneme adhesins +
erythrocyte receptors
– RONs + AMA-1
• Parasite Entry
– reorganization of submembrane
cytoskeleton
– PVM formation
– shedding of merozoite surface
proteins (eg, MSP-1)
– moving junction
• Force generation involves actin
and unique Apicomplexan
membrane associated myosin
• TRAP necessary for invasion
and gliding motility
• actin-myosin motor =
glideosome
Besteiro et al 2011, Cell.
Microbiol. 13,797
Merozoite invasion:
a complex and ordered process
• Initial Binding
• merozoite surface proteins (eg. MSP-1)
• Reorientation (AMA-1)
• Microneme Discharge and Junction Formation
• receptor-ligand interactions (adhesive proteins)
• Rhoptry Discharge and Vacuole Formation
• clearing of host membrane proteins
• moving junction formation (RONs/AMA-1)
• Parasite Entry
• mediated by actin-myosin ‘glideosome’
• shedding of merozoite surface
• Closure of PVM and Erythrocyte Membrane
• Merozoite invasion
involves specific
interactions with the
host erythrocyte.
• The actively growing
parasite places metabolic
and other demands on
the host cell.
• Ultrastructural modifications are evident in the
infected erythrocyte.
Erythrocyte Modifications
Affecting Permeability
• In 48 hours parasite produces 20-30
merozoites
•  uptake of anions, sugars, amino acids,
organic cations
• New permeability pathways induced in host
erythrocyte
• Parasite modifies host cell by exporting
proteins into host erythrocyte
P. falciparum expresses ‘knobs’ on the surface of infected
erythrocytes. Knobs mediate cytoadherence to endothelial cells.
Several Parasite Proteins Are Associated with Knobs
• KAHRP and PfEMP2 are believed to interact with the
submembrane cytoskeleton of the host erythrocyte
• reorganization of the membrane skeleton may result in
knob formation
• PfEMP1 crosses the erythrocyte membrane and is
exposed on the surface
KAHRP = knob associated histidine rich protein
EMP = erythrocyte membrane protein
PfEMP-1 Structure
• family of ~60 var genes
• mitotic recombinations continuously
produce new variants
• conserved intracellular C-terminus
• acidic terminal segment (ATS)
• binds cytoskeleton + KAHRP
• transmembrane domain
• variable extracellular domain
composed of modules
• 2-7 copies of Duffy-binding like
(DBL) domains
• 5 sequence types (a, b, g, d, e)
• 0-2 cys-rich interdomain (CIDR)
regions
• participates in cytoadherence
Possible Host Receptors
• CD36
• Ig super-family (eg,
ICAM-1)
• endothelial protein C
receptor
• chondroitin sulfate A
• E-selectin
• thrombospondin
• hyaluronic acid
• Rosetting Receptors
• CR-1
• glycosaminoglycan
• blood group A
Binding Sites
Domain Receptor
CIDR
DBLa
DBLb
DBLg
CD36
rosetting
ICAM-1
CSA
A high rate of antigenic variation is
observed on the erythrocyte surface
Roberts et al (1992) Nature 357:689
• agglutinating anti-sera used to define antigenic types
• antigenic variants obtained from a cloned parasite line
A switch rate of 2% per
generation in absence of
immune pressure.
• clones also exhibited
different ICAM1/CD36
binding phenotypes
Antigenic switching is accompanied by
changes in binding phenotype
Binding Phenotypes Can Be Selected In Vivo
Beeson et al (1999) JID 180:464
Chondroitin sulfate A (CSA) is a complex carbohydrate
found on the surface of endothelial cells in the placenta.
A Specific PfEMP1 Variant
Binds to CSA
•
•
•
•
VAR2CSA binds to CSA
Found in most parasite strains
Member of domain cassette (DC) 2
(defined combination of domains always
found together)
Members of DC8 and DC13
associated with binding to brain
endothelial cells and severe malaria
Claessens et al (2012) PNAS 109:E1772
Differential expression of var genes in organs
Montgomery et al (2007) Mol. Microbiol. 65, 959-967
Expression of Particular var Genes
May Correlate with Disease
Manifestations and Virulence
Var Gene Expression
• one var gene is expressed at a time
(allelic exclusion)
• specific expression site
in nucleus
• repressor proteins bind
promoters of nonexpressed variants
• switching mechanism?
Borst and Genest (2006) Nature 439, 926
SUMMARY
• parasite modifies host via exported proteins
– permeability changes
– knobs + PfEMP-1
• PfEMP-1 participates in cytoadherence
• immune evasion accomplished through
antigenic switching (var gene family)
• some var genes may correlate with specific
disease manifestations and virulence
• maintain chronic infections