1. Cell Biology of Plasmodium
Mark F. Wiser
( like the biochemistry,) the cell and molecular biology of Plasmodium will be similar to other eukaryotes.
But there are unique featues
many of these unique features of the malarial parasite will relate it being an intracellular parasite and to its very intimate interaction with the host and vector
for example ...

2. Merozoite invasion involves specific interactions with the host erythrocyte.
The actively growing parasite places metabolic and other demands on the host cell.
For example, the parasite recognizes and specifically invades host cells and in particular erythrocytes.
within the erythrocyte the parasite then continues to interact with the host erythrocyte and drastically alters many properties of the erythrocyte
metabolic changes
ultrastructural changes, most notable is the appearance of knobs which promote binding to endothelial cells
Today's lecture will concentrate on these two aspects--invasion and modification--of the interaction between the malaria parasite and the host erythrocyte.
During the 1st half, I will discuss the process of invasion on the cellular and molecular levels.
During the 2nd half, I will discuss how the parasite modifies host erythrocyte with particular reference to knobs and cytoadherence.
Ultrastructural modifica-tions are evident in the infected erythrocyte.

3. Plasmodium Invasive Stages
ookinete (motile)
mosquito gut epithelial cells
sporozoite (motile)
mosquito salivary glands
hepatocytes
merozoite (non-motile)
erythrocytes
as mentioned in the first lecture, Plasmodium exhibits specialized invasive stages: merozoite, sporozoite, ookinete
all characterized by apical organelles and the ability to penetrate host cells
ookinete and sporozoite are also capable of movement
for this lecture I will focus on the invasion of erythrocytes by the merozoite
more is known
more relevant to pathology
however, I will bring in examples from the other stages as well as from other apicomplexa
apicomplexa are related parasites all characterized by having invasive stages with apical organelles
OK, lets look at the merozoite ....

4. Apical Organelles found in invasive stages of apicomplexans
participate in invasion process
Small around 3 microns
Characterized by apical organelles
Has a subpellicular microtubule cytoskeleton and inner membrane complex
And all the other organelles
Released from the infected erythrocyte ....

5. Steps in Merozoite Invasion
on the morphological level merozoite invasion can be broken down into 4 steps:
initial interaction: random (but in blood stream so immediate0 and probably reversible
Obviously involves proteins on the surface of the merozoite
Best characterized is msp-1 (you may hear more later)
the parasite then reorients so that the apical end is juxtaposed with the host membrane
a junction then forms between the parasite and host
parasite enters coming to lie within PV, but not a phagocytosis
whole process takes approximately 20 seconds
Lets look at this deformation and reorientation a bit more ….

6. 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
a deformation of the erythrocyte is observed as the parasite reorients
the mechanisms of reorientation is not known
but recently some insight has been gained
AMA-1 seems to be involved
the reason though, obviously involve the location of the apical organelles at one end of the parasite
show rhoptry
so, what are these apical organelles ....
*Apical Membrane Antigen-1
Mitchell et al (2004) Inf. Imm. 72, 154.

7. 3 types of apical organelles have been described, although dense granules are not always apical
all are secretory organelles--membrane bound and release their contents
an interesting experiment in Toxoplasma--another Apicomplexan
micronemes released first coincident with initial contact
rhoptries immediately after that
dense granules are last, usually after parasite entry and continue for an extended period
since micrones first: What are the contents?

8. 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)
proteins localized to micronemes are related to adhesive proteins
in merozoites are 2 proteins
in sporozoites, ookinetes and other apicomplexa is a family of related proteins
relation involves a common domain that function is cell-cell or cell-matrix interactions
lets concentrate on the merozoite proteins.
*Thrombospondin family characterized by von Willebrand factor type A domain. Functions in cell-cell and cell-matrix interactions.

9. proteins are found in micronemes of merozoites had been previously identified as ligands for host erythrocyte receptors
these proteins involved in receptor ligand interactions (lock and key)
both are related—especially the receptor binding domain
not highly conseverd—but key amino acid residues (aromatic and cys residues) that are important for protein structure
in addition, they integral membrane proteins
receptor binding activity localized to specific domain
lets go back to the morphological level ...

10. Tham et al (2012) Tr. Parasitol. 28:23
Several receptor ligand combinations involved in attachment of merozoties. Redundancy? Increased avidity of binding? Two families EBA (= Duffy). Rh = reticulocyte binding like homologue.
Tham et al (2012) Tr. Parasitol. 28:23

11. receptor-ligand interactions junction formation
microneme secretion
receptor-ligand interactions
junction formation
these proteins are imbedded in the micronemes
after release from micronemes an extracellular domain is exposed
Calcium might be trigger (data from Toxoplasma)
fusion of vesicle with membrane exposes binding domains
at the same time electron dense junctions are formed
go to board and draw release and receptor-ligand interactions
Electron micrograph from Aikawa et al (1978) J. Cell Biol. 77:72

12. Events correlated with entry
clearance of erythrocyte membrane proteins
host membrane invagination
parasitophorous vacuolar membrane (PVM) formation
discharge of rhoptries
at this junction, erythrocyte membrane proteins are cleared
ery. memb. has 2-D cytoskeleton
this cytoskeleton is rigid and does not allow for envagination
membrane evagination and formation of vacuolar membrane
junction becomes ring-like
parasite moves into this forming vacuole
lets look more at vacuolar membrane formations--looks like a connection, at higher magnification ….
junction becomes an annulus (ring)

13. 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

14. Rhoptries are likely involved in PVM formation
it appears that the contents of the rhoptries are involved in forming the PVM
membraneous whorls are observed also
suggests that substantial amounts of PVM are derived from rhoptry contents

15. Junction Formation Parasite Entry
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

16. TRAP necessary for invasion and gliding motility
motility is based on actin myosin cytoskeletal systems
myosin is known as a motor protein--it can move along actin filaments
if a motor is attached to the ligand it would drag it through the lipid bilayer
filaments arrange so that the motor moved toward the posterior of the parasite would result in forward movement
Little more complex, current models ...
TRAP necessary for invasion and gliding motility
actin-myosin motor = glideosome

17. Besteiro et al 2011, Cell. Microbiol. 13,797

18. the PVM and erythrocyte membrane will seal thus completing entry

19. a complex and ordered process
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

21. Merozoite invasion involves specific interactions with the host erythrocyte.
The actively growing parasite places metabolic and other demands on the host cell.
as mentioned previously the 2nd half will concentration on modifications
the parasite modifies the erythrocyte--these modifications are probably necessary for parasite survival, for example …
Ultrastructural modifica-tions are evident in the infected erythrocyte.

22. Erythrocyte Modifications Affecting Permeability
In 48 hours parasite produces 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

23. many ultrastructural changes seen
most notorious are the knobs
the knobs are electron dense protuberances found on the erythrocyte membrane of infected cells
knobs likely involved in a process called cytoadherence as illustrated on next slide ...

24. P. falciparum expresses ‘knobs’ on the surface of infected erythrocytes. Knobs mediate cytoadherence to endothelial cells.
these knobs are believed to mediate cytoadherence--or binding to endothelial cells of the capillaries
this results in sequestration of the mature parasites in the deep tissues
This sequestration has two major advantages: 1) metabolic low O2 tensions, and 2) spleen advoidence
this sequestration is also responsible for the increased pathology associated with Pf.
for the remaining lecture I will discuss the molecular and cellular biology of knobs and this process of cytoadherence
lets first look at knobs ….

25. 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
proteins synthesized by the parasite are transported to the host erythrocyte membrene
some of these proteins have been localized to the knobs
in particular KAHRP and PfEMP2 are believed to interact with the submembrane cytoskeleton
as discussed earlier the cytoskeleton is responsible for cell shape
a reorganization of this cytoskeleton could result in knob formation
neither of these proteins are exposed on the surface
PfEMP1 is exposed on the erythrocyte surface
interestingly it has an acidic domain at its C-terminus, or the portion on the cytoplasmic side. the KAHRP is very basic. Binding has been demonstrated., suggesting that PfEMP1 is anchored into the knob through interactions with KAHRP
the exposure of PfEMP1 suggests that it could mediate binding to endothelial cells
the gene for PfEMP-1 has been cloned and sequenced ...
KAHRP = knob associated histidine rich protein
EMP = erythrocyte membrane protein

26. 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
there is not just one PfEMP-1 gene, but it is a multigene family, called var, with an estimated members
as shown on the previous slide, one end of the protein (the C-terminus) probably interacts with the erythrocyte submembrane skeleton
this intracellular domain is conserved
there is a domain with transmembrane characteristics
the extracellular domain is variable, but contains recognizable motifs
1-5 copies of Duffy-binding like domains.
exhibit similar cys and hydrophobic residues even though not highly conserved
there is also cys-rich interdomain region
because of its location speculated to be a ligand for cytoadherence
a ligand suggests the presence of a receptor--many have been identified ...

27. 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
numerous possible receptors identified
most widely studied is CD kDa glycoprotein found on monocytes, platelets and endothelial cells
many var gene products bind to CD36, widely used for in vitro studies
ICAM most likely to be involved in cerebral pathology
some binding sites mapped to domains on PfEMP-1
receptor/ligand interactions usually specific????

29. 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.
study of Roberts et al may provide some insight into what is going on
they defined antigenic types with a panel of agglutinating anti-sera which recognize erythrocyte surface antigens (presumably PfEMP-1)
studies done before PfEMP-1 cloned
they took a cloned parasite line (A4) and isolated several clones
most of these clones exhibit a different antigenic type from parental as well as from each other
a switch rate of 2% per generation
they also looked at the in vitro binding to CD36 and ICAM-1 and the different clones exhibit different phenotypes
for example, look at C28 ...
clones also exhibited different ICAM1/CD36 binding phenotypes

30. Antigenic switching is accompanied by changes in binding phenotype
the parental line A4 bound CD36 and ICAM-1 equally well
C28 exhibited a marked preference for CD36
therefore, antigenic types exhibit different binding phenotypes
selection of C28 on ICAM-1 resulted in a greatly increased capacity to bind ICAM and new antigenic type
share one more experiment ...

31. Binding Phenotypes Can Be Selected In Vivo
Beeson et al (1999) JID 180:464
Beeson et al looked at numerous isolates and their abilities to bind to ICAM, CD36 and CSA
CSA is a complex carbohydrate found on placental cells
isolates from placental sources exhibited a higher level of binding to CSA than peripheral isolates from either mother or children
may be related to problems associated with malaria and pregnancy, especially in primigravids
Pf. more severe in pregnant women, especially 1st pregnancy
abs against CSA binding ligands may protect in subsequent pregnancies
immune response to particular PfEMP-1 molecules will lead to parasite elimination
through switching, parasite will express new PfEMP-1 with new antigenic characteristics and binding phenotypes
a particular var gene is expressed in parasites selected on CSA (EMBO 17, 5418)
var genes that bind to CSA do not bind CD36
this particular gene has 7 DBL and DBL#3 binds to CSA (pnas 96, 12743)
Chondroitin sulfate A (CSA) is a complex carbohydrate found on the surface of endothelial cells in the placenta.

32. 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)

33. Members of DC8 and DC13 associated with binding to brain endothelial cells and severe malaria
Claessens et al (2012) PNAS 109:E1772

34. Differential expression of var genes in organs
This phenomenon is not restricted to the placenta in that there is a dominant expression of particular var genes in the various tissues (Figure, from Montgomery 2007). This tissue specific expression of particular var genes implies that different tissues are selecting out different parasite populations based on the particular PfEMP1 being expressed on the surface of the infected erythrocyte.
Figure, from Montgomery Shows the proportion of the various types of PfEMP1 (designated as groups 1-6) expressed in different tissues (brain, lung, heart and spleen) from 3 different patients. PM30 died of severe malaria anemia. PM32 was diagnosed with both cerebral malaria and severe anemia. PM55 was diagnosed with only cerebral malaria.
Rottmann et al 2006, Infect. Immun. 74: 3904 (certain var transcripts are more abundant in patients with severe malaria than in patients with uncomplicated malaria
Montgomery et al (2007) Mol. Microbiol. 65,

35. Expression of Particular var Genes May Correlate with Disease Manifestations and Virulence

36. Ag variation allows for escape from immune system
switch will alter binding phenotype though

37. Var Gene Expression
one var gene is expressed at a time (allelic exclusion)
specific expression site in nucleus
repressor proteins bind promoters of non-expressed variants
switching mechanism?
Borst and Genest (2006) Nature 439, 926

38. 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
parasite modifies the host cell to make it a better home
the process of cytoadherence which leads to sequestration is probably a key factor in the increased virulence of Pf.
in this regard a parasite proteins expressed on the surface of Pf. infected erythrocytes has been identified.
pfemp1 probably mediates the cytoadherence
antigenic variation will allow for avoidance of the immune system
coincident with ag switching is modification of cytoadherent phenotype in regards to receptor specificity
however, other variant surface antigens on Pf. have been identified--their roles not known
variant surface antigens are also found in other species--which do not exhibit the strong cytoadherence and sequestration
therefore, surface antigens may play other roles and cytoadherence is only a 2o process
(Saul proposes to maintain chronic infection (PT 15:455))