Cell Biology of Plasmodium Mark F. Wiser

Merozoite invasion involves specific interactions with the host erythrocyte. The actively growing parasite places metabolic and other demands on the host cell. Ultrastructural modifica- tions 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

Bannister et al (2003) J Cell Sci 116, 3825

Steps in Merozoite Invasion

accompanied by erythrocyte deformation AMA-1 implicated* apical membrane antigen-1 binds erythrocytes antibodies inhibit invasion and reorientation antibodies do not inhibit initial attachment Reorientation *Mitchell et al (2004) Inf. Imm. 72, 154.

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 *Thrombospondin family characterized by von Willebrand factor type A domain. Functions in cell-cell and cell-matrix interactions.

Tham et al (2012) Tr. Parasitol. 28:23

Electron micrograph from Aikawa et al (1978) J. Cell Biol. 77:72 microneme secretion receptor-ligand interactions junction formation

Events correlated with entry clearance of erythrocyte membrane proteins host membrane invagination parasitophorous vacuolar membrane (PVM) formation junction becomes an annulus (ring)

Tonkin et al 2011, Science 233,463 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

Rhoptries are likely involved in PVM formation

Junction Formation –microneme adhesins + erythrocyte receptors –RON2 + AMA-1 Parasite Entry –reorganization of submembrane cytoskeleton –PVM formation –shedding of merozoite surface proteins –moving junction Force generation involves actin and unique Apicomplexan membrane associated myosin

TRAP necessary for invasion and gliding motility acto-myosin motor = glideosome

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

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 (RON2/AMA-1) Parasite Entry mediated by acto-myosin ‘glideosome’ shedding of merozoite surface Closure of PVM and Erythrocyte Membrane Merozoite invasion: a complex and ordered process

Merozoite invasion involves specific interactions with the host erythrocyte. The actively growing parasite places metabolic and other demands on the host cell. Ultrastructural modifica- tions are evident in the infected erythrocyte.

Permeability to metabolites is increased in the infected 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

family of ~60 var genes 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 (  ) 0-2 cys-rich interdomain (CIDR) regions participates in cytoadherence PfEMP-1 Structure

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 Possible Host Receptors

Roberts et al (1992) Nature 357:689 agglutinating anti-sera used to define antigenic types antigenic variants obtained from a cloned parasite line A high rate of antigenic variation is observed on the erythrocyte surface clones also exhibited different ICAM1/CD36 binding phenotypes A switch rate of 2% per generation in absence of immune pressure.

Antigenic switching is accompanied by changes in binding phenotype

Binding Phenotypes Can Be Selected In Vivo Chondroitin sulfate A (CSA) is a complex carbohydrate found on the surface of endothelial cells in the placenta. Beeson et al (1999) JID 180:464

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)

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

Differential expression of var genes in organs Montgomery et al (2007) Mol. Microbiol. 65,

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 non- expressed 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