Cell Biology of Plasmodium

1. Cell Biology of Plasmodium Mark F. Wiser http://www.tulane.edu/~wiser/malaria/

2. 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.

3. Plasmodium Invasive Stages ookinete (motile) • mosquito gut epithelial cells sporozoite (motile) • mosquito salivary glands • hepatocytes merozoite (non-motile) • erythrocytes

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

5. Steps in Merozoite Invasion

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 *Mitchell et al (2004) Inf. Imm. 72, 154.

8. Proteins 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.

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

11. microneme secretion • receptor-ligand interactions • junction formation 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 • 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 Tonkin et al 2011, Science 233,463

14. Rhoptries are likely involved in PVM formation

16. Parasite Entry • entry requires force • cytochalasin inhibits invasion, but not attachment/junction • cytochalasin resistance maps to actin gene in Toxoplasma • unique Apicomplexan membrane associated myosin Micrograph from Bannister et al (1986), Parasitology 92:291

17. force can be generated by actin/myosin • TRAP necessary for invasion and gliding motility

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

20. Merozoite invasion: a complex and ordered process • Initial Binding • merozoite surface proteins (eg. MSP-1) • Reorientation (AMA-1) • Microneme Discharge and Junction Formation • Ca2+ signal? • 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 actin-myosin ‘glidesome’ • Closure of PVM and Erythrocyte Membrane

22. 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.

23. Permeability to metabolites is increased in the infected erythrocyte.

25. P. falciparum expresses ‘knobs’ on the surface of infected erythrocytes. Knobs mediate cytoadherence to endothelial cells.

26. 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

27. PfEMP-1 Structure • 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 domains • 5 sequence types (a, b, g, d, e) • 1-2 cys-rich interdomain regions • all have DBL1a + CIDR • participates in cytoadherence

28. Possible Host Receptors • CD36 • Ig super-family • ICAM-1 • VCAM-1 • PECAM-1 • E-selectin • thrombospondin • chondroitin sulfate A • hyaluronic acid • Rosetting Receptors • CR-1 • glycosaminoglycan • blood group A

30. 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

31. Antigenic switching is accompanied by changes in binding phenotype

32. Beeson et al (1999) JID 180:464 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.

33. 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)

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

35. Differential expression of var genes in organs Montgomery et al (2007) Mol. Microbiol. 65, 959-967

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) • other variant surface antigens? • rif and stevor in P. falciparum • in other species  cytoadherence • maintain chronic infections?