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 ... 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. 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.

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 ....

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 ....

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 ….

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.

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?

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.

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 ...

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

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

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)

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 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

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

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

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

the PVM and erythrocyte membrane will seal thus completing entry

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

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.

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

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 ...

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 ….

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

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 40-50 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 ...

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 CD36--88 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????

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

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 ...

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.

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 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 2007. 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, 959-967

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

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

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 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))