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 theinvasion of erythrocytes by the merozoite more is known more relavant 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

Merozoite invasion involves specific interactions with the host erythrocyte. The actively growing parasite places metabolic and other demands on the host cell. cell and molecular biology of Plasmodium will be similar to other eukaryotes. many of the 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, 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 molecualr 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.

Steps in Merozoite Invasion meroite invasion can be broken down into 4 steps: initial interaction: random (but in blood stream so immediate0 and probably reversible 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 presumaably the initial interaction involves the merozoite surface ….

Merozoite reorientation is accompanied by erythrocyte deformation. a deformation of the erythrocyte is observed as the parasite reorients the mechanisms of reorientation is not known the reason though, obviously involve the location of the apical organelles at one end of the parasite show rhoptry

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?

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 functions in cell-cell and cell-matrix interactions.

proteins are found in micronemes these proteins involved in receptor ligand interactions integral membrane proteins after release from micronemes an extracellular domain is exposed receptor binding activity localized to specific domain go to board and draw release and receptor-ligand interactions at morphological level at the same time ...

receptor-ligand interactions junction formation microneme secretion receptor-ligand interactions junction formation an electron dense junction forms between merozoite and erythrocyte connection with microneme release Electron micrograph from Aikawa et al (1978) J. Cell Biol. 77:72

Events correlated with entry clearance of erythrocyte membrane proteins host membrane invagination vacuolar membrane formation 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 requires force lets look more at vacuolar membrane formations--looks like a connection, at higher magnification …. junction becomes an annulus

Are rhoptries 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

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? Microneme Discharge and Junction Formation receptor-ligand interactions Ca2+ signal? Rhoptry Discharge and Vacuole Formation clearing of host membrane proteins Parasite Entry mediated by actin-myosin MSP-1 proteolysis and shedding of surface coat? 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. cell and molecular biology of Plasmodium will be similar to other eukaryotes. many of the 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, 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 molecualr 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.

UPTAKE AND PERMEABILITY The malaria parasite has a high metabolic rate and has a large demand for small molecular metabolites that will serve as precursors for the synthesis of nucleic acids, proteins and lipids. The erythrocyte has a rather sluggish metabolism and limited transport capabilities, but infected erythrocytes exhibit a substantial increase in permeability to low molecular weight solutes. Metabolites need to cross the PVM and the parasite plasma membrane. A channel on the PVM has been implicated in the acquistion of nutrients. Others have proposed a direct connection to the host plasma via a 'parasitophorous duct‘. Presumably the parasite plasma membrane has transporters which are typical of other eukaryotes.

REDOX METABOLISM A bi-product of metabolism and respiration are reactive oxygen intermediates (ROI) such as superoxide, hydroxyl radical and hydrogen peroxide. In particular, the digestion of oxy-hemoglobin results in the production of ROI. These ROI can damage lipids, proteins and nucleic acids and therefore need to be oxidized to oxygen and water. Parasite enyzmes involved in redox metaboism have been identified. Superoxide dismutase (SOD), catalase, and glutathione peroxidase are involved in the detoxification of ROI. Oxidized glutathione is recycled by glutathione reductase and the reducing equivalents of NADPH are probably generated through the pentose phosphate cycle.

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.

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 the acidic domain (C-terminus) interacts with the basic KAHRP and cytoskeletal proteins 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 ...

Human malaria: adaptations to the parasite 1) Sickle Cell- single point mutation- abnormal shape of a percentage of RBC will not allow parasite development RBC have bumps on surface- stick to capillary walls, loss of potassium, parasites inside die, damaged cells removed Only benefit is to heterozygous individuals: double dominant are susceptible double recessive often die from anemia Example of strong evolutionary pressure to respond to a parasite 2) G-6-Phosphate dehydrogenase deficiency: results in reduced parasitemias 3) Duffy Blood group: double recessive- completely resistant to P. vivax. parasite cannot find receptors to enter RBC Found in 80% of W. African black population

Future of malaria management New drugs New insecticides Greater involvement by governments in vector control and monitoring Habitat manipulation to reduce mosquito populations Involve people in their own primary health care Transgenic mosquitoes- resistant to Plasmodium sp.

Malaria Cases Number of deaths / year Deaths/day # Jumbo Jets day 3,000,000 8,200 20 2,000,000 5,500 14 HOW MANY HAVE DIED IN THIS 1 HOUR LECTURE?