1. Mechanisms of Malarial Infections
PHM Fall 2018
Instructor: Ms. Maya Latif
Coordinator: Dr. J. Henderson
Mechanisms of Malarial Infections
PHM142
September 18, 2018
By: Chelsea Chow
Naomi Lewin
Himani Patel
Cynthia Selvanathan

2. OVERVIEW: What is Malaria?
Parasitic infection of red blood cells by some Plasmodium species
P. falciparum, P. vivax, P. ovale, P. knowlesi, P. malariae
Plasmodium sporozoites transmitted through female Anopheles mosquito bites (25 spp.)
216 million cases and deaths (2016, 94 countries)
Especially common in sub-Saharan Africa and young children
Clinical Presentation: may be classified as uncomplicated or severe
Fever, anemia + jaundice, swelling of liver and/or spleen
General discomfort/aches, diarrhea, chills, tiredness
Cerebral malaria, ARDS, AKI/acidosis/hypoglycemia, coma, seizure
Ashley et al., 2018, Lancet
White et al., 2014, Lancet
World Health Organization, 2017

3. Life Cycle of Malaria
Anopheles mosquitoes feed on human blood and transmit Plasmodium sporozoites
Sporozoites circulate to and invade hepatocytes, where they reproduce asexually and form merozoites
Merozoites are released into circulation and collide with, and then invade red blood cells
Merozoites reproduce asexually in red blood cells, eventually causing cell lysis to release merozoites which can invade new red blood cells
Some merozoites transform into gametocytes for uptake by Anopheles mosquitoes
Gametocytes reproduce sexually in Anopheles mosquitoes to produce sporozoites
Cowman et al., 2017.
Ashley et al., 2018, Lancet

4. Hepatocyte Invasion Sporozoites transverse the dermis
using SPECT & TRAP proteins
Sporozoites enter liver through fenestrations
Sporozoites transverse cells using proteins SPECT1/2 & CelTOS
CSP & TRAP on sporozoite surface attach to HSPG lining hepatocyte surface
Sporozite switches from “migratory” to “invasive” mode
Replication into merozoites
When a mosquito bites the skin, the sporozoites are delivered to the dermis. They transverse the dermis cells using SPECT proteins and glide down using TRAP proteins. When they get to the bloodstream they travel to the liver and enter through fenestrations in the endothelium.
There is a barrier of Kupffer cells, liver macrophages, before the sporozoites can reach the hepatocytes. They are able to transverse the Kupffer cells using the proteins SPECT1, SPECT2 and CelTOS. As well, hepatocytes are lined with HSPG (heparin sulfate proteoglycans). The sporozoite surface proteins CSP and TRAP attach to HSPG. The sporozoites then switch from migratory to invasive mode, where they invade hepatocytes by transvering the membrane. Then the sporozoites are able to replicate in the hepatocytes, and become merozoites which will enter the bloodstream.
Acharya et al. 2017, Front Microbiol.

5. Immune System Evasion The liver is an immunoprivileged organ
Sporozoites alter the cytokine profile and MHC expression of Kupffer cells
CSP on sporozoites suppress NFκB signalling of hepatocytes
Sporozoites form a parasitophorous vacuole in hepatocytes to avoid lysosome
Merozoites exit liver by covering themselves with hepatocyte-derived membrane
Of course, the parasites must overcome the immune system in order to effectively infect the person and be able to replicate.
Firstly, the liver is an immunoprivilged organ, meaning there is no strong immune responses in the liver.
The sporozoites are able to alter the cytokine profile and MHC expression of Kupffer cells so that they are not able to clear the parasite.
Sporozoites also suppress the NFkB signalling of hepatocytes using CSP protein, so the inflammatory response is suppressed.
As well, the parasites form a a parasitophorous vacuole inside the hepatocyte, which keeps them isolated from the lysosome to avoid the cell from being able to defend itself by apoptosis or autophagy.
Lastly, merozoites avoid the Kuppfer cells in the liver by covering themselves in hepatocyte-derived membranes when they are exiting the liver.
Gomes et al, 2016, Front Microbiol.

6. Mechanism of Erythrocyte Invasion
Merozoites are polarized cells
Attachment: merozoite surface proteins
Pore formation:
Between merozoite and RBC through a PfRh5/PfRipr/CyRPA complex
Provides secure apical attachment to the RBC
Tight Junction Formation/Invagination:
Merozoite inserts RON2 into RBC membrane
AMA1 ligand on merozoite creates tight junction with RON2 receptor
Tight junction creates a depression in the RBC
Tight junction moves via parasite’s actinomyosin motor
Merozoite surface proteins (MSPs), present along the entire merozoite surface, are strong candidates for invasion ligands that mediate primary encounter with an RBC, either directly or indirectly → essentially act as a mechanism for initial binding onto the red blood cell
Once bound, the merozoite creates a small pore at from its apical end into the RBC through a PfRh5 complex, providing it secure attachment
Cowman et al. 2017, Cell Host Microbe
Paul et al. 2016, Curr Opin Hematol

7. Mechanism of Erythrocyte Invasion
“Sheddase”, a serine protease, cleaves surface coat found at the moving junction of the merozoite
Completion: a rhomboid protease cleaves any adhesive proteins
Reseals the membrane
Creates a parasitophorous vacuole
Inside the RBC, merozoites mitotically proliferate
RBC lysis → reinfect new RBCs (~1-2min)
Small % of merozoites commit to gametocytogenesis
Curr Opin Hematol
Cowman et al. 2017, Cell Host Microbe
Paul et al. 2016, Curr Opin Hematol

8. Cytoadherence Infected RBCs attach to endothelium, other RBCs
or immune cells.
Advantages:
Escape clearance by spleen
Microaerophilic environment ideal for maturation
Mechanism/mode:
IRBCs present PfEMP1 on their surface
PfEMP1 can bind to different endothelial cell (EC) receptors: CD36, ICAM-1, PECAM-1 & EPCR for adhesion & start of sequestration
Binding ECs also activates proinflammatory and procoagulant responses, reduces barrier function, and impairs vasomotor tone
Causes clinical manifestations of malaria:
Microcirculatory obstruction & hypoxia, metabolic disturbances & multi-organ failure
Cytoadherence is the unique ability of IRBCs to stick to the endothelium (called sequestration) and to other RBCs and immune cells (called rosetting/clumping). This is advantageous for the parasite because it creates a microaerophilic environment that is ideal for its reproduction and by sequestering/clumping, it can avoid being cleared by the spleen. The way in which it does this is by the merozoite producing and presenting PfEMP1 (plasmodium falciparum erythrocyte protein 1) onto the RBC’s surface. PfEMP1 can bind to several endothelial cell receptors like CD36, ICAM1, PECAM1 and EPCR. Binding to these receptors can also lead to proinflammatory and procoagulant responses, permeability in the endothelial membrane and impairment of the vasomotor tone. All these changes ultimately lead to the sever clinical manifestations of malaria such as microcirculatory obstruction, hypoxia, metabolic disturbances and multi-organ failure.
Ho & White, 1999, Am J Physiol
Aird et al. 2014, Blood

9. Clonal Antibody Variation
PfEMP1 is an important target of the host-immune response
Each parasite has 60 var genes that encode for PfEMP1
Staying one step ahead of the immune system:
Immune response to one type of PfEMP1 develops
Parasite switches expression to a different var gene/PfEMP1
Parasites avoid established adaptive immune response by expressing variant PfEMP1
One interesting way by which the parasite evades the immune system is through a process known as clonal antibody variation. PfEMP1 is presented on the IRBC’s surface and can get recognized as an antigen by the immune system. However, each parasite has a set of 60 var genes which each encode for a different PfEMP1. Essentially, even if the immune system recognizes one PfEMP1 antigen and produces antibodies against it, the parasite can always switch the expression to a variant PfEMP1, thus avoid being cleared by the immune response.
Aird et al. 2014, Blood
Flick & Chen, 2004 Mol Biochem Parasitol

10. Summary
Plasmodium parasites transmitted through Anopheles mosquitoes, invade hepatocytes and erythrocytes while effectively evading the immune system
Hepatocyte Invasion
Attach to hepatocyte HSPG through CSP and TRAP
Transversal of cells through SPECT and CelTOS
Erythrocytic Invasion
Attachment to RBC through merozoite surface proteins and pore formation
Tight junction formation through RON2 and AMA1
Invagination of the RBC via actinomyosin motor
Parasitophorous vacuole formation and cleavage of adhesive proteins and surface coat
Cytoadherence is the ability of IRBCs to stick to the endothelium and other circulating cells, causing sequestration & clumping that results in clinical manifestations of malaria.
Expression of PfEMP1 proteins can be varied to avoid the adaptive immune response
Edit this slide when all info posted

11. References
Acharya, P. et al. (2017). Host–Parasite Interactions in Human Malaria: Clinical Implications of Basic Research. Frontiers in Microbiology, 8,
Aird, WC. et al. “Plasmodium Falciparum Picks (on) EPCR.” Blood, vol. 123, no. 2, American Society of Hematology, Jan. 2014, pp. 163–67, doi: /blood
Ashley, EA., Pyae Phyo, A., Woodrow, CJ. (2018). Malaria. Lancet, 391, 1608–21.
Cowman, AF. et al. (2017). The Molecular Basis of Erythrocyte Invasion By Malaria Parasites. Cell Host Microbe, 22, 2. Doi: /j.chom
Flick, Kirsten, and Qijun Chen. “Var Genes, PfEMP1 and the Human Host.” Molecular and Biochemical Parasitology, vol. 134, no. 1, Mar. 2004, pp. 3–9,
Gomes, PS. et al. (2016). Immune Escape Strategies of Malaria Parasites. Frontiers in Microbiology, 7,
Ho, M., and White, NJ. “Molecular Mechanisms of Cytoadherence in Malaria.” The American Journal of Physiology, vol. 276, no. 6 Pt 1, June 1999, pp. C ,
Paul, AS., Egan, ES., Duraisingh, MT. (2016). Host-parasite interactions that guide red blood cell invasion by malaria parasites. Curr Opin Hematol, 22, 3. doi: /MOH
White, NJ. et al.(2014). Malaria. Lancet,
World Health Organization. World malaria report Geneva, 2017.