Apicoplasts - malaria Flashcards
Malaria immune evasion
Evade recognition
Evade innate immunity
Modulating adaptive immunity
Malaria - evading recognition
Intracellular location Antigenic variation Tandem repeats High polymorphism Functional redundancy Reduced immunogenicity
Malaria pathogenesis
Know the asexual cycle
Basic symptoms and causes
Syndromes
Placental malaria
Malaria pathogenesis - asexual cycle
Invasion by merozoites, digestion of Hb, morphological changes, asexual replication and rupture of RBC.
Malaria pathogenesis, asexual cycle, morphological changes.
More spherical, formation of knobs.
400 proteins exported by parasite.
pfEMP1 cluster under membrane knobs.
Basic malaria symptoms
Fever, headache, nausea, vomiting, general malaise.
Cause of fevers in malaria.
Rupture –> release of toxins (GPI lipid tethering + hemozoin) –> production of inflammatory mediators and cytokines (C5a, TNF, IL-1B, IL-8) –> effect on hypothalamus –> natural synchronisation.
Severe malaria syndromes
Importance of sequestration
Cerebral malaria
Severe malarial anaemia
Metabolic acidosis.
Severe malaria syndromes - importance of sequestration.
Disappear from peripheral circulation, avoid splenic clearance and immune attack.
Need for ntigenic variation.
Mechanism of transport of proteins not fully understood
Cerebral malaria
Importance
Causes
Causes of severe cerebral malaria
Cytoadherence Inflammatory response Damage to the BBB Role of NO Localisation to brain
Causes of severe malarial anemia
Destruction of RBCs
Decreased productions of RBCs
Co-infections
Causes of metabolic acidosis
The shock model
End products of parasite metabolism
Decreased elimination through impaired hepatic blood flow.
Cerebral malaria importance
Paediatric cerebral malaria: particularly bad and common in children under 5 in sub-Saharan Africa.Mostly P. falciparum.
Modelling cerebral malaria
Difficult to model. Observation in humans depends on post-mortem findings and malarial retinopathy. Animal models have limitations. 3 main contributing factors? Vascular probably important due to correlation between vascular changes in retinopathy and severity of CM.
Cytoadherence in malaria - cause.
PfEMP1 on surface – rosetting, endothelial cytoadhesion.
Cytoadhesion in malaria - effects.
Endothelial activation leading to increased vasoconstriction
Endothelial activation in cytoadherence
Due to upregulation of proinflammatory response and release of cytokines such as TNF-a and IL-1B which act on endothelial cells.
Inflammatory response cerebral malaria
TNFa
The mouse model
Effects
The inflammatory response TNF-a
Cerebral malaria.
TNF-a associated with disease severity.
The inflammatory response observed in the mouse model. Cerebral malaria.
Hyper-inflammation observed in mouse model, and TNF-a and IFNy known to be important.
Dysregulation of TNF-a commonly associated (although in murine models, injection of TNF-a does not mimic CM).
Cerebral malariaThe inflammatory response - effects
Angiopoitin 2
Upregulation of CAMs
Inflammation important in loss of BBB integrity.
Damage to the BBB cerebral malaria
Loss of tight junctions
Effect of inflammation.
Damage to the BBB cerebral malaria - inflammation
Due to inflammation, activation of endothelium and occlusion of microvasculature –> apoptosis of cells of BBB –> haemorrhagic lesions into parenchyma. Edema. –> myelin loss, axonal damage
Cerebral malaria. The inflammatory response - upregulation of CAMs
Inflammation (TNF-a) leads to upregulation of CAMs, which increase sequestration. Especially in cerebellum, may explain motor coordination deficits in children post-CM.
Cerebral malaria. The inflammatory response - angiopoietin 2.
Angipoietin 2 sensitises endothelium to inflammation, and is dysregulated in many cases severe malaria. Increasing Ang2/Ang1 ratio is associated with CM, can predict severity.
Receptors important for pfEMP1.
ICAM-1 for initial rolling and CD31, CD36, CSA, E-selecting, TSP and VCAM-1, among others for cytoadherence.
Cerebral malaria - loss of tight junctions.
Focal loss of tight junctions appear to occur at points of sequestration
Role of NO in cerebral malaria - highly controversial.
Associated both with pathogenesis and protection.
Differing levels are important.
In mice, treatment with NO decreased endothelial activation and reduced sequestration.
Role of NO in cerebral malaria - differing levels
o High levels alter neurotransmission, cause vasodilation
o Low levels lead to endothelial dysfunction.
o Generally low in disease, possibly due to scavenging effects of free haemoglobin.
Cerebral malaria - brain localisation.
- pfEMP1 receptors that are more common in brain – some found, but not yet associated with disease severity.
- Importance of BBB
Cerebral malaria - potential modulators.
NO - successful in mice.
Possibly cerebrovasculature modulators could be important for adjunctive therapies e.g. PPARy agonists, calcium channel blockers, erythropoietin etc.
Metabolic acidosis - the shock model
The shock model: Reduced circulating blood volume and reduced oxygen carrying capacity (hypovolemic shock) –> anaerobic metabolism –> lactate and keto acid production and impaired metabolism –> acidosis
Placental malaria
Pathogenesis
Immunity
Placental malaria pathogenesis
Var2csa bind chondroitin sulphate A and hyaluronic acid. Little role for rosette formation.
Accumulation of infected erythrocytes in intervillous space.
Intervillous infiltrates of monocytes.
Hemozoin deposits in fibrin.
Placental malaria immunity
Can develop, so placental malaria mostly a problem in primagravidae.
Changes in cellular immunity in pregnancy could be important.
Malaria - evading recognition, antigenic variation.
Altering expression
Generating diversity
Malaria - antigenic variation, altering expression.
Basics
Silencing of all except one var gene.
Switching of active genes.
Malaria - antigenic variation, altering expression, BASICS.
60 var genes, monallelic expression. Frequent switching. Strain specific anti-pfEMP1 Abs can only block cytoadherence when pfEMP1 is the same.
Malaria - antigenic variation, altering expression, SILENCING
Location near telomeres
Reversible histone modifications
Upstream of histone modifications.
Malaria - pfEMP1 silencing, location.
Most are located near telomeres (also VSG in T. brucei)
Some are in chromosome central positions.
All are tethered to nuclear periphery.
Malaria - pfEMP1 silencing, tethering to periphery.
This tethering occurs via a var intron region that interacts with nuclear proteins.
Malaria - pfEMP1 silencing, HISTONE MODIFICATIONS
Very key. Histone deacetylases Histone methyltransferases Histone lysine demethylases. Recruited by telomere binding proteins.
Malaria - pfEMP1 silencing, histone deacetylases
Different ones act on different variant gene families.
Deacetylation probably leads to establishment of histone 3 lysine 9 trimethylation by pfSETvs. This overall recrutes heterochromatin protein 1 to silent genes.
Sir2A and Sir2B
Sir2A and Sir2B have complementary actions repressing different var genes
H3K9me3
histone 3 lysine 9 trimethylation
pfSETvs knockout
transcription of almost all var genes.
Knockout of histone methyltransferases in falciparum.
transcription of almost all var genes.
Malaria pfEMP1 silencing - UPSTREAM of HISTONES.
var intron may act as silencing element. If paired with var promoter, latter is silent, but if a promoter is unpaired it will be expressed, even if it is integrated into a silent var gene cluster.
Malaria - antigenic variation, altering expression.
Switching of active genes.
Basics
Mechanism
Altering rate of switching.
Malaria - switching of active genes.
BASICS
A single gene is selectively activated in the early phase of blood stage development. A var gene which is activated is not expressed in the late blood stage, but is ‘poised’ for re-expression in the next cycle .
Adjacent var genes on this telomere are not transcribed due to boundary elements.
Malaria - switching of active genes.
MECHANISM
Not fully understood.
Limiting activation factor hypothesis
Active area hypothesis
Enhancer element hypothesis.
Malaria. Mechanism for switching of active var genes.
Limiting activation factor hypothesis.
Var genes compete for limiting activation factor.
Malaria. Mechanism for switching of active var genes.
ACTIVE AREA HYPOTHESIS.
Movement of telomeric end to active area (how defined?) leads to remodeling and allows transcription. Nuclear actin associated with var gene introns could provide mechanical framework for spatial organization. Position could be involved in ‘poising’.
Malaria. Mechanism for switching of active var genes.
Active area hypothesis - EVIDENCE
An exceptional case where more than one var gene was active showed that both copies were at the same site (RNA FISH analysis).
Malaria. Mechanism for switching of active var genes.
ENHANCER ELEMENT HYPOTHESIS
H-element discovered to mediate monallelic activation of olfactory receptor genes, but an enhancer element with a similar role has not been found in plasmodium.
Malaria - rate of switching of active var genes.
Inactivation occurs at a low frequency, resulting in switching of active gene.
No genes modifying rate have yet been found.
Plasmodium var genes - generating diversity.
Frequent recombination.
Needs to maintain function.
Plasmodium var genes - generating diversity.
FREQUENT RECOMBINATION.
Occurs due to location
Occurs during mitosis
May involve multiple var genes, or several recombinations between two.
In theory, 2.4 million novel var genes generated in an infected individual but needs to maintain function
Plasmodium var genes - generating diversity. Frequent recombination. ROLE OF LOCATION.
Increased due to location near telomeres, where this is frequent.
Plasmodium var genes - generating diversity. Frequent recombination. WHEN IN CELL CYCLE
During mitosis: as demonstrated by experiment with dilution cloning and whole genome sequencing at each step.
Plasmodium var genes - generating diversity. NEED TO MAINTAIN FUNCTION.
DBLs and CIDRs.
Cross-overs.
Plasmodium var genes - generating diversity. Need to maintain diversity. DBLs and CIDRs
Var genes classed by class of DBL (Duffy binding like) and CIDRs (cysteine rich interdomain region). These need to be functionally conserved, although they can be diverse in sequence. Different classes appear to catalyze cytoadherence to different host proteins. Recombination preferentially happens within the same class.
Plasmodium var genes - generating diversity. Need to maintain diversity. Cross-overs
low sequence identity (on average 63%) but crossovers are error-free, with resulting sequence IN FRAME and domain architecture conserved (prob since recombination is between the same class). Cross-overs generally occur at an ‘identity block’
Plasmodium var genes - generating diversity. Need to maintain diversity. Cross-overs - identity blocks.
A short section with perfect sequence identity, within section of generally elevated sequence homology.
Kinases in apicoplasts
Important as signalling mediators.
Involve both Ca++ dependent protein kinases and secreted protein kinases.
Apicoplast - calcium dependent protein kinases.
Action
Regulation of host cell attachment and invasion, gliding motility and parasite egress.
In plasmodium is also involved in gametogenesis, gliding of ookinetes and switching from gliding to invasion.
Kinases in apicomplexa - secreted kinases.
Plasmodium FIKK family and Tg ROPK family secreted into host cell.
Tg rhoptry proteins alter immune response.
Alter immune response. Many predicted to be kinases or pseudokinases. No orthologues of rhoptry kinases in malaria parasites.
Molecular mechanisms of pseudokinases poorly understood.
ROP proteins crossing the cellular membrane.
Spike in conductivity of membrane suggests break in membrane which is resealed, but what mediates this is not understood, although there are several candidates. ROPs carry localization signals
ROP localisation signals
ROP16 has a nuclear localization signal. RAH domain targets to PVM.
Plasmodium FIKKs export
PEXEL motif on FIKKs lead to predicted export into RBC. But not automatic: at least one stays in parasite despite PEXEL motif.
2 thought to be involved in remodeling RBC cytoskeleton.
Purpose of FIKKs
In liver stages, alter host cell signaling, downregulating NFkB and preventing apoptosis.
Apicomplexan egress
Potential therapeutic target.
Mechanism
Signalling in egress
Permeabilisation in egress.
Apicomplexan egress. MECHANISM
o Signal must be given – intrinsic egress cue
o Activation of motility and secretes egress effector proteins
o Requires breaching of multiple barriers including PVM, host cytosolic structure like cytoskeleton and host plasma membrane.
o Permeabilisation of PVM a few minutes prior to egress was calcium and parasite dependent.
Apicomplexan egress - signalling in egress.
K+ and Ca++ (involved in motility) important.
Abscisic acid, ABA.
Apicomplexan egress - signalling in egress. ABA
induces production of cyclic ADP-ribose and hence Ca++ release from intracellular pool (prob ER), triggering secriont of microneme proteins. ABA is probably synthesized in apicoplast given plant lineage.
Apicomplexan permeabilisation.
Toxoplasma - pore forming protein.
Plasmodium - proteases key
Possible role for host calpain proteases too.
Plasmodium proteases in permeabilisation in egress.
PfSUB1, a subtlilisin may have a role in this.
Protease inhibitors demonstrate that proteases are necessary.
Targets of these inhibitors are yet to be determined.
Pore forming protein in Tg in permeabilisation in egress.
TgPLP1. Could weaken the membrane, or could form conduit for other proteins to get through to disrupt cytosol and host membrane. Some plasmodium PLPs have been mooted as potentially having the same role in PV escape in liver stages.
Malaria - evading recognition
Intracellular location Antigenic variation High polymorphism Functional redundancy Reduced immunogenicity
Malaria - modulating the immune system.
Effects on dendritic cells Macrophages T cells. T regs B cells.
Plasmodium immunity - different stages.
Pre-erythrocytic
Erythrocytic
Pre-erythrocytic immunity
High antibody levels needed to neutralize sporozoites.
CD8+ T cells lyse infected hepatocytes and destroy the parasites using interferon based mechanisms.
Erythrocytic immunity
Abs
Pro-inflammatory response controls parasitaemia and contribution to pathology.
Erythrocytic immunity - Abs.
- Inhibit merozoite invasion of erythrocytes
- Prevent adhesion of parasitized RBCs
- Opsonise parasitized RBCS
Malaria - evading recognition, HIGH POLYMORPHISM.
• Highly polymorphic, many variants of same gene in a population. Diallelic with multiple variants of each allelic class.
MSPs, AMA1
Malaria - evading recognition, high polymorphism, MSPs
- MSPs exposed to antibodies for longest as they are involved in initial stages of invasion.
- Some MSPs under a clonally variant expression system, so different parasites are immunologically different.
Malaria, evading recognition. Functional redundancy
If a single protein was responsible for any step of invasion, antibodies would give sterile immunity, but actually several proteins of each family can do each step.
Expression is complex.
EBA and Rh proteins partially compensate for knockout of other.
Essential/non-redundant = AMA1/RON2, Rh5-basigin
Apicomplexans
Plasmodium, toxoplasma, eimeria
Protein export in plasmodium
Membranes to cross
Motifs for crossing.
Motifs for protein export in plasmodium.
PEXEL/HT
PNEPs
PEXEL/HT proteins.
This motif mediates export if present after signal peptide.
PEXEL is a binding site for PI3P and is cleaved by aspartic protesase in the ER. This triggers export, but exact role uncertain.
Possibly PI3P binding leads to segregation in vesicle targeted to the plasma membrane.
PNEP proteins
Do not have PEXEL motif. Have structural similarities but no shared sequence similarities or N terminal processing.
pfEMP1 export
Has PEXEL sequence, but is not cleaved at it, so function uncertain.
Plasmodium immunity - general
Repeat exposure to different strains leads to slow development of partial immunity in endemic areas, with decreasing risk of severe clinical syndromes.
High risk groups.
Plasmodium immunity - high risk groups.
Young children, first time exposure adults, primagravidae.
Malaria immunity in high transmission areas.
Different stages.
Type of immunity.
Types of cells.
Delay in immunity
Malaria immunity in high transmission areas. - different stages.
Little immune response to skin and liver stages. But inoculation with irradiated sporozoites leads to development of sterile immunity in humans as well as in model systems.
Malaria immunity in high transmission areas.- types of immunity
Sterile immunity does not develop: control of parasitaemia levels and limitation of pro-inflammatory immune response does.
Malaria immunity in high transmission areas - types of cells.
CD4 T cells is important in controlling peak parasitaemia
IFNy is essential for clearing infections.
B cell responses are also vital in clearance and elimination of the parasite.
Malaria immunity in high transmission areas - delay in immunity.
Antigenic variation
Immunomodulation
Malaria immunity in high transmission areas - delay in immunity, immunomodulation.
Genetic differences in cell surface receptors
Efficiency of antigen processing
Nature of memory cells.
Efficiency of T cell responses.
Malaria immunity in high transmission areas - immunomodulation, genetic differences in CELL SURFACE RECEPTORS.
- Differences in KIR responses lead to different strength of NK cell responses.
- NK cells important contributors to IFN-y synthesis in early stages, and also lyse iRBCs.
Malaria immunity in high transmission areas - immunomodulation, Efficiency of ANTIGEN PROCESSING.
MHC class Dendritic cells differentially affected.
Malaria immunity in high transmission areas - immunomodulation, NATURE OF MEMORY B CELLS
- Induction and maintenance of responses inefficient
- Atypical memory B cells are inhibitory. Host skewed towards making these will be more susceptible to future infections. Appear like exhausted, but exact functionality is unclear.
- Possible induced by interactions between plasmodium products and B cells e.g. pfEMP1.
Malaria immunity in high transmission areas - immunomodulation, efficiency of T CELL RESPONSES.
• Markers of exhaustion also present here.
Malaria immunity in high transmission areas - immunomodulation, Efficiency of antigen processing, MHC class.
Can be modulated by the parasite according to some reports. Genetic factors from host side probably affect how much MHC class I is down regulated and maturation is delayed.
Malaria immunity in high transmission areas - immunomodulation, Efficiency of antigen processing, dendritic cells.
- Overall pattern
* Specific Effects
Why young children are high risk for malaria.
Protection when breast feeding.
First exposure after weaning.
Later exporsures.
Malaria in children - breast feeding.
Breast-feeding infants have maternal antibodies: these lead to infection being cleared. During infection, T and B cells are primed.
Malaria in children - first exposure after weaning.
There are no maternal antibodies.
Primed B cells produce a few, so clearance isn’t fast enough and high parasitaemia levels can build up.
Primed T cells stimulate macrophages to pro-inflammatory responses, with high levels of TNF-a produced, contributing to severe cerebral malaria.
Malaria in children - later repeat exposures.
More antibodies are produced faster. T regs have developed, preventing overproduction of pro-inflammatory cytokines.
Malarial immunity high risk groups - first exposure adults.
Previous exposure to different antigens.
First exposure.
Subsequent exposures.
Malarial immunity high risk groups - first exposure adults.
Previous exposure to different antigens.
Previous exposure to a different antigen, possible soil-dwelling mycobacteria leads to cross-reactive T cells. B cells are naïve.
Malarial immunity high risk groups - first exposure adults; first exposure.
Bcells naive –> Development of antibody response to give clearance is slow, leading to high parasitaemia levels.
T cells primed –> stimulate macrophages to produce pro-inflammatory cytokines.
Overall high parasitaemia and proinflammatory response –> high risk of cerebral malaria and systemic shock.
Malarial immunity high risk groups - first exposure adults; subsequent exposures.
Development of Treg responses and antibody responses leads to minimal symptoms and clinical immunity.
Malarial immunity high risk groups - primagravidae
Upregulated expression of var2csa leads to sequestration in the placenta.
Models of immunity to plasmodium.
- Many plasmodium species. Several for mice.
- Mouse models are good, but not perfect. None of the mouse parasites produce the same range of clinical syndromes as P. falciparum, although several different ones can be used to model different aspects of it.
- Human experiments involve inoculating humans with low doses of drug sensitive parasites, or giving low doses of iRBC, but these do not exactly mimic inoculation in nature. Or the complex seasonal patterns of transmission in many areas.
AMA1-RON2 - essential and non-redundant invasion protein.
Several roles in invasion, but especially important in moving junction formation. AMA1 receptor complex inserted into RBC membrane.
Although each AMA1 protein is essential to its parasite, much variation across strains. Maintains functional role despite many immunologically distinct variants.
AMA1-RON2. evidence essential.
Gene deletion experiments –> essential
Rh5
Rh5 predicted to be secreted rather than anchored. Involved in moving junction, thought to be essential as difficult to knockout.
Identified in P. falciparum but not in P. vivax.
Blocking effect of antibodies appears to work across many strains.
Also: reduced antigenicity.
Plasmodium evading recognition - reduced antigenicity
Anti-Rh5 antibodies are highly effective but low seropositivity in infected individuals; appears to have mechanisms to decrease immunogenicity. Could include o Limited levels of expression o Limited levels of exposure o Active immunomodulation.
Malaria immunity in high transmission areas - immunomodulation, dendritic cells. OVERALL PATTERN
Variable in different species, so difficult to model.
DC sub-populations may be differentially affected.
Effects on DC cells appear to increase as infection progresses.
Malaria immunity in high transmission areas - immunomodulation, dendritic cells, EFFECTS.
On DC cells:
Impairment of DC function. Inhibition of maturation. Downregulation Class II MHC, decreased trafficking to surface.
Wider effects
• Alters polarization of immune response.
• DCs also act as accessory cells to promote IFN-y secretion by NK cells. Requires multiple contact dependent and cytokine mediated signals.
Malaria immunity in high transmission areas - immunomodulation, dendritic cells, CAUSE.
Possibly due to iRBC adhesion, or due to hemozoin production (hemozoin has similar effects when applied).
Malaria immunity -immunomodulation, macrophages
P. falciparum encodes homologue of macrophage inhibitor factor. In liver stage, invade kupffer cells as well as hepatocytes, and down regulate MHC class II (classI?) and IL-12.
Malaria immunity -immunomodulation, T cells
Apoptosis
Chronic exposure.
Malaria immunity -immunomodulation, T cells, APOPTOSIS.
Apoptosis of immune cells may contribute to poor memory responses.
Malaria immunity -immunomodulation, T cells, CHRONIC EXPOSURE.
Chronic exposure leads to dysfunction.
• Induction of exhaustion has been reported in mice models.
• Possibly due to altered peptide ligands: these can decrease threshold for activation, induce anergy or skew towards pro-inflammatory response.
Malaria immunity - role of Tregs.
Problems if too many (persistent parasitaemia) or too few (overwhelming pro-inflammatory response) .
Malaria immunity -immunomodulation, Tregs.
• Increased frequency of Tregs in malaria infected individuals and susceptible ethnic groups.
Malaria immunity -immunomodulation, observation ofB cells.
Atypical, apparently exhausted memory B cells observed
Atypical memory B cell expansion in chronic exposure. Express inhibitory receptors and replace typical and effective memory B cells.
Plasmodium vaccine development
Vaccine design
Attempted vaccines.
Challenges
Alternative approaches.
Plasmodium vaccine design
Type of immunity
Accelerating acquisition of immunity
Good protein targets in subunit vaccines
Neutralising plasmodium immunomodulation.
Plasmodium vaccine design, type of immunity.
Mimic natural immunity, or induce different immunity to try to induce sterile immunity? Focus on cell mediated responses or antibody based? If former, what role do these play in pathology?
Plasmodium - attempted vaccines.
Whole parasite vaccines.
Subunit vaccines.
Plasmodium - whole parasite vaccines.
o Irradiated sporozoite vaccines.
o More recently, genetically or chemically attenuated parasites.
o Whole parasite blood stage vaccines in development.
o Appear generally to depend on CD8+ T cell responses rather than antibody.
Plasmodium - subunit vaccines.
o Require adjuvant
o Many would appear to need to be multicomponent – high cost. RTS,S vaccine – short lived protection in 50% of cases.
o Sporozoite protein CSP largely targeted. For a long time has been the most hopeful, but success rate not great. Rh5 now more hopeful?
Pre-erythrocytic vaccines.
o High antibody levels needed to neutralize sporozoites.
o CD8+ T cells lyse infected hepatocytes and destroy the parasites using interferon based mechanisms.
o Immunity incomplete.
Challenges to developing malaria vaccines.
Species and strain specific suboptimal immunity, and incomplete knowledge of antimalarial immunity.
Evasion of immunity (antigenic variation, polymorphism, redundancy, reduced immunogenicity and immunomodulation).
Alternative approaches to malarial vaccine development
Vaccinate against placental malaria.
Vaccinate to prevent transmission.
Alternative approaches to malarial vaccine development - vaccinating against placental malaria.
Only var2csa binds chondroitin sulphate A and causes placental pregnancy associated death. Selectively expressed in pregnant hosts, due to unique regulatory transcriptional mechanisms that are upregulated in pregnancy. Women rapidly acquire immunity (1 or 2 pregnancies).
Using var2csa in a subunit vaccine could protect this high-risk group.
Alternative approaches to malarial vaccine development - vaccinating to prevent transmission.
• Difficult to get past safety constraints since for individuals, the risk of side effects will outweigh the benefits, as the benefits are at a community level.
• Low uptake potentially
• A vaccine producing sterile immunity by targeting other stages would have the same effect on transmission.
Raise antibodies to sexual stage in mosquito. These would be taken up with blood meal, preventing fertilization or motility of ookinete.
How does Tg lifecycle allow global distribution?
Primary host w. wide distribution.
Chronically infect primary host –> consistent shedding.
Intermediate host –> propagates indefinitely.
Infects almost any mammal.
Changes behaviour of secondary hosts to increase predation.
General mechanisms of motility
Flagella, cell deformation, gliding motility.
The glideosome
General
Apical complex
Invasion
The glideosome - general
Powered by actin cytoskeleton
Dependence on..
Types of motility
Secretion of proteins.
The glideosome - general - powered by actin.
- Cytochalasin D disrupts actin polymerization. At low conc host cells insensitive, but parasites sensitive, and invasion is blocked, so must be parasitic in nature.
- Parasites selected to be able to invade in low cytochalasin D have mutations in the actin gene.
The glideosome is dependent on…
The parasite actin cytoskeleton. Myosin class XIV (myosin ATPase inhibitor inhibits) Discharge and processing of adhesion proteins, depletion abrogates gliding.
The glideosome - types of motility.
Circular gliding, upright twirling and helical rotation.
The glideosome - the apical complex.
Structures
Mechanism
The glideosome, the apical complex, the structures.
Apical polar ring Secretory organelles Conoid Myosin motors Microneme proteins.
The glideosome, the apical complex, the structures. APICAL POLAR RING.
Ring for attachment of subpellicular microtubules, which radiate out under hte pellicle for about 2/3 of the length of the parasite. Varying numbers.
Numbers of ubpellicular microtubules.
3-4 in plasmodium merozoites and about 60 in plasmodium ookinetes.
The glideosome, the apical complex, the structures. SECRETORY ORGANELLES.
Micronemes
Rhopteries
Dense granules.
Microneme protein are used in …
motility; on the whole, the more motile an organism is, the more micronemes you expect to see.
MIC2 particularly important in tg.
Rhopteries function
for invasion; numerous in invasive stages e.g. plasmodium merozoite stage
The glideosome, the apical complex, the structures. CONOID.
Made of tubulin polymer.
The glideosome, the apical complex, the structures. MYOSIN MOTORS
Anchored to an inner membrane complex by gliding associated proteins, but move relative to actin towards plus end.
Microneme protein adhesion.
Diverse adhesion domains which attach to host cell proteins via EGF-like, lectin like, von Willebrand-like and apple domains. Thrombospondin like domain TSR has many homologues.
Examples of microneme proteins with TSR domains.
TRAP proteins of plasmodium, TgMIC2 of toxoplasma, EtMIC1 of eimeria, among others; TSR regions interact with heparin sulphate proteoglycans.
Function of microneme proteins.
Act as bridges between host proteins/extracellular matrix and the parasite (via cytoplasmic tail vital for motility; exact attachment unknown, but maybe via aldolase).
Many oligomerise
Oligomerisation of microneme
Allows correct folding, efficient trafficking to the microneme, fine tuned receptor specificity, and improved valency and avidity when binding host cells. Oligomerisation with rhoptery proteins important in invasion
Microneme proteins
Function
Adhesion (TSR)
Oligomerisation
Release.
Microneme protein release
not random; ring-like for TRAP proteins in plasmodium, punctuate pattern for Eimeria MIC5 and TgMIC2.
The glideosome apical complex - mechanism
Overview
Regulation
The glideosome apical complex - mechanism OVERVIEW
Microneme proteins translocate posteriorly during motility, due to action of actinomyosin motor, which movement propels the parasite forward. When they reach the posterior end they are released by proteolytic cleavage (hence trail).
The glideosome apical complex - mechanism REGULATION.
o Phosphorylation and palmitoylation of certain proteins in the complex may regulate glideosome activity. Transient phosphorylation may also be important in assembly.
o Motility increased by low environmental K+, leads to increased parasitic Ca++.
The glideosome - invasion.
Initial motility Initial attachment Motility across cell surface Apical attachment Rhoptry secretion and discharge Invasion Closure and separation
The glideosome - invasion. INITIAL ATTACHMENT.
Surface receptors are GPI anchored surface antigens: tgSAGs, pfMSP-1. The glide about surface looking for optimal invasion site.
Anti-SAG Abs, or SAG KO have poor invasion.
The glideosome - invasion. APICAL ATTACHMENT.
Highly polarised Calcium dependent MIC accumulation on apical surface. Conoid extrusion Receptors.
The glideosome - invasion. Apical attachment, CALCIUM DEPENDENCE.
Ca++ dependent deployment of MICs
tg: dependent on abscisic acid synthesis, in pf mediated through activation of PLC
pf sporozoites down to cAMP regulation as well.
The glideosome - invasion. Apical attachment, MIC accumulation on apical surface.
If active penetration is inhibited, parasites arrested in apicaly attached state. Probably mediated by
MIC1 and MIC3 in tg.
Erythrocyte binding like proteins and reticulocyte binding like Rhs in plasmodium. Rh5 binds basigin.
Duffy antigen and duffy binding protein P vivax?
The glideosome - invasion. Apical attachment, CONOID EXTRUSION
Apical tip even closer to the host membrane.
Host specificity in Eimeria.
Invasion
Motility
Conditions
Host specificity in Eimeria. Invasion.
Avian eimeria parasites have lower site specificity during only invasive stages than they do in the host organism, so specificity must rely on more than invasion (although in some of these experiments, apparent invasive ability into unexpected cells might be due to multiple passage)
Host specificity in Eimeria. Motility.
Receptor molecules used in motility act as prelude to invasion. Microneme protein 3 might be important – recent work.
Host specificity in Eimeria. Conditions.
Conditions (e.g. pH, enzymes) in gut might potentiate invasion.
Rhoptry secretion
Signal
Moving junction.
Rhoptry secretion signal
Binding of microneme proteins leads to signalling for rhoptry release.
Rhoptry secretion - moving junction.
rhoptry neck (RON2) proteins are secreted into the RBC, and associate with microneme derived protein AMA1 (tg and pf) and other RON proteins to create ring-like structure of binding interface (tg), with similarities to mammalian tight junctions.
Moving junction
During invasion moves around parasite as a circumferential ring. Similar to tight junctions.
Rhoptry secretion - moving junction activity
Sieve
Invagination
Driving force.
Rhoptry secretion - moving junction activity, invagination.
One model for eimeria suggests insertion of amphipathetic molecule onto cytoplasmic leaflet of membrane causes it to expand, leading to invagination
Rhoptry secretion - moving junction activity, sieve.
removes some parasite and host proteins from membranes. Most MIC proteins excluded, but AMA1 and SAGs are included. Probably prevents lysosomal trafficking.
Rhoptry secretion - moving junction, driving force.
Either
MJ loosely attached to host and parasite membranes, parasite driven forward through it due to interactions between motors and other proteins e.g. MIC2.
Or MJ has a role in propulsion of the parasite through, and is directly connected to the motors, with MIC2 being sieved out as it goes.
Getting to the host cells
Transcellular migration of sporozoites across hepatocytes.
Toxoplasma does not go through cells, but via paracellular route using ICAMs.
Malaria stats per year.
500,000 deaths per year with around 500m cases per year and 3.2bn at risk of infection —> mostly in Africa (90% of cases) and mostly in
Malaria plasmodium species
over 170 but most infect birds, only 25 infect primates and 5 infect humans
Malaria species
Falciparum cause the most morbidity (and is most studied so will be the focus of this essay)
Other spp = vivid, oval, malaria and knowlesi (mostly in monkeys though - zoonosis)
Malaria vectors.
Malaria = female anopheles mosquito spp (30-40 commonly transmit malaria) —> A. gambiae best known for transmitting falciparum
Malaria rosetting.
iRBCS adhere to normal RBCs through blood group AGs, CD36, CR1 and HS —> increases severity of disease
Simplified hypotheses for cerebral malaria.
Mechanical hypothesis.
Humoral hypothesis.
Poising
Var gene txn ceases in late blood stage but epigenticaly marked for re-activation during next erythrocytic cycle = poised
Var gene structure.
Consist of Exon1 coding polymorphic sequences forming extracellular domain, Exon2 coding semi-conserved intracellular domain connected by single highly conserved intron
Malaria evading recognition, tandem repeats
Provide immunodominant B cell epitopes, stimulating antibody production. Even with high levels of these antibodies, immunity is low, suggesting that they don’t provide good protection.
The presence of polymorphic repeats in antigens that are not targets of protective immunity, affects affinity maturation of antibodies and thus masks the critical epitopes. Tandem repeats also induce T cell-independent B cell activation by cross-linking their surface immunoglobulins and suppressing antibody responses to important adjacent regions.
