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The main aim of this dissertation is to look at the way in which the parasite Toxoplasma gondii unicellular eukaryote can manipulate the cell signalling mechanism of its host CNS in vertebrates and what biochemical and molecular pathways are manipulated and affected. Toxoplasmosis is an opportunistic parasite that is capable of producing disease in immune deficient hosts. In the immune-competent host, it is either found in a latent form or acute form and causes a self-limitingdisease. The species Toxoplasma gondii is part of the phylum apicomplexa, like other well-known protozoan parasites such as Plasmodium Falciparum that causes malaria.
The aspect I am most interested in for this paper is its ability to manipulate host signalling e.g. deploying varying parasite kinases and phosphatase, subverting host cells ability for apoptosis and degrading host cell function (Laliberté J, 2008 Jun).
Protein kinases regulate cellular processes throughout the body by adding phosphate groups to target proteins (phosphorylation). The parasite protein kinases are released by secretory organelles, these include rhoptrys, micronemes and dense granules, the parasite kinases include rhoptry kinases that are named from the area where they are released and pseudokinases that are believed to have weak or no catalytic activity. Phosphatases are groups of enzymes that hydrolyse phosphate groups the effects of these parasite phosphatases secreting by T.gondii is still being explored.
Apoptosis is the bodys own way of controlling programmed cell death or degradation, this ability can be altered by T.gondii to aid its own survival by modulating host cell apoptosis (Lüder CG, 2005) this is meant that is can induce host cell apoptosis when needed e.g. when a cell is full of infected tachyzoites and resisting it when the host cell is first infected to protect the infected cell till it reaches maturity. This will play are role in the degradation of the host cell functions and manipulating the host cell to the parasite advantage to increase its own survivability.
Critical analyses of the different mechanism in which the host cell signalling is manipulated will be looked at, including the manipulation of cell signalling in invertebrates. This will be broken down into smaller questions, I will give a detailed background into host immune response to infection and cell signalling in vertebrates, and will address what biochemical factors are guiding and causing such behaviours, also we will focus on answering, what interaction are there between the pathogen and host and what changes are occurring in the host cell in response to infection. I will deal with what mechanisms are common between vertebrates and rodents and what are unique in selected cases. Chapter five will question the biochemical alterations to signalling in the CNS that arise from infection. Chapter six will break down what signalling mechanisms are affected and how this affects the host. Certain parasites are able to infect neurons and in turn inflame the central nervous system, also invading host cells and co-opting cell signalling mechanism and altering the cell machinery, alter neurotransmitter and hormone communication within the host for its own purposes.
Even simple parasites have the ability to infect many different organisms, using very complex mechanisms at their disposal to survive and flourish. Evidence will be used to answer the question on which biochemical and molecular pathways are manipulated, to see how the organism T.gondii achieves the manipulation of the host within the CNS.
In Figure 1 the structure of the tachyozoites is shown, this is important in understanding how T.gondii can alter the host cells. The conoid is located near the apical end of the organism and is believed to be associated with how the parasite can enter host cells. The main secretary organelles are found near the apical end and consist of the micronemes, rhoptries and dense granules and are all used to aid the invasion process into the host cell. The microneme have been found to help the parasite in movement and facilitating the way in which it is able to bind to the host cell. The Rhoptry proteins are released during the invasion of the cell once the parasitphorous vacuole has been formed and are detectable with the membrane of this vacuole. The dense granule proteins are released during and after the formation of the parasitphorous vacuole to change the environment to more favourable conditions for the parasite and aid in replication and intracellular survival.
The apicoplast is a four membrane organelle similar to a plastid, the majority of protein encoding is done by a single nucleus, and the cell also contains a single mitochondria, golgi body and rough endoplasmic reticulum.
Toxoplasma Gondi is an endoparasite that can infect most cells with a nucleus and can infect all invertebrates. The way in which it is transmitted to a host is via oocysts containing sporoziotes from cat feces, cyst tissue containing bradyzoites from infected meat that are ingestion into the host body. Once within the body the oocysts and tissue cyst transform into tachyozoites that multiply and eventually form tissue cysts within the CNS and muscle converting again into tissue cysts. This is believed to be due to the body's immune response.The parasite can enter a cell (dendriticcell, macrophage) by phagocytosis and created a parasitophorous vacuole to protect itself from the host. The parasite uses the host cell and alters the cell functions to be able to faster replicate and sustain infection.
The apicomlexan parasite T.gondii is used as a model organism as it is easily grown in-vivo to the cyst stage within infected tissue and used to infect a varying degree of hosts, like many pathogens it is most devastating in immunocompromised hosts and is usually self limiting in hosts with effective immune systems. When the host has been infected the host immune response fights the infection by producing antibodies against the T.Gondii antigens. The body's cell mediated immune response is believed to control the infection using cytotoxic T cells that release IFN-y cytokine. The rapidly growing tachyzoites begin to change back into the slower growing bradyzoites. Cysts containing bradyzoites begin to develop in the cell tissue such as muscle and brain and remain until they rupture and release large amounts of parasite tachyzoites that will replicate and lyse the host cells causing organ damage and the host immune response causing inflammation to the body. Due to the presence of particular cellular receptor that allow the parasite to attack the neurons,astocytes and microglia and cells within the eye, most of the damage is caused within the brain(encephalitis) and eyes (Toxoplasmic retinopathy).
HOST IMMUNE RESPONSE TO INFECTION AND CELL SIGNALLING
To first understand how the host cells within the body are being affected by T.gondii, I will be discussing the body first response to the infection and various cells signalling mechanism that are important to the host immune response. Once the host is infected by the T.gondii tachyzoites or cyst tissue an immune response within the body is triggered and requires the presence of an antigen-presenting cell such as a macrophage or dendritic cell used in conjunction with a B cell or T cell. The antigen-presenting cell will present an antigen on its cell surface to a B cell which will in turn activate the production of antibodies that will target and bind to the antigen. Once the antibodies have binded to the antigens on infected cell, this causes a signal to be produced that will mark the infected cells to be destroyed by macrophages by ingesting them.
The presence of antibodies within the host creates a complement cascade that effects the signalling to other immune functions, these can include the lyses of infected cell presenting antibodies. Antibodies can also signal natural killer cells and macrophages to kill viral or bacterial-infected cells. . Once the infected cell has been phagocytised they will present MHC markers to show to the extracellular environment what proteins are inside the cell and cause T cells induces the cell's self destruct mechanism. If the APC presents the antigen to the T cell they will become activated and replicate. CD4+ helper T cells will secrete Cytokines and interleukins that up regulate growth factors and pass on signals for more immune related cells to come to the infected area and also create a pro inflammatory response to allow more blood flow to the infected area, this is crucial in maintaining a continuous immune response and recruiting NK cells, macrophages and CD8 Killer T cells. CD8+ killer T cells will hunt for infected target cells that are specifically expressing the antigen presented by the APC. Once the tachyzoites have entered the CNS they will infect neurons, astrocytes and microglial cell (see Fig 3). CD8+ killer T cells and CD4+ helper cells as well as macrophages and NK cells are able to enter the CNS during the infection, T cells are able to produce IFN-y which has been shown to be a major factor preventing further activation of Bradyzoites cyst cells (Imtiaz A. Khan, 1999)T.gondii is believed to have the ability to infect dendritic cells and macrophages and hide within the host cell while crossing the blood brain barrier straight into the Brain (N. Courret, 2006).The parasite T.gondii has evolved certain adaptation that allows it to avoid and manipulate the innate immune system and this will be looked at next.
HOST-PATHOGEN INTERACTION - WHAT CHANGES OCCUR IN THE HOST CELL IN RESPONSE TO INFECTION?
T.gondii tachyozoites will actively seek out cells to infect by penetrating through the host cell wall or by phagocytosis. Tachyozoites will attach themselves to the outer membrane of the host cell using the apical end exocytosising adhesive proteins from the secretory organelle (microneme) this will aid in the recognition and adhesion and create a stable binding between the host and parasite. Using gliding motility the junction between the host cell wall and the parasite is moved posteriorly, at the parasite apical end a vacuole is formed which develops further with the discharge of lytic substances from the rhoptry. The rhoptry secretions are members of the ROP2 family,PP2c-hn, ROP16 and ROP18. Rhoptry kinase ROP16 and the rhoptry protein phosphatase 2C (PP2C-hn) carry a nuclear localisation signal (Gilbert et al., 2007), which mediates the trafficking to the host nucleus, ROP18 interacts with the parasitophorous vacuole and the vacuole membrane remains continuous with the ingress of the parasite. No host cell protein diffuses in the parasitic vacuole during this process. The vacuole membrane is a composite between host cell membrane lipids and rhoptry content. After internalisation the dense granules are discharged into the parasitophorous vacuole and move to distinct exocytosis sites to accumulate, along with the rhoptry proteins creating transmembrane pores that are involved in the nutrient of the parasite. Once the Parasitophorous vacuole has been formed it will move from the outer cell to a location nearer the host nucleus migrates along microtubules position near the host cell nucleus. Research has been undertaken that has found that ROP16travels to the cell's nucleus and is able to block the signal that would lead to a rapid immune response from an intracellular pathogen such a T.gondii (Saeij et Al, 2007).The parasitophorous vacuole membrane associates with the host mitochondria and endoplasmic reticulum. The reason for this is believed to be the relationship between the mitochondria and a rhoptry protein that is embedded in the Parasitophorous vascular membrane giving access via T.gondii antigens to the MHC complex of the host cell, allowing activation of host transcription factors (Kamerkar s, 2012).
Once a chronic infection is established within the host and the Tachyozoites have disseminated into the CNS and cyst tissue is formed and the tachyozoites become bradyzoites.The T. gondii cyst wall membrane, is made up of glycoproteins, and is important in maintaining the structural and nutrient needs of the parasite while mitigating host immune system detection. The acute immune response clears all of the infected cells leaving bradyzoites viable cells left that are able to effectively avoid the immune system as they are not visible to CD8+ killer T cells in intracellular cyst form. Cyst location within the brain concentrates in the cerebral cortex, basal ganglia, hippocampus and amygdala, also infection skeletal muscle and neurons, Microglia, astrocytes.
WHAT MECHANISMS ARE COMMON AND WHAT ARE UNIQUE?
T.gondii has also produced strange effects in rodents including the way in which it manipulates the defensive behaviours or rats so they are unafraid of cats, this is believed to be achieved by altering the way in which cat odour's are perceived within the rats that are infected with cysts within the brain, the rats will approach the odour's that they would usually avoid. This is believed to be an evolutionary process that has evolved to allow the T.gondii to continue it sexual life cycle within the cat. This host response is only found in rodents and it is only the odour of cats that the rats are attracted to. It is reported that toxoplasma infection can alter neural activity within the limbic area of the brain that is involved in defensive behaviour in response to cat odours (Patrick K. House, 17 Aug 2011), the action of T.gondii is an action in which he innate fear response in rats is compromised allowing T.gondii to return to its primary host.
Evidence that suggests that Toxoplasma Gondi increases the level of dopamine on mice. The study examines in-vivo Toxoplasma Gondi bradyzoites infecting the brain, and expressing tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis. The parasite also has tyrosine hydroxylase encoded within its genome. The study also shows that dopamine levels were increased within the body by 350 per cent which is a significant increase and could lead to neurological a disorder, what is interesting is that the study shows it is possible for infected cells to increase the levels of dopamine. The rate of infection and an increase in dopamine levels provides us with a directly proportional relationship between both (PRANDOVSZKY, E. et al. 2011).
None of the above mechanisms are found as yet within mammalian host so I believe that these are unique mechanisms to Rodents, but as further research is undergone I believe that similar mechanism will be found.
WHAT BIOCHEMICAL ALTERATIONS TO SIGNALLING IN THE CNS ARISE FROM INFECTION?
T.gondii also has the ability to affect the MHC molecules, The MHC molecules are protein complex's that will present antigens to T cells to aid in the immune response. These molecules are broken down into many classes but the one we are most interested is the MHC class 2 molecule that present antigens of infected cells that have either been phagacytosed invaded host cells. Since T.gondii infects host cells intracellular expression of infected cells is important and this isdone by the MHC class 2 pathways. T.gondii can interfere with the IFN-y induced MHC class 1 and class 2 expressions, this mechanism is believed to affect a variety of cells including rodents primary rat astrocytes and microglia and human-derived glioma cells (Lüder et al. 1998). This shows that the inhibition of MHC class 2 expressionsin cells isa general mechanism and happens in rodents and mammals. Since T.gondii is able to interfere with the expression of MHC 2 of APC this down regulation will have a knock on effect on the CD4 helper T cell and diminish the effectiveness of the immune response. Evidence showing that the mechanism by which T.gondii down regulates the MHC 2 expression is achieved by direct interference with the host cell. (Lüder et al. 1998)
T.gondii can also affect cell death which is important as infected cells and damaged cell can be effectively destroy themselves by regulation of lymphocyte enabled death.
Apoptosis is initiated by the binding of death ligands or TNF-a to a death receptor pathway, infection of a host cell presenting Antigens also attracts NK cells and CD8-T cells that release Granzymes. The important factor for T.gondii is the host cell remains intact while it is replicating and can modulate apoptosis of the host cell to stop cell death, after infection the immune response to T. Gondi is considerably reduced by the level of apoptosis in different leucocyte populations. Khan et al. (1996)
Accepted 27 March 1999
Available online 1 April 2002
WHAT SIGNALLING MECHANISMS ARE AFFECTED AND HOW DOES THIS AFFECT THE HOST?
T.Gondii is believed to play an important part in reducing the hosts immune response by stopping the production of pro-inflammatory cytokines (. IFN-Î³, IL-12, TNF-Î±) and anti-inflammatory cytokines (TGF-Î²), cytokines are very important in controlling the mediation of the host inflammatory response to infection. During acute toxoplasmosis, IL-10 fulfils a dual role in the suppression of the host's cellular immune response as observed in both humans and in mice. First, it inhibits the IFN-Î³ production and possibly the proliferation of T lymphocytes,i.e. antagonizes the development of a potentially protective Th1 immune response, such T-cell-dependent immunosuppression exerted by IL-10 primarily appears to avoid overwhelming inflammation which eventually leads to the host's death , IL-10 may also deactivate macrophages,thereby diminishing IFN-Î³-induced toxoplasmacidal activity and facilitating intracellular parasite survival (Bogdan and Nathan 1993). These results provide clear evidence that IL-10-induced immunosuppression following infection with T. gondii is beneficial for both the parasite and the host and favours a stable parasite-host relationship. In addition to IL-10, TGF-Î² is an important deactivator of macrophages (Langermans et al. 2001). Toxoplasma must allow cytokines to trigger enough of an immune response to keep its own numbers in check and ensure its own proliferation. If large amounts of cytokines are produced they will cause an overwhelming immune response that could damage host or eliminate the parasites. Toxoplasma sends ROP16 to access the communication pathways withinthe immune cells, enabling T.gondii to lower the production of cytokines.
WHAT ARE SECRETORY PROTEINS AND HOW ARE THEY USED IN MANIPULATING HOST CELL?
T.gondii is able to secrete and inject proteins from specialized secretory organelles called rhoptries which are a critical part in aiding processes such as cell invasion and modulation of the host cell immune response. The rhoptries are broken down in structure and function into two compartments, the first is the apical duct that contains the rhoptry neck (RON) proteins and are involved in driving the T.gondii invasion. The second is the posterior bulb which contains rhoptry proteins (ROP) that are involved in co-opting the host processes and can modulate the parasites growth and virulence once in injected into host cells.
Rhoptry secretion is initiated after contact with the host cell but the reason for this process is still unknown, rhoptry proteins will move to the parasite plasma membrane and fuse with the surface, RONS are delivered to the parasites surface and will aid in the formation of the moving junction. During apical attachment rhoptry proteins are immediately released. ROP18 and ROP16 are the most well studied active kinases that are capable of phosphorylation.
ROP18 is expressed to protect the infected cell, T.gondii that don't express ROP18 on the surface membrane will be killed by the body's immune system as the infected cell will become interferon activated, interferon will up regulate IRGs(GTPases) causing the degradation of the cell. When ROP18 is present within an infected cell it can phosphorylate the IRG proteins and can modulate the GTPase function causing the infected cell to remain intact (Fentress, 2011).
ROP16 which is also a rhoptry kinase is secreted into the cytosol and moves to the host cell nucleus and can modulate the host STAT3 and STAT6 signalling, and IL12 production. This was shown by (Ong YC Et al, 2010) .Using recombinant wild-type and kinase-deficient ROP16, that in vitro ROP16 has intrinsic tyrosine kinase activity and is capable of directly phosphorylating the key tyrosine residue for STAT6 activation. STAT3 is involved in the mediation and expression of different genes in response to cell stimuli, controlling cell growth and apoptosis. STAT6 is required for mediated response to IL-4 which in turn will control differentiation of CD4+ T cells to a Th2 phenotype. When the STAT 6 pathway is affected by ROP16 this will cascade causing Cytokine Inhibition and Arginase-1-Dependent Growth Control.
Included in the secretory proteins are so called pseudokinase (ROP2,ROP4,ROP7 and ROP8), in comparison to the above ROP18 and ROP16 they lack important residues in the catalytic loop and have also lost the glycine loop that stabilizes the Î±Î²-phosphate of adenosine triphosphate causing them not to be able to complete phosphorylation reaction but could help in providing substrate and stabilizing the infected cell. (Fleckenstein, 2012)Has shown that pseudokinases still have the ability to aid in the more active rhoptry proteins although have no ability to phosphorylate. ROP5 pseudokinases have been found to aid ROP18 by binding to the IRG proteins, keeping them inactive and open so ROP18 can disable them, this shows that the pseudokinases still have the ability to regulate or become co-factors.
T.gondii like other Protozoan parasite has long been neglected in research but will only require a breakthrough that will be able to target the mechanisms by which the parasite survives. The ability of the parasite to reducing the hosts immune response by stopping the production of pro-inflammatory cytokines, interfere with the IFN-y induced MHC class 1 and class 2 expression, modulate apoptosis of the host cell to stop cell death, and its ability to effectively avoid the immune system in intracellular cyst form and the manipulation of the innate fear response in rats are serious reason why more research is needed. Immune invasion is relevant as the transmission to the final host (cat) depends on the parasites ability to cause acute infection. The list of mechanism discussed in this dissertation are not complete and the Biochemical and molecular mechanisms in which T.gondii is able to subvert the host immune systems are still trying to be understood and fresh research has shown that itdirectly interferes with signalling cascades within the host. T.gondii is a unique parasite in that it employsmechanisms that only reduce the host immune response and don't destroy it creating a balance so it can proliferate a longer lasting parasite-host relationship eventually ending in itgetting to its primary host.
The major concern with T.gondii infection is its ability to remain within the host cells as cyst tissue and cause a latent infection at a later time, e.g. if the host is immunocompromised. Due to the high number of people infected world wide and 7-34 percent of people in the UK having been infected with T. gondii according to (HPA, 2012). A new strategy will be required to develop anti-parasitic agents that are able to cross the blood brain barrier of the infected host and enter the parasite cyst wall within cell tissue. If the parasite could be removed from infectedcell tissue a requirement to do the least amount of damage would be a major concern due to the repercussions of destroying neurons, astocytes, microglia and cells within the eyes. Scientist are starting to understand the complexities of the signalling and regulatory processes involved in parasitic infection and the molecular events in T cell controlled infection. New novel targets will be found to exploit the asexual stages within the host as new techniques are developed in imaging and molecular engineering, this will enable a better understanding about the parasite host relationship. Several new compounds have been identified that are capable of inhibiting T. gondii tachyzoites in vitro and also show signs of anti-bradyzoite properties (Doerig, 11 March 2004). A major difference between protozoan parasites is the different protein kinases they secrete and this could be a main target to inhibit to stop the parasite development. New anti-parasitic drugs based on the inhibition of protein kinase are split into two paths, the first consisting of a blanket approach screening chemicallibraries on recombinant enzymes and has yielded several kinases that could be future targets. The second approach I believe it better due to also gaining knowledge on the molecular mechanism used by T.gondii, this is the identification ofmolecular targets of kinase inhibitors that display antiparasitic traits. This approach has produced a new developments regarding PKG as a new drug target against T.gondii, and purvalanol B a purine based CDK inhibitor which affects new targets in protozoan parasites. Resent research on the structure of Plasmodium protein kinase complexes will allow the design of new drugs that will inhibit kinase mechanisms.
Table 1: The response of CNS-resident cells to Toxoplasma gondii infection.
Brain cell type
Parasites can encyst in neurons
Infection induces cytokine and chemokine production; stimulated neurons are unable to inhibit parasite growth
Neurons containing parasite cysts avoid scrutiny by CD8+ T cells
Murine Nramp1âˆ’/âˆ’ models are affected in stress response and mortality following Toxoplasma gondii infection
Microglial cells are preferentially infected, but most effectively inhibit parasitic growth within CNS cells
Upon Toxoplasma infection, microglia produce IL-1 beta, IL-10, and tumor necrosis factor-alpha
Murine model infection induce an upregulation of CD200R & CD200, which control CNS inflammation
Infection downregulates MHC class II expression
Toxoplasmic encephalitis induces IL-12p40, iNOS, IL-1beta, TNF-alpha largely due to CD8+ T cell interaction. MHC classes I and II, ICAM-1, and leukocyte function-associated antigen-1 are also upregulated
Toxoplasmic encephalitis induces vascular cell adhesion molecule, ICAM-1, and MHC classes I and II. Induction depends on IFN-gamma receptor
Infection induces ICAM-1, IL-6, and MCP-1
Induction levels vary depending on parasite strain
Astrocytes are preferentially infected compared to neurons
Intracellular infection reduces expressed MHC II
Interferon-gamma-activated indoleamine 2,3-dioxygenase (IDO) induction inhibits parasite growth
IFN- gamma induced parasite growth inhibition is independent on reactive oxygen intermediates
Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) is induced by infection
Autophagy may be involved in the elimination of the degraded parasite material from the astrocyte host cell cytoplasm
IGTP is required for IFN-gamma-induced inhibition of parasite growth
Toxopasma gondii genetic darabase
Figure 1.The structure of Toxoplasma gondii in tachyozoites form.
Figure 2.The most common form of infection by T. gondii is via the ingesting of infected meat containing tissue cysts, ingestion of oocysts in contaminated cat feces (www.dpd.cdc.gov).