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Toxoplasma gondii

By Levi Clancy for Student Reader on

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Toxoplasma gondii is an obligate intracellular parasite that grows and divides inside a host cell and has no extracellular lifecycle. Toxoplasma gondii's importance rests upon its worldwide distribution and ability to infect almost any mammalian or avian cell.

Toxoplasma gondii has a haploid genome, making it amenable to loss of function mutations (achievable by chemical mutagenesis). Toxoplasma gondii can also undergo homologous recombination, making possible full knockouts of certain genes. The Toxoplasma gondii genome has been sequenced, making gene identification easier. Felines (where its sexual stage occurs) are Toxoplasma gondii's definitive host; it lacks a vector.

Humans can contract this by eating under cooked meat or cat feces. Toxoplasma gondii has a worldwide distribute, with 15-30% of Americans infected and over 70% of Europeans.

Many people are already infected but because they are healthy they are asymptomatic. Toxo cause a lifelong chronic infection that is never cleared. Toxoplasma gondii only causes serious problems in immunocompromised individuals and unborn children when their mother gets a primary infection. Toxo is the leading cause of death among AIDS patients. As an apicomplexan parasite, Toxoplasma gondii is often used as a model system because it is much easier to work with than other apicomplexan parasites such as plasmodium.

Toxoplasma has many unusual organelles.

The apicoplast or plastid have some features in common with the chloroplast and like the mitochondria and chloroplast it originated from an endosymbiotic event. The important organelles to know are the micronemes, the rhoptries, and the dense granules as these all appear to be secretory organelles that originated from the golgi (not from endosymbiosis) and they are all involved in host cell invasion. Conoid is a microtubule based tip on the parasite body (don’t worry about this one too much). Another structure to keep in mind is the inner membrane complex which is also involved in host cell invasion.

Toxoplasma gondii life cycle

Humans and other animals are intermediate hosts. Cats are the definitive host as they harbor the Toxoplasma gondii sexual cycle.

The sexual stage allows for the creation of diversity and possibly more virulent strains.

ReleaseCat releases infective oocysts.
IngestionIntermediate host ingests infective oocysts.
Asexual stage

Toxo undergoes an asexual stage in humans with 2 forms: 1) tachyzoite (which is similar to but not exactly the same as the trophozoite so don’t mix these up) 2) bradyzoite (which is a tissue cyst similar but again not the same as the cysts formed by Giardia and Entamoeba).

DifferentiationInfective oocysts becomes a tachyzoites.
InvasionTachyzoites may invade host cells.
DormancyTachyzoites may become dormant pseudocysts containing bradyzoites.
PseudocystsTazhyzoites may become pseudocysts activated during an immune system breakdown.
Toxoplasma gondii can invade many tissues, including muscle, brain, eye and intestinal epithelium, and is even found free in the blood in heavy infections. Each tachyzoite forms an independent parasitophorous vacuole inside the host cell. Inside this vacuole, the parasite divides mitotically (via endodyogeny) with up to 64 parasites within a single vacuole.

Unabated tachyzoite replication bursts the cell and releases tachyzoites that can infect new host cells. An active immune response induces a halt in division and the parasite encases itself in a membrane to become a pseudocyst. Within a pseudocyst are dormant bradyzoites that can survive for decades. They reactive when the immune system is compromised.

Toxoplasmosis arises in immunosuppressed patients (cancer, transplants, HIV) and also in fetuses when the mother becomes infected during pregnancy. Congenital toxoplasmosis causes hydrocephalus, chorioretinitis, cerebral calcification, seizures and severe developmental delays.

Host cell invasion

As an intracellular parasite, Toxoplasma gondii must avoid lysosomal destruction.

It accomplishes this by active invasion into a parasite vacuole that filters out host cell transmembrane proteins which prevents the vacuole from being targeted to the lysosome (this filtering is accomplished by rhoptry proteins). Invasion also involves an actin-based gliding motility which requires the secretion of adhesive proteins from the micronemes


Micronemes secrete adhesive proteins that anchor in the parasite plasma membrane to mediate the initial attachment of the parasite to the host cells.

The primary microneme proteins are myosin and aldolase. Myosin is anchored in the inner membrane complex and drives host cell invasion by the parasite. Aldolase mediates the connection between microneme proteins and the actin-myosin motor.

Micronemes fuse with the parasite plasma membrane to release microneme proteins onto the parasite's surface. At this point the microneme proteins can interact with the host cell. These are transient surface proteins that only exist on the surface during invasion.

As the parasite invades the host cell, a protease cleaves microneme adhesion proteins. This releases them from the parasite plasma membrane. Microneme protein, aldolase and protease activity is collectively the glideosome.

The release of microneme proteins is regulated by calcium. This was identified by treatment with a calcium ionophore and a separate treatment with a calcium chelator; supernatant (secreted proteins) was compared to cell lysate (total protein) using SDS-PAGE and immunoblots.

IonophoreTreatment with a calcium ionophore caused an increase in microneme proteins in the supernatant. Thus, an increase in secretion occurred.
ChelatorTreatment with a calcium chelator caused a decrease in microneme proteins in the supernatant. Thus, a decrease in secretion occurred.

Perforin-like proteins

Toxoplasma gondii contains a perforin-like protein localized to the micronemes. Perforin-like proteins are involved in pore formation.

Researchers thought that perhaps this protein is involved in parasite egress (bursting) from the host cell. Knockout studies revealed that perforin-like protein was not essential for egression in vitro. However, egression was slower in its absence. When induced by calcium to egress, wild-type parasites took ~2 minutes while mutants took ~20 minutes.

However, in vivo this perforin-like protein is essential. The delay in bursting out of the host cell makes the parasite vulnerable to the host immune system, and infection does not take hold.

Rhoptry proteins

Rhoptry proteins form the moving junction, which is the ring that forms around the parasite cell through which the parasite squeezes into the host cell.

Rhoptry proteins also act a molecular sieve to filter out host cell transmembrane proteins from the parasite vacuole to keep the vacuole from being targeted to the lysosome.

Rhoptry body proteins (ROPs)

Rhopty body proteins (ROPs) modulate host cell function. They are found in the bulbous body of the rhoptries.

Once secreted into the host cell, they are localized to specific areas (ie, the nucleus) where they exert their function.

This is determined via a microarray experiment.

InfectedHuman cells are cultured with T. gondii.
UninfectedAnother batch of human cells are cultured alone.
Isolate mRNAmRNA is isolated from each cultures.
Make cDNAcDNA is prepared from the mRNA by adding fluorescent nucleotides. The infected sample may use red nucleotides and the uninfected sample may use green nucleotides. It is important to use differently colored nucleotides.
HybridizationcDNA from both samples is hybridized to a microarray chip containing every human gene.

A yellow spot means the gene is expressed equally in both infected and uninfected cells. A red spot means the gene is induced in infected cells. If the spot is green then the gene is repressed in infected cells.

Typical of a general response, proinflammatory genes are upregulated. Some other changes were induced by Toxoplasma gondii infection but they were unremarkable.

Rhoptry neck proteins (RONs)

Rhoptry Neck Proteins (RONs) are involved in formation of the moving junction, and are a molecular sieve that removes host cell transmembrane proteins from the parasite vacuole.

The moving junction is the interface between the host and parasite cells during invasion. This same moving junction and sieve activity occurs in Plasmodium as well. RONs are found in the narrow neck of the rhoptries.

When rhoptries were first discovered, they were isolated and then a proteomics approach (much like that used for hydrogenosomal proteins) was used to determine their function.

SeparationFirst the protein contents of the rhoptries were separated out on a gel via SDS-PAGE.
IsolationEach band of protein was cut out of the gel.
DigestionProteins were digested with trypsin.
SpecrometryMass spectrometry determined each protein's amino acid sequence.
AnalysisThe sequences were analyzed to determine the function of each protein.

Dense granules

Dense granules are involved in remodeling the parasite vacuole so the parasite can live, grow and divide within it.

Toxoplasma gondii proteins accessing the host cell

This method reveals the location of T. gondii proteins:

Cyto DCyto D disrupts actin, stopping invading parasites.
AntibodiesA fluorescent antibody for the protein of interest will reveal if its present in the host cell.
Rhoptry proteins access the host cell via evacuoles. One rhoptry protein is targeted to the nucleus. Also, a rhoptry kinase (ROP16) is secreted to the host cell nucleus and modulates host STAT3 signaling and IL12 production. Some ROP proteins secreted into the host cell likely modulate host cell pathways.

It is unclear how parasite proteins get inside the host cell -- the current model is called kiss and spit. In this model, the parasite breaches the host cell membrane and injects proteins into the host cell. There is a spike in conductivity before invasion, indicating a possible breaching of the host cell.