[Above: Toxoplasma strains differ in how they manipulate immune signaling pathways. Fluorescence microscopy images of macrophages infected with Toxoplasma (green). The activated STAT6 transcription factor (in pink, phosphorylated-STAT6) is in the host nucleus (blue DNA of the macrophage) poised to induce the transcription of hundreds of host genes related to immune function, but only the parasite strain on the left (the “type I” strain) is able to activate STAT6, while the strain on the right (“type II”) is unable to do so. The parasite kinase, ROP16, is injected into the host cell and is responsible for this phenotype; a mutation in ROP16 of the type II strain renders it unable to maintain STAT6 activation.]

The Global Prevalence of Toxoplasma gondii

The intracellular apicomplexan parasite (What is a parasite?), Toxoplasma gondii is considered by and large the most successful parasite of warm blooded animals. Earning its title as the “ubiquitous parasite” it infects birds, sea and land mammals and can be found anywhere from the tropics of South America to the Svalbard islands of northern Norway. Toxoplasma is an orally acquired parasite that enters the body through the small intestine and disseminates to the brain, heart and muscle tissues. At these sites Toxoplasma becomes dormant and forms a cyst wall around itself protecting the parasite against the attacks of the immune system and anti-parasitic drugs. This protective structure, called the tissue cyst, is infectious and allows the parasite to survive the digestive environment of the next host should it be consumed unsuspectingly by another hungry animal. Once infected, most hosts are chronic carriers for life. An estimated 2 billion people are currently infected in this way, and most are unaware of the time they were initially exposed. Symptoms during initial exposure can range from mild flu like symptoms to no symptoms at all. Toxoplasma has therefore mastered the art of stealth and immune evasion. We think by studying these tactic new understandings about our immune system may be learned.

The Flexible Life Cycle of Toxoplasma gondii Contributes to its Global Prevalence

Toxoplasma’s broad host range and massive amplification during its sexual cycle contributes to the global prevalence and survival of Toxoplasma. Members of the cat family (Felidae) are the only known definitive host, meaning this is where the parasite undergoes genetic recombination and sexual reproduction. Following infection in the cat, for example after a cat makes a kill and consumes a mouse or bird infected with tissue cysts, within 3 to 14 days the cat will shed up to 100 million infectious oocyts in its feces. Oocysts are formed only in feline small intestinal epithelial cells and encompass the newly formed progeny following Toxoplasma mating. The oocyst wall is extremely resilient to chemical and mechanical stress, such that oocysts are commonly washed and stored in bleach or sulfuric acid. Oocysts can remain infectious for years in aquatic or soil environments, spew into the oceans through sewage runoff and have been responsible for infecting and causing death of sea otters off the coast of California. Shedding of oocysts into the environment is a transmission strategy shared by all coccidian parasites, like Eimeria the causative agent of animal coccidiosis. Where Toxoplasma is unique, is that the parasite has little species restriction for a productive infection in the next host. As far as we know if the next species is warm blooded then an infection occurs.Life cycle 2

[Above: Life cycle of Toxoplasma. Only the cat is the definitive host that sheds millions of environmentally stable oocysts, and most animals serve as intermediate hosts. Arrows represent the routes of transmission and parasite development. Only oocysts and tissue cysts are infectious. Not diagrammed: Oocysts are also infectious to cats and vertical transmission in intermediate hosts.]

Toxoplasma can also spread between intermediate hosts through carnivorous activity. You or I may become infected by eating under cooked meat bearing tissue cysts. Theoretically, this mode of transmission can occur indefinitely without need for the cat, again another unique adaptation of the life cycle. Chronic infection in rodents make them less afraid of cat odor, thus facilitating being preyed upon by the definitive feline host. Thus, the rules of transmission for the Toxoplasma life cycle are flexible- any warm blooded animal that eats a tissue cyst or oocyst becomes infected. The key point is that transmission depends on forming tissue cysts or oocysts, which depends upon keeping the infected host alive until transmission is achieved. In other words, Toxoplasma would rather not kill you but infect you.

Toxoplasmosis- congenital, ocular and encephalitis in the immune suppressed

However, infection by Toxoplasma is not always asymptomatic. Toxoplasmosis is one of the 5 most common foodborne illness in the US, producing roughly 4000 hospitalizations a year due to infection. The CDC considers toxoplasmosis as a neglected parasitic disease in the US, and Toxoplasma is one of the original “TORCH” pathogens known to cause infection in utero. If one is infected by Toxoplasma during pregnancy, mother to child transmission (a.k.a. “vertical transmission”) can occur leading to congenital toxoplasmosis (CT). Vertical transmission rates and the severity of CT correlates with the trimester of infection. On average a third of infections during pregnancy will result in vertical transmission, and symptoms in the new born can range from having inflammation in the retina and posterior vasculature of the eye (chorioretinitis), brain calcifications, cognitive developmental delays, abnormal head size (seen below) and seizures. Miscarriages and still born births are also caused by Toxoplasma. An estimated 190,000 cases of CT occur yearly, leading to 1,200,000 DALYs. To prevent Toxoplasma infection during pregnancy, a list of recommended precautionary measures can be found here.

Toxoplasmosis

Ocular toxoplasmosis (OT) is the most common manifestation of congenital infection, and may not develop until later in life. OT can also be acquired as an adult, and is the leading cause of posterior uveitis worldwide. Inflammation in the posterior eye can lead to permanent scaring of the retina (seen above) which causes visual impairment. Finally, in immune compromised individuals, such as those with HIV, reactivation of the dormant form of Toxoplasma leads to uncontrolled infection and tissue necrosis (seen above as light rings in a CAT scan). The encephalitis that ensues can be life-threatening if left untreated.

Different strains, different diseases

Strain differences

An interesting geographic association is observed between parasite strains (“Types” or “Haplogroups” I through XVII, representing “Clades” A-F), prevalence of human infection, severity of human infection and mouse virulence (over simplified above). Why these associations exist is the subject of ongoing investigation, but is likely related to unique parasite adaptations to specific species found in these locales. In other words, a productive infection in the South American capybara may select for Toxoplasma strains that (unintentionally) cause severe disease in other species. Whatever the reason, it is known that congenitally infected Brazilian children or South American patients with ocular toxoplasmosis present a more severe form of eye disease than those in other locales, and strains endemic to South America seem to be responsible. Severe congenital infection in North America is also linked to certain parasite genotypes. Although it has been difficult to definitively show that disease severity in humans is caused by a particular parasite strain type, in mice this has been clearly demonstrated. The Molecular Koch’s postulate, criteria used to identify disease causing genes in a pathogen, has been fulfilled for several Toxoplasma virulence factors (see below) and explain why certain strains produce a lethal infection in mice while others do not. Whether the discovered virulence factors promote disease in humans or other animals is unknown.

Host-Parasite Battles Inside the Cell

Host and parasite survival hinges on the outcome of a battle that occurs inside the host cell. Host cell invasion by Toxoplasma is an active, parasite-driven process that leads to the formation of a specialized non-fusogenic compartment, termed the parasitophorous vacuole (PV). The invasion process is accompanied by a sequential discharge of parasite proteins from apical secretory organelles called micronemes, rhoptries and dense granules. Proteins secreted from the micronemes are involved in the initial attachment and invasion, while dense granule and rhoptry proteins convert the host cell into a suitable environment for parasite growth by modulating a variety of host processes. It is perhaps not surprising that many of the most mutated (“polymorphic”) proteins in the Toxoplasma genome are secreted factors that interact with the host cells of various species. Over the past 15 years it has become increasingly clear that many of these secreted effectors manipulate host immune defense mechanisms and determine differences in parasite virulence.Virulence Effectors

One form of host manipulation is through intercepting and changing host cell communication (or “cell signaling”). As alluded to in the featured image, a single mutation in a secreted rhoptry protein (ROP16) determines whether Toxoplasma strains can activate the immune communication pathway called the “STAT6 signaling pathway”. Communication through this pathway is paramount in the host immune response to worm infections, and Toxoplasma either turns this pathway on or off depending on the strain type and ROP16 version (allele) it encodes. We found that mice have less intestinal inflammation when Toxoplasma encodes the active version of ROP16. Toxoplasma encodes hundreds of effectors that are derived from these secretory organelles and we only know the function of a handful of them. Here is a cursory list of some of the host communication pathways targeted by Toxoplasma through secreted factors (“GRA” is short for dense granule, “ROP” is short for rhoptry): GRA15 activates NF-kB, GRA24 activates the p38 MAPK pathway, GRA6 activates NfAT4, GRA16 activates p53, ROP38 activates the ERK pathway, and GRA25 induces expression of chemokines that recruit macrophages to the site of the infection. Through mutations or changes in gene expression particular combinations of active and inactive effectors are encoded by different parasite strains. Thus, a little P53 activation, less MAPK and NF-kB signaling, but strong ERK by one strain could be perfect to establish a chronic infection in the capybara, but disastrous in mice or humans- or so the hypothesis goes. The magnitude of the inflammatory responses to the same infection can be quite different between different species or even different individuals of the same species (see page on Toxoplasma induced ileitis). It is in this context that various parasite strains have adapted to or co-evolved with different intermediate hosts possessing unique genetics and propensities to inflammation.

Another form of subversion is through evasion of killing mechanisms elicited by the immune response. Host control of many intracellular pathogens depends on the ability of the immune system to make the cytokine IFNγ. IFNγ activates effector mechanisms for intracellular elimination of Toxoplasma, including induction of reactive nitrogen intermediates, tryptophan degradation and autophagy. Additionally in mice, IFN-γ induces the expression of two classes of GTPases, the immunity-regulated GTPases (IRGs) and guanylate-binding proteins (GBPs), which are important host resistance factors to pathogens that reside in vacuoles, like Toxoplasma, Chlamydia, and Mycobacterium species. When Toxoplasma infects a cell previously stimulated with IFN-γ, the absence of regulatory GMS IRGs on the parasitophorous vacuole membrane (PVM) leads to PVM localization of the GKS class of effector IRGs and many GBPs, causing PVM destruction and rapid parasite death. The Toxoplasma rhoptry kinases ROP18 (also here) and ROP17, the dense granule GRA7, and the ROP5 pseudokinases are secreted into the host cell upon invasion and subsequently traffic back to the PVM, where they cooperatively inhibit the localization and function of IRGs (here and here) and GBPs. Differences in virulence between the clonal lineage strains (i.e. Type I, II and III) are the result of polymorphisms in the ROP5 (and here) and ROP18 effectors (and here), with smaller contributions from GRA15 and ROP16. The type I strain and South American strains encode virulent alleles of the ROP5 and ROP18 effectors, while the type II and III strains encode combinations of less virulent alleles that render them more susceptible to IRG-mediated killing and therefore less virulent in laboratory mice (the subspecies of house mouse Mus musculus domesticus). However, the mouse IRG locus is extremely diverse, rivaling that of the MHC, and mouse subspecies from different geographic locations have unique IRG haplotypes (or different gene sets) that promote resistance to virulent Toxoplasma strains. For example, subspecies of mice indigenous to Southeast Asia, Mus musculus castaneus, do not die from type I infection (as a comparison, one parasite of this strain kills the Mus musculus domesticus mouse) and instead form a life-long chronic infection because of a unique IRG, Irgb2-b1. Perhaps this is why the virulent type I strain is frequently isolated in Asia. Thus, a balanced infection can be achieved by certain Toxoplasma strains in various species across the globe.

From the cumulative work of many labs, I hope you can see that by analyzing parasite strain differences, using their virulence factors and studying the downstream consequences of such manipulation one could increase our understandings of the basic principles of immunology and discover new treatments for toxoplasmosis or inflammatory related diseases.