Abstract
Toxoplasma gondii is a natural intracellular protozoal pathogen of mice and other small mammals. After infection, the parasite replicates freely in many cell types (tachyzoite stage) before undergoing a phase transition and encysting in brain and muscle (bradyzoite stage). In the mouse, early immune resistance to the tachyzoite stage is mediated by the family of interferon-inducible immunity-related GTPases (IRG proteins), but little is known of the nature of this resistance. We reported earlier that IRG proteins accumulate on intracellular vacuoles containing the pathogen, and that the vacuolar membrane subsequently ruptures. In this report, live-cell imaging microscopy has been used to follow this process and its consequences in real time. We show that the rupture of the vacuole is inevitably followed by death of the intracellular parasite, shown by its permeability to cytosolic protein markers. Death of the parasite is followed by the death of the infected cell. The death of the cell has features of pyronecrosis, including membrane permeabilisation and release of the inflammatory protein, HMGB1, but caspase-1 cleavage is not detected. This sequence of events occurs on a large scale only following infection of IFNγ-induced cells with an avirulent strain of T. gondii, and is reduced by expression of a dominant negative mutant IRG protein. Cells infected by virulent strains rarely undergo necrosis. We did not find autophagy to play any role in the key steps leading to the death of the parasite. We conclude that IRG proteins resist infection by avirulent T. gondii by a novel mechanism involving disruption of the vacuolar membrane, which in turn ultimately leads to the necrotic death of the infected cell.
Highlights
The mouse is a natural intermediate host for Toxoplasma gondii, an apicomplexan parasite whose definitive host is the cat
Irga6-ctag1-EGFP was transfected into IFNc-induced mouse embryonic fibroblasts (MEFs) that were infected with T. gondii ME49 strain
We can characterise this series, as follows: (1) the accumulation of immunity-related GTPases (IRGs) proteins on the parasitophorous vacuole, which begins on some vacuoles as early as 2 minutes after entry of the parasite and typically reaches a maximum between 30 minutes and one hour later, (2) rupture of the IRGloaded parasitophorous vacuole membrane (PVM), a process which occurs suddenly and is completed in a few minutes, (3) the permeabilisation of the T. gondii plasma membrane documented by entry of fluorescent cytosolic protein markers, which occurs as a sudden event between 20 and 40 minutes after PVM disruption, (4) the necrotic death of the cell, documented by sudden loss of fluorescent cytosolic protein markers and release of the chromatin modelling protein, HMGB1, from the necrotic cell
Summary
The mouse is a natural intermediate host for Toxoplasma gondii, an apicomplexan parasite whose definitive host is the cat. Following the development of a strongly IFNc-dependent primary immunity, rapidly replicating tachyzoites convert to the slowly-replicating bradyzoite stage and encyst in brain and muscle without causing severe symptoms, there to await completion of the infection cycle following ingestion by a cat at some later time [3,4,5]. This relatively benign course of infection is, drastically altered by disruption of genes encoding key components of the interferon-gamma (IFNc)-response pathway [6,7,8]. Genomic disruption of individual members of this gene family causes normally avirulent T. gondii strains to behave as highly virulent pathogens, killing infected mice as early as 10 days after primary infection [11,12]
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