Abstract
Toxoplasma gondii is the most common protozoan parasitic infection in man. Gamma interferon (IFNγ) activates haematopoietic and non-haematopoietic cells to kill the parasite and mediate host resistance. IFNγ-driven host resistance pathways and parasitic virulence factors are well described in mice, but a detailed understanding of pathways that kill Toxoplasma in human cells is lacking. Here we show, that contrary to the widely held belief that the Toxoplasma vacuole is non-fusogenic, in an immune-stimulated environment, the vacuole of type II Toxoplasma in human cells is able to fuse with the host endo-lysosomal machinery leading to parasite death by acidification. Similar to murine cells, we find that type II, but not type I Toxoplasma vacuoles are targeted by K63-linked ubiquitin in an IFNγ-dependent manner in non-haematopoetic primary-like human endothelial cells. Host defence proteins p62 and NDP52 are subsequently recruited to the type II vacuole in distinct, overlapping microdomains with a loss of IFNγ-dependent restriction in p62 knocked down cells. Autophagy proteins Atg16L1, GABARAP and LC3B are recruited to <10% of parasite vacuoles and show no parasite strain preference, which is consistent with inhibition and enhancement of autophagy showing no effect on parasite replication. We demonstrate that this differs from HeLa human epithelial cells, where type II Toxoplasma are restricted by non-canonical autophagy leading to growth stunting that is independent of lysosomal acidification. In contrast to mouse cells, human vacuoles do not break. In HUVEC, the ubiquitinated vacuoles are targeted for destruction in acidified LAMP1-positive endo-lysosomal compartments. Consequently, parasite death can be prevented by inhibiting host ubiquitination and endosomal acidification. Thus, K63-linked ubiquitin recognition leading to vacuolar endo-lysosomal fusion and acidification is an important, novel virulence-driven Toxoplasma human host defence pathway.
Highlights
Host cells invaded by intracellular pathogens have to mount a rapid recognition and cellautonomous defence program to curb the replication of the intruder [1]
Toxoplasma gondii is an intracellular parasite that can invade nucleated cells of any warmblooded animal into a compartment known as a parasitophorous vacuole (PV)
The production of gamma interferon (IFNγ) drives the restriction and killing of Toxoplasma. It is not fully known how the parasite inside the PV is eliminated in human cells, its fate depends on the cell type into which it invades
Summary
Host cells invaded by intracellular pathogens have to mount a rapid recognition and cellautonomous defence program to curb the replication of the intruder [1]. The cytokine gamma interferon (IFNγ) can stimulate cell-autonomous defence in immune or non-immune cells and is produced early during infection with many intracellular pathogens, including the protozoan parasite Toxoplasma gondii [2]. Human Toxoplasma infections are estimated at 30% and the parasite can infect all warm-blooded animals. Human infections are mostly asymptomatic, but the parasite establishes a lifelong chronic infection in the form of cysts in brain and muscle tissue. Ocular disease is a complication for both the immunocompetent and immunocompromised, while serious illness and death are possibilities in the immunocompromised and the developing foetus of pregnant women. Toxoplasma strains in North America and Europe are mostly of the types I, II and III, with type I strains classified as highly virulent with an LD100 of 1 parasite in mice, and type II and III strains being less virulent in mice, with an LD50 greater than 1000 parasites/mouse [3,4]
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