Schultz, A. J. | bioRxiv Science

bioRxiv2021

Toxoplasma gondii Toxolysin 4 contributes to efficient parasite egress from host cells

Egress from host cells is an essential step in the lytic cycle of T. gondii and other apicomplexan parasites; however, only a few parasite secretory proteins are known to affect this process. The putative metalloproteinase Toxolysin 4 (TLN4) was previously shown to be an extensively processed microneme protein, but further characterization was impeded by the inability to genetically ablate TLN4. Herein we show that TLN4 has the structural properties of an M16 family metalloproteinase, that it possesses proteolytic activity on a model substrate, and that genetic disruption of TLN4 reduces the efficiency of egress from host cells. Complementation of the knockout strain with the TLN4 coding sequence significantly restored egress competency, affirming that the phenotype of the {Delta}tln4 parasite was due to the absence of TLN4. This work identifies TLN4 as the first metalloproteinase and the second microneme protein to function in T. gondii egress. The study also lays a foundation for future mechanistic studies defining the precise role of TLN4 in parasite exit from host cells. IMPORTANCEAfter replicating within infected host cells, the single celled parasite Toxoplasma gondii must rupture out of such cells in a process termed egress. Although it is known that T. gondii egress is an active event that involves disruption of host-derived membranes surrounding the parasite, very few proteins that are released by the parasite are known to facilitate egress. In this study we identify a parasite secretory protease that is necessary for efficient and timely egress, thus laying the foundation for understanding precisely how this protease facilitates T. gondii exit from host cells.

microbiology↗

bioRxiv2019

A role for Toxoplasma gondii chloroquine resistance transporter in bradyzoite viability and digestive vacuole maintenance

Toxoplasma gondii is a ubiquitous pathogen that can cause encephalitis, congenital defects, and ocular disease. T. gondii has also been implicated as a risk factor for mental illness in humans. The parasite persists in the brain as slow growing bradyzoites contained within intracellular cysts. No treatments exist to eliminate this form of parasite. Although proteolytic degradation within the parasite lysosomal-like vacuolar compartment (VAC) is critical for bradyzoite viability, whether other aspects of the VAC are important for parasite persistence remains unknown. An ortholog of Plasmodium falciparum CRT has previously been identified in T. gondii (TgCRT). To interrogate the function of TgCRT in chronic stage bradyzoites and its role in persistence, we knocked out TgCRT in a cystogenic strain and assessed VAC size, VAC digestion of host-derived proteins and parasite autophagosomes, and viability of in vitro and in vivo bradyzoites. We found that whereas parasites deficient in TgCRT exhibit normal digestion within the VAC, they display a markedly distended VAC and their viability is compromised both in vitro and in vivo. Interestingly, impairing VAC proteolysis in TgCRT deficient bradyzoites restored VAC size, consistent with a role for TgCRT as a transporter of products of digestion from the VAC. In conjunction with earlier studies, our current findings suggest a functional link between TgCRT and VAC proteolysis. This work provides further evidence of a crucial role for the VAC in bradyzoite persistence and a new potential VAC target to abate chronic Toxoplasma infection.\n\nIMPORTANCEIndividuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of experiencing reactivated disease that can result in progressive loss of vision. No effective treatments exist for chronic toxoplasmosis due in part to a poor understanding of the biology underlying chronic infection and a lack of well validated potential targets. Here we show that a T. gondii transporter is functionally linked to protein digestion within the parasite lysosome-like organelle and that this transporter is necessary to sustain chronic infection in culture and in experimentally infected mice. Ablating the transporter results in severe bloating of the lysosome-like organelle. Together with earlier work, this study suggests the parasites lysosome-like organelle is vital for parasite survival, thus rendering it a potential target for diminishing infection and reducing the risk of reactivated disease.

microbiology↗