<i>Toxoplasma gondii</i> Toxolysin 4 Contributes to Efficient Parasite Egress from Host Cells

Abstract

Egress from host cells is an essential step in the lytic cycle of <i>T. gondii</i> 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 <i>TLN4</i>. Here, 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 <i>TLN4</i> reduces the efficiency of egress from host cells. Complementation of the knockout strain with the <i>TLN4</i> coding sequence significantly restored egress competency, affirming that the phenotype of the Δ<i>tln4</i> parasite was due to the absence of TLN4. This work identifies TLN4 as the first metalloproteinase and the second microneme protein to function in <i>T. gondii</i> egress. The study also lays a foundation for future mechanistic studies defining the precise role of TLN4 in parasite exit from host cells. <b>IMPORTANCE</b> After replicating within infected host cells, the single-celled parasite <i>Toxoplasma gondii</i> must rupture out of such cells in a process termed egress. Although it is known that <i>T. gondii</i> 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, laying the foundation for understanding precisely how this protease facilitates <i>T. gondii</i> exit from host cells.