Pore-forming proteins have been classically associated with host invasion from the bacterial perspective and cell lysis from the host perspective. However, we identified a new role for Toxoplasma gondii perforin-like protein 1 (PLP1) in rapid parasite exit from host cells (Kafsack et al., Science 2009). This places PLP1 at a unique junction combining vacuolar escape and cell lysis. I found that PLP1 is sufficient for membrane disruption and has a conserved mechanism of pore-formation. The highly conserved, central MACPF domain and the beta-sheet rich C-terminal domain were crucial for membrane permeabilization. Loss of the unique N-terminal extension reduced lytic activity and delayed egress, but did not significantly diminish virulence, suggesting that a small amount of lytic activity is still sufficient for pathogenesis. Both N- and C-terminal domains have membrane-binding activity, with the C-terminal domain being critical for function. This dual mode of membrane association may promote PLP1 activity and parasite egress in the diverse cell types in which this parasite replicates.
Since the parasite must lyse the host cell upon egress and preserve intact host membranes upon invasion, I sought to identify mechanisms regulating lytic activity during egress and invasion. Using a hemolysis assay, I detected maximal PLP1 activity at low pH and minimal activity at neutral pH. Although the compartment in which the parasite replicates was presumed to be neutral pH, I found that pH-disrupting agents suppressed PLP1 activity during egress and reduced parasite exit. Modest decreases in vacuolar pH were observed when the parasite was exposed to egress cues and late in the replication cycle prior to native egress. Furthermore, low pH augments PLP1 activity during cell invasion, resulting in damage to host cells. These results support a model by which PLP1 activity is controlled by a pH switch: PLP1 is highly active in the low pH-environment during host cell exit, and is suppressed at neutral pH during host cell invasion. This work has uncovered novel structural and functional features of PLP1 pore formation. As parasite egress is a vulnerable point in the lytic cycle, further investigation of this process may identify potential targets for future therapeutics.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99994/1/mroiko_1.pd
