Functional dissection of Toxoplasma gondii perforin-like protein 1 reveals a dual domain mode of membrane binding for cytolysis and parasite egress

Abstract

The recently discovered role of a perforin-like protein (PLP1) for rapid host cell egress by the protozoan parasite Toxoplasma gondii expanded the functional diversity of pore-forming proteins. Whereas PLP1 was found to be necessary for rapid egress and pathogenesis, the sufficiency for and mechanism of membrane attack were yet unknown. Here we further dissected the PLP1 knock-out phenotype, the mechanism of PLP1 pore formation, and the role of each domain by genetic complementation. We found that PLP1 is sufficient for membrane disruption and has a conserved mechanism of pore formation through target membrane binding and oligomerization to form large, multimeric membrane-embedded complexes. The highly conserved, central MACPF domain and the β-sheet-rich C-terminal domain were required for activity. Loss of the unique N-terminal extension reduced lytic activity and led to a delay in rapid egress, but did not significantly decrease virulence, suggesting that small amounts of lytic activity are sufficient for pathogenesis. We found that 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.

FIGURE 1.

Loss of PLP1 leads to a primary egress defect and a secondary invasion defect. A, 30-h vacuoles were treated with vehicle (dimethyl sulfoxide (DMSO), white bars) or ionophore (A23187, black bars) for 2 min and >100 vacuoles in >10 fields of view scored by immunofluorescence as occupied or unoccupied vacuoles. B and C, naturally egressed (B) or mechanically liberated (C) parasites were tested for invasion defects by red/green invasion assay; light gray bars = attached parasites, dark gray bars = invaded parasites. Fifteen fields of view were counted per strain per experiment. Graphs display the mean ± S.D. (error bars) of three independent experiments (*, p < 0.05 by one-tailed Student's t test compared with RH).

FIGURE 2.

PLP1 is sufficient for lytic activity and binds and oligomerizes on host membranes. A, host cells were incubated for 10 min at 37 °C with buffer alone, 100 nm recombinant M2AP, PLP1, or LLO and propidium iodide, which stains nuclei of cells with membrane damage. Immunofluorescence was performed for PLP1. The PLP1 antibody cross-reacted with LLO, but not M2AP. B, lytic activity was observed by hemolysis assay with varying concentrations of recombinant protein. Graph displays the mean ± S.D. (error bars) of triplicate wells and is representative of three independent experiments. C, sucrose gradient membrane flotation of cell debris after parasite egress revealed PLP1 and dense granule protein 4 (GRA4) signal associated with lighter density (membrane) fractions. Endogenous microneme protein 5 (MIC5) was not detected due to lack of membrane binding activity. Recombinant MIC5 added to the cell debris sample was associated with the higher density fractions, also indicative of no membrane binding. Low density, high molecular mass PLP1 signal is absent in the PLP1 knock-out sample. D, membrane-associated PLP1 is resistant to alkaline treatment, indicative of membrane integration. E, in parasite lysate (L), PLP1 migrated rapidly on SDS-agarose, whereas in the HSP (host cell debris), PLP1 signal occurred in multiple, slowly migrating complexes (arrowheads). F, treatment of HSP with HFIP, which disrupts hydrophobic interactions, led to PLP1 migrating at the same molecular mass as in parasite lysate (L), along with some smaller processed forms (arrowheads). Molecular mass markers are indicated in kDa.

FIGURE 3.

Generation of plp1 knock-out and complementation strains for domain deletion analysis and immunofluorescence of complemented strains. A, the plp1 knock-out was generated in the RHΔku80Δhxg strain by replacing the PLP1 locus with the HXG drug selection marker. The plp1ko was complemented at the endogenous locus by double homologous recombination and negative selection. B, PCR on parasite genomic DNA demonstrated plp1 locus replacement with HXG in the knock-out and HXG replacement with PLP1 cDNAs in complemented strains. C, PLP1 domain structure and domain deletions tested in this study are shown. D, immunofluorescence with a polyclonal PLP1 antibody showed some PLP1 complementation constructs co-localized with MIC2, whereas other constructs were poorly detected. Scale bars, 2 μm. E, parasite strains investigated in this study are listed, including the genotype, common name used throughout this paper, and the presence or absence of spheres in postegressed cultures. Spheres were observed by bright field examination of egressed cultures, and images were acquired on bright field at a magnification of ×200 on a Zeiss Axio inverted microscope.

FIGURE 4.

PLP1 domain deletion constructs are expressed by the parasites and secreted from the micronemes. A, parasite lysate immunoblotted with a polyclonal (αPLP1) or affinity-purified (αNterm, αCterm) PLP1 antibodies. Arrowheads indicate faint bands specifically detected by the antibodies. The schematic below indicates domains recognized by the antibodies. B, microneme secretion induced with 1% ethanol and the secreted fraction immunoblotted for PLP1 and MIC2. C, immunoblot of secreted fractions demonstrating that PLP1 is processed in the N-terminal domain, indicated by the presence of multiple bands detected by the PLP1 antibody in constructs with the N-terminal domain and absent in the plp1ko. Arrowheads indicate specific bands for single domain constructs. Molecular mass markers are indicated in kDa.

FIGURE 5.

Phenotypic analysis of PLP1 domain deletion strains reveals intermediate or nonfunctional complementation. A, 50 parasites per well were allowed to form plaques for 7 days. B, plaque area was measured by determining the average diameter in two dimensions and approximating a circle. C, egress was tested following ionophore (A23187) treatment by immunostaining vacuoles for dense granule protein 7 (GRA7) and parasites for surface antigen 1 (SAG1) and quantifying vacuoles containing parasites (occupied) and parasite-free vacuoles (unoccupied). D, time course of parasite egress is shown as in C with indicated times for each strain. E, PVM permeabilization was determined by treating infected cells with cytochalasin D prior to ionophore (A23187) treatment and fixation; infected cells were identified by bright field, and PVM permeabilization was indicated by release of the PV-targeted fluorescent protein dsRed into the host cytosol. F, lytic activity determined from PVM permeabilization was normalized to WT parasites. Graphs depict the average ± S.D. (error bars) of three independent experiments. *, p < 0.05 by one-tailed Student's t test. G, mouse survival after infection. Ten Swiss-Webster mice per strain were injected intraperitoneally with the indicated numbers of parasites and monitored for morbidity and mortality.

FIGURE 6.

Functional analysis of PLP1 domain deletions reveals differences in membrane binding and oligomerization. A, membrane flotation of host cell debris was acquired from complemented strains immunoblotted for PLP1. Arrowheads indicate specific bands. B, membrane flotation of recombinant N- and C-terminal domains used red blood cell ghosts (RBCg) and was immunoblotted for the His₆ epitope tag. C, host cell debris from complemented strains was TCA-precipitated, treated with HFIP or buffer, and immunoblotted for PLP1. D, SDS-AGE and PLP1 immunoblotting of WT comp and MACPF+Cterm lysate (L) and HSP were performed. Marker is Coomassie Blue-stained mouse muscle lysate; the indicated sizes are in kDa. Arrows indicate monomers in lysate.