TgPLP1 is secreted from the micronemes in a calcium-dependent manner. (A) Immuno-localization of TgPLP1 (red) and the microneme marker MIC2 (green) in WT, epitope-tagged plp1HA, and plp1ko strains. (B) Secretion of TgPLP1, MIC2, and constitutively secreted GRA1 in response to a 3min treatment with the calcium ionophore A23187 (A), 20min with the calcium chelator BAPTA-AM (B), or appropriate solvent control (C). Actin included as control for inadvertent lysis. (C) Immunoblot of TgPLP1 in WT, plp1ko, and plp1ko/PLP1myc strains.

Evidence of pore formation by TgPLP1 (A) Ionophore-induced permeabilization by immobilized WT or plp1ko tachyzoites. Stage 0: PV marker (GRA1, blue) remains contained within the PVM, no microneme release (SUB1, green), no labeling of host cell nucleus by PI, (red or outlined when unlabeled). Stage 1: Microneme contents have been secreted onto the parasite surface (green, see also inset; arrowhead = parasite apex), PV markers have diffused throughout the host cell but host cell nucleus remains unlabeled. Stage 2: Host nucleus is now PI positive, indicating permeabilization of the HPM. Stage 3: Microneme proteins have diffused throughout the host cytoplasm. Cells infected by plp1ko remain in stage 0 or display microneme secretion without PV marker release (stage 1′). (B) Quantification of induced PVM and HPM permeabilization for WT, plp1ko, or plp1ko/PLP1myc. Error bars are SEM (n = 6) and asterisk indicates P < 0.05 (Student’s t-test). (C) Rapid release of DsRed from the vacuole in WT (top) and plp1ko/PLP1myc (bottom), but not plp1ko (middle). Time stamp (h:min:sec) indicates time following calcium ionophore addition. (D) Average time to DsRed release within a 10min observation period. Error bars are SEM (n ≥ 30). (E) Virulence in outbred mice following inoculation of escalating tachyzoite numbers.

plp1ko egress phenotypes (A) Parasite cultures 48h post-infection. (B) Transmission electron micrograph of spherical structures shows remnants of host cell structures (HS), the intravacuolar network (IVN), and numerous tachyzoites encased by the parasitophorous vacuolar membrane (PVM) and host cell plasma membrane (HPM). (C–D) Ionophore-induced egress from WT or plp1ko vacuoles. IFA colors: Parasites, red; PVM, green; host nuclei, blue. (E) Time to motility activation or egress following addition of calcium ionophore in WT, plp1ko, plp1ko/PLP1myc, and MBE1.1. Error bars are SEM (n ≥ 30) and asterisk indicates P < 0.05 (Student’s t-test). (F) Egress delay of secondary vacuoles following egress of an initial vacuole from cells multiply-infected with WT, plp1ko, or both.