PMC

EOP of Iie⁻ and Iid⁻ mutants. Parasites were allowed to infect HFF monolayers for 24 h, after which extracellular parasites were removed and the plaques were allowed to develop for 4 more days. Mutants defective in IIE also showed a reduction in the efficiency of plaquing.

Quantitation of egress-independent host lysis using a ⁵¹Cr release assay. HFF monolayers were infected with either RHΔ, MBE1.1, MBE3.3, or MBD2.1 and were loaded with ⁵¹Cr. Cells were pretreated with either DMSO (a), cytochalasin D (b), or BAPTA-AM (c). A23187 was then added to all cultures, and the counts per minute (CPM) obtained from ⁵¹Cr release was measured and normalized to 10⁴ parasitophorous vacuoles.

The IIE phenotype of wild-type (RHΔ) and two Iie⁻ mutants (MBE1.1 and MBE3.3). (a) A time course of egress using 1 μM A23187 in HBSS_c. (b) IIE as a function of A23187 concentration. Egress was measured after 2 min of exposure to the ionophore. Monolayers for both panels a and b were scored for the percentage of lysed vacuoles per field.

A23187 induction of parasite elongation in intracellular Iie⁻ mutants. Infected cells were treated with DMSO (left) or 1 μM A23187 (right) in HBSS_c and were fixed after 2 min at 37°C. The morphological phenotype is observed as an extension of the apical end (pointing out from the center of the vacuoles). Intracellular MBE1.1 parasites demonstrate this phenotype, while those of the MBE3.3 strain remain rounded.

Relationship between the IIE and IID phenotypes. (a) MBE1.1, but not MBE3.3, is resistant to IID. Extracellular parasites were exposed to A23187 and assayed for the formation of plaques. EOP was defined as the percentage of the PFU arising from parasites incubated with A23187 versus the DMSO-treated control. (b) Iid⁻ phenotypes of MBD1.1 and MBD2.1, two independent mutants isolated in the Iid⁻ selection. Assays were performed as in panel a. (c) IIE in the Iid⁻ selected mutants. MBD1.1 is Iie⁻, while MBD2.1 is Iie⁺. The IIE assay was performed as described for Fig. a.

Saponin induces egress in a Ca²⁺-dependent manner. Intracellular parasites were induced to exit by treatment with either 0.005% saponin (a) or 1 μM A23187 (b) at 4°C for 20 min. After an additional 2 min of incubation at 37°C, the cells were fixed and stained. The influence of extraparasitic [Ca²⁺] on each treatment was examined by using either HBSS_c (1 mM Ca²⁺) or HBSS_nc (5 mM EGTA and no added Ca²⁺).

The effect on EOP of pretreating extracellular parasites with either A23187 or BAPTA-AM. Extracellular parasites were pretreated with either 1 μM A23187 or 50 μM BAPTA-AM in HBSS_c for 30 min at 37°C and were plated to determine the EOP expressed as the percent relative to treatment with DMSO alone. The data are pooled from three independent trials. The two Iid⁺ strains (RHΔ and MBE3.3) showed the expected drop in EOP after exposure to A23187. Upon treatment with BAPTA-AM, RHΔ, MBE1.1, and MBE3.3 showed about a 90% drop in EOP while MBD2.1 was hypersensitive to this treatment, with a drop of over 99%.

IID is not due to a freely diffusible lytic activity. A strain of RHΔ that was transformed with a SAG1-driven β-galactosidase reporter (24; RHΔ-βgal) was used as the Iid⁺ strain and mixed with RHΔ, MBE1.1, MBE3.3, or MBD2.1 in a 1:1 ratio. The extracellular parasites were incubated in 1 μM A23187 in HBSS_c at 37°C for periods of 0 to 60 min, diluted, and plated on HFF monolayers for the development of plaques. After 5 days, the monolayers were fixed and stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) to distinguish the plaques from each strain. After counting the blue plaques, the monolayer was counterstained with crystal violet to score all the remaining “colorless” plaques. (a) Control showing the expected drop in EOP of the Iid⁺ RHΔ-βgal strain in the four coincubation assays from panel b (scoring for blue plaques). (b) The effect of coincubation with RHΔ-βgal on the IID phenotype of the four strains.

Ionophore-induced membrane permeabilization by intracellular Iie⁺ parasites. Infected cells were pretreated with cytochalasin D to block parasite motility, and A23187 was added before fixing the monolayer. Immunofluorescence was used to examine the membrane integrity of host cells infected with Iie⁺ and Iie⁻ strains. A rabbit anti-SAG1 antibody was used before permeabilizing the monolayer with detergent so as to stain all the parasites that are extracellular or within permeabilized host cells. A mouse anti-SAG1 MAb was used in the presence of Triton X-100 to permeabilize all membranes and allow all the parasites to be stained regardless of their location or the state of the host cell. The two antibodies were detected with different fluorescent dyes, rabbit antibodies with Texas red and mouse antibodies with FITC, such that all parasites stained with FITC but only those that were within permeabilized host cells also stained with Texas red and thus appear yellow. (a) The effect of A23187 on the membrane integrity of host cells infected with Iie⁺ (RHΔ and MBD2.1) and Iie⁻ (MBE1.1 and MBE3.3) strains. Cells infected with RHΔ and MBD2.1 are permeabilized despite the absence of parasite motility. This permeabilization is not observed in cells infected with MBE1.1 and MBE3.3. (b) The A23187-induced permeabilization by the Iie⁺ RHΔ strain is inhibited by pretreating the infected monolayer with BAPTA-AM.