Acidic pH induces parasite egress and pH shifts occur upon egress induction, and late in the replication cycle.

<p>(A) Parasite egress quantified by immunofluorescence microscopy. 30 h vacuoles were treated with high K⁺ buffer ±15 µM digitonin at 37°C for 3 min prior to fixation and egress enumeration. *<i>p</i><0.05 vs. pH 8.4 by student's <i>t</i>-test. Graph indicates the average and standard deviation of 3 independent experiments. (B) Superecliptic pHuorin signal is quenched at low pH. HFF cells were inoculated with increasing doses of superecliptic pHluorin expressing <i>plp1ko</i> parasites (<i>plp1ko</i>sepH) and fluorescence was observed 30 h post-infection in live cells or cells detergent lysed at pH 5.4 or 7.4. (C, D) A moderate decrease in pH occurs during induced egress. HFF inoculated with <i>plp1ko</i> parasites expressing superecliptic pHluorin for 30 h before treatment with DMSO or A23187 with or without 20 mM NH₄Cl (panel C) or 40 µM DCCD (panel D). Results indicate the average and standard deviation of triplicate wells and are representative of 3 independent experiments for NH₄Cl and 2 independent experiments for DCCD. * <i>p</i><0.05 vs. DMSO by student's <i>t</i>-test. (E) pH changes during parasite replication. Fluorescence signal without or with 20 mM NH₄Cl was followed for <i>plp1ko</i> expressing superecliptic pHluorin over the course of intracellular replication. The graph indicates the mean and standard deviation of triplicate wells and is representative of two independent experiments with independent clones. * <i>p</i><0.05 vs. 0 mM NH₄Cl by student's <i>t</i>-test.