Acidification Activates <i>Toxoplasma gondii</i> Motility and Egress by Enhancing Protein Secretion and Cytolytic Activity

<div><p>Pathogenic microbes rely on environmental cues to initiate key events during infection such as differentiation, motility, egress and invasion of cells or tissues. Earlier investigations showed that an acidic environment activates motility of the protozoan parasite <i>T. gondii</i>. Conversely, potassium ions, which are abundant in the intracellular milieu that bathes immotile replicating parasites, suppress motility. Since motility is required for efficient parasite cell invasion and egress we sought to better understand its regulation by environmental cues. We found that low pH stimulates motility by triggering Ca²⁺-dependent secretion of apical micronemes, and that this cue is sufficient to overcome suppression by potassium ions and drive parasite motility, cell invasion and egress. We also discovered that acidification promotes membrane binding and cytolytic activity of perforin-like protein 1 (PLP1), a pore-forming protein required for efficient egress. Agents that neutralize pH reduce the efficiency of PLP1-dependent perforation of host membranes and compromise egress. Finally, although low pH stimulation of microneme secretion promotes cell invasion, it also causes PLP1-dependent damage to host cells, suggesting a mechanism by which neutral extracellular pH subdues PLP1 activity to allow cell invasion without overt damage to the target cell. These findings implicate acidification as a signal to activate microneme secretion and confine cytolytic activity to egress without compromising the viability of the next cell infected.</p></div

pH neutralization suppresses parasite egress.

<p>(A–D) Parasite egress quantified by immunofluorescence microscopy. Wild type (RH) parasites were allowed to replicate for 30 h (A23187, Zaprinast) or 35 h (DTT) prior to pre-treatment with or without inhibitor followed by vehicle (DMSO, buffer) or egress inducer (2 µM A23187, 5 mM DTT, 250 µM Zaprinast) with or without inhibitor for 2 min. Immunofluorescence was performed for parasites (SAG1) and parasitophorous vacuole (GRA7) and occupied vacuoles were quantified. (*<i>p</i><0.05, student's <i>t</i>-test compared to A23187/DTT/Zaprinast alone). Graphs reflect the average and standard deviation of 3 independent experiment; * <i>p</i><0.05 by student's <i>t</i>-test vs. A23187/DTT/Zaprinast with no inhibitor.</p

PLP1 displays pH-dependent lytic and membrane binding activity.

<p>(A) pH dependent hemolysis. 100 nM recombinant PLP1 or LLO or 2 IU SLO was incubated with sheep erythrocytes before measuring the 450 nm absorbance of the supernatant. Results indicate the average and standard deviation of triplicate wells and the graph is representative of 3 independent experiments. (B) PLP1 binding to membranes is pH dependent. Sheep erythrocyte ghosts were incubated with recombinant PLP1, washed with cold PBS, pelleted, and input and bound fractions were analyzed by immunoblot for PLP1. (C) PLP1 and its Cterm domain display pH dependent membrane binding measured by flotation during sucrose density centrifugation. Recombinant mature, Nterm, and Cterm PLP1 were incubated with sheep erythrocyte ghosts as for B, and the reaction mixture was subjected to sucrose density gradient membrane flotation. Fractions were collected from the top of the sucrose gradient and analyzed by immunoblot. Images are representative of 3 independent experiments.</p

Low pH promotes parasite motility and microneme secretion.

<p>(A) Videomicroscopy analysis of pH dependent parasite motility. Parasites were purified in high K⁺ buffer (pH 8.4) and analyzed before and after exchanging to the same buffer at pH 7.4 or 5.4. Graph indicates the average and standard deviation of 3 independent experiments. *<i>p</i><0.05 vs. pH 7.4 by student's <i>t</i>-test. (B) Immunoblot analysis of pH dependent microneme secretion. Parasites were purified in Endo buffer and switched to Endo buffer of the indicated pH, incubated for 2 min at 37°C and placed on ice. Blots of secreted material were probed with antibodies for MIC10, MIC4 or GRA1 as indicated. (C) Low pH induces more microneme secretion than ethanol stimulation and PLP1 is secreted and processed in a pH-dependent manner. Blots of secreted material were probed with antibodies for PLP1 and GRA4 as indicted. Also shown is a blot of PLP1 from cell pellets (cellular fraction) obtained by centrifugation after secretion. (D) Low-pH induced microneme secretion is blocked by the Ca²⁺ chelator, BAPTA-AM, as observed by immunoblot of PLP1, MIC2, MIC10, and MIC4. GRA1 secretion is not affected by BAPTA-AM. (E) Low pH induces microneme secretion in the presence of high K⁺ concentration. Arrowheads for panels B, D and E indicate products of SUB1 proteolysis.</p

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.</p

A Novel High Throughput Invasion Screen Identifies Host Actin Regulators Required for Efficient Cell Entry by <i>Toxoplasma gondii</i>

<div><p><i>Toxoplasma gondii</i> critically relies on cell invasion as a survival strategy to evade immune clearance during infection. Although it was widely thought that <i>Toxoplasma</i> entry is parasite directed and that the host cell is largely a passive victim, recent studies have suggested that host components such as microfilaments and microtubules indeed contribute to entry. Hence to identify additional host factors, we performed a high-throughput siRNA screen of a human siRNA library targeting druggable proteins using a novel inducible luciferase based invasion assay. The top 100 hits from the primary screen that showed the strongest decreases in invasion were subjected to confirmation by secondary screening, revealing 24 proteins that are potentially involved in <i>Toxoplasma</i> entry into host cells. Interestingly, 6 of the hits appear to affect parasite invasion by modifying host cell actin dynamics, resulting in increased deposition of F-actin at the periphery of the cell. These findings support the emerging notion that host actin dynamics are important for <i>Toxoplasma</i> invasion along with identifying several novel host factors that potentially participate in parasite entry.</p></div

Categorization of the validated hits.

<p>Categorization of the validated hits.</p

Acidic pH increases invasion but results in host cell damage.

<p>(A) Low pH promotes parasite attachment and invasion. HFF cells were pulse invaded for 2 min with parasites in DMEM, or DMEM-like buffer at pH 5.4 or 7.4. *<i>p</i><0.05 vs. DMEM by student's <i>t</i>-test. Graph indicates the average and standard deviation of 3 independent experiments. (B) PLP1- and pH-dependent damage to host cells. HFF cells were preloaded with calcein-AM and WT and <i>plp1ko</i> parasites were allowed to settle on host cells in Endo buffer, which was subsequently switched to DMEM-like buffer of the indicated pH and incubated at 37°C for 10 min. Calcein signal in the supernatant was measured by fluorometry. Graph indicates average and standard deviation of triplicate wells and is representative of 3 independent experiments. *<i>p</i><0.05 vs. WT pH 7.4 by student's <i>t</i>-test.</p

pH-neutralizing agents reduce egress-associated membrane permeabilization.

<p>(A–D) RH (WT) or <i>plp1ko</i> infected cells were incubated for 30 h prior to treatment with 1 µM CytD with or without inhibitor for 3 min followed by 1 µM CytD with vehicle (DMSO) or ionophore (2 µM A23187) with or without inhibitor plus 12.5 µg/ml propidium iodide (PI) for 3 min. Cells were fixed and stained with DAPI. Infected cells were determined by brightfield and PI positivity by nuclei fluorescent in the red channel. Results indicate average and standard deviation of 3 independent experiments for WT and 2 independent experiments for <i>plp1ko</i>. * <i>p</i><0.05 by student's <i>t</i>-test.</p

A schematic showing the location of 24 validated host-factors that appear to play roles in host cell entry by <i>Toxoplasma</i>.

<p>Arrows indicate enhancement of expression or activity whereas T-lines indicate inhibition of expression or activity. Dashed lines indicate hypothetical interactions. Relationships are based on the findings herein or from the literature. See text for additional descriptions.</p