The invasion pore induced by Toxoplasma gondii

Abstract

Obligate intracellular parasites invade host cells to survive. Following host cell contact, the apicomplexan Toxoplasma gondii injects proteins required for invasion into the host cell. Here, electrophysiological recordings of host cells acquired at sub-200 ms resolution allowed detection and analysis of a transient increase in host membrane conductance following exposure to Toxoplasma gondii. Transients always preceded invasion but parasites depleted of the moving junction protein RON2 generated transients without invading, ruling out a direct structural role for RON2 in generating the conductance pathway or restricting the diffusion of its components. Time-series analysis developed for transients and applied to the entire transient dataset (910,000 data points) revealed multiple quantal conductance changes in the parasite-induced transient, consistent with a rapid insertion, then slower removal, blocking, or inactivation of pore-like conductance steps. Quantal steps for RH had a principal mode with Gaussian mean of 0.26 nS, similar in step size to the apicomplexan protein translocon EXP2. Without RON2 the quantal mean was significantly different (0.19 nS). Because no invasion occurs without poration, the term 'invasion pore' is proposed.

Figure 1.

Each invading T. gondii WT RH strain tachyzoite produces a single electrical transient. a) DIC microscopy of Toxoplasma invasion of COS1 cell under visible patch pipette (upper panel); 10-μm scale bar. Four parasites (P1-P4) invaded during recording of a whole-cell patch-clamp electrophysiology experiment. White arrowheads indicate visible constrictions observed when parasites penetrate the host cell during invasion (middle panel: P1 and P2 invasions; lower panel: P3 and P4 invasions; 3-μm scale bar panels P1-P4). b) Multiple transients were detected from the electrophysiological recording obtained under the whole-cell configuration (−60 mV holding potential) during the invasions of the four parasites. c) Expanded time record of the transients (T1-T4) shown in b) for a clearer visualization of the wave form.

Figure 2.

T. gondii invasion pore formation does not require RON2, and thus does not require complete moving junction formation. A) Phenotype of untreated WT and KD-RON2 parasites evaluated using electrophysiology (G) or calcium assay (Ca²⁺) data sets. Percentages of conductance and calcium transients (% of n parasites presented to host cells). Errors are Wilson, two-tailed upper and lower 95% confidence intervals. The percentage of transients observed in KD-RON2 (G) and KD-RON2 (Ca²⁺) are not significantly different from WT (G) (two-proportion z-test, z=0.8389, 1.227, p=0.40, 0.22, respectively). b) Conductance waveforms for WT (blue) and KD-RON2 strains (red) (mean, solid lines; 95% CI, shadings; n=25, 30, respectively).

Figure 3.

a) Representative Change Point Analysis (CPA) of a transient waveform induced by a WT parasite. Time dependent changes in conductance are represented by the gray dotted lines (CP Time) over a selected duration of 1 second. Characteristic features of the transient including the change point means (CP Mean, red), Duration, and Residual conductance (black arrows) are indicated. b) Representative PWC analysis of the same transient in a) showing the identified conductance levels (clusters) in blue. Expanded portions of the rising phase (100 ms) and a larger section of the transient (200 ms) are shown in the expanded plots. The changes in conductance between identified level sets (D) are indicated. The conductance changes from each set of transients were analyzed and used to identify the primary conductance change.

Figure 4.

The conductance transients induced by WT and KD-RON2 tachyzoites differ in peak duration but not in transient duration nor peak or residual conductance. a-d). Violin plots comparing the waveform parameters obtained for WT and KD-RON2 parasites: a) peak conductance, b) residual conductance, c) transient duration, and d) peak conductance duration. Note, in 4d the peak durations are different; bootstrap differences in the mean, alpha=0.05; ANOVA, p=0.034 without Box-Cox transformation; following Box-Cox transformation and Welch’s T-test for unequal variance, p=5.69E-17.

Figure 5.

Evidence for quantal conductance levels from time series analysis of individual transient recordings. Piece wise constant (PWC) filtering was used to analyze the time-dependent conductance changes observed during individual transients. Examples of three a) WT-induced and b) KD-RON2 transients analyzed using PWC filtering to calculate the distributions of conductance level changes. c) Density distribution functions of combined transient conductance level changes (DLevel G) produced by WT and KD-RON2 strains (n=25 and 30; respectively). d) Peak normalized density of data presented in c).

Figure 6.

Model for invasion pore formation during parasite invasion. Invasion pore formation is triggered by rhoptry exocytosis (see companion paper, Male and Kegawa et al., 2024). Electrophysiological analysis supports multiple rather than single pore formation on the host cell membrane. Pore formation occurs prior to moving junction formation, but differences in the dominant quantal values induced by WT and KD-RON2 parasites suggest that RON2 contributes to the poration process or the passage of rhoptry contents through the pore.