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Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress [1]

['My-Hang Huynh', 'Department Of Microbiology', 'Immunology', 'University Of Michigan Medical School', 'Ann Arbor', 'Michigan', 'United States Of America', 'Vern B. Carruthers']

Date: 2022-07 

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during ionomycin or zaprinast induced egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead to an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.

Toxoplasma gondii is a single celled intracellular parasite that infects many different animals, and it is thought to infect up to one third of the human population. This parasite must rupture out of its replicative compartment and the host cell to spread from one cell to another. Previous studies indicated that a decrease in pH occurs within the replicative compartment near the time of parasite exit from host cells, an event termed egress. However, it remained unknown whether the decrease in pH is directly tied to egress and, if so, what is responsible for the decrease in pH. Here we used a fluorescent reporter protein to directly measure pH within the replicative compartment during parasite egress. We found that pH decreases immediately prior to parasite egress and that this decrease is linked to parasite disruption of membranes. We also identified a family of transporters that release acidic products from parasite use of glucose for energy as contributing to the decrease in pH during egress. Our findings provide new insight that connects parasite glucose metabolism to acidification of its replicative compartment during egress from infected cells.

Herein, we utilize a ratiometric pHluorin (RatpH) [ 16 ] to directly quantify PV pH in individual infected cells. Expression of RatpH in the PV consistently revealed a decrease in PV pH immediately preceding induced or natural egress. Using parasites lacking PLP1 we identified a role for this pore forming protein in PV acidification and Ca 2+ signaling within the host and parasite prior to PV rupture. We also identified formate-nitrite transporters (FNTs) as contributing to the acidification of the PV during egress, likely via co-transporting protons with the glycolytic products lactate and pyruvate. However, disruption of FNTs did not completely abrogate the release of lactate and pyruvate or eliminate the drop in PV pH, suggesting the involvement of other unidentified transporters or products that also contribute to PV acidification during egress.

Early work showed that tachyzoite motility is pH-dependent, and that alkaline conditions inhibit motility whereas acidic buffers induce motility [ 8 ]. It was later shown that this pH-dependent motility is likely due to the effect of pH on microneme secretion, wherein low pH activates microneme secretion, and also leads to parasite egress [ 9 ]. Concomitantly, pH neutralization or treatment with DCCD was found to suppress tachyzoite egress. This study also noted that a drop in PV pH was associated with late-stage infection ~30 h post-inoculation, a decrease that was partially reversed upon treatment with the weak base NH 4 Cl. The low pH associated with egress was correlated with an increase in PLP1 activity. More specifically, in erythrocyte hemolysis assays, recombinant PLP1 lytic activity was shown to be most active between pH 5.4 and 6.4, a pH range for which PLP1 binding to erythrocyte ghost membranes was also enhanced [ 9 ]. T. gondii PLP1 is not unique in this respect, as several other pathogenic cytolytic proteins have been found to be more active at low pH, including Listeria monocytogenes listeriolysin O [ 10 , 11 ], Leishmania amazonensis a-leishporin [ 12 ], Trypanosoma cruzi TC-TOX [ 13 ], and Bacillus thuringiensis Cyt1A [ 14 ]. Also, acidification of transient PVs containing Plasmodium yoelii sporozoites was suggested to promote parasite escape in a PyPLP1-dependent manner [ 15 ]. For TgPLP1, increased activity at low pH is consistent with the observed lower pH of the PV beginning at ~30 h post-infection when parasites are beginning to egress [ 9 ]. However, the study was carried out using superecliptic pHluorin, which measures relative pH [ 16 ] rather than absolute pH. Also, the measurements were made on a population of infected cells over a relatively long period of time. Thus, this earlier work failed to capture changes in pH occurring during egress from individual cells.

Most of the studies involving egress have been performed using chemical inducers such as ionomycin or zaprinast, which increase cytosolic Ca 2+ to elicit egress. A proposed general model for the activation of egress is as follows: (1) addition of inducer initiates parasite Ca 2+ signaling (reviewed in [ 2 , 3 ]); (2) host cytosolic Ca 2+ levels transiently increase by a mechanism that is not well understood [ 4 ]; (3) the parasite internalizes host-derived Ca 2+ through a nifedipine-sensitive Ca 2+ channel [ 4 ]; and (4) the internalized Ca 2+ increases parasite Ca 2+ levels to above a threshold [ 4 ], which activates the secretion of the micronemes, including the perforin-like protein 1 (PLP1), and facilitates rupture of the parasitophorous vacuole membrane (PVM) for egress. During spontaneous or natural egress, diacylglycerol kinase 2 (DGK2) was shown to be a plausible candidate for an intrinsic signal [ 5 ]. DGK2 is secreted into the PV and generates phosphatidic acid as a signaling molecule. DGK2-defective parasites were selectively defective in natural egress but were able to respond to chemical inducers. Conversely, egress is negatively regulated by the cAMP-dependent protein kinase A catalytic subunit 1 (PKAc1) through suppression of cyclic GMP (cGMP) cytosolic Ca 2+ signaling, as demonstrated by premature egress of PKAc1-deficient parasites [ 6 , 7 ]. These studies also implicated PV acidification as a determinant for the early egress of PKAc1-deficient tachyzoites based on treatment with a P-type ATPase inhibitor (dicyclohexylcarbodiimide, DCCD) [ 7 ] or neutralization with NH 4 Cl [ 6 ].

As an obligate intracellular pathogen, Toxoplasma gondii critically relies on efficiently completing each step of its lytic cycle for successful propagation and survival. In recent years, a greater focus has been applied to the egress step, wherein the parasites exit the host cell as a necessary prelude to infecting a neighboring cell. This increased attention has elucidated a well-orchestrated and complex cascade of events involving several molecular players, including activation of protein kinases by calcium (Ca 2+ ) or cyclic guanine monophosphate, release of proteins from apical microneme organelles, and activation of the glideosome motility machinery [ 1 ]. As with most other parasites belonging to the phylum Apicomplexa, T. gondii parasites replicate inside a membrane bound parasitophorous vacuole (PV), from which it must escape before leaving the host cell to initiate another round of the lytic cycle.

Results

PV pH decreases immediately prior to induced and natural egress Green fluorescent protein (GFP) has two excitation peaks at 410 nm and 470 nm. This property has been exploited to create, via amino acid substitutions, pH sensitive variants termed pHluorins [16]. Ratiometric pHluorin (RatpH) responds to a decrease in pH with reduced fluorescence from excitation at 410 nm and increased fluorescence from excitation at 470 nm. To measure the pH of the T. gondii PV, we expressed a codon-optimized RatpH in RH parasites (RH-RatpH). We then tested the ability of RatpH to respond to changes in pH by incubating RH-RatpH infected cells in buffers of defined pH containing nigericin to equilibrate H+ across membranes (Fig 1A). Obtaining ratiometric images by excitation at 410 nm and 470 nm (Fig 1B) allowed the generation of a calibration curve for pH (Fig 1C). These findings show that RatpH is a robust indicator of pH when expressed in the PV and thus is suitable for measuring changes in PV pH during egress. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Measurement of pH in T. gondii PVs expressing ratiometric pHluorin. A) Equilibration of vacuoles to the surrounding buffer of known pH values with nigericin. B) Vacuole ratio images at 410/470 nm that are false colored according to the scale shown to the right. C) Calibration curve from the ratio 410/470 nm values for each pH value (mean ± S.D); R2 = 0.9992. Scale bar, 5 μm. D) Reductions in PV pH during egress observed by live imaging of ionomycin- induced RH-RatpH parasites. The upper image series is from excitation at 410 nm to visualize the time of egress i.e., when pHluorin first leaves the PV due to rupture of the PVM. The lower series is of ratio images according to the pH scale bar shown on the right. // indicates a gap in time. E) The magnitude of pH changes varies between PVs. Tracings of pH values of RH-RatpH PVs induced with DMSO (a) or ionomycin (b, c, d). Tracing d represents the vacuole shown in 1D. Image acquisition was paused following frame 5 (25 sec), inducer was added, and acquisition was started again. Green data points indicate the time when pHuorin first leaves the PV, which is designated as the time of egress. F) A significant reduction in PV pH occurs after ionomycin or zaprinast induction of egress. Data points represent changes in PV pH starting from baseline to a drop greater than 0.05 following induction with either ionomycin or zaprinast. Numbers in squares within the graph indicates the numbers of PVs enumerated. A Kruskal-Wallis test with Dunn’s multiple comparison was performed. **** p≤0.0001. Bars indicate the median. ns, not significant. G) A drop in PV pH occurs during natural egress. Representative PVs late in replication (~50 h) and pH tracings associated with egress. Scale bar, 10 μm. H) Quantification of pH changes in PVs during natural egress. Bar indicates the mean. https://doi.org/10.1371/journal.ppat.1010139.g001 To determine if the expression of RatpH in RH affected parasite egress or fitness, we compared RH and RH-RatpH parasites for extent of egress and time to egress in response to ionomycin or zaprinast and their growth in a co-culture competition assay. We found no difference in the percentage of parasites that egressed within 5 min of induction (S1A Fig) or in the time to egress (S1B Fig). Furthermore, there was no difference in relative abundance of each strain following serial co-culture for 5 passages (S1C Fig). To initially quantify changes in pH during egress we performed live ratiometric imaging of RH-RatpH infected cells in Ca2+ containing buffer upon adding the Ca2+ ionophore ionomycin, which induces egress by equilibrating calcium across membranes. Collecting a series of ratio images following ionomycin treatment showed that a decrease in PV pH occurs prior to rupture of the PVM, which was indicated by the release of RatpH from the PV in non-ratioed images (Fig 1D). Tracings from individual PVs showed that in contrast to a consistent pH observed upon treatment with vehicle (DMSO) (Fig 1E, a), PV pH decreased 10–30 sec prior to PV rupture in infected cells treated with ionomycin (Fig 1E, b,c,d and S1 Video). To quantify the decrease in PV pH from the tracings, we took the lowest pH value, which typically occurred near the time of PV rupture (identified by pHluorin release) and subtracted it from the pH value that was the highest immediately prior to the descent, as described more extensively in the Materials and Methods. We found that although the magnitude of the decrease was variable, all the PVs we observed showed a drop in PV pH prior to PV rupture (Fig 1F). Analysis of 35 PVs recorded over multiple experiments showed a mean decrease of 0.35 pH units, corresponding to a ~3-fold increase in [H+] within the PV. We also observed a similar decrease in PV pH upon inducing egress with zaprinast (Fig 1F), which activates parasite protein kinase G upstream of Ca2+ signaling [17], a mechanism distinct from that of ionomycin. To determine if a decrease in pH occurs during natural egress, we imaged RH-RatpH infected cells at ~48–52 h post-infection for 20 min/field of view. We observed a decrease in PV pH in 10 out of 12 egress events, with a mean decrease of 0.17 pH units (Fig 1G and 1H and S2 Video). Taken together, our findings suggest that PV pH decreases immediately prior to induced and natural egress. Due to the challenges of capturing natural egress events, all subsequent experiments were performed with induced egress.

Ionic environment modulates the vacuolar pH response Earlier work suggested that a loss of cytoplasmic K+ from the host cell triggers egress of T. gondii tachyzoites through activation of phospholipase C and a subsequent increase in cytoplasmic Ca2+ [18]. A more recent study proposed that the decrease in K+ accelerates egress but is not a trigger [4]. While Ca2+ is necessary for activation of egress, the absence of extracellular Ca2+ delayed but did not inhibit parasite egress [2]. pH measurements presented thus far have been obtained in Ringer’s buffer (155 mM NaCl, 3 mM KCl, 1 mM Ca2+, 1 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, 3 mM NaH 2 PO 4 , pH 7.4). To test the effect of Ca2+ and K+ in the extracellular media on the vacuolar pH drop, infected cells were incubated in Ringer’s or 145 mM K+ (termed high K+) to mimic the [K+] of the host cell cytosol, each with and without Ca2+. Buffer compositions used in this study are listed in Table 1. PPT PowerPoint slide

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TIFF original image Download: Table 1. Ion composition of buffers used in this study. https://doi.org/10.1371/journal.ppat.1010139.t001 Egress from infected cells in Ringer’s buffer was induced with zaprinast (Fig 2A) or ionomycin (Fig 2B) with or without cytochalasin D (CytD) treatment to determine whether immobilizing the parasites affects the decrease in PV pH after induction. Since CytD did not markedly affect PV pH, to facilitate imaging it was included in all subsequent experiments unless otherwise noted. Whereas removal of Ca2+ from Ringer’s significantly attenuated the drop in PV pH after induction with zaprinast or ionomycin, removal of Ca2+ from the high K+ had no effect. In the presence of Ca2+, incubation in high K+ showed a trend toward attenuating the drop in PV pH after induction with zaprinast and a significant attenuation following ionomycin induction. When Na+ in the extracellular buffer is replaced with choline to maintain the total concentration of monovalent cations, with or without Ca2+, there was no effect on the pH drop observed (Fig 2C). A role for chloride was assessed by replacing it with glutamate or sulfate (-Chloride buffer) composed of potassium glutamate, sodium glutamate, calcium glutamate, and magnesium sulfate. The absence of chloride also had no effect on PV acidification (Fig 2C). Together these findings imply that extracellular Ca2+ is necessary for normal acidification of the PV during egress and that the loss of K+ from host cells also influences PV pH. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Composition of surrounding buffer mildly abates PV pH changes. A) and B) Extracellular buffer without Ca2+ tempers the drop in pH with zaprinast (A) or ionomycin (B) induction. RH-RatpH PVs, treated with or without CytD, incubated in Ringer’s buffer or High K+ with or without Ca2+ and induced with DMSO, ionomycin, or zaprinast. ns, not significant. C) Na+ and Cl- do not affect pH changes. Extracellular buffer with Na+ replaced with choline or all Cl- replaced with glutamate, with or without Ca2+. Numbers in squares within all graphs indicate the numbers of vacuoles enumerated. A Kruskal-Wallis test with Dunn’s multiple comparison was performed for all graphs. Bars indicate the median. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. https://doi.org/10.1371/journal.ppat.1010139.g002

PLP1 influences acidification of the PV and calcium signaling in the parasite Parasites deficient in PLP1 were previously shown to be substantially delayed in induced egress, with a proportion of the parasites unable to leave the vacuole [19]. Since PLP1 is a pore-forming protein, we reasoned that it could facilitate ion flux associated with acidification of the PV during egress. To address this, we introduced RatpH into PLP1 deficient parasites (Δplp1-RatpH). As expected, Δplp1-RatpH showed a delay in egress, with parasite egress occurring substantially later than the onset of motility (Fig 3A). We found that although Δplp1-RatpH vacuoles from which the parasite was able to egress exhibited a drop in pH with ionomycin or zaprinast induction, this decrease was significantly attenuated relative to RH-RatpH (Fig 3B). Whereas the measurements thus far were performed on the entire PV, we noted that some regions of the PV show greater acidification. Thus, we also measured the minimum pH in each PV prior to egress. This analysis showed that the mean difference in the lowest regional PV pH for non-induced (DMSO) and induced (zaprinast or ionomycin) RH-RatpH parasites was ~0.7 pH units, while for Δplp1-RatpH parasites, it was ~0.4 pH units (Fig 3C). The analysis of lowest regional pH also confirmed an attenuation of PV acidification during induced egress of Δplp1-RatpH parasites. PPT PowerPoint slide

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TIFF original image Download: Fig 3. PV acidification is attenuated in Δplp1 parasites. A) Representative tracings of pH values in Δplp1-RatpH from live imaging. Arrow indicates the time point that inducer was added. The yellow data points indicate the time point where motility of the tachyzoites began and the green points indicate the time of PV rupture. B) Δplp1 PVs show a significant attenuation of PV acidification after induction with ionomycin or zaprinast. Each point represents the change in pH within an individual PV. RH-RatpH data sets are the same as those presented in Fig 1F. Values in squares within the graph indicates the numbers of PVs enumerated. RH, RH-RatpH; Δplp1, Δplp1-RatpH. C) Punctate foci in individual vacuoles reach lower pH than whole vacuole. Ratio images of RH-RatpH or Δplp1-RatpH vacuoles were analyzed in ImageJ using the Process Plugin to find min-max values (v.1.00). Data points indicate the lowest pH measured within a PV. RH, RH-RatpH; Δplp1, Δplp1-RatpH. Kruskal-Wallis tests with Dunn’s multiple comparison were performed for B) and C). * p≤0.05, ** p≤0.01, **** p≤0.001, **** p≤0.0001. Bars indicate the median. D) Paralyzed WT parasites undergo PV acidification. Representative traces of RH-RatpH parasites untreated or immobilized with cytochalasin D (CytD) or mycalolide B (MycB), followed by ionomycin or zaprinast induction. RH, RH-RatpH; Δplp1, Δplp1-RatpH. E) Representative traces of Δplp1-RatpH parasites untreated or immobilized with CytD or MycB, followed by ionomycin or zaprinast induction. F,G) Kinetics of parasite Ca2+ signaling relative to PV acidification. Representative traces of relative Ca2+ levels (left axis) and pH (right axis) of RH-RatpH-RGECO PVs (F) and Δplp1-RatpH-RGECO PVs (G) following zaprinast induction. a.u., arbitrary units. H) Enumeration of 41 RH-RatpH-RGECO and 28 Δplp1-RatpH-RGECO PVs shows an increase in the time from addition of inducer to PV rupture in Δplp1-RatpH-RGECO. A two-tailed student’s t-test was performed, **** p≤0.0001. https://doi.org/10.1371/journal.ppat.1010139.g003 While PLP1-deficient parasites are defective in egress, some Δplp1 parasites are capable of egressing due to the motility of the parasites that eventually break through the PVM and plasma membrane of host cells. To more selectively study the role of PLP1 and motility in the pH drop associated with egress, RH-RatpH or Δplp1-RatpH parasites were paralyzed with either CytD or mycalolide B (MycB), which prevent actin polymerization or severs F-actin, respectively. As observed earlier in this study (Fig 2A and 2B), PVs of immobilized RH-RatpH parasites induced with ionomycin or zaprinast still displayed a significant drop in pH associated with pHluorin release (Fig 3D). However, PVs of immobilized Δplp1-RatpH showed no decrease in pH and they failed to release pHluorin for the entire duration of the experiment (up to 20 min) in all 225 vacuoles observed (Fig 3E). Taken together, these findings suggest that PLP1 influences PV pH and that the absence of PLP1 or motility precludes PV acidification and rupture. Since Ca2+ signaling triggers the secretion of microneme proteins including PLP1 and it activates motility, we sought to define the timing of parasite cytosolic Ca2+ and its relationship to changes in PV pH during egress by stably expressing a red genetically encoded Ca2+ indicator RGECO in the cytosol of RH-RatpH and Δplp1-RatpH parasites. Ca2+ and PV pH measurements were obtained from motility competent parasites so that we could define the timing of Ca2+ and pH dynamics in relation to PV rupture and egress. RH-RatpH-RGECO parasites displayed a rapid initial increase in cytosolic Ca2+ after zaprinast induction and well before the drop in PV pH and egress (Fig 3F and S3 and S4 Videos). To determine if the order of signal acquisition during imaging (i.e., Ca2+ then pH or pH then Ca2+, at each time point) influenced the results, we compared Ca2+ and pH dynamics by collecting data in both orders and found no difference (S2 Fig). The decrease in PV pH often corresponded to a second peak of Ca2+, the presence of which has been reported previously as a potentiation of the initial peak via Ca2+ influx from the host cell and the media [2]. Consistent with a prior report [4], Δplp1-RatpH-RGECO parasites usually did not have a potentiation peak (Fig 3G). Also, whereas RH-RatpH-RGECO parasites ruptured the PVM 60 sec after induction on average, Δplp1-RatpH-RGECO parasites took more than twice as long (average of 132 sec) to rupture the PVM (Fig 3H). From these findings we conclude that zaprinast induction initiates parasite Ca2+ signaling followed by the potentiation of the Ca2+ signal and PV acidification, and that the timing of PV acidification and rupture is delayed in parasites lacking PLP1.

PLP1 contributes to the initiation of egress by dictating Ca2+ influx into host cells Previous studies have shown that a transient rise in host Ca2+ occurs prior to parasite egress [4]. To confirm this elevation of host Ca2+ and analyze its temporal relationship with Ca2+ signaling in the parasite and acidification of the PV, we infected RGECO-expressing host cells with RH-RatpH-RGECO parasites, thereby allowing us to monitor host Ca2+, parasite Ca2+, and PV pH by imaging reporters that are spatially (host RGECO vs parasite RGECO) or spectrally (RGECO vs pHluorin) distinct. Upon zaprinast induction we observed an initial peak of parasite Ca2+ like previous experiments, which was later followed by coincident elevation of host and parasite Ca2+ and PV acidification prior to PV rupture (Fig 4A and 4B). To confirm this, we loaded infected host cells with the synthetic Ca2+ indicator Cal-590-AM and again noted the coincident elevation of host Ca2+ and PV acidification and rupture (Fig 4C, left panel and S5 Video). Our earlier results indicated that extracellular Ca2+ in the medium is necessary for normal PV acidification. This prompted us to examine how the absence of extracellular Ca2+ in the medium affects the elevation of host Ca2+ during egress by adding zaprinast in Ca2+ free medium. We found that the absence of extracellular Ca2+ blunted the elevation of host Ca2+ (Fig 4C, right panel), suggesting that the elevation of host Ca2+ is due to influx of Ca2+ from the medium. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Host cytosolic calcium increases prior to PV rupture. A) Still images of time-course of HeLa-RGECO host Ca2+ levels and PV rupture (loss of pHluorin from PV) following zaprinast induction. // indicates a gap in time. Scale bar, 10 μm. Dashed red outline indicates the region of the host cell used to analyze the RGECO fluorescence in B), and the dashed white outline indicates the PV used to analyze pH (pHluorin) and tachyzoite Ca2+ (RGECO). B) Tracings of host and tachyzoite Ca2+ levels (left axis) and PV pH (right axis) of vacuole shown in A. A vertical line denotes the time point after which host Ca2+ levels increase and near when the PV pH decreases. Blue trace, parasite Ca2+; black trace, PV pH; purple trace, host cell Ca2+. a.u., arbitrary units. C) Representative tracings of an HFF host cell preloaded with Cal-590-AM, infected with RH-RatpH-RGECO parasites, and induced with zaprinast in Ringer’s buffer with or without Ca2+. A vertical line denotes the time point after which host Ca2+ levels increase and when the PV pH decreases. D,E) Cal-590-AM-loaded HFF cells infected with RH-RatpH (D) or Δplp1-RatpH (E), following treatment without or with CytD. Measurements of host Ca2+ (left axis) and PV pH (right axis). Purple trace, host Ca2+; black trace, PV pH; orange trace, host Ca2+ with CytD-treated parasite, blue trace, PV pH with CytD-treated parasite. Yellow data point indicates the start of motility (in E) and green data points indicate PV rupture (in D and E). F) Time from host Ca2+ (Cal-590 fluorescence) increase to PV pHuorin release in host cells infected with RH-RatpH (29 vacuoles) or Δplp1-RatpH (16 vacuoles). A two-tailed student’s t-test was performed. ns, not significant. https://doi.org/10.1371/journal.ppat.1010139.g004 Previous work noted that PLP1 deficient parasites show muted Ca2+ signaling and no elevation of host cell Ca2+ when they attempt to egress naturally (i.e., after removing an inhibitory treatment) unless they ruptured the cell via motility [4]. These findings suggest that during natural egress, PLP1 or motility is required for Ca2+ signaling in both the parasite and host cell. However, the study did not test immobilized parasites to determine if PLP1 is necessary for host Ca2+ signaling in the absence of motility-dependent PV rupture and egress. To determine if PLP1 and/or motility are required for host Ca2+ signaling and when such signaling occurs relative to PV rupture, we measured host Ca2+ with Cal-590-AM in cells infected with RH-RatpH without or with CytD treatment or with Δplp1-RatpH without or with CytD treatment. As noted earlier, cells infected with motility competent RH-RatpH showed an elevation of host Ca2+ prior to PV rupture, as did cells infected with CytD immobilized RH-RatpH (Fig 4D). This indicates that when PLP1 is present, motility is not necessary for the elevation of host Ca2+ prior to PV rupture. Cells infected with motility competent Δplp1-RatpH also showed an elevation of host Ca2+ before PV rupture (Fig 4E). Notably though, elevation of host Ca2+ occurred 15–30 sec prior to PV rupture, and motility initiated ~30–60 sec before the elevation of host Ca2+. Taken together, this suggests that in the absence of PLP1, motility can stimulate elevation of host Ca2+ before PV rupture and egress. Remarkably, cells infected with immobilized Δplp1-RatpH parasites showed no host Ca2+ signaling (12 infected host cells examined) (Fig 4E). Since the presence or absence of PLP1 is the only variable in this comparison, these findings indicate that PLP1 is necessary for host Ca2+ signaling. Also, elevation of host Ca2+ always preceded PV rupture (Fig 4F), suggesting that parasite motility or secretion of PLP1 triggers an elevation of host Ca2+ before rupture of the PV. Taken together with the requirement for extracellular Ca2+, our findings indicate that PLP1 (or motility) plays an early role in egress by directing the influx of Ca2+ into infected host cells. Because the uptake of host-derived Ca2+ by the parasite potentiates parasite Ca2+ signaling [2], and PV acidification coincides with such potentiation, PLP1-dependent Ca2+ signaling appears to be linked to PV acidification.

The PV and host cytosol acidify simultaneously Although the PVM contains pores that allow the diffusion of small molecules <1,300 Da including ions [2,20,21], we nevertheless attempted to determine the directionality of acidification by transiently expressing ratiometric pHluorin in the cytosol of HeLa cells followed by infection with RH-RatpH parasites (S3A and S3B Fig). Ratio images following induction with ionomycin in the provided example and others seem to indicate a pH change first occurring in the PV, followed by acidification in the host cell (S3C Fig). However, pH measurements of the entire PV and a section of the host cytosol show an indistinguishable pattern of the pH changes (blue and green tracings in S3D Fig). Thus, we conclude that a distinction between the drop occurring first in either the PV or host cannot be made. Nevertheless, from these experiments we ruled out that ionomycin or zaprinast treatment directly affects the cytosolic pH of host cells by treating and analyzing uninfected cells (S3D and S3E Fig).

Several plasma membrane proton transporters are not required for acidification of the PV Next, we sought to identify the basis for acidification of the PV by initially focusing on proton transporters with the potential to be expressed on the parasite surface. A V-type H+ ATPase (V-ATPase) is localized on the plasma membrane as well as to the plant-like vacuole (PLV)/Vacuolar compartment (VAC, used hereafter) organelle. The V-ATPase was shown to function in maintaining cytoplasmic pH and the acidic pH of the VAC and immature rhoptries [22]. To test if the V-ATPase contributes to acidification of the PV, we transiently expressed RatpH in the inducible knockdown parasite strain of VHA1 (iVHA), a critical subunit of V-ATPase, generated in Stasic et al. [22]. We confirmed down-regulation of VHA1 protein levels following ATc treatment by western blotting for the HA tag appended to VHA1 (Fig 5A). We found that parasites lacking VHA1 (iΔvha1-HA +ATc) showed a drop in PV pH after egress induction that was indistinguishable from those expressing VHA1 (iΔvha1-HA) (Fig 5B and 5C). These findings suggest that VHA1 does not play a role in PV acidification during egress. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Known plasma membrane-localized V-ATPase and sodium-hydrogen exchangers do not affect vacuolar pH changes during egress. A) Western blot of inducible VHA1 (iΔvha1-HA) +/- anhydrotetracycline (ATc) treatment for 32 h. Blots were probed with Ms anti-HA to detect VHA1-HA; Rb anti-MIC2 was a loading control. B) Two representative pH tracings from C) of iΔvha1-HA parasites transiently transfected with RatpH, incubated +/–ATc, and induced with ionomycin. The green data points indicate the time point that pHluorin leaves the vacuole. C) Knockdown of iΔvha1-HA does not affect the magnitude of a pH change. ns, not significant. D-F) Sodium-proton exchangers do not contribute to vacuolar pH changes. Parasite strains Δnhe1 (D), Δnhe3 (E), and NHE4.AID + IAA (F) transiently transfected with RatpH were induced with ionomycin and pH changes measured by live-imaging. Numbers in squares within graphs indicate the numbers of vacuoles enumerated. Kruskal-Wallis tests with Dunn’s multiple comparison was performed for all graphs. * p≤0.05, ** p≤0.01. ns, not significant. Bars indicate the median. https://doi.org/10.1371/journal.ppat.1010139.g005 T. gondii expresses four sodium/proton exchangers: NHE1 is on the plasma membrane [23], NHE2 is associated with the rhoptries [24], NHE3 is associated with the VAC [25], and NHE4 is predicted to be on the plasma membrane or Golgi, depending on the prediction program used for the analysis [26]. Since it was unlikely that a rhoptry proton exchanger would affect PV acidification during egress, NHE2 was excluded from testing. We tested available knockout strains that lack NHE1 [23] or NHE3, [25], which we confirmed by PCR (S4 Fig). Upon transiently expressing RatpH, we found that Δnhe1 and Δnhe3 both showed normal acidification of the PV during induced egress (Fig 5D and 5E). To assess the potential contribution of NHE4 to pH changes in the PV during egress, we used CRISPR-Cas9 to append an auxin-inducible degron (AID) to the C-terminus of NHE4 (NHE4.AID) in RHΔku80 parasites expressing the auxin receptor (TIR1) for AID-based protein degradation (RHΔku80/TIR, TIR hereafter) [27]. Immunofluorescence localization of the HA tag downstream of the AID indicated that the tagged NHE4 was found primarily in the region anterior to the nucleus, reminiscent of Golgi staining (S5A Fig). Addition of auxin (indoleacetic acid, IAA) effectively reduced expression of NHE4.AID to a level below detection based on western blotting (S5B Fig). NHE4.AID parasites showed an acidification of the PV that was indistinguishable from that of RH or TIR parasites (Fig 5F). Altogether, this showed that NHE1, NHE3, and NHE4 do not contribute to the vacuolar acidification observed during egress.

FNT3 does not play a role in PV acidification Although FNT3 appears to be expressed almost exclusively in the sporozoite stages of the parasite based on data in Toxodb, compensation of FNT1 and FNT2 transport by upregulation of FNT3 expression in the absence of FNT1 and FNT2 was a possibility. To measure potential changes in FNT3 expression by reverse transcriptase quantitative PCR, we tested two independent primer sets to the FNT3 mRNA and used actin as the housekeeping gene for normalization. No significant change in FNT3 transcript level was observed in the absence of FNT1 and FNT2 (S8 Fig). To more conclusively rule out a role for FNT3 in the residual decrease of PV pH observed in iFNT1Δfnt2, we deleted FNT3 to generate an iFNT1Δfnt2Δfnt3 strain (S6A Fig). We found that deletion of FNT3 in the absence of FNT1 and FNT2 did not further exacerbate the attenuation of PV acidification during egress, and that a residual drop of PV pH is still observed (Fig 6F). Despite the attenuation of a pH change in the PV during egress, neither the iFNT1Δfnt2 nor the iFNT1Δfnt2Δfnt3 parasites were defective in their ability to egress upon induction with zaprinast in a static (single 2 min time point) egress assay (Fig 6G). The absence of these FNTs also had no effect on intracellular replication (S9 Fig). Taken together our findings suggest that FNT1 and FNT2 contribute to acidification of the PV during egress, acidification is not critical for induced egress, and other transporters likely exist for residual acidification of the PV in parasites lacking the FNTs.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010139

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