Targeting an exocytosis reporter to exonemes

To observe exoneme exocytosis in live parasites, we utilized a pH-sensitive variant of green fluorescent protein (GFP) known as superecliptic pHluorin or SEP [20,21] that is widely used to monitor the exocytosis and pH of subcellular compartments in several organisms including P. falciparum [22,23]. SEP fluorescence is quenched in acidic environments (pH < 6) and is fluorescent in neutral pH environments [20,21,24,25] (Fig 1A). Secretory vesicles are acidified as they are generated and trafficked from the Golgi. Thus, we hypothesized that SEP will not be fluorescent within acidic secretory vesicles, like exonemes, but will fluoresce when exonemes fuse to the plasma membrane and SEP is released into the neutral pH environment of the PV during exocytosis (Fig 1A).

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Fig 1. Dual-fluorescent NF54^attB parasites to monitor exoneme exocytosis and PVM rupture.

(A) The schematic shows Plasmepsin X (PMX) tagged with super-ecliptic pHluorin or SEP. SEP is non-fluorescent at pH 6.0 or less, but it is fluorescent at pH 7. (B) Schematics of the targeting (pMGPMX-SEP; Top) and (pbPM2-EXP2-mRuby3; bottom) plasmids, the expected recombination product, and representation of the primer pairs used to analyze an integration. (C) PCR diagnosis of mRuby3 fusion to the PfEXP2 locus in P. falciparum NF54^attB (left) and PMX^SEP in the PfEXP2^mRuby3(right) parasites (primers sequences shown in S1 Table). (D) Western blot of parasite lysates isolated from P. falciparum NF54^attB, PfEXP2^mRuby3, and PfEXP2^mRuby3/PMX^SEP parasites probed with anti-EXP2 and anti-GFP antibodies. Results from one representative experiment of three are shown. (E) A representative image of live PfEXP2^mRuby3/PMX^SEP schizonts (48 hpi). Images from left to right are PMX^SEP (gray), EXP2^mRuby3 (magenta), fluorescence merge, and differential interference contrast or phase-contrast (DIC) Scale bar = 5 μm.

https://doi.org/10.1371/journal.ppat.1014214.g001

Our current models for P. falciparum egress suggest that exoneme exocytosis occurs in the final minutes of egress [12,19]. To determine the relationship between exoneme exocytosis and PVM rupture, we wanted to simultaneously monitor both these processes in live parasites. Therefore, we used CRISPR/Cas9 gene editing to generate a double-labeled parasite mutant, where we tagged exported protein 2 (EXP2), a protein located in the PVM with mRuby3 (PfEXP2^mRuby3) [26] and the exoneme protein, PMX [10,13–15], with SEP (PfEXP2^mRuby3/PMX^SEP; Fig 1B and 1C). These data show that we successfully tagged both endogenous EXP2 and PMX gene loci with mRuby3 and SEP, respectively (Fig 1C and 1D). Since PMX is essential for the completion of the intraerythrocytic lifecycle, these data also suggest that fusion of SEP to PMX did not inhibit its biological function (Figs 1C, 1D and S1). The expression of EXP2 protein fused with mRuby3 and PMX fused with SEP was detected by western blot and showed the expected sizes, including the correct maturation species for PMX (Fig 1D). Live imaging of late schizonts showed PMX^sep fluorescence as it was likely secreted into the PV, delineated by the EXP2^mRbuy3 labeled PVM (Fig 1E), indicating the tagging did not change the subcellular localization of either protein. Together, these data showed that tagging the pmx locus with SEP did not affect its maturation or function.

Blocking Protein Kinase G signaling inhibits exoneme exocytosis

Using the PfEXP2^mRuby3/PMX^SEP parasites, we wanted to observe the spatio-temporal dynamics of exocytosis and PVM rupture. Studies using fixed parasites in immunofluorescence assays have suggested that exocytosis of egress-specific vesicles requires PKG signaling [3,5,9,10]. Therefore, we hypothesized that exoneme exocytosis will be blocked by inhibiting PKG using specific inhibitors like compound 1 (C1) [8]. We incubated synchronized PfEXP2^mRuby3/PMX^SEP schizonts at 44 hours post-invasion (h.p.i) with 1.5 µM C1. After 4 hours of incubation, the schizonts were washed and either incubated again with or without C1 and observed via live fluorescence microscopy for 30 minutes (Fig 2).

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Fig 2. PKG inhibition blocks exoneme exocytosis.

(A) Quantification of PfEXP2^mRuby3/PMX^SEP parasite egress in the presence of C1 (+C1; black) or absence of C1 (-C1; magenta) during the 1800-second recording period. Each dot represents data from one time-lapse imaging experiment (n = 5 recordings from 3 biological replicates for PfEXP2^mRuby3/PMX^SEP parasites with C1, n = 6 recordings from 3 biological replicates for PfEXP2^mRuby3/PMX^SEP parasites without C1, error bars = SEM; **p < 0.001 by unpaired two-tailed t test) (B) A representative image from live imaging of synchronized PfEXP2^mRuby3/PMX^SEP schizonts in the presence of C1. (C) A representative image from live imaging of synchronized PfEXP2^mRuby3/PMX^SEP schizonts after C1 removal. Parasite egress occurs, and free-merozoites are scattered in the extracellular space (DIC). (D) Corrected total cell fluorescence (CTCF) of PMX^SEP quantified from time-lapse images of synchronized PfEXP2^mRuby3/PMX^SEP schizonts incubated with (black; n = 86, 3 biological replicates) or without (magenta; n = 68, 4 biological replicates) C1. ****p-value<0.0001, unpaired t-test. error bars = SEM. (E) Corrected total cell fluorescence (CTCF) of PMX^SEP quantified from snapshots of live synchronized PfEXP2^mRuby3/PMX^SEP (48 h.p.i) schizonts incubated with ML10 (black; n = 65, 3 biological replicates) or ML10 and NH₄Cl (magenta; n = 73, 3 biological replicates) C1. ****p-value <0.0001, unpaired t-test. error bars = SEM. (F) A representative image from live imaging of synchronized PfEXP2^mRuby3/PMX^SEP schizonts in the ML10 only (Top) and ML10 plus NH₄CL (bottom). Scale bar = 5 μm.

https://doi.org/10.1371/journal.ppat.1014214.g002

We observed that in the presence of C1, PfEXP2^mRuby3/PMX^SEP schizonts did not egress during the 30-minute window of observation (Fig 2A and S1 Movie). The PVM was intact in these schizonts during this time period (Fig 2B). These data show that blocking exocytosis via PKG inhibition also inhibits PMX^SEP fluorescence (Fig 2B), indicating that the environment within unsecreted exonemes is acidic resulting in quenching of SEP fluorescence (Fig 2B). In some schizonts incubated with C1, we observed SEP fluorescence, which may be due to exocytosis of exonemes prior to C1 addition (Fig 2). However, none of these schizonts were able to rupture the PVM or egress during the observation period (Fig 2A).

In the absence of C1, we observed punctate PMX^SEP fluorescence within the PVM, indicating PMX^SEP was exposed to neutral pH and suggesting that exoneme exocytosis had occurred (Fig 2C and S2 Movie). The time-lapse images are snapshots at a single timepoint and as the vesicle pH is neutralized upon fusion to the parasite plasma membrane, resulting in a punctate PMX^SEP fluorescent signal observed from the neutralized exoneme (Fig 2C and S2 Movie). We observe multiple vesicles with PMX being secreted from vesicles into the PV (Fig 2C and S2 Movie). The PMX^SEP signal rapidly decreases as it dilutes into the much larger PV compartment. These data suggest that exoneme exocytosis may occur all over the schizont and may not be limited to a single exit site or specific cellular location within the merozoite. However, another possibility is that the vesicles are released from specific locations within the merozoite and the Brownian motion of merozoites results in the appearance that exocytosis occurs at several sites on the membrane. In the absence of C1, exoneme exocytosis occurred first, then the PVM ruptured in most PfEXP2^mRuby3/PMX^SEP schizonts followed by the release of free merozoites (Fig 2C). The corrected total cell fluorescence for each schizont observed via live video microscopy was integrated across several time points (Fig 2D). These data show that in the presence of C1, PMX^SEP fluorescence signal is substantially reduced suggesting that exocytosis does not occur when PKG is inhibited (Fig 2D).

Incubating PfEXP2^mRuby3/PMX^SEP schizonts with ML10, another PKG inhibitor [27], also resulted in loss of PMX^SEP fluorescence (Figs 2E, 2F and S2). One possibility we considered is that the reduction in PMX^SEP fluorescence signal is a result of direct interference from the PKG inhibitors (C1 or ML10). Thus, we aimed to neutralize the acidic pH within exonemes, in the presence of PKG inhibitors, which should also lead to an increase in PMX^SEP fluorescence without exocytosis. Ammonium chloride is a weak base that neutralizes the pH in acidic intracellular compartments, including in P. falciparum infected RBCs [28,29]. PfEXP2^mRuby3/PMX^SEP schizonts were incubated with ammonium chloride and ML10 showed increased PMX^SEP fluorescence unlike those incubated with only ML10 (Fig 2E and 2F). Thus, these data show that exonemes are acidic secretory vesicles, and that their exocytosis is dependent upon PKG signaling.

Spatiotemporal relationship between exoneme exocytosis and PVM rupture

The spatiotemporal relationship between exoneme exocytosis and merozoite egress has been determined based on studies that utilized fixed parasites and live imaging [12,30]; however, live imaging with specific genetic expression of proteins targeting exoneme and PVM is needed. More recently, live imaging approaches have been utilized to investigate the morphology of the PVM during egress [11,19]. These data show that a calcium-dependent signal causes the rupture of the PVM followed by RBC membrane breakdown to release the merozoites [11,19]. However, a central part of the egress model, the signal-dependent exocytosis of exonemes has not yet been observed by live microscopy. Therefore, we wanted to define the relationship between signal-dependent exocytosis of exonemes and PVM rupture.

To determine the relationship between signal-dependent exocytosis of exonemes and PVM rupture, synchronized PfEXP2^mRuby3/PMX^SEP schizonts (44 h.p.i) were prepared as described previously and incubated with C1 for 4 hours. After removing C1, we observed the schizonts via time-lapse imaging for 30 mins with 30 sec intervals (Fig 3 and S3 Movie). As others have reported [11,19], we observed irregularly shaped PVM at the start of our observation (Fig 3A). About 8 minutes prior to egress, the PVM adopted a rounded shape (Fig 3) and this was followed rapidly (~1 minute) by the first observable break in the PVM (Fig 3B). The schizonts egressed about 7 minutes after the PVM rupture (Fig 3C). Our observations agree with prior studies that also utilized reversible PKG inhibitors to establish the timeline of egress starting from PVM rounding [11,19]. Thus, despite differences in parasite lines and preparation across labs, as well as microscopy settings, the observed timeline of the egress cascade is consistent.

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Fig 3. Exoneme exocytosis prior to egress.

(A) Representative time lapse images (captured at 30 sec intervals) during egress of PfEXP2^mRuby3/PMX^SEP merozoites. Minus time indicates time points before merozoite egress. PVM (magenta) morphology changes from an irregular shape to a rounded shape. In most schizonts, PVM rupture (white arrows) occurred in the next frame or two (30 to 60 sec) after PVM rounding. Merozoites free from membranes indicate egress. Several exoneme exocytosis (gray) events were observed from the start of the recording until merozoite egress. Scale bar = 5 μm. (B) The estimated time between PVM rounding to rupture (Mean ± SEM = 53.919 ± 5.321 sec) and (C) the time between PVM rupture to merozoite egress (Mean ± SEM = 445.946 ± 25.276 sec). n = 74, 7 biological replicates, double-labeled parasites, error bars = SEM, time-lapse recordings of parasite egress is a 30-sec interval.

https://doi.org/10.1371/journal.ppat.1014214.g003

Since our images were taken using 30 sec imaging intervals, we wanted to observe the exact temporal relationship between PVM rounding and PVM rupture as well as observe PVM rupture at a higher temporal resolution. Therefore, we utilized 4 sec and 2 sec time-lapse imaging to observe egressing PfEXP2^mRuby3/PMX^SEP schizonts (Fig 4). Similar to our prior observations (Fig 3), we observed that the PVM morphology changed from an irregular shape to a more rounded shape (Figs 4A, 4B, and S3A-S3C and S4 and S5 Movies). The diameter (~4.3 μm) and circumference (~13.6 μm) of the PVM in all rounded schizonts were similar across biological replicates (Fig 4B and 4C).

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Fig 4. PVM rupture occurs at a single site.

(A) Representative time lapse images captured from 4-sec (top) and 2-sec intervals (bottom), the white arrow indicates two ruptured ends of the PVM (B) The diameter of rounded PVM from 2-sec interval time lapse images (Mean ± SEM = 4.31 ± 0.08 μm), (C) circumference (Mean ± SEM = 13.54 ± 0.264 μm), (D) the duration of PVM rounding (Mean ± SEM = 26 ± 6.706 sec; n = 13, 4 biological replicates). (E) The pore size changes between the first time point PVM breakdown (T1), Mean ± SEM = 2.88 ± 0.191 μm, and the second time point (T2), Mean ± SEM = 3.33 ± 0.226 μm (each color represents one schizont imaged every 2 sec, n = 13, 4 biological replicates, error bars = ± SEM, paired t-test with ** p-value ≤ 0.001). The measurement data are shown in S3 Fig. Scale bar = 5 μm.

https://doi.org/10.1371/journal.ppat.1014214.g004

Within a few seconds (~25 sec) after the schizont rounds, the PVM ruptures (Fig 4D), though we observed some variation in the time taken for PVM rupture after rounding (Fig 4D). In some schizonts, it took nearly a minute to observe PVM rupture after rounding, while in other schizonts PVM rupture occurred a few seconds (<10 sec) post-rounding (Fig 4D). These data support the hypothesis that PVM rounding is a critical morphological step for PVM rupture to occur (Fig 4D) [11,19]. Regardless of the time interval of imaging, PVM rupture always occurred at a single site after rounding (Fig 4). The size of this initial rupture was consistent across several biological replicates (~2.8 μm; Figs 4E and S3D-S3F). We never observed a second site of PVM rupture during egress and the initial rupture widened in subsequent time-lapse images allowing the merozoites to escape the PVM (Fig 4E). These data suggest that the PVM rupture may be regulated and likely occurs at a specific site on the membrane (Fig 4).

Exoneme exocytosis begins several hours prior to merozoite egress

Our studies using reversible PKG inhibitors showed that exoneme exocytosis was already occurring in PfEXP2^mRuby3/PMX^SEP schizonts at the start of our observation (Figs 2-4). Therefore, we wanted to establish a time-line for signal-dependent exoneme exocytosis and study this process without the use of any inhibitors. Using highly synchronized PfEXP2^mRuby3/PMX^SEP parasites (44 h.p.i), we monitored exoneme exocytosis until merozoites egressed from these schizonts (Fig 5 and S6 Movie).

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Fig 5. Exoneme exocytosis begins 3 hours prior to egress.

(A) Representative time lapse images of PfEXP2^mRuby3/PMX^SEP merozoite exocytosis and egress. The arrow indicates the first image where fluorescence signals were observed. Time points prior to merozoite egress (0 min) are indicated. (B) The average time of exoneme exocytosis observed in PfEXP2^mRuby3/PMX^SEP schizonts prior to egress. (n = 13 schizonts from 4 biological replicates, Mean ± SEM = 196.53 ± 8.35 min). (C) Representative time lapse images of PfEXP2^mRuby3/PMX^SEP merozoite in the presence of ML10. Time point shows every 30 min over 5 hours recording. Scale bar = 5 μm.

https://doi.org/10.1371/journal.ppat.1014214.g005

We observed several naturally egressing PfEXP2^mRuby3/PMX^SEP schizonts over four biological replicates and at the start of the recording we did not observe any PMX^SEP fluorescence in any of these schizonts indicating that exoneme exocytosis had not yet started (Fig 5). Parasite viability was assessed using the movement of schizonts within the infected RBC as well as by hemozoin movement within the food vacuole and only viable schizonts that egressed over this time period were analyzed further (Fig 5). Moreover, the changes in PVM morphology as assessed by PfEXP2^mRuby3 fluorescence was another measure of parasite viability over the observation time period (Fig 5A and S6 Movie).

In all PfEXP2^mRuby3/PMX^SEP schizonts, we observed punctate PMX^SEP fluorescence within the PVM starting several hours (~3 hours) prior to merozoite egress (Fig 5B). Once the punctate PMX^SEP fluorescence started, these events were observed continuously until the merozoites egressed from the infected RBC (Figs 5A and S4A-S4B and S6 Movie). These data suggest that the signals that kickstart exoneme exocytosis occur at least 3 hours prior to egress (Fig 5). To determine if the scale of exocytosis changes during these 3 hours prior to egress or if exocytosis peaks at a specific time before egress, we normalized PMX^SEP fluorescence for each schizont (S5 Fig). The normalized PMX^SEP fluorescence shows no clear pattern of exocytosis over these 3 hours, with some schizonts showing steady increase in exocytosis until egress (5/13 schizonts), while others show a steady and constant normalized PMX^SEP fluorescence until egress (4/13 schizonts) and yet others showing randomly changing PMX^SEP fluorescence until egress (4/13 schizonts; S5 Fig). It is possible that gathering more examples may reveal a pattern or that there may not be a clear pattern for exocytosis prior to egress.

To confirm that the PMX^SEP fluorescence we observed at 44 h.p.i was due to exoneme exocytosis, we incubated similarly isolated PfEXP2^mRuby3/PMX^SEP schizonts (44 h.p.i) with the PKG inhibitor ML10 over the same time period (Figs 5C and S4C-S4D and S7 Movie). We did not observe any PMX^SEP fluorescence suggesting that SEP was already present in the acidic exonemes (Figs 5C and S4C-S4D and S7 Movie). Furthermore, if the PMX is present in acidic vesicles at 44 h.p.i, we reasoned that we should be able neutralize the pH of these vesicles in these schizonts using a weak base like ammonium chloride (Fig 6A-6B). Exocytosis was prevented in these schizonts using the PKG inhibitor, ML10. We observed that PMX^SEP fluorescence increased in ammonium chloride-treated parasites compared to untreated schizonts (Fig 6A-6B). Thus, these data demonstrate that PMX^SEP is present in mature acidic secretory vesicles at 44 h.p.i, and the observed PMX^SEP fluorescence at this time is due to exoneme exocytosis (Fig 5). Together, these data suggest that a critical concentration of egress proteases may be needed in the PV to activate merozoite egress, which may take several hours to achieve (Fig 5). Alternatively, there may be a second signal that activates the egress protease cascade and PVM rounding [11,19].

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Fig 6. A Protein kinase G inhibitor does not inhibit secretory vesicle acidification.

(A) Representative images from live imaging of synchronized 44 h.p.i PfEXP2^mRuby3/PMX^SEP schizonts in the presence of ML10 (top) or ML10 as well as NH₄Cl (bottom). (B) Corrected total cell fluorescence (CTCF) of PMX^SEP quantified from images of synchronized 44 h.p.i PfEXP2^mRuby3/PMX^SEP schizonts in ML10 alone (black; n = 88, 3 biological replicates) or ML10 and NH₄Cl (magenta; n = 96, 3 biological replicates). ****p-value<0.0001, unpaired t-test. error bars = SEM. (C) Representative time lapse images of synchronized 40 h.p.i PfEXP2^mRuby3/PMX^SEP acidification schizont. Snapshots of 5 consecutive time points are shown. (D) Representative time lapse images of synchronized 40 h.p.i PfEXP2^mRuby3/PMX^SEP schizonts incubated with ML10. Snapshots of 5 consecutive time points are shown. (E) Corrected total cell fluorescence (CTCF) of PMX^SEP quantified from images of synchronized 40 h.p.i PfEXP2^mRuby3/PMX^SEP schizonts incubated without ML10 (black; n = 27, 3 biological replicates) or with ML10 (magenta; n = 23, 3 biological replicates). Unpaired t-test, ns = non-significant. error bars = SEM. Scale bar = 5 μm. Time-lapse recordings of acidification is a 5-sec interval.

https://doi.org/10.1371/journal.ppat.1014214.g006

Since we observed PMX in already acidified vesicles at 44 h.p.i, we wanted to determine when exonemes are acidified during schizogony. The process of vesicle acidification is intrinsic to the secretory pathway and independent of PKG signaling. In this case, PKG inhibitors would not inhibit PMX^SEP fluorescence within vesicles that were not yet acidified. Therefore, we assessed PMX^SEP fluorescence in 40 h.p.i PfEXP2^mRuby3/PMX^SEP schizonts (Fig 6C and S8 Movie). We found several schizonts that briefly exhibited PMX^SEP fluorescence before the signal disappeared (S8 Movie). The loss of PMX^SEP fluorescence could either be due to exocytosis of these fluorescent vesicles or because these vesicles were being acidified. As we show (Fig 2) and others have shown [3,5,8,9], vesicle exocytosis depends upon PKG signaling. We reasoned that if the loss of fluorescence was due to vesicle acidification, then inhibiting PKG will have no effect on PMX^SEP fluorescence. Therefore, we incubated 40 h.p.i PfEXP2^mRuby3/PMX^SEP schizonts with the PKG inhibitor ML10. As we expected, PMX^SEP fluorescence was not inhibited by the PKG inhibitor ML10 (Fig 6D-6E and S9 Movie). Thus, these data show that in 40 h.p.i PfEXP2^mRuby3/PMX^SEP schizonts, vesicles were undergoing acidification. We did not observe any PMX^SEP fluorescence in schizonts again until later during schizogony (~44 h.p.i, Fig 5).

A recent study showed that P. falciparum V-type ATPases may localize to secretory vesicles in merozoites [31] and therefore, we wanted to determine if these V-type ATPases function to maintain the acidic pH within exonemes. To test this, we incubated PfEXP2^mRuby3/PMX^SEP late schizont with ML10 to prevent exocytosis as well as with the V-type ATPase-specific inhibitor Bafilomycin A1 (BaF). We reasoned that incubation with ML10 will prevent changes in PMX^SEP fluorescence due to exocytosis and the addition of BaF will inhibit the V-type ATPase leading to neutralization of the vesicle pH that will result in PMX^SEP fluorescence (S6 Fig). No exocytosis or egress was observed in ML10-treated PfEXP2^mRuby3/PMX^SEP parasites (S6A Fig). Surprisingly, we did not see an increasing fluorescence during the 15-min time-lapse videos in ML10-treated PfEXP2^mRuby3/PMX^SEP parasites incubated with BaF (S6B Fig). There was no discernible difference in PMX^SEP fluorescence upon incubation with BaF (S6B Fig). These data suggest that the mechanisms maintaining the acidic pH within exonemes are insensitive to BaF (S6 Fig). The data suggest there may be other BaF-insensitive proton pumps or proton exchangers that may play a role in maintaining the pH within exonemes.

PfERC is required for exoneme exocytosis

Calcium signals have been suggested to be essential for exoneme exocytosis but the mediators of this calcium signal within the secretory pathway have not yet been identified [12,19]. We had previously identified that the P. falciparum endoplasmic reticulum-resident calcium-binding protein PfERC (PlasmoDB ID: PF3D7_1108600), EF-hand calcium-binding protein, is required for merozoite egress and regulates the maturation of PMX [32]. This protein belongs to a CREC family of proteins (calumenin, reticulocalbin 1 and 3, ERC-55, and Cab) [33] and one member of this family is shown to interact directly with a protein to facilitate exocytosis [34]. However, the specific role of PfERC in exoneme egress remains unknown and we wanted to determine if PfERC functions in signal-dependent exocytosis of exonemes.

Our data showed that PfERC functions in the egress proteolytic cascade but is not required for ER calcium homeostasis or for microneme exocytosis [32]. Since egress proteases are stored within exonemes and released via exocytosis into the PV during egress, interfering with either exoneme biogenesis or exocytosis may result in loss of protease activity or function. Therefore, we hypothesized that PfERC is required for egress protease function because it may function in exoneme biogenesis or exocytosis.

To test this hypothesis, we first generated PfERC conditional mutants using the glucosamine (GlcN) responsive ribozyme system (Fig 7A) [32,35]. The addition of GlcN activates the glmS but not M9 ribozyme, resulting in cleavage of mRNA that leads to mRNA degradation and protein knockdown (Fig 7B). The endogenous locus of PfERC in the NF54 P. falciparum parental strain was fused to three copies of the hemagglutinin (HA) tag and either the inducible glmS (termed PfERC-glmS) ribozyme or the inactive M9 (termed PfERC-M9) ribozyme (Fig 7A) [32,35]. Clonal PfERC-glmS and PfERC-M9 parasite lines were generated from independent transfections (S7A and S7B Fig). The PMX locus in these clonal PfERC-glmS and PfERC-M9 parasite lines was then tagged with SEP to generate double mutants (termed PfERC-glmS/PMX^SEP and PfERC-M9/PMX^SEP; S7C and S7D Fig). In these PfERC-glmS/PMX^SEP and PfERC-M9/PMX^SEP double mutants, PMX expression was also regulated by the tetR aptamer system [32,36] (S7 Fig).

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Fig 7. PfERC is required for exoneme exocytosis in live parasites.

(A) Schematic shows the mechanism of the condition that knocks down the glmS ribozyme system. glmS is an inactive ribozyme that is transcribed but not translated with the mRNA of a protein of interest. Adding glucosamine (GlcN) leads to phosphorylation within the cell to glucosamine-6-phosphate (GlcN-6P). GlcN-6P binds to the transcribed mRNA, and the glmS ribozyme is activated and cleaves itself from the mRNA. This leads to disassociation of the mRNA from its poly(A) tail and results in the degradation of target-specific mRNA. The resulting decline in mRNA levels leads to reduced protein levels and, thus to loss of gene expression. As a control, we generated parasite lines containing a mutated version of the glmS ribozyme, called M9, which cannot cleave itself upon binding of GlcN. (B) Representative of western blot of parasite lysates isolated from PfERC-glmS/PMX^SEP and PfERC-M9/PMX^SEP schizonts, PfERC-glmS schizonts grown in the presence of GlcN for 48 h and probed with anti-HA antibodies (top panel), anti-GFP antibodies (middle panel), and anti-GAPDH (loading control; bottom panel). (C) Representative images from Glc-treated PfERC-glmS/PMX^SEP and PfERC-M9/PMX^SEP schizonts from live fluorescence microscopy. Phase contrast (top) and fluorescence (bottom) images. (D) Corrected total cell fluorescence (CTCF) integrated over the entire video (n = 10 recordings from 3 biological replicates for Glc-treated PfERC-M9/PMX^SEP, n = 11 recordings from 3 biological replicates for Glc-treated PfERC-glmS/PMX^SEP, ****p-value<0.0001, unpaired t-test). Scale bar = 5 μm.

https://doi.org/10.1371/journal.ppat.1014214.g007

Parasites were prepared as previously to obtain highly synchronized PfERC-glmS/PMX^SEP and PfERC-M9/PMX^SEP ring stage parasites (2 h.p.i). The 2 h.p.i ring stage parasites were incubated with 7.5 mM GlcN for 48 h. The addition of GlcN led to a reduction in PfERC expression in PfERC-glmS/PMX^SEP schizonts, while there was no reduction in PfERC expression in PfERC-M9/PMX^SEP schizonts grown under identical conditions (Fig 7B). These data confirmed that in these double mutants the addition of GlcN led to knockdown of PfERC only in PfERC-glmS/PMX^SEP parasites (termed -PfERC) but had no effect on PfERC expression in PfERC-M9/PMX^SEP parasites (termed +PfERC; Fig 7B). Our prior work had shown that PfERC knockdown led to a block in PMX activity [32,36]. The addition of GlcN to PfERC-glmS/PMX^SEP and PfERC-M9/PMX^SEP parasites also resulted in loss of PMX processing (Fig 7B).

Next, we wanted to test if PfERC knockdown had any effect on exoneme exocytosis. As before, we treated ring stage (2 h.p.i) PfERC-glmS/PMX^SEP and PfERC-M9/PMX^SEP parasites with GlcN and then these GlcN treated schizonts (44 h.p.i) were incubated with C1 for 4 hours to prevent egress, allow schizonts to fully develop and become egress competent. At 48 h.p.i, C1 was washed off from these schizonts and we observed these parasites using wide-field live fluorescence microscopy (Fig 7C). Time-lapse images were collected every 45 seconds for 30 minutes (Fig 7C). During the 30-min video, GlcN-treated PfERC-MP/PMX^SEP schizonts were able to egress (Fig 7C and S10 Movie). Several fluorescent events were observed in PfERC-M9/PMX^SEP, while almost no fluorescence events were observed in GlcN-treated PfERC-glmS/PMX^SEP (Fig 7C and S11 Movie). The corrected total cell fluorescence for each observed schizont within the field was integrated over 30 minutes (Fig 7D). Incubating PfERC-M9/PMX^SEP with GlcN does not have an effect on exoneme exocytosis or egress (Fig 7D). In contrast, incubating PfERC-glmS/PMX^SEP with GlcN results in loss of exoneme secretion and blocks egress (Fig 7D). It is suggested that PfERC is required for exoneme exocytosis (Fig 7C and 7D).

Plasmid construction

Genomic DNA was isolated from NF54^attb P. falciparum cultures using a QIAamp DNA Blood Kit (Qiagen), and all constructs were confirmed by sequencing. Briefly, PCR products were inserted into the respective plasmids using the sequence- and ligation-independent cloning (SLIC) method. A mixture of an insert, a cut vector, and T4 DNA polymerase was incubated for 2.5 minutes at room temperature and then for 10 minutes on ice. A bacterial transformation uses a heat shock at 42 °C for 35 sec. All primers utilized in this study are in S1 Table. Two plasmids, pbPM2-EXP2-mRuby [26] and pAIO2-EXP2 gRNA [46], were gifts from Dr. Josh Beck, Iowa State University.

SEP was amplified from pHmK-C1 (Addgene) [25] using primers P10/P11. The pPMX-V5-Apt-pMG74 plasmid [32] was modified to replace V5 tag with THE SEP amplicon between PsPXi and AfIII cut site, resulting in a pPMX-SEP-Apt-pMG74 plasmid. A PMX guide RNA targeting the 30 bp upstream pmx stop codon was annealed using P12/P13. The annealed fragment was inserted into the plasmid pUF1-Cas9, which contains the DHOD resistance gene, resulting in the plasmid pUF1-Cas9-PMX.

Primers P14/15 were used to amplify an 843 bp fragment of 5′ homology PfERC C-terminus (excluding the stop codon) from P. falciparum NF54^attb genomic DNA. Amplicons were inserted into pHA-SDEL-glmS and pHA-SDEL-M9 using restriction sites SacII and AfeI [32]. A 831-bp 3′ homology PfERC 3UTR (beginning at 13 bp downstream PfERC stop codon) was amplified by primers P16/P8 and was inserted into pHA-SDEL-glmS and pHA-SDEL-M9 containing 5 ′ homology PfERC C-terminal region at restriction sites HindIII and NheI, resulting in pPfERC-HA-SDEL-glmS or pPfERC-HA-SDEL-M9 [32]. A gRNA targeted at 27 bp upstream of the erc stop codon. The PfERC gRNA was annealed using P17/P18 and inserted into the plasmid pUF1-Cas9-guide, resulting in the plasmid pUF1-Cas9-ERC.

Parasite culturing and transfection

All transfections were done in duplicate. For transfections, an uninfected RBC pellet was electroporated with a matching pair of 20 μg of donor plasmid and 50 μg of CRISPR/Cas9 gRNA plasmid using a Bio-Rad Gene Pulser Xcell Electroporation System. The parasite line was then added to the transfection mixture and cultured in RPMI medium supplemented with Albumax I (Gibco) and the required drugs described previously [32,47–50]. All primers used are in S1 Table.

To generate PfEXP2^mRuby3 parasites, 20  μg of the pbPM2-EXP2-mRuby donor plasmid was linearized with AflII overnight, and 50 μg of the pAIO-EXP2-gRNA were co-transfected into NF54^attb parasites [51]. At 24 h after transfection, 2.5 μg/ml of Blasticidin-S (BSD) was added to select for pbPM2-EXP2-mRuby expression [26,42]. The parasite population was cloned by limiting dilution. A PCR diagnostic for integration was performed using primers P1/P3 to amplify a 2.1 kb fragment corresponding to the correct integrant.

For generating PfEXP2^mRuby3/PMX^SEP parasites, 20  μg of pPMX-SEP-Apt-pMG74, a donor plasmid, was linearized with EcoRV overnight, and 50 μg of pUF1-Cas9-PMX gRNA plasmid were co-transfected into PfEXP2^mRuby3 parasites [13,32]. Transfected parasites were grown in 0.5 μM anhydrous tetracycline (aTc) (Cayman Chemical) and 2.5 μg/ml of Blasticidin-S (BSD) [32,42]. At 48 h post-transfection, Cas9 expression was selected by drug cycling [52,53] in the presence and absence of 1 μM DSM1 for 4 days until parasites were detected in the culture. Next, the parasite population was cloned by limiting dilution. A PCR diagnostic for integration was performed using primers P4/P6 to amplify a 2.8 kb fragment corresponding to the correct integrant.

For generating the PfERC-glmS and PfERC-M9 parasites, a mix of two plasmids (20 μg of a pPfERC-HA-SDEL-glmS or pPfERC-HA-SDEL-M9 donor plasmid and 50 μg of pUF1-Cas9-ERC guide plasmid) was co-transfected into NF54^attb parasites. To select for Cas9 expression,1 μM DSM1 was applied to the transfected culture as previously described [32]. Clonal parasites were subjected to PCR diagnosis using primers P7/P8 and P7/P9 to amplify a 2.2-kb and 1.1-kb fragment, respectively, for correct integration [32].

To generate PfERC-glmS/PMX^SEP and PfERC-M9/PMX^SEP parasites, 20  μg of pPMX-SEP-Apt-pMG74, a donor plasmid, was linearized with EcoRV overnight, and a 50 μg of pUF1-Cas9-PMX gRNA plasmid were co-transfected into PfERC-glmS or PfERC-M9 parasites [13,32]. Transfected cultures were grown in 2.5 μg/ml of blasticidin S (BSD) [32,42]. To select a donor plasmid, 0.5 μM anhydrous tetracycline (aTc) (Cayman Chemical) was applied at 48 h post-transfection. A PCR diagnostic for integration was performed using primers P4/P6 to amplify a 2.8 kb fragment corresponding to the correct integrant.

Growth assay

Asynchronous parasites were placed in 6 well-plates at 0.3% parasitemia at 2% hematocrit in triplicate and grown for 6 days. For growth assessment, samples were taken three technical replicates every 48 hours. For each technical replicate, 5 μL of culture from each well was resuspended in 200 μl of 8 μM Hoechst (ThermoFisher Scientific) in PBS. Parasites were incubated shaking in the solution for 20 min at room temperature before being assessed via flow cytometry on The Quanteon (Agilent) analyzer. Flow cytometry analysis was done using FlowJo (Tree Star, Inc) and data were graphed using GraphPad Prism (Prism 10 for macOS Version 10.2.2 (341).

Western blotting

Western blotting was performed using P. falciparum lysates as described previously [32] with minor modifications. Highly synchronized late-schizont parasite culture pellets were treated with ice-cold 0.05% saponin–phosphate-buffered saline (PBS) for 15min, followed by sonication. The sonicated lysates were collected for detection of proteins separated via a western blot assay. The antibodies used in this study were rabbit anti-HA (715500; Invitrogen) (1:1000), mouse anti-GFP (JL-8; Takara) (1:3000), rabbit anti-EXP2 (from H.Ke) (1:10000), and mouse anti-GAPDH (1.4; Millipore Sigma)(1:2000). The secondary antibodies used were IRDye 680CW goat anti-rabbit IgG and IRDye 800CW goat anti-mouse IgG (Li-COR Biosciences) (1:20,000). The Western blotting images were processed using Odyssey Clx Li-COR infrared imaging system software (Li-COR Biosciences).

Time-lapse imaging of live P. falciparum

To prepare highly synchronized parasites, late schizont parasites were incubated with C1 or ML10 to prevent egress, then washed off the compound. After allowing parasite egress and invasion for 2 hours, we perform 5% sorbitol on the culture to collect 0–2 h.p.i rings. The rings were grown in media supplemented with mixed gas in a 37 °C shaker incubator.

Late-stage schizont parasites (44 h.p.i) were isolated on a Percoll gradient (Genesee Scientific). Percoll pellet was incubated in the PKG inhibitors either 1.5 μM compound 1 (C1) {4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine} [8,18] or 25 nM ML10 (BEI Resources, catalog no. NR-56525, www.beiresources.org) [54] at 37 °C in a CO2 incubator. After 4 h incubation, the egress inhibitor was washed off twice using pre-warmed 37^oC cRPMI media. After washing off the egress inhibitor, 200–500 μL schizont-stage parasite culture was spun down at 3000 rpm for 30-sec, and the supernatant was removed. The pellet was gently resuspended with warm (37^oC) RMPI media, and the tube was spun down at 3000 rpm for 1 min. After removing the supernatant, the pellet was gently resuspended with 200–500 μL warm (37^oC) media before being placed onto a 35 mm glass-bottom dish coated with poly-L-lysine for imaging.

The 30-minute time-lapse video was performed using a DeltaVision II microscope with autofocus. The PfEXP2^mRuby3/PMX^SEP schizonts were imaged with a Live cell filter set CFP, YFP, GFP, and mCherry. To minimize photodamage, the laser power level was kept at 10% (or less). A 488-nm-wavelength laser was used to excite PMX^SEP, and a 561-nm-wavelength laser was used to excite PfEXP2^mRuby3. Time-lapse images were taken every 30 sec up to 30 min at 37 °C in an imaging chamber supplemented with CO2.

The 5-h time-lapse video was imaged using a Zeiss LSM 980 Confocal system using an inverted Axio Observer 7 microscope stand with transmitted (HAL), UV (HBO), and laser (405 – 730 nm) illumination sources. A 488-nm-wavelength laser (10% power) was used to excite PMX^SEP, and a 561-nm-wavelength laser (2% power) was used to excite PfEXP2^mRuby3. The microscope system uses Zen 3.3 (Blue) acquisition software with autofocus. The dark incubation chamber with regulated CO2 (5%), temperature (37 °C), and humidity was used. Images were captured every 5 minutes for 5 hours.

To treat parasites with BaF, highly synchronized PfEXP2^mRuby3/PMX^SEP parasites were prepared as described. The 44 h.p.i schizont pellet was incubated with ML10 for 2–4 h. Before imaging, 100 nM BaF (Cayman Chemical) was added to the culture and incubated for 10 min.

Imaging acidification of exonemes

The 40 h.p.i PfEXP2^mRuby3/PMX^SEP schizonts were separated using the Percoll gradient (Genesee Scientific). The schizont pellet was either incubated in media supplemented with and without 25 nM ML10 (BEI Resources, catalog no. NR-56525, www.beiresources.org) [54] at 37 °C in a CO2 incubator. For imaging, 200–500 μl of ML10-schizont culture was placed onto a 35 mm glass-bottom dish coated with poly-L-lysine. The 15-minute time-lapse video was performed using a DeltaVision II microscope with autofocus. The 40 h.p.i PfEXP2^mRuby3/PMX^SEP schizonts were imaged with a Live cell filter set CFP, YFP, GFP, and mCherry. The laser power level was kept at 32% (or less). A 488-nm-wavelength laser was used to excite PMX^SEP. Time-lapse images were taken every 5 sec up to 15 min at 37 °C in an imaging chamber supplemented with CO2.

Ammonium chloride treatment of live P. falciparum

Ammonium chloride treatment was carried out at two time points: 44 h.p.i and 48 h.p.i PfEXP2^mRuby3/PMX^SEP schizont parasites. For both timepoints, schizonts were separated using the Percoll gradient (Genesee Scientific). The schizont pellet was incubated media supplemented with 25 nM ML10 (BEI Resources, catalog no. NR-56525, www.beiresources.org) [54] at 37 °C in a CO2 incubator. For the control, 200–500 μL ML10-schizont culture was placed onto a 35 mm glass-bottom dish coated with poly-L-lysine for imaging. For ammonium chloride treatment, 200–500 μL ML10-schizont culture was resuspended with ammonium chloride at a final concentration of 20 mM in ML10 supplemented medium. Ammonium chloride was incubated for 5–10 minutes before imaging.

Image processing and analysis

ImageJ (Fiji version 2.0.0-rc-68/1.52h) was used to crop images, adjust brightness and intensity, overlay channels, and prepare montages. The adjustments to brightness and contrast were made only for display purposes. The fluorescence intensity of each cell was calculated using the calculation for corrected total cell fluorescence (CTCF) = integrated density–(area of selected cell × mean fluorescence of background readings) [55,56]. The data for calculation were obtained only from the SEP fluorescence channel as follows: 1) From the Analyze menu on ImageJ, set measurements were selected with the area, min, max gray values, and mean gray values. 2) The area of the cell of interest was manually selected using a freehand tool. 3) The selected area was measured using Measure. 4) Step 2–3 was repeated for every time point. 5) For background readings, an outline was drawn on the space outside the cell of interest. 6) The measure menu was used to measure fluorescence as described above. 7) The fluorescence intensity of each cell was calculated using Excel (Microsoft Excel for Mac Version 16.103 (25110922)). Plots and statistical analysis (2-sided unpaired Student t-tests) were done using GraphPad Prism (Prism 10 for macOS Version 10.2.2 (341).