CRISPR/Cas9-Based Knockout of GNAQ Reveals Differences in Host Cell Signaling Necessary for Egress of Apicomplexan Parasites

The coordinated release of apicomplexan parasites from infected host cells prior to reinvasion is a critical process for parasite survival and the spread of infection. While Toxoplasma tachyzoites and Plasmodium blood stages induce a fast disruption of their surrounding membranes during their egress from host cells, Plasmodium liver stages keep the host cell membrane intact and leave their host cell in host cell-derived vesicles called merosomes.

minutes later the host cell plasma membrane (HCM) is also disrupted (1)(2)(3). During Plasmodium release from hepatocytes, the vacuole is similarly ruptured; however, the HCM stays intact for several hours, enabling the formation of host cell-derived vesicles termed merosomes that transport parasites from the liver to the bloodstream. Only then does the HCM-derived merosomal membrane rupture, whereby hepatic merozoites are released to infect erythrocytes (4,5).
Egress is a highly regulated process, and several parasite proteins involved have been identified in Toxoplasma and Plasmodium, including proteases, kinases, poreforming proteins, as well as phospholipases (reviewed in references 6 and 7). Much less is known about the contribution of host cell factors to this process. However, RNA interference (RNAi)-based knockdown and antibody-mediated depletion previously showed that a host signaling cascade dependent on guanine nucleotidebinding protein subunit alpha q (GNAQ) is necessary for the egress of T. gondii tachyzoites and P. falciparum blood stage parasites from host cells. In this cascade, putative parasite G-protein-coupled receptor (GPCR) ligands overstimulate host GPCRs, leading to the GNAQ-mediated activation of protein kinase C, which weakens the host cell cytoskeleton by the phosphorylation of adducin. This in turn induces calcium influx through the mechanosensitive cation channel TRPC6 that activates host calpain-1 to proteolyze the host cytoskeleton, enabling parasite release (8,9).
CRISPR/Cas9-based knockout of GNAQ. We aimed at testing whether GNAQmediated host cell signaling also contributes to Plasmodium liver stage egress and therefore used CRISPR/Cas9 technology to generate GNAQ-deficient knockout (KO) cells. In order to minimize the risk of off-target mutations, we applied the CRISPR/Cas9 paired nickase approach, which leads to two single-strand nicks situated close to each other on the genomic DNA (10). Two guide RNAs (gRNAs) targeting GNAQ in exon 2 were designed (Fig. 1A) and expressed together with Cas9 in HeLa cells. These cells were chosen because they fully support T. gondii tachyzoite as well as Plasmodium berghei liver stage development (11,12) and are particularly well suited for imaging-based analysis of liver stage egress (13). Two single clonal cell lines (named KO1 and KO2) were obtained, and the absence of GNAQ was confirmed by Western blotting (Fig. 1B). Furthermore, successful manipulation of the GNAQ locus was confirmed in these two clones by genomic PCR followed by sequencing of the mutated region in exon 2 (see Fig. S1 in the supplemental material).
GNAQ-mediated signaling is involved in the egress of T. gondii. We next infected wild-type (WT) and GNAQ-KO cells with T. gondii tachyzoites. Upon the fixation of infected cells at 24 h postinfection (hpi), counting of the number of parasites per vacuole showed, as expected, that GNAQ-KO cells fully supported T. gondii growth compared to WT cells (Fig. 1C). Interestingly, the determination of the rate of natural egress by fixation and counting of infected cells at 36 and 50 hpi revealed a statistically significantly higher percentage of unruptured vacuoles in the two GNAQ-KO lines than in WT cells (Fig. 1D). These findings confirmed previous RNAibased results showing that GNAQ-mediated signaling is important for the egress of T. gondii (8).
P. berghei liver stage egress occurs independently of GNAQ. To study Plasmodium liver stage development in these cells, we infected WT and GNAQ-KO cells with transgenic, fluorescent P. berghei sporozoites and measured parasite size at 48 hpi. No change of size was observed ( Fig. 2A), confirming that GNAQ deficiency does not affect the growth of liver stage parasites up to this time point.
Upon the formation of merozoites, parasites rupture the PVM, leading to their release into the host cell cytoplasm and the detachment of the infected host cell, which typically occurs at between 55 and 60 hpi (4,14). Since in vitro cell detachment is considered a robust marker for liver stage egress, we next counted the number of detached cells. Remarkably, no major difference between WT and GNAQ-KO cells was observed (Fig. 2B), suggesting that, in contrast to T. gondii tachyzoites, P. berghei liver stage egress is independent of host cell GNAQ. The formation of detached cells was even greater in KO2 cells than in WT cells, which is presumably derived from a clonal effect and not related to the loss of GNAQ, as it was observed in only one of the two KO cell lines. To study P. berghei liver stage egress in these cells in more detail, we used a previously established assay based on quantitative live-cell time-lapse microscopy that analyzes PVM rupture (14). Differences in neither the percentage of parasites undergoing PVM rupture nor the timing of this process were visible ( Fig. 2C to E), further supporting the notion that P. berghei liver stage exit does not depend on GNAQ.
Of relevance, P. berghei liver stages induce a breakdown of the host cell actin cytoskeleton during their release from host cells (13). We thus expressed the filamentous actin marker green fluorescent protein (GFP)-utrophin (15) in WT and GNAQ-KO cells  and analyzed the egress of liver stages in these cells by live-cell time-lapse microscopy. In line with data from the other egress assays, the actin cytoskeleton also collapsed during parasite egress from GNAQ-deficient cells, similar to the observations in WT cells ( Fig. 2F; Movies S1 and S2), indicating that GNAQ KO also does not affect the remodeling of the host actin cytoskeleton.
Discussion. After the rupture of the PVM, liver stage-derived Plasmodium merozoites leave the infected host cells packed in merosomes. The HCM-derived merosomal membrane needs to remain intact for several hours until merosomes have reached the bloodstream. At this point, merozoites are released to infect erythrocytes, thereby initiating the pathogenic blood phase of infection (4,5). This strategy contrasts with the lytic egress of T. gondii tachyzoites and Plasmodium blood stage merozoites, whereby the PVM and HCM are disrupted in quick succession, leading to fast parasite exit and reinvasion of neighboring cells (1)(2)(3). In this study, we provide evidence that differences in GNAQ-mediated host cell signaling between these two different egress strategies might exist (Fig. 2G). We observed an increased percentage of T. gondii tachyzoite stage parasites that did not egress from their PV in GNAQ-KO cells in comparison to WT cells, while PV rupture and the subsequent formation of detached cells of P. berghei liver stages were not affected by KO of GNAQ. Of note, the T. gondii egress phenotype in our GNAQ-deficient HeLa cells was not as strong as that previously reported for U2OS cells, in which GNAQ was knocked down by RNAi (8). This could be due to either differences in the host cell type or the fact that KO cells generated by CRISPR/Cas9 have more time to upregulate compensating factors than RNAi-based knockdown cells, and this might well affect the observed phenotype. Importantly, natural egress in T. gondii is triggered by a combination of factors derived from the parasite (i.e., diacylglycerol kinase 2 activity) as well as from the host (i.e., host calcium raise or potassium drop) (7,16). Due to this redundancy, the absence of one important host cell factor is more likely to affect egress efficiency rather than to completely abolish it (7).
Liver stage egress consists of PVM rupture and the induction of merosome formation and is ultimately terminated by the rupture of merosomes in the bloodstream, leading to merozoite release (4). While PVM rupture and merosome formation appear to be unaffected by KO of GNAQ, the potential importance of GNAQ in merosome rupture currently cannot be excluded and needs further investigation.
P. falciparum merozoites egress spontaneously from red blood cells in in vitro cultures, whereas P. berghei merozoites are not able to do so (17,18). This, together with some transcriptional variation (19), points to certain differences in the egress process between both species. Thus, for a complete understanding of GNAQ function during apicomplexan release and to see if the differences in GNAQ dependence are a general (C to E) P. berghei liver stage PVM rupture is not affected by the absence of GNAQ. WT and GNAQ-KO cells were infected with sporozoites, and the percentage of merozoite-forming parasites that ruptured the PVM and the time difference between the successful formation of merozoites and PVM rupture were measured by quantitative live-cell imaging. The influx of GFP into the PV was used as a measure of PVM rupture. Imaging was started at ;55 hpi and lasted for 12 h. (C) Time between the formation of merozoites and PVM rupture. Each line represents the time difference between successful merozoite formation (beginning of the line) and PVM rupture (end of the line) and corresponds to one analyzed parasite. Continuous lines indicate parasites that did not rupture the PVM at all, which were not considered for the determination of the average PVM rupture time in panel E. Shown are combined data from three independent imaging experiments per cell line. (D) Percentage of merozoite-forming parasites that ruptured the PVM. The percentage of PVM rupture was determined in each of the three independent imaging experiments, in which the number of parasites that successfully developed to merozoites within the first 6 h of imaging was set to 100% in each experiment. Based on these, the percentage of parasites that successfully ruptured the PVM was calculated. Shown are means 6 SD. (E) Elapsed time from merozoite formation to PVM rupture determined for all parasites that ruptured the PVM. Displayed are means 6 SD. For all statistical analyses between WT and GNAQ-KO cells, one-way ANOVA followed by a Holm-Sidak multiple-comparison test was performed. All statistically significant differences are indicated (*, P , 0.05). (F) Egress-associated breakdown of the host actin cytoskeleton is comparable in WT and GNAQ-deficient cells. Cells expressing the filamentous actin marker GFP-utrophin (green) were infected with P. berghei parasites constitutively expressing mCherry (red), and their egress was studied by live-cell time-lapse microscopy. Representative images before and after egress are shown. Bars, 10 mm. See also Movies S1 and S2 in the supplemental material. (G) Comparison and GNAQ dependence of the egress strategies of T. gondii tachyzoites and P. falciparum blood stages versus P. berghei liver stages. The PVM is displayed in red. feature of Plasmodium liver stage egress, it remains to be shown if the observations made here on P. berghei liver stages are also true for other Plasmodium species, including P. falciparum.
While the kinetics of egress are to some extent driven by parasite effector proteins that differ between species and stages, our study indicates that differences in host cell signaling might also be important. Future studies are necessary to work out the molecular basis of these differences and if other host cell factors differentially regulate the egress of apicomplexan parasites.
Methods. (i) Ethics statement. All experiments were conducted in strict accordance with the guidelines of the Swiss Tierschutzgesetz (TSchG) (Animal Rights Laws) and approved by the ethical committee of the University of Bern (permit number BE109/13).
(ii) Experimental animals. Mice used in the experiments were between 6 and 10 weeks of age. BALB/c mice were obtained from Harlan Laboratories or bred in the central animal facility of the University of Bern. Mosquito feeds were performed on mice anesthetized with ketamine hydrochloride-medetomidine hydrochloride (Ketavet-Domitor; Pfizer), and all efforts were made to minimize suffering.
(v) Generation and confirmation of GNAQ-deficient HeLa cells. The CRISPR/Cas9 nickase system described previously (10) was used to knock out GNAQ in HeLa cells. CRISPR gRNA pairs were designed and selected using the Benchling CRISPR gRNA design tool to target exon 2 of the GNAQ gene. Cloning of the 2 gRNA oligonucleotides GCAGGCCATGATCAGAGCCA and GATGTTCTGATACACCAGCT into the plasmid pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) (Addgene plasmid 42335, supplied by Feng Zhang) was performed according to the protocol of the Zhang laboratory (21). HeLa cells were transfected with 5 mg of the two pX335 plasmids, each encoding one gRNA sequence. Three days after transfection, transfected cells were plated into 96-well plates to obtain clonal cell lines. Single colonies were expanded and screened for the absence of GNAQ by Western blot analysis. Two clones that showed no GNAQ signal were further analyzed on the genomic DNA level. Genomic DNA was isolated from these cells using QuickExtract DNA extraction solution 1.0 (Epicentre), and the regions of interest were amplified by PCR using primers GNAQ-Ex2-fw (AGACTGAGATGG CACTGTGG) and GNAQ-Ex2-rev (TTGCTGAACATCCAAATATGCC). PCR products were subcloned into pJet1.2 (Thermo Fisher Scientific), and 15 plasmids from each cell line were sequenced.
(vi) SDS-PAGE and Western blot analysis. A total of 0.25 Â 10 6 HeLa cells were transferred to an Eppendorf tube and spun down in a tabletop centrifuge. The supernatant was removed, and cells were resuspended in 40 ml of water. Ten microliters of 5Â SDS sample buffer was added, and after vortexing, the samples were heated at 95°C for 5 min. After cooling down, 1 ml of Benzonase per tube was added, and the samples were incubated for another 5 min at 37°C. Samples were then resolved by SDS-PAGE and transferred to nitrocellulose membranes (LI-COR). The membranes were blocked in 5% milk in Tris-buffered saline-Tween (TBS-T), followed by incubation with the following primary antibodies that were diluted in TBS-T containing 5% milk: rabbit anti-GNAQ (catalog number sc-393; Santa Cruz) (1:1,000) and chicken anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog number AB2302; Millipore) (1:5,000). After three washes in TBS-T, membranes were incubated with the following similarly diluted secondary antibodies: goat anti-mouse-800CW (LI-COR) (1:10,000) and goat anti-chicken-680RD (LI-COR) (1:10,000). Subsequently, membranes were washed another three times with TBS-T and scanned on a LI-COR Odyssey imaging system.
(vii) Analysis of T. gondii development and egress. T. gondii tachyzoites were grown in human foreskin fibroblast (ATCC CRL-1634) monolayers in Dulbecco's modified Eagle's medium (Gibco) supplemented with 5% fetal calf serum, 2 mM glutamine, and 25 mg/ml gentamicin (37°C with 5% CO 2 ). As previously described (16), for the study of parasite development and natural egress, 1 Â 10 5 HeLa cells were plated in 24-well plates and allowed to attach overnight. A total of 1 Â 10 5 freshly egressed RH parasites were allowed to invade for 3 to 5 min before extensive washing in order to obtain partially synchronous cultures as previously described (22). Intracellular parasite growth was measured at 24 hpi. Natural egress of the parasites was monitored at 36 and 50 hpi by immunofluorescence analysis.
(viii) Immunofluorescence analysis. T. gondii-infected cells on coverslips were fixed with 4% paraformaldehyde (PFA)-0.05% glutaraldehyde (GA) for 10 min prior to quenching in 0.1 M glycine-phosphate-buffered saline (PBS). Cells were then permeabilized with 0.2% Triton X-100-PBS (PBS-Triton) and blocked in the same buffer supplemented with 2% bovine serum albumin (BSA) (PBS-Triton-BSA). Cells were incubated with anti-GRA3 (a kind gift from J. F. Dubremetz) (1:20) and anti-GAP45 (23) (1:10,000) primary antibodies diluted in PBS-Triton-BSA for 1 h, followed by PBS-Triton washes (3 times for 5 min each). Cells were incubated with secondary antibodies (Alexa Fluor 488-or Alexa Fluor 594-conjugated goat anti-mouse or goat anti-rabbit IgGs, both diluted 1:1,000) in PBS-Triton-BSA. Nucleus staining was performed by incubating the cells with 49,6-diamidino-2-phenylindole (DAPI) (50 mg/ml in PBS) prior to the final wash step (3 times for 5 min each). Coverslips were mounted in Fluoromount G (Southern Biotech) on glass slides and stored at 4°C in the dark. Images were acquired on the LSM700 confocal microscope (Zeiss) at the Bioimaging Core Facility of the Faculty of Medicine, University of Geneva. Image processing was done using ImageJ.
(ix) Size measurement of P. berghei liver stages and analysis of detached cells. A total of 5 Â 10 4 HeLa cells per well were seeded in 24-well plates and infected the next day with sporozoites. At 48 hpi, the parasite size (area) was determined by density slicing using ImageJ, and infected cells were counted. At 65 hpi, the number of detached cells in the supernatant was counted. The percentage of detached cells was then calculated by dividing the number of detached cells in the supernatant by the number of infected cells at 48 hpi.
(x) Microscopy of P. berghei liver stage egress. Late liver stage development and detachment of infected host cells were analyzed by live-cell time-lapse imaging as previously described (13). For this, an LSM880 microscope (airyscan mode) with a Zeiss Plan-Apochromat 63Â/1.4 oil objective was used for confocal microscopy. Alternatively, a Leica DM12000B microscope with a Leica Plan-Apochromat 63Â/1.2 water immersion objective was used for wide-field microscopy. For multiposition time-lapse imaging, ZEN 2.1 software or Leica Application Suite 2.6 was used. During imaging, cells were kept in a CO 2 incubator at 37°C. Quantification of PVM rupture in WT and GNAQ-deficient host cells was performed as previously described (14). Image processing was done using ImageJ.
Data availability. All data generated or analyzed during this study are included in this article (and the supplemental material).