A genome-wide crispr screen in Toxoplasma Identifies Essential Apicomplexan Genes
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(B) Timeline for the generation of mutant populations and subsequent selection in the presence or absence of FUDR. Times at which parasites were passaged (P) are indicated. (C) Heatmap showing the phenotype score of genes at different time-points following transfection of the library into wild-type (wt) or Cas9-expressing parasites. (D) Relative abundance of sgRNAs following growth of the population in the presence or absence of FUDR. Mean log 2 (normalized abundance) for each sgRNA in three independent experiments; sgRNAs against UPRT (blue). (E) Phenotype score calculated for each gene comparing growth ± FUDR. Mean ± SEM for n = 3 independent experiments; UPRT (blue). (F) Comparison of phenotypic scores in untreated samples after three (P3) or six (P6) passages. Pearson’s correlation coefficient (r) is shown. 4 Cell 167, 1–13, September 22, 2016 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019 Figure 3. A Genome-scale Screen Measures the Contribution of Each Parasite Gene to Fitness in Human Fibroblasts (A) Diagram of T. gondii chromosomes with genes colored according to phenotype. (B and C) Significantly enriched (B) or depleted (C) gene sets identified by GSEA. Genes belonging to each category (gray) are plotted according to their rank in the screen, relative to the maximum enrichment score (red) and zero phenotype (green). Phenotype scores for a given set were compared to the entire set by a Kolmogorov-Smirnov test (FDR corrected) to calculate the p values. (D) T. gondii genes rank-ordered based on their phenotype. Genes previously reported are highlighted, indicating whether they are dispensable (yellow), or indispensable as inferred from overexpression (blue) or another method (red). Dotted line represents the median phenotype score for the dispensable genes. Mean ± SEM for n = 4 independent experiments. The two groups are compared in a box plot where whiskers indicate the most extreme data within 1.5 times the interquartile range from the boxed quartiles (right). (E) Correlation of phenotype scores to gene expression based on maximum RPKM values. The distribution of phenotypes in each expression quartile is plotted in the violin graph. Bars indicate the group median. (F) Analysis of selective pressure for 5,897 syntenic genes found in all three coccidian genomes (Tg, T. gondii; Nc, N. caninum; Hh, H. hammondi). Histogram shows the distribution of d N /d S values, highlighting the top and bottom third. Genes binned according to d N /d
show higher phenotype scores for genes under purifying selection (orange). Bars indicate the group median. The distributions were compared using a Kolmogorov-Smirnov test. (G) Correlation between phenotype scores and depth of conservation. The phylogenetic relationship between T. gondii and the other genomes used in the analysis is illustrated by the dendrogram, with number of genomes in each taxon indicated. The proportion of T. gondii genes and, within this, the proportion that are functionally annotated, are shown for each category. The distribution of phenotype scores for each category is plotted. Bars indicate the group median. See also
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Tables S1 , S2 , and S3 . Cell 167, 1–13, September 22, 2016 5 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019
displayed Ty expression in 5%–20% of parasite vacuoles ( Fig-
ures 4 B and 4C). Several proteins displayed characteristic struc- tures including secretory vesicles like the micronemes (ICAPs 1 and 12), organelles like the mitochondrion (ICAPs 2, 3, 6, 8, 9, 11, 14, and 15), and compartments like the nucleolus (ICAP7) and conoid (ICAP16) ( Figure 4 C). Overlap with a known marker of the mitochondrion ( MacRae et al., 2012 ) confirmed the localiza- tion of the putative mitochondrial ICAPS ( Figure 4 D). Both micro- nemal proteins co-localized with the microneme markers MIC8 ( Kessler et al., 2008 ) and PLP1 ( Kafsack et al., 2009 ) ( Figure 4
E). ICAP1 was shown to be essential for regulated exocytosis during the preparation of this work ( Bullen et al., 2016 ), confirming our predictions regarding its importance and localization. To assist the functional characterization of ICAPs, we engi- neered parasites that stably expressed both Cas9 and a nuclear YFP marker (H2B-YFP) ( Hu et al., 2004 ) for fluorescence micro- scopy. This strain exhibited a high rate of sgRNA-mediated gene disruption, comparable to the strain used in the screen ( Figure S3 ). As controls, we selected several genes known to be either indispensable (MYOA and CDPK1) or dispensable (SAG1, PLP1, and MYOC) and five uncharacterized genes pre- dicted to be dispensable by the screen (controls 1–5; Figure 5 A).
Figure 4. Subcellular Localization of Indispensable Conserved Apicomplexan Proteins (A) CRISPR was used to introduce a C-terminal Ty tag into the endogenous locus of individual genes through homologous recombination (HR) following the indicated timeline. (B) List of successfully tagged indispensable conserved apicomplexan proteins (ICAPs), numbered according to their phenotype scores from lowest (ICAP1) to highest (ICAP17). (C) 3 days after transfection, intracellular parasites were fixed and stained for Ty (green), ACT1 (red), and DAPI (blue). Asterisk denotes fixation in methanol instead of formaldehyde. Scale bar, 10 mm. The diagram illustrates the relative position of various organelles within the parasite. (D and E) Colocalization ICAPs (green) with a mitochondrial marker (MYS; red) (D) or micronemal proteins (MIC8 or PLP1; red) (E). Nuclei stained with DAPI (blue) are shown in the merged image. Scale bar, 10 mm. See also
Table S2 . 6 Cell 167, 1–13, September 22, 2016 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019 Figure 5. Functional Characterization of Indispensable Conserved Apicomplexan Proteins (A) Position of analyzed genes within the phenotypic ranking of all T. gondii genes. Known indispensable (red) or dispensable (yellow) genes used as controls are indicated. The ICAPs (blue) and uncharacterized genes predicted to be dispensable (green) are numbered in ascending order according to their rank. Mean ± SEM for n = 4 independent experiments. (B and C) Gene disruption observed at a population level three days after transfection with various control constructs. Disruption of the target locus is observed by Surveyor assay comparing, for each locus, the specific sgRNA to an irrelevant sgRNA against the dispensable gene MYOC (B). Loss of the target proteins (red) is observed in samples treated with the targeting sgRNA but not the control, while the loading controls (green) remain unchanged (C). (D and E) Plaque assays performed immediately following transfection with sgRNAs targeting ICAPs or control genes (D). The number of plaques observed for disruption of each gene relative to the sgRNA against SAG1 (E). Mean ± SEM for n = 2 independent experiments; *, FDR-adjusted p < 0.1 relative to the control. (F) Secondary screen for genes involved in invasion. The period of intracellular growth prior to phenotypic changes was extended by forced release and passaging the day after transfection. Invasion was assayed after the subsequent lysis, and calculated relative to PLP1 disruption. Mean ± SEM for n = 2 independent experiments; *, FDR-adjusted p < 0.1 relative to the control. Representative immunofluorescence images are shown. See also Figure S3 and Table S2
. Cell 167, 1–13, September 22, 2016 7 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019 Figure 6. CLAMP Mediates T. gondii Invasion and Is Essential for the P. falciparum Asexual Cycle (A) Neighbor-joining tree showing the phylogenetic relationships of CLAMP homologs in diverse apicomplexans. Bootstrap values for 10,000 trials are displayed. (B) Inferred topology of CLAMP highlighting transmembrane domains (orange) and the proline-rich domain (green). See also Figure S4 . (C–E) CLAMP-mNeonGreen localization during egress and invasion. Intracellular parasites expressing CLAMP-mNeonGreen were stimulated to egress with A23187 (C). Relative fluorescence across the length of each parasite (dotted line) is plotted for the four time-points shown. Lines are polynomial regressions ± 95% CI (D). The localization was also monitored during invasion (E). The position of the moving junction is indicated with paired open arrows. Solid arrowhead indicates a punctum of mNeonGreen at the posterior of the parasite appearing immediately after invasion. Time is expressed in minutes:seconds following addition of the compound (C and D) or initiation of invasion (E). Scale bar, 10 mm. (F) Diagram of the DiCre/CLAMP strain showing how after rapamycin (rapa) treatment the reporter locus switches from expressing KillerRed to expressing YFP, and CLAMP mRNA degradation is induced. (G and H) A 2-hr treatment with rapa is sufficient to induce CLAMP degradation as demonstrated by immunofluorescence microscopy 24 hr later (G) or immunoblotting 2 days later (H). The parental strain (DiCre) is included as a control. (legend continued on next page) 8 Cell 167, 1–13, September 22, 2016 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019 Examining populations of parasites three days after transfection with guides against selected controls showed high rates of on- target mutations ( Figure 5
B) and significant loss of the target pro- teins (
Figure 5 C). Therefore, we can study the effect of a given sgRNA without isolating clonal populations, allowing us to analyze a large set of candidate genes in arrayed, secondary screens. As an initial measure of gene function, we analyzed plaque formation immediately after transfection with each specific sgRNA construct. Plaques are formed as infection originating from single parasites spreads to adjacent cells clearing a portion of the monolayer, thereby reflecting parasite viability and competency over several lytic cycles. This resulted in reproducible plaque numbers for all sgRNAs against genes known or predicted to be dispensable ( Figures 5 D and 5E). In contrast, sgRNAs against known essential proteins and most of the ICAPs led to formation of small or significantly fewer plaques ( Figures 5 D and 5E). These experiments confirm our screen’s results and identify several previously un- characterized genes predicted to be essential for the T. gondii lytic cycle. Invasion of host cells is a central feature of the apicomplexan life cycle. To identify unknown components of the invasion machinery, we investigated the role of ICAPs in this process. The effect of each sgRNA was measured 3 days post-transfec- tion relative to the disruption of PLP1, which is known to be dispensable for invasion ( Kafsack et al., 2009 ) ( Figure 5
F). MYOA and CDPK1 served as positive controls based on their documented phenotypes ( Lourido et al., 2010; Meissner et al., 2002
). A quarter of the genes tested appeared to impact parasite invasion ( Figure 5 F). Although this assay could be affected by de- fects in extracellular survival, slow protein turnover, or sgRNA ef- ficiency, it provides a rapid means to identify candidate invasion factors. ICAP12 had the strongest effect on invasion, which, in light of its micronemal localization, motivated a more detailed characterization. ICAP12 Is an Invasion Factor Conserved Throughout the Apicomplexa ICAP12 orthologs were present in all available apicomplexan ge- nomes, and their alignment recapitulated the known relationship between the species ( Figure 6 A). Topology prediction supports a model with four transmembrane domains and cytoplasmic N and C termini ( Figure 6 B). The transmembrane domains and extracel- lular loops are more conserved than the C-terminal proline-rich domains ( Figure S4 ). No related sequences could be identified outside the Apicomplexa. However, hidden Markov model- based searches suggest structural similarity between ICAP12 and the mammalian tight-junction proteins claudin-15 and claudin-19. Based on these features and its localization, we named ICAP12 ‘‘claudin-like apicomplexan microneme protein’’ (CLAMP).
We tagged the endogenous CLAMP locus with mNeonGreen to study its localization in vivo. The fusion protein concentrated at the apical end of parasites, consistent with micronemal local- ization ( Figure 6 C). Micronemes are secreted in response to increased cytosolic Ca 2+ , which mediates parasite motility, egress, and invasion of host cells ( Carruthers et al., 1999 ). We monitored CLAMP localization following stimulation with the Ca 2+ ionophore A23187 and observed increased apical fluores- cence prior to egress ( Figures 6 C and 6D; Movie S1
). Following incubation with host cells, formation of CLAMP foci could be de- tected at the posterior of most parasites ( Figure S5 A). We also observed active formation of such foci during invasion ( Figure 6 E, arrowheads; Movie S2 ). This relocalization of CLAMP is similar to what has been observed for other membrane-tethered microne- mal proteins ( Carruthers and Sibley, 1999; Garcia-Re´guet et al., 2000
). To directly examine CLAMP function, we used a conditional gene-silencing method ( Pieperhoff et al., 2015 ). In a strain ex- pressing a rapamycin-dimerizable version of the Cre recombi- nase (DiCre), we modified the endogenous CLAMP locus to include a C-terminal hemagglutinin (HA) tag and a floxed 3 0 UTR followed by four U1-binding sequences (DiCre/CLAMP; Figure 6 F). Rapamycin treatment triggers excision of the 3 0 UTR and U1-mediated mRNA degradation. The strain also carries a reporter that switches expression of KillerRed for YFP upon Cre activation. A 2-hr rapamycin treatment during initial infection efficiently downregulated CLAMP expression (green), as measured by immunofluorescence 24 hr later ( Figure 6 G) or
by immunoblot 2 days later ( Figure 6
H). To determine the impact of CLAMP on the lytic cycle, we examined plaque formation following treatment with rapamycin. The treatment did not affect the parental strain (DiCre; Figure 6 I). However, CLAMP silencing blocked plaque formation, and the few plaques remaining likely represent the 5%–10% of parasites that do not undergo recom- bination (DiCre/CLAMP; Figure 6
I). To determine the precise defect associated with CLAMP loss, we examined several stages of the lytic cycle. We tested whether CLAMP downregulation might affect microneme secretion, which can be experimentally triggered by ethanol treatment ( Carruthers et al., 1999 ). Rapid shedding of micronemal adhesins from the parasite surface allows quantification of secretion by measuring protein accumulation in the supernatant. Comparing the relative abundance of secreted MIC2—a micronemal adhesin—demonstrates that CLAMP silencing has no effect on microneme secretion ( Figure 6 J). We also observed normal motility and egress when intracellular parasites were treated with A23187 ( Figure 6 K) or the phosphodiesterase inhibitor zaprinast ( Movie S3
). Following stimulated egress, parasites (I–L) The DiCre/CLAMP strain or its parental stain (DiCre) was treated as above. Parasites were harvested and phenotypically assayed for plaque formation (I), microneme secretion (J), egress (K), or invasion (L). Secretion was measured as the percentage of total MIC2 present in the parasites (J). Egress was induced with A23187 and compared to a vehicle control (DMSO) over the same period (K). All results are means ± SEM for n = 3 independent experiments; **p < 0.005 relative to the untreated DiCre strain. (M) Diagram of the PfCLAMP cKD showing how removing aTc allows the TetR-DOZI regulator to bind and suppress expression. (N) Growth curves of the parental strain (left) or the cKD (right) ± aTc. Means ± SD for n = 3 technical replicates. See also Figure S5 D for an independent replicate. See also
Figures S4 and
S5 and
Movies S1 , S2 , S3 , and S4 . Cell 167, 1–13, September 22, 2016 9 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019 frequently reinvade adjacent host cells. However, CLAMP silencing rendered reinvasion attempts unsuccessful ( Movie S4
thrusting motion and deformation. To directly measure this defect, CLAMP was silenced during the growth cycle prior to the assaying invasion. Knockdown of CLAMP had a profound ef- fect on invasion, reducing the number of intracellular parasites by 80% ( Figure 6
L). These results implicate CLAMP in the cellular events immediately preceding invasion of host cells. CLAMP Is Essential during the Asexual Cycle of Malaria To test whether the essentiality of CLAMP extends to other apicomplexans, we constructed a conditional knockdown (cKD) of its ortholog in P. falciparum. We tagged the endogenous locus of PfCLAMP with a FLAG epitope tag and ten tandem ap- tamer sequences, which bind the Tet repressor protein (TetR) when transcribed ( Ganesan et al., 2016 ). TetR is expressed as a fusion with the translational repressor (DOZI) in the same strain, which suppresses expression of the aptamer-tagged transcript unless anhydrotetracycline (aTc) is added to the media ( Fig- ure 6
M). We confirmed correct integration of the construct into the PfCLAMP locus by sequencing ( Figures S5 B and S5C). To test the effect of CLAMP repression on the asexual cycle of
contained or lacked aTc. The different conditions had no effect on the growth of the parental strain ( Figure 6
N, left). In contrast, withdrawal of aTc from the PfCLAMP cKD led to a rapid and complete block in the asexual cycle ( Figure 6
N, right). These re- sults demonstrate the essentiality of CLAMP in a second api- complexan species. DISCUSSION We present the first genome-wide functional analysis of an api- complexan. Using CRISPR/Cas9, we targeted all annotated pro- tein-coding genes in the T. gondii genome to generate mutant populations for screens based on positive or negative selection. This method enabled rapid identification of genes that mediate infection of human fibroblasts or confer drug sensitivity. Our results agree with published observations, follow expected genomic trends, and provide new robust predictions that identify several essential proteins conserved throughout the phylum. We also demonstrate that this method can easily identify mutants resistant to the antiparasitic compound FUDR. For such special cases where gene disruption can mediate resistance, our method will facilitate the identification of drug-resistance path- ways and provide a complementary approach to mapping spon- taneous resistance mutations ( Flannery et al., 2013 ). Essential apicomplexan adaptations represent ideal targets for therapeutic or prophylactic interventions. However, genes involved in such pathways are difficult to identify. Based on re- sults from our genome-wide screen, we characterized 17 ICAPs. Individually disrupted, most ICAPs could be shown to indepen- dently contribute to growth in fibroblasts. None of the ICAPs have defined domains or resemble proteins outside the phylum, yet most of them localized to distinct subcellular structures, assuming no detrimental effect of epitope tagging on localiza- tion. Of the eight that localized to the mitochondrion, only ICAP3 and ICAP14 have predicted signal peptides that might have suggested their compartmentalization. Nonetheless, the preponderance of mitochondrial ICAPs and their conservation Yüklə 8 Mb. Dostları ilə paylaş:
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