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CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor - PubMed

CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor

Ying Xu et al. Nature. 2022 Oct.

Abstract

Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.

© 2022. The Author(s), under exclusive licence to Springer Nature Limited.

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Conflict of interest statement

Competing Interests

The authors declare no conflicts of interest.

Figures

Extended Data Fig. 1.
Extended Data Fig. 1.. Characterizing pTc toxicity in mammalian and insect cells.

a-b, U2OS, 5637, and HeLa cells were exposed to pTc for 24 h. Representative images were from one of three independent experiments and shown in (a). The percentage of rounded cells and CR50 values was quantified and plotted in (b). c-d, S2 cells were treated with the indicated dose of pTc for 4 days. Representative images were from one of three independent experiments and shown in (c). The percentage of enlarged cells and CE50 values was quantified and plotted in (d). Data were analyzed from the total number of images indicated in the Source data from three experiments and shown as mean ± SD or the best-fit value ± 95% CI. Scale bar = 50 μm.

Extended Data Fig. 2.
Extended Data Fig. 2.. Top-ranked genes identified in genome-wide CRISPR screens.

a, List of most prevalent 38 sgRNAs following the last round of selection with pTc or mTc. b, Prevalence of all sgRNAs targeting Vsg, Mop, FIG4, or VAC14 across three rounds of pTc or mTc screens were quantified and plotted according to their corresponding gRNA abundances.

Extended Data Fig. 3.
Extended Data Fig. 3.. RNAi knock-down validation of top hits from CRISPR screens.

a, Schematic of the experimental steps. Briefly, S2 cells were bathed in 50 nM dsRNAs targeting the top hits from pTc CRISPR screen # 1 round 1 and allowed to recover for 3 days. Next, cells were treated with 100 pM pTc for 2 days, re-seeded and grown for an additional 7 days. DNA was stained by Hoechst dye and cells were analyzed by flow cytometry. b, Comparison of untreated cells (red) versus those treated with 100 pM pTc (blue) revealed an increase in DNA content after pTc treatment (left). Comparison of pTc treatment following bathing in vsg dsRNA (green) versus LacZ control dsRNA (gold) showed that vsg dsRNA pre-bathing abrogated pTc-induced increase in DNA content. Gate representing DNA content of 80–90% of untreated cells was set. There was no pre-gating on cell sizes as pTc treatment causes changes in cell sizes and shapes. c, The indicated top hits from pTc CRISPR screens were knocked down in S2 cells using dsRNA and then exposed to pTc as described in (a). Percentage of cells with normal Hoechst levels were plotted. d, Comparison of untreated cells (No Toxin) versus those treated with 100 pM pTc revealed increase in cell size (quantified using the forward scatter/side scatter profile, SSC-A versus FSC-A). Comparison of pTc treatment following bathing in vsg dsRNA (DRSC25227 and DRSC10219) versus LacZ control dsRNA showed that vsg dsRNA pre-bathing abrogated pTc-induced cell size enlargement. e, The indicated top hits from pTc CRISPR screens were knocked down in S2 cells using dsRNA and then exposed to pTc as described in (a). The percentage of enlarged cells was plotted, which gave similar results to DNA-content measurement in (c).

Extended Data Fig. 4.
Extended Data Fig. 4.. Characterization of Vsg KO S2 cells.

a-c, S2 cells expressing one of the three indicated sgRNAs before or after treatment with 130 pM pTc for 2 weeks were harvested with the sequence at the gRNA targeting sites analyzed by deep sequencing. The frequency of deletions of the indicated size were plotted (insertions represented less than 1% and were ignored). Non-frame-shift mutations (potentially retaining Vsg receptor activity, red) versus frameshift mutations (blue) are shown. d, Vsg KO2, Vsg KO3, and the control S2 cells expressing scrambled sgRNA were exposed to two Tc toxins (Xn-XptA1, 3.5 nM; PI-TcdA4, 3 nM) for 4 days. Both contain the same TcdB2-TccC3 as pTc but utilize distinct A subunits (XptA1 from X. nematophila and TcdA4 from P. luminescens, respectively). Both toxins induced cell enlargement on Vsg KO cells and showed no difference on potency on control cells versus Vsg KO cells. Data were analyzed from the total number of images indicated in the bar graphs from three experiments and shown as mean ± SD.

Extended Data Fig. 5.
Extended Data Fig. 5.. TcdA1 binds to Vsg-ECD and Vsg-ECD can reduce binding of TcdA1 to cells.

a, S2 cells stably expressing GFP-Vsg (GFP was inserted after Vsg signal peptide, SP-GFP-Vsg cells) were mixed with the control S2 cells. Cells were incubated with Alexa Fluor-647-conjugated TcdA1 (pentamer, 25 nM) at room temperature for 1 h and then subjected to flow cytometry analysis. S2 cells that express GFP-Vsg showed elevated binding of TcdA1 compared with the control S2 cells. b, Vsg-ECD-Fc produced in S2 cells shown in SDS-PAGE gels with Coomassie blue staining, which showed an apparent molecular weight of ~ 130 kDa, much larger than its predicted molecular weight, indicating that the protein is heavily glycosylated. Representative images were from one of three independent experiments. c, Biotinylated TcdA1 interacted with purified Fc-tagged Vsg-ECD, but not the control IgG1-Fc. For gel source data, see Supplementary Figure 1. Representative images were from one of three independent experiments. d, BLI analysis showing that TcdA1 did not bind to empty probes, and Morganella morganii TcdA4 from mTc and a Tc toxin (YenTc) from Yersinia entomophaga showed no binding to immobilized Vsg-ECD-Fc. e, Pre-incubation of 15 nM TcdA1 pentamer with 1 μM Vsg-ECD for 2 h in 4 °C reduced TcdA1 binding to U2OS cells transfected with Vsg. Representative images were from one of three independent experiments. Scale bar = 100 μm.

Extended Data Fig. 6.
Extended Data Fig. 6.. Alignment of Vsg, Vsg orthologs, and an artificial receptor.

a, The protein sequences of the indicated Vsg orthologs were aligned in Mega-X through the CLUSTWS method (Drosophila melanogaster: NP_729535.1; Aedes aegypti: XP_021708011.1; Anopheles gambiae: XP_001688320.1; Tribolium castaneum: XP_972460.1; Homo sapiens: NP_006007.2; Galleria mellonella: XP_026748881.1; Spodoptera frugiperda: KAF9794377.1). b, Percentages of T/S residues within the PTS region in Vsg (residues 41–134 in D. melanogaster Vsg, between two arrows) and the corresponding region in other Vsg homologs and the artificial receptor were counted and listed.

Extended Data Fig. 7.
Extended Data Fig. 7.. Expression of Vsg orthologs in U2OS cells and rescue of Vsg KO S2 cells with Vsg and aVsg.

a, GFP fused Vsg, gVsg, sVsg, TMEM123, or CD164 were transfected into U2OS cells (GFP was inserted after the signal peptide of Vsgs). Cells were fixed and immunostaining was carried out using an anti-GFP antibody without permeabilization of cell membranes. Representative images from one of three independent experiments were shown. Scale bar = 20 μm. b, Vsg KO2 S2 cells were transiently transfected with plasmids encoding blue fluorescent protein (BFP) or BFP plus Vsg or agVsg. Cells were then incubated with 25 pM pTc for 20 h, fixed, and stained with Alexa Fluor-647-conjugated phalloidin. BFP marked transfected cells. The percentages of transfected cells with toxin-induced actin clustering phenotype were plotted. Representative images from one of three independent experiments were shown. Scale bar = 50 μm. c, Vsg KO2 S2 cells (expressing GFP from the Vsg gRNA plasmid) were transiently transfected with BFP or BFP + agVsg plasmids, then treated with 30 pM pTc for 3 days. Cells were recorded directly under confocal. The percentages of transfected cells to become enlarged were plotted. Representative images from one of three independent experiments were shown. Scale bar = 50 μm. Data were analyzed from the total number of images indicated in the bar graphs from three experiments and shown as mean ± SD.

Extended Data Fig. 8.
Extended Data Fig. 8.. O-glycosylation contributes to pTc binding and mass spectrometry analysis of O-glycans on Vsg and sVsg.

a, Vsg-ECD treated with O-glycosidase and PNGase F showed reduced molecular weight. b, Rounding of U2OS cells transiently expressing Vsg-GFP fusion proteins and Vsg-GFP with two N-glycan site mutations (N40T and N121S) via transient transfection. Cells were exposed to pTc for 24 h. The percentage of rounded GFP-positive cells was quantified and plotted. Data were analyzed from the total number of images indicated in the bar graphs from three experiments and shown as mean ± SD. c, Characterization of TcdA1 binding to equal amounts of immobilized Vsg-ECD, Vsg-ECD treated with PNGase F, and Vsg-ECD treated with O-glycosidase, using the BLI assay. d, Vsg-ECD and sVsg-ECD were purified as Fc-tagged proteins in S2 cells. β-eliminated glycans were permethylated and analyzed using mass spectrometry. Mass peaks were manually assigned to the indicated O-glycan moieties. e, The O-glycosylaton sites in Vsg-ECD and sVsg-ECD were predicted using NetOGlyc (

http://www.cbs.dtu.dk/services/NetOGlyc/

) and marked in red.

Extended Data Fig. 9.
Extended Data Fig. 9.. Vsg expression in D. melanogaster tissues.

a, Schematic illustration of the GFP-Vsg gene fusion in fly strain 50812. The protein-trap constructs P{PTT-GA} carry an Avic\GFP vital fluorescent protein-trap marker. GFP is inserted into the first intron of vsg, resulting in a fusion of GFP between residues 24 and 25 of Vsg. b, GFP-Vsg (green) was detected in larval ovary tissue and co-localized with phalloidin labeled ring canal structures (red). It was also detected in ring canals across the cell membrane marked by Hts staining (red) in larval lymph gland, eye disks and brain tissues. Hoechst dye marks the nuclei (blue). Scale bar = 10 μm. c, GFP-Vsg was detected in hemocytes and fat body cells. Hoechst dye marks the nuclei (blue). The edge of a fat body cell is marked. Scale bar = 10 μm. Representative images were from one of three independent experiments.

Extended Data Fig. 10.
Extended Data Fig. 10.. Hemocytes from Vsg KO1 fly are resistant to pTc.

a-b, Hemocytes from the control strain (nos-Cas9;attP2) or Vsg KO1 D. melanogaster were exposed to the indicated concentrations of pTc for 18 h. Cells were fixed and stained with phalloidin (red) and Hoechst (blue). Representative images (a), and quantification of the percentage of cells with actin clustering phenotype were shown in (b). Scale bar = 10 μm. c-d, Hemocytes from a Vsg KO fly line (Hml-Gal4,UAS-EGFP; KO2) and a rescue line that expresses aVsg in hemocytes (Hml-Gal4,UAS-EGFP/UAS-avsg; KO2) were exposed to 100 pM pTc for 18h. Cells were fixed and stained with phalloidin (red). Representative images (c), and quantification of the percentage of cells with actin clustering were shown in (d). Scale bar = 20 μm. e-f, Hemocytes from Control or Vsg KO2 D. melanogaster were treated with pTc (25 pM, 18 h) and then co-incubated with fluorescently labelled E. coli bioparticles (red) for 30 min. Cells were then fixed and stained with Alexa Fluor-488-conjugated phalloidin (green) (e). E. coli bioparticles per cell were counted and plotted in (f). Scale bar = 10 μm. Data were analyzed from the total number of images indicated in the bar graphs from three experiments and shown as mean ± SD.

Fig. 1.
Fig. 1.. Genome-wide CRISPR–Cas9-mediated screen identifies host factors for pTc and mTc.

a, Schematic of the pooled screen using pTc or mTc toxins in the D. melanogaster PT5/S2R+ cell line. b, Genes identified in two independent pTc screens (screen #1-high and screen #2 as described in Methods and Source Data) ranked based on the robust rank aggregation (RRA) score (analyzed with MAGeCK). The top-ranking genes were color-coded and grouped. c, Comparison of the genes identified in the pTc and mTc screens.

Fig. 2.
Fig. 2.. Vsg is a receptor for pTc.

a-b, The sensitivity to pTc of vsg KO S2 cell lines or control S2 cell lines expressing scrambled sgRNAs were assessed by exposure to a titration of pTc for 4 days. Representative images with 15.6 pM pTc treatment were shown in (a), cell nuclei were marked with Hoechst dye (blue). The percentages of enlarged cells were quantified and plotted over pTc concentrations in (b). c, Cell sizes were recorded and plotted over time after exposure to pTc (30 pM) for Vsg KO S2 cells versus the control S2 cells expressing a scrambled sgRNA. d, Cells were incubated with pTc and ADP-ribosylation of actin was detected by immunoblot analysis of cell lysates. Tubulin served as a loading control. For gel source data, see Supplementary Figure 1. e-f, Sensitivity of U2OS cell lines expressing TMEM123, CD164, or Vsg to pTc were assessed by exposure to a titration of pTc for 24 h. Representative images with 1.44 nM pTc treatment are shown in (e). The percentages of rounded cells were quantified and plotted (f). The toxin concentration resulting in 50% cell rounding is defined as CR50. g, Ectopic expression of Vsg-GFP fusion proteins in U2OS cells via transient transfection mediated binding of TcdA1 to transfected cells (~96% TcdA1-bound cells are GFP positive, n=165). Biotinylated TcdA1 was labeled with Alexa Fluor 555-conjugated streptavidin. Cells transfected with GFP served as a control. h, Characterization of TcdA1 binding to immobilized biotin-conjugated Vsg-ECD using BLI. i, Preincubation of Alexa Fluor-488-conjugated TcdA1 (pentamer, 20 nM) with Vsg-ECD (1 μM) for 2 h in 4 °C reduced TcdA1 binding to S2 cells, detected by confocal microscopy (upper panel; cells were labeled with Hoechst in blue, TcdA1: green) or flow cytometry (lower panel). Representative images and sensorgrams were from one of three independent experiments. Data were analyzed from the total number of images indicated in the Source data from three experiments and shown as mean ± SD, CR50 in panel (f) displayed as the best-fit value ± 95% confidence interval (CI)). Scale bars = 50 μm.

Fig. 3.
Fig. 3.. Vsg orthologues from mosquitos and beetles can serve as pTc receptors.

a, The Maximum-likelihood tree of Vsg orthologs in the indicated insect species. b-c, Vsg and the indicated Vsg orthologs, each fused with GFP, were expressed in U2OS cells via transient transfection. Cells were exposed to 2.5 nM pTc for 24 h (b). The percentage of rounded GFP-positive cells was quantified and plotted in (c). d-e, U2OS cells stably expressing aVsg, tVsg, or sVsg through lentiviral transduction were exposed to pTc. Representative images with exposure to 1.44 nM pTc (d). Percentage of rounded cells was recorded and plotted in (e) together with CR50 values. f-g, Two agVsg KO mosquito Sua5B cell lines and the control Sua5B cell line expressing a scrambled sgRNA were exposed to pTc. Representative images with exposure to 312.5 pM pTc (f). The percentage of enlarged cells was quantified and plotted in (g) together with CE50 values. h, Schematic drawing of Vsg truncation mutants. i-j, The indicated Vsg mutants fused with GFP were expressed in U2OS cells. Cells were exposed to pTc (2.5 nM) and representative images were collected (i). Quantification of the percentage of rounded cells among transfected cells was shown in (j). k, O-glycan moieties in Vsg-ECD and sVsg-ECD identified by mass spectrometry analysis were shown as relatively profile intensity in a pie chart. Representative images were from one of three independent experiments. Data were analyzed from the total number of images indicated in the Source data or bar graphs from three experiments and shown as mean ± SD or the best-fit value ± 95% CI. Scale bar = 50 μm.

Fig. 4.
Fig. 4.. Vsg KO flies show reduced sensitivity to pTc and P. luminescens infection.

a, Schematic illustration of vsg knock out strategy and the genotyping results of two different vsg KO strains. Representative images were from one of three independent experiments. b-c, Hemocytes from control (CTRL, nos-Cas9;attP2), vsg KO, or a wild-type strain (w1118) of D. melanogaster were exposed to the indicated concentrations of pTc for 18 h. Cells were fixed and stained with phalloidin (red) and Hoechst dye (blue). Representative images were shown in (b), and percentages of cells with actin clustering were quantified and plotted in (c). Scale bar = 10 μm. d, Hemocytes from a vsg KO fly line (Hml-Gal4,UAS-EGFP; KO2) and a rescue line that expresses aVsg in hemocytes (Hml-Gal4,UAS-EGFP/UAS-avsg;KO2) were exposed to 20 pM pTc for 18 h. Cells were fixed and stained with phalloidin (red). Scale bar = 20 μm. e-f, Hemocytes from control (w1118) or vsg KO2 D. melanogaster were treated with pTc (25 pM or 50 pM, 18 h) and then co-incubated with pHrodo™ Red labeled E. coli bioparticles (red) for 30 min. Cells were then washed, stained with Hoechst (blue), and imaged (e). Internalized E. coli fluorescence intensity per cell was counted and plotted (f). Scale bar = 20 μm. g-h, Control (nos-Cas9;attP2) and vsg KO D. melanogaster were infected with the control bacterium, Yersinia entomophaga, through pricking with an insect pin. Survival curves shown in (g). Flies were collected and homogenized, and bacterial loads (colony formation units) were assessed by serial dilution plating (h). i-j, Control (nos-Cas9;attP2) and vsg KO D. melanogaster were infected with P. luminescens through pin prick. Survival curves (i) and bacterial loads (j) were shown. Data in (c), (d) and (f) were analyzed from the total number of images indicated in the Source data or bar graphs from three experiments and shown as mean ± SD or the best-fit value ± 95% CI. Data in (h) and (j) were analyzed from the total number of flies indicated in the Methods section. Center values represent the median. The box plots in (h) and (j) show all points. The bounds are minimum to maximum values, 25th and 75th percentiles, respectively. Two-tailed test was used for comparing between Control-microbe and vsg KO-microbe group in (h) and (j). Log-rank (Mantel-Cox) test was used for comparing between Control-microbe group and vsg KO-microbe group in (i). p = 0.29 (h), p = 0.008 (i) and p = 0.003 (j). **p < 0.01

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References

    1. Lacey LA et al. Insect pathogens as biological control agents: Back to the future. J Invertebr Pathol 132, 1–41 (2015). - PubMed
    1. Labaude S & Griffin CT Transmission Success of Entomopathogenic Nematodes Used in Pest Control. Insects 9 (2018). - PMC - PubMed
    1. Bowen D et al. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129–2132 (1998). - PubMed
    1. Roderer D & Raunser S Tc Toxin Complexes: Assembly, Membrane Permeation, and Protein Translocation. Annu Rev Microbiol 73, 247–265 (2019). - PubMed
    1. Waterfield NR, Bowen DJ, Fetherston JD, Perry RD & ffrench-Constant RH The tc genes of Photorhabdus: a growing family. Trends Microbiol 9, 185–191 (2001). - PubMed

Additional References

    1. Li W et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol 15, 554 (2014). - PMC - PubMed
    1. Viswanatha R et al. Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos. Nat Commun 12, 6825 (2021). - PMC - PubMed
    1. Jones SA & Hurst MR Purification of the Yersinia entomophaga Yen-TC Toxin Complex Using Size Exclusion Chromatography. Methods Mol Biol 1477, 39–48 (2016). - PubMed

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