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A CRISPR Screen Using Subtilase Cytotoxin Identifies SLC39A9 as a Glycan-Regulating Factor - PubMed

  • ️Tue Jan 01 2019

A CRISPR Screen Using Subtilase Cytotoxin Identifies SLC39A9 as a Glycan-Regulating Factor

Toshiyuki Yamaji et al. iScience. 2019.

Abstract

Subtilase cytotoxin (SubAB) is a virulence factor produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains. The toxin recognizes sialoglycans for entry and cleaves an endoplasmic reticulum chaperon, binding immunoglobulin protein, to cause cell death. However, no systematic screening has yet been performed to identify critical host factors. Here, we performed a genome-wide CRISPR/Cas9 knockout screen for SubAB-induced cell death and identified various sialoglycan-related and membrane-trafficking genes. Analysis of glycan-deficient cells demonstrated that not only N-glycans but also O-glycans serve as SubAB receptors. In addition, SLC39A9, which is a predicted zinc transporter, as well as KDELRs and JTB, were required for SubAB to induce maximal cell death. Disruption of the SLC39A9 gene markedly reduced both complex-type N-glycans and core 1 O-glycans, and the O-glycan reduction was attributed to the reduction of core 1 synthase (C1GalT1). These results provide insights into the post-transcriptional regulation of glycosyltransferases by SLC39A9, as well as sialoglycan species as SubAB receptors.

Keywords: Biochemistry; Biological Sciences; Cell Biology; Microbiology; Molecular Biology.

Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

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Figures

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Graphical abstract
Figure 1
Figure 1

Identification of SubAB Resistance Genes in a Genome-wide CRISPR Screen (A) Identification of sgRNAs enriched in the screen. Fold enrichment represents the average of two independent experiments. Genes, including at least one sgRNA enriched in duplicate, are aligned in descending order of fold enrichment. Orange and green bars indicate that multiple sgRNAs were enriched for a gene, whereas blue bars indicate that a single sgRNA was enriched for a gene. The full raw dataset is shown in Data S1, S2, and S3. (B) Reproducibility of SubAB resistance conferred by individual sgRNAs. Each sgRNA was transduced into HeLa cells. Untransfected cells were excluded using puromycin selection, and successfully transfected cells were then treated for 24 h with SubAB at the indicated concentration (boxes) and further cultured for 4 days in the absence of the toxin. Viability was estimated using an 3-(4,5-Dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and is expressed as the percentage of the MTT value (OD570) in the absence of SubAB. The percentages shown are the mean percentages ± SD obtained from four independent experiments. The dotted line indicates the viability of mock-transfected cells treated with 0.3 ng/mL SubAB. The Holm-Bonferroni corrected t test was used for multiple comparisons. Asterisks denote statistical significance. (C) Fold enrichment of six sgRNAs in glycan-related and membrane-trafficking genes enriched in the SubAB screen compared with that of an STx screen (Yamaji et al., 2019). The heatmap is representative of individual sgRNA enrichment (sg1–6) in two independent experiments (groups #1 and #2). See also Figures S1 and S2, and Data S1, S2, and S3.

Figure 2
Figure 2

Both N- and O-glycans Serve as SubAB Receptors (A) SubAB sensitivity in glycan-gene KO cells. Cells were treated with SubAB as described in Figure 1B at the indicated concentration. Viability was estimated as described for Figure 1B and is expressed as the mean percentage of the MTT value (OD570) in the absence of SubAB. The percentages shown are the mean percentages ± SD obtained from three independent experiments. (B) BiP cleavage in glycan-gene KO cells. Cells were incubated with SubAB at the indicated concentration of SubAB for 12 h. SubAB-induced BiP cleavage was determined by immunoblots using anti-BiP monoclonal antibodies. GAPDH served as a loading control. Quantification of BiP uncleaved (78 kDa) by SubAB was performed by densitometry. The percentages shown are the mean percentages ± SD obtained from three independent experiments. The Bonferroni-corrected t test was used for multiple comparisons. *p < 0.017. (C) Surface binding of SubAB on glycan-gene KO cells. Cells were stained with (blue lines) or without (black lines) Alexa 488-labeled SubAB (Alexa 488-SubAB) and analyzed using FACS. Yellow-green lines in all panels indicate staining in parent cells. (D) Detection of SubAB-binding proteins in glycan gene KO cells. Biotinylated cell surface proteins prepared from the indicated cells were immunoprecipitated with heat-inactivated (HI) or native (N) SubAB as described in the Transparent Methods section. SubAB-binding proteins were detected with streptavidin-horseradish peroxidase (HRP). Data are representative of at least three separate experiments. See also Figure S2 and Data S4.

Figure 3
Figure 3

SLC39A9 Regulates the Receptor Expression (A) SubAB sensitivity in KDELR2, JTB, and SLC39A9 KO cells and their revertant cells. HA-tagged SLC39A9 was used in SLC39A9 revertant cells. Cells were treated with SubAB at the indicated concentrations. Viability was estimated as described for Figure 1B and is expressed as mean percentages ± SD obtained from three independent experiments. The Bonferroni corrected t test was used for multiple comparisons. *p < 0.0083. (B) BiP cleavage in KDELR2, JTB, and SLC39A9 KO cells. Cells were incubated with the indicated concentration of SubAB for 12 h. SubAB-induced BiP cleavage was determined as described for Figure 2B. Experiments were repeated three times with similar results (upper panel). Data are the mean percentages ± SD obtained from three independent experiments. The Bonferroni corrected t test was used for multiple comparisons. *p < 0.017. (C) Surface binding of SubAB on KDELR2, JTB, and SLC39A9 KO cells. Cells were stained with (blue lines) or without (black lines) Alexa 488-labeled SubAB (Alexa 488-SubAB) and analyzed using FACS. Yellow-green lines in all panels indicate staining in parent cells. A magenta line indicates staining in cDNA-restored cells. (D) Detection of SubAB-binding proteins in KDELR2, JTB, and SLC39A9 KO cells. In the left image, biotinylated cell surface proteins prepared from the indicated cells were immunoprecipitated and detected as described for Figure 2D. Data are representative of at least three separate experiments. In the right image, L1CAM proteins in whole lysates were detected with anti-L1CAM antibodies. See also Figure S3.

Figure 4
Figure 4

Alteration of N-glycans in SLC39A9 KO Cells Quantitative analysis of N-glycan species shown in KDELR2, JTB, and SLC39A9 KO cells, and their revertant cells. A schematic of N-glycan biosynthesis is shown in the upper section. Red boxes indicate glycan structure numbers as shown in Data S5. Pink boxes indicate statistically increased glycans, and gray boxes indicate statistically decreased glycans in SLC39A9 KO cells as illustrated in the graphs below. Glycans N3, N4, N5, and N7 may take one of two structures; therefore they are distinguished as N3-1, N3-2, N4-1, N4-2, etc. The graphs show the expression levels of respective N-glycan species compared among the indicated cells (#1–7): mean expression levels ± SD obtained from eight independent experiments. The full raw data set is shown in Data S5. The Bonferroni corrected t test was used for multiple comparisons. *p < 0.025; **p < 0.0083. See also Figures S3 and S4, and Data S5.

Figure 5
Figure 5

Alteration of O-glycans in SLC39A9 KO Cells Quantitative analysis of O-glycan species in the indicated cells shown in Figure 4. A schematic of O-glycan biosynthesis is shown in the upper section. Expression levels of respective O-glycan species were compared among the indicated cells (#1–7): mean expression levels SD obtained from eight independent experiments. The full raw dataset is shown in Data S5. The Bonferroni corrected t test was used for multiple comparisons. *p < 0.025; **p < 0.0083. See also Figure S3 and Data S5.

Figure 6
Figure 6

Loss of SLC39A9 Reduces C1GalT1 Proteins (A) Quantitative real-time PCR of C1GalT1 and MAN2A1 mRNAs in parent cells, SLC39A9 KO cells, wild-type SLC39A9-expressing SLC39A9 KO cells, and H155R mutant-expressing SLC39A9 KO cells. Relative mRNA levels of C1GalT1 and MAN2A1 are expressed as the percentage of the value in parent cells and represent the mean percentages ± SD obtained from three independent experiments. (B) Western blot analysis for C1GalT1, C1GalT1C1, and MAN2A1 proteins in parent cells, SLC39A9 KO cells, wild-type SLC39A9-expressing SLC39A9 KO cells, and H155R mutant-expressing SLC39A9 KO cells. The amounts of proteins were expressed as a percentage of the intensity of bands in parent cells: mean percentages ± SD. obtained from three independent experiments. The Bonferroni corrected t test was used for multiple comparisons. *p < 0.017. See also Figure S5.

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References

    1. Bassik M.C., Kampmann M., Lebbink R.J., Wang S., Hein M.Y., Poser I., Weibezahn J., Horlbeck M.A., Chen S., Mann M. A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. Cell. 2013;152:909–922. - PMC - PubMed
    2. Bassik, M.C., Kampmann, M., Lebbink, R.J., Wang, S., Hein, M.Y., Poser, I., Weibezahn, J., Horlbeck, M.A., Chen, S., Mann, M., et al. (2013). A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. Cell 152, 909-922. - PMC - PubMed
    1. Blackburn J.B., Lupashin V.V. Creating knockouts of conserved oligomeric Golgi complex subunits using CRISPR-mediated gene editing paired with a selection strategy based on glycosylation defects associated with impaired COG complex function. Methods Mol. Biol. 2016;1496:145–161. - PMC - PubMed
    2. Blackburn, J.B., and Lupashin, V.V. (2016). Creating knockouts of conserved oligomeric Golgi complex subunits using CRISPR-mediated gene editing paired with a selection strategy based on glycosylation defects associated with impaired COG complex function. Methods Mol. Biol. 1496, 145-161. - PMC - PubMed
    1. Blondel C.J., Park J.S., Hubbard T.P., Pacheco A.R., Kuehl C.J., Walsh M.J., Davis B.M., Gewurz B.E., Doench J.G., Waldor M.K. CRISPR/Cas9 screens reveal requirements for host cell sulfation and fucosylation in bacterial type III secretion system-mediated cytotoxicity. Cell Host Microbe. 2016;20:226–237. - PMC - PubMed
    2. Blondel, C.J., Park, J.S., Hubbard, T.P., Pacheco, A.R., Kuehl, C.J., Walsh, M.J., Davis, B.M., Gewurz, B.E., Doench, J.G., and Waldor, M.K. (2016). CRISPR/Cas9 screens reveal requirements for host cell sulfation and fucosylation in bacterial type III secretion system-mediated cytotoxicity. Cell Host Microbe. 20, 226-237. - PMC - PubMed
    1. Bonifacino J.S., Hierro A. Transport according to GARP: receiving retrograde cargo at the trans-Golgi network. Trends. Cell Biol. 2011;21:159–167. - PMC - PubMed
    2. Bonifacino, J.S., and Hierro, A. (2011). Transport according to GARP: receiving retrograde cargo at the trans-Golgi network. Trends. Cell Biol. 21, 159-167. - PMC - PubMed
    1. Bulldan A., Malviya V.N., Upmanyu N., Konrad L., Scheiner-Bobis G. Testosterone/bicalutamide antagonism at the predicted extracellular androgen binding site of ZIP9. Biochim. Biophys. Acta. 2017;1864:2402–2414. - PubMed
    2. Bulldan, A., Malviya, V.N., Upmanyu, N., Konrad, L., and Scheiner-Bobis, G. (2017). Testosterone/bicalutamide antagonism at the predicted extracellular androgen binding site of ZIP9. Biochim. Biophys. Acta 1864, 2402-2414. - PubMed