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Kinase-independent synthesis of 3-phosphorylated phosphoinositides by a phosphotransferase

Nature Cell Biology volume 24, pages 708–722 (2022)Cite this article

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Abstract

Despite their low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival in host organisms. We demonstrate that PtdIns(3,4)P2 is a major product generated in host cells by the effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4,5)P3 and PtdIns(3,4)P2 from PtdIns(4,5)P2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signalling to gain entry and even provoke oncogenic transformation.

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Fig. 1: Rapid and sustained PtdIns(3,4)P2 synthesis during Salmonella entry and maturation.
Fig. 2: SopB is necessary and sufficient for PtdIns(3,4)P2 biosynthesis in mammalian cells.
Fig. 3: Class I PI3Ks and class II PI3K-C2α are not required for PtdIns(3,4)P2 synthesis during Salmonella entry.
Fig. 4: PtdIns(3,4)P2 generation in S. cerevisiae that are devoid of PI3K activity.
Fig. 5: PtdIns(4,5)P2 levels correlate inversely with SopB-mediated PtdIns(3,4)P2 formation.
Fig. 6: SopB requires a PLC-sensitive inositide to generate PtdIns(3,4)P2.
Fig. 7: In vitro reconstitution reveals SopB phosphotransferase activity.
Fig. 8: Time-resolved HPLC–MS analysis of SopB phosphotransferase-phosphatase activities.

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Data availability

Data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

Custom macros used in this study have been deposited on GitHub and are available for download at the manuscript repository (https://github.com/walpoleg/Walpole-et-al-Nature-Cell-Biology-2022).

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Acknowledgements

We thank S. Emr (Department of Molecular Biology and Genetics, Cornell University) for sharing plasmids and yeast strains for this study. We thank K. Lau and P. Paroutis (The Imaging Facility, The Hospital for Sick Children) for technical training and discussions of analyses. Models (Figs. 1c,2d,3a,3h,8i and Extended Data Figs. 3a,3b,4b,8e) were created with BioRender.com. G.F.W.W. is supported by a Vanier Canada Graduate Scholarship from the Canadian Institutes of Health Research (CIHR) and an MD/PhD Studentship from the University of Toronto. J.H.B. is supported by CIHR grant no. FDN-154329. A.D. is funded by grant no. R01GM132565 from the National Institutes of Health. G.R.V.H. is funded by grant no. 1R35GM119412-01 from the National Institutes of Health. S.G. is supported by CIHR grant no. FDN-143202, and G.D.F. is supported by CIHR project grant no. PJT165968 and the Natural Sciences and Engineering Research Council (NSERC) of Canada.

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Authors and Affiliations

  1. Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada

    Glenn F. W. Walpole, Fernando Montaño-Rendón, Zetao Liu, Hongxian Zhu, John H. Brumell & Sergio Grinstein

  2. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Glenn F. W. Walpole, Zetao Liu, Sergio Grinstein & Gregory D. Fairn

  3. Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Jonathan Pacheco & Gerald R. V. Hammond

  4. Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Ontario, Canada

    Neha Chauhan, Sergio Grinstein & Gregory D. Fairn

  5. Signalling Programme, Babraham Institute, Cambridge, UK

    Jonathan Clark, Karen E. Anderson, Len R. Stephens & Phillip T. Hawkins

  6. Molecular Medicine Program, Hospital for Sick Children, Toronto, Ontario, Canada

    Yazan M. Abbas

  7. Clinical Microbiology, Health Sciences North–Horizon Santé-Nord, Sudbury, Ontario, Canada

    Danielle Brabant-Kirwan

  8. Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada

    Fernando Montaño-Rendón, John H. Brumell, Sergio Grinstein & Gregory D. Fairn

  9. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Hongxian Zhu & John H. Brumell

  10. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA

    Alexander Deiters

  11. Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada

    Gregory D. Fairn

Authors
  1. Glenn F. W. Walpole
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  2. Jonathan Pacheco
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  3. Neha Chauhan
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  4. Jonathan Clark
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  5. Karen E. Anderson
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  6. Yazan M. Abbas
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  7. Danielle Brabant-Kirwan
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  8. Fernando Montaño-Rendón
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  9. Zetao Liu
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  10. Hongxian Zhu
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  11. John H. Brumell
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  12. Alexander Deiters
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  13. Len R. Stephens
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  14. Phillip T. Hawkins
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  15. Gerald R. V. Hammond
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  16. Sergio Grinstein
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  17. Gregory D. Fairn
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Contributions

G.F.W.W., S.G., G.R.V.H. and G.D.F. conceived experiments and developed methods. G.F.W.W. conducted experiments and analysed the resulting data. J.P. conducted photoactivation assays and analysed the resulting data with G.R.V.H. K.E.A. and J.C. performed HPLC–MS measurements and analysed the resulting data with L.R.S. and P.T.H. D.B.-K. performed invasion efficiency assays. N.C., Y.M.A., F.M.-R., Z.L., H.Z., J.H.B. and A.D. made and/or provided critical reagents. G.F.W.W. wrote the original draft of the manuscript. All authors reviewed and edited the manuscript.

Corresponding authors

Correspondence to Sergio Grinstein or Gregory D. Fairn.

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The authors declare no competing interests.

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Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Rapid and sustained PtdIns(3,4)P2 synthesis during Salmonella entry and maturation.

(a) Model of PtdIns(3,4)P2 biosensors based on single, double-, or triple-tandem carboxy-terminal PH domains from TAPP1 (PLEKHA1). NES, nuclear export signal. Right, gel electrophoresis of PCR amplicons generated by primers that house the open reading frames. (b) Cells expressing cPHx1, cPHx2, or cPHx3 were infected for 10 min with wild-type Salmonella prior to staining the PM with CellMask. Representative maximum intensity projections (main) and a corresponding confocal section of the invasion ruffle (bottom panels) are presented. (c) Confocal imaging of cells (three examples in I, II, and III) expressing cPHx3 during invasion by wild-type Salmonella. Bottom vertical panels are expanded from the white box region and correspond to the minute-by-minute time series. cPHx3 is also presented in a grey inverted lookup table (RGB intensity 0=white, 255=black). (d) As in (c), three examples (I, II, and III) of cells expressing the PtdIns(3,4,5)P3 sensor aPHx2 during invasion by wild-type Salmonella. (e) Cells serum-starved for 3 h were infected by Salmonella. Extracellular bacteria were removed, and cells returned to serum-free medium containing gentamycin. PM cPHx3 intensities were quantified in the following number of cells: 59 (control), 60 (WT, 30 min), 42 (∆sopB, 30 min), 58 (WT, 60 min), 53 (∆sopB, 60 min), 75 (WT, 120 min), 48 (∆sopB, 120 min), 66 (WT, 240 min), and 36 (∆sopB, 240 min) across n=3 independent experiments. Data are trial means ± s.e.m. (foreground) overlaid on cell measurements (background). Data from Fig. 1c are presented with ∆sopB infections. ****P < 0.0001; **P = 0.0054 (UI-vs-WT60), **P = 0.0044 (WT120-vs-∆sopB120); ns, not significant. (f) Cells serum-starved for 3 hours, were exposed to Salmonella (wild-type or ∆sopB) for 10 min. Extracellular bacteria were removed, and cells were returned to serum-free medium with gentamycin. Lysates were collected at the indicated time points and analysed on parallel membranes for pAKT (S473) or pAKT (T308) prior to stripping and re-probing for total AKT or GAPDH (loading control). Representative immunoblots are presented from n=2 independent experiments. UI, uninfected. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 PtdIns(3,4,5)P3 analysis during Salmonella invasion or during optogenetic activation of SopB.

(a) Model of the PtdIns(3,4,5)P3 biosensor NES-EGFP-aPHx2 derived from tandem ARNO PH domains (2G splice variant, I303E mutation). NES, nuclear export signal. (b) Cells were exposed to invasive RFP-expressing wild-type or isogenic ∆sopB Salmonella and the PM was stained with CellMask prior to imaging. Maximum intensity projections (main) and single confocal sections of the invasion ruffle are presented at right for each. (c) Quantification from (b). Normalized intensity of aPHx2 in the PM of invasion sites from n=3 independent experiments analysing 71 (WT) and 63 (∆sopB) invasion sites. *P = 0.0294. (d) Normalized intensity of aPHx2 in the PM during optogenetic activation of SopBWT-464TAG or SopBC460S-464TAG. Filtered baseline-corrected time-lapse data are mean ± s.e.m. of individual cell measurements from 3 independent experiments quantifying n=29 cells (WT) and n=13 cells (C460S). (e) AUC calculations of aPHx2 intensities from (d). Data are median, box (25th-75th) and whisker (10th-90th) percentiles of n=29 cells (WT) and n=13 cells (C460S) from 3 independent experiments. P = 0.4415, ns. (f,g) SopB-mediated synthesis of PtdIns(3,4)P2 is not accompanied by a robust PtdIns(3,4,5)P3 response. Representative confocal sections of aPHx2 and cPHx3 localization during optogenetic activation of WT or C460S SopB. Left, corresponding linear RGB intensity scales are presented. The indicated times are prior to (t - 30 s) or after illumination with 405 nm light to photolyze hydroxycoumarin lysine in SopB. See corresponding quantification of (f) presented in Fig. 2g. Source numerical data are available in source data.

Source data

Extended Data Fig. 3 Sequence determinants of SopB-mediated PtdIns(3,4)P2 generation.

(a) Bacterially injected SopB localizes to the vacuolar membrane and PM invaginations. HeLa cells were exposed to ∆sopB (control) or ∆sopB + SopB-c-myc Salmonella for 10 min prior to labelling the PM (WGA) and immunostaining against c-myc and Salmonella. Inset panels are expanded from the hashed box region. Representative maximum intensity projections are from n=3 independent experiments. (b) Anti-myc fluorescence is not contaminated by non-secreted SopB or anti-Salmonella immunostaining. ∆sopB + SopB-c-myc Salmonella were centrifuged onto poly-L-lysine coated coverslips. Bacteria were processed as in (a) and imaged with equivalent acquisition settings. The representative maximum intensity projection is from n=3 independent experiments. (c) Heterologous SopB coalesces within puncta that abut cortical membranes. Full-length SopB constructs were co-transfected with cPHx3 and imaged live. Amino-terminally tagged SopB was enriched along cytosolic reticular structures; amino- and carboxy-terminal EGFP fusions also enriched on the basal footprint of cells. Representative confocal sections are presented for each construct, from more than three similar experiments. CellMask served as a PM marker. (d) SopB requires amino acids 68-172 and 520-554 for PtdIns(3,4)P2 generation. The indicated SopB plasmids (top panels) were co-transfected with cPHx3 (bottom panels) and imaged live in HeLa cells. Representative confocal sections are presented. Note that membrane-targeting of SopB is disrupted following the deletion of amino acids 68-172 while PM-targeting is preserved following the deletion of amino acids 520-554. (e) CRISPR–Cas9-mediated deletion of Cdc42. Total lysates from control sgRNA- or Cdc42-specific sgRNA-treated Henle 407 cells were immunoblotted against Cdc42. Alpha tubulin served as a loading control. (f) Cdc42 regulates SopB targeting but is not strictly required for PtdIns(3,4)P2 generation. Representative maximum intensity projections are presented of SopB (1-561)-EGFP localization in parental wild-type Henle (top) or Cdc42 KO Henle cells (bottom). Inset images depict localization of cPHx3 (confocal section). cPHx3 was markedly enriched in the PM with a concomitant decrease in cytosolic fluorescence in (mean ± s.e.m.) 94.1 ± 2.92% of parental WT and 88.5 ± 7.28% of Cdc42 KO cells across n=3 independent experiments. (g) Quantification of SopB-EGFP puncta per cell from n=3 independent experiments analysing the following number of Henle 407 cells: 91 (WT) and 59 (Cdc42 KO). Data are trial means ± s.e.m. (foreground) overlaid on cell measurements (grey, background). **P = 0.0014. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 3-phosphorylated phosphoinositides promote bacterial invasion of host cells.

(a) Cells were exposed to wild-type or ∆sopB Salmonella for 10 min and extracellular bacteria were removed by extensive washing before returning to growth medium for an additional 20 min. Extracellular and intracellular bacteria were differentially stained. Data are mean ± s.e.m. from n=3 independent experiments quantifying ≥300 infected cells per strain. *P = 0.0333. (b,c) PtdIns(3,4)P2 and/or PtdIns(3,4,5)P2 promote bacterial invasion. (b) Model of PTEN-catalysed reactions: membrane-associated PTEN dephosphorylates both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 at the 3-position of the inositol ring. (c) Cells transfected with EGFP (control) or the A4 mutant of PTEN were left untreated or pre-treated with LY294002 (10 μM, 30 min) prior to invasion. Twofold excess of ∆sopE/sopE2 bacteria was used to obtain sufficient infected cells. Data are mean ± s.e.m. (normalized to respective control) from n=3 independent experiments quantifying ≥300 infected cells per strain. WT: **P = 0.0022, ***P = 0.0007; ∆sopE/E2: *P = 0.0125, **P = 0.0039. (d) Cells were co-transfected with cPHx3 and PM-targeted catalytic subunit of class IA PI3K (p110α-CAAX). Images were acquired immediately before (left) or 3 min after addition of DMSO (vehicle, right). (e) HeLa cells expressing vector control or p110α-CAAX were treated for 20 min with DMSO, wortmannin (100 nM), PI-103 (500 nM), or GDC-0941 (500 nM) prior to collection of cell lysates and immunoblotting using phospho-AKT (S473) and pan AKT antibodies. The immunoblot presented is representative of n=3 independent experiments. Corresponding quantification, Fig. 3c. (f) Confirmation of anti-PI3K-C2α polyclonal antibody labelling of heterologously-expressed PI3K-C2α. HeLa cells transfected with PM-targeted myc-PI3K-C2α were co-stained for the myc epitope tag and PI3K-C2α. Panels right depict enlargement of hashed box regions (I, II), where the non-transfected cells (I) were overexposed to visualize endogenous staining. (g) Class II PI3K-C2α is not enriched at the site of Salmonella invasion. Confocal sections of uninfected and wild-type Salmonella infected HeLa cells stained for endogenous PI3K-C2α. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 5 Lysine residues within the carboxyl-terminus of SopB support optimal phosphotransferase activity.

(a) Alignment of SopB amino acids 504-554 with other PtdIns(4,5)P2 and PtdIns(3,4,5)P3, 5-phosphatases. Basic residues in SopB are highlighted red along with conserved basic residues in the other phosphatases; additional conserved residues are highlighted purple, and regions of highest similarity are boxed (grey). Notably, residues that share sequence similarity with Mus musculus Synaptojanin-1 (SYNJ1) 534-584 fall outside of the SYNJ1 5-phosphatase domain and have not been implicated in its catalytic activity. Local homology between SopB and INPP5B or OCRL failed to be identified by NCBI BLAST. (b) Mutation of lysines 525 and 528 blunt PtdIns(3,4)P2 generation by SopB. Wild-type, K525A, K528A, and C460S SopB amino-terminally tagged with EGFP (bottom panels) were transiently expressed with cPHx3 (inverted grey, main panels). The PM was stained with CellMask before imaging live. Representative confocal sections are presented for each. Note that expression of the K528A mutant led to notable perturbation of cellular morphology including rounding, cortical membrane ‘crumpling’, and blebbing. (c) Normalized intensity of PM cPHx3 from (b) was quantified across n=3 independent experiments (WT, 78 cells; K525A, 81 cells; K528A, 86 cells; C460S, 57 cells). Data are trial means ± s.e.m. (foreground) overlaid on cell measurements (grey, background). ****P < 0.0001. (d) cPHx3 intensities from K528A- and C460S SopB-transfected cells (red box, panel c) plotted on an expanded axis. Subtle PM-enrichment of cPHx3 was evident in K528A-transfected cells relative to the C460S-transfected mutant. Data are trial means ± s.e.m. (foreground) overlaid on cell measurements (grey, background). **P = 0.0017. (e,f) Lysine 525 and 528 of SopB are structurally predicted to neighbour the C(X)5R motif. The primary sequence of SopB (residues 1-561) was analysed by RoseTTAFold and the resulting highest ranked model is presented in the surface filling view (grey). The location of lysine 525 and 528 (green) and the C(X)5R motif of SopB (blue) are annotated. Source numerical data are available in source data.

Source data

Extended Data Fig. 6 Generation of PtdIns(3,4)P2 by the S. flexneri effector IpgD does not require class I or class II PI3Ks.

(a) Sequence alignment of the phosphate-binding (P-loop) sequence from SopB (Salmonella enterica) and IpgD (Shigella flexneri). Residue 439 of IpgD encodes the cysteine of the C(X)5R motif. (b) IpgD reduces PM PtdIns(4,5)P2 levels in a C(X)5R-dependent manner. Heterologous expression of EGFP-IpgD (WT or C439S) and PH-PLCδ1 (inverted grey, main panels). The PM was stained with CellMask prior to imaging live. (c) Quantification of PH-PLCδ1 intensity in the PM from (b) across n=3 independent experiments quantifying 82 cells (IpgDWT) and 74 cells (IpgDC439S. Data are trial means ± s.e.m. (foreground) overlaid on cell measurements (grey, background). ****P < 0.0001. (d) Live cell imaging of heterologously-expressed EGFP-IpgD (WT or C439S) and NES-mCherry-cPHx3 following PM staining with CellMask. Cells were treated with the indicated inhibitors (PI-103, 500 nM; wortmannin, 100 nM) for 20 min prior to and throughout imaging. (e) PM cPHx3 intensity was quantified from (d) across 3 independent trials (IpgDWT, 70 cells; IpgDC439S, 62 cells; IpgDWT(PI-103), 68 cells; IpgDWT(Wortmannin), 76 cells). Data are trial means ± s.e.m. (foreground) overlaid on cell measurements (grey, background). ****P < 0.0001. (f) PM PtdIns(3,4)P2 synthesis in S. cerevisiae. Galactose-inducible empty vector (control), IpgDWT, or IpgDC439S were induced for 2 hours in yeast that expressed cPHx3. Trypan Blue staining demarcates the cell wall and non-viable yeast. (g) Yeast from (f) were scored for plasmalemmal cPHx3 localization across n=4 independent experiments analysing the following number of yeast: control, 881 cells; IpgDWT, 1165 cells; IpgDC439S, 938 cells. Data are mean ± s.e.m. ****P < 0.0001. Source numerical data are available in source data.

Source data

Extended Data Fig. 7 SopB requires an INPP5E-sensitive plasmalemmal inositide for the generation of PtdIns(3,4)P2.

(a) Inhibition of PtdIns(3,4)P2 generated during invasion by pre-recruitment of INPP5E. HeLa cells expressing mRFP-FKBP-INPP5E (WT or D556A) together with PM-targeted Lyn(11)-FRB and the biosensor cPHx1 (grey) were treated with 1 µM rapamycin for 2 min before addition of wild-type Salmonella (BFP, red) for an additional 10 min. The PM was stained (CellMask) and cells fixed before imaging. Maximum intensity projections (main panels) and confocal sections of the boxed region (bottom panels) are presented. Re-localization of mRFP-FKBP-conjugates to the PM is depicted in the bottom panels. (b) Normalized PM intensity of cPHx1 was quantified from part (a) across n=2 independent trials (INPP5EWT, 90 cells; INPP5ED556A, 85 cells). Data are trial means ± s.e.m. (foreground) overlaid on cell measurements (grey, background). **P = 0.0019. (c) Comparison of PtdIns(4,5)P2 depletion by chemically induced recruitment of PLCβ3 and INPP5E. Normalized PM intensity of PH-PLCδ1 was quantified from n=3 independent experiments (control, 55 cells; PLCβ3, 56 cells; INPP5E, 74 cells). Data from Fig. 6a,b are re-plotted with INPP5E (acquired in parallel) and support that INPP5E results in a modest PtdIns(4,5)P2-depletion relative to PLCβ3. Data are trial means ± s.e.m. (foreground) overlaid on cell measurements (grey, background). ***P = 0.0002, *P = 0.0477. (d) PtdIns(4,5)P2-depletion by INPP5E recruitment depends on its catalytic activity. Comparison of HeLa cells co-transfected as in (a) but expressing the biosensor PH-PLCδ1-EGFP. Representative confocal sections are presented following 5-min rapamycin treatment (1 µM) and PM staining with CellMask. (e,f) Chemically induced INPP5E recruitment modestly increases PM PtdIns(4)P. HeLa cells expressing mRFP-FKBP-(control), -PLCβ3, or -INPP5E (wild-type) together with PM-targeted Lyn(11)-FRB and EGFP-2xP4M were imaged live 5 min after treatment with 1 µM rapamycin and staining the PM (CellMask). (e) The normalized PM intensity of 2xP4M was quantified from n=3 independent experiments (control, 55 cells; PLCβ3, 41 cells; INPP5E, 53 cells). Data are trial means ± s.e.m. (foreground) overlaid on cell measurements (grey, background). **P = 0.0020. (f) Representative confocal sections of each channel are presented. Source numerical data are available in source data.

Source data

Extended Data Fig. 8 Time-resolved HPLC–MS analysis of SopB phosphotransferase-phosphatase activities.

(a) Inorganic phosphate release assessed following treatment of liposomes with 2.5 µg recombinant SopBWT or SopBC460S. Liposome composition was (mol%) POPS:PtdIns(4,5)P2 (90:10) and 2.5 nmol PtdIns(4,5)P2 was provided per reaction. At the indicated time points, reactions were terminated by addition of 50 mM NEM. Data are mean ± s.e.m. of duplicate wells. Time 0 min corresponds to a no enzyme control. (b) Separation of PtdIns(3,4)P2 and PtdIns(4,5)P2 regio-isomers in a sample treated with SopBWT. An example HPLC–MS trace derived from LUVs treated with 0.5 µg (71.5 nM) SopBWT for 5 min. Note the appearance of PtdIns(3,4)P2 at ≈20.6 min. (c) Model of the PtdIns(3,4,5)P3 biosensor designed with tandem Bruton’s Tyrosine Kinase (BTK) PH domains (bPHx2). Each component was separated by flexible, serine- and glycine-rich linker sequences. NES, nuclear export signal. (d) Heterologous expression of wild-type SopB induces PM-translocation of bPHx2. Representative confocal micrographs of mCherry-tagged bPHx2 (inverted grey) co-transfected with EGFP-SopBC460S or EGFP-SopBWT. (e) Hypothesized PPIns conversions catalysed by SopB and potential modulation by host phosphatases. In the presence of PtdIns(4,5)P2 in vitro, SopB generates the species PtdIns(3,4,5)P3, PtdIns(3,4)P2, and PtdIns(3)P. The latter species are hypothesized to arise, at least in part, by sequential dephosphorylation of PtdIns(3,4,5)P3. In vivo, Salmonella infection favours the accumulation of PtdIns(3,4)P2 likely due to the high basal activity of host 5-phosphatases (INPP5B, SYNJ1/2, SHIP1/2, and others) that rapidly convert PtdIns(3,4,5)P3 to PtdIns(3,4)P2. It remains unclear if the rapid clearance of PtdIns(3,4)P2 following fission of the Salmonella-containing vacuole from the PM is due to the intrinsic activity of SopB or to the activity of host 4-phosphatases (INPP4A/B). Nonetheless, SopB is sufficient to give rise to PtdIns(3)P in vitro from PtdIns(4,5)P2, arguing for a second –likely minor– pathway to generate this inositide in addition to Vps34-mediated synthesis on the bacterial vacuole (Mallo et. al., 2008). Finally, phosphatidylinositol may arise by direct dephosphorylation of the 4- and 5-positions of PtdIns(4,5)P2 by SopB, or indirectly by dephosphorylation of products of the phosphotransferase reaction. Source numerical data are available in source data.

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Supplementary information

Reporting Summary

Peer Review File

Supplementary Table

Table 1. Plasmids used in this study. Table 2. Reagents used in this study. Table 3. Antibodies used in this study. Table 4. sgRNA oligonucleotides used in this study.

Supplementary Video 1

Live-cell imaging of cPHx3 (grey, left) expressed in HeLa cells during invasion by RFP-expressing Salmonella (red on left). Frames were acquired at intervals of 30 s and are presented as maximum intensity projections of stacks of serial confocal images with a 12 f.p.s. playback. cPHx3 was pseudo-coloured (right) to ease visualization, according to the inset calibration bar. See corresponding insets in Fig. 1a.

Supplementary Video 2

Time-lapse of fluorescence imaging of cPHx3 (grey, left) expressed during invasion by RFP-expressing Salmonella (red, left) delivered at high (>10) multiplicity of infection. Frames were acquired at intervals of 1 min and single confocal sections are presented at 6 f.p.s. playback. cPHx3 was pseudo-coloured (right) to ease visualization according to the inset calibration bar. See additional examples in Extended Data Fig. 1a.

Supplementary Video 3

Time-lapse of fluorescence imaging of aPHx2 (grey) expressed during invasion by wild-type RFP-expressing Salmonella (red on left). Frames were acquired at intervals of 1 min and are presented at a 6 f.p.s. playback. Merged confocal sections are presented on the left and aPHx2 is pseudo-coloured on the right, as indicated by the inset calibration bar. Additional examples are provided in Extended Data Fig. 1d.

Supplementary Video 4

Confocal time-lapse of NES-iRFP-cPHx3 (grey) during optogenetic activation of EGFP-SopBWT-464-TAG. The 405-nm illumination begins at frame 15 (30 s) and is maintained for the duration of the video (5:29). Captures were made at intervals of 2.3 s and are present at a 60 f.p.s. playback. See the corresponding panels and quantification in Fig. 2.

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Walpole, G.F.W., Pacheco, J., Chauhan, N. et al. Kinase-independent synthesis of 3-phosphorylated phosphoinositides by a phosphotransferase. Nat Cell Biol 24, 708–722 (2022). https://doi.org/10.1038/s41556-022-00895-y

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