Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-κB and restricts

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1 In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 2 ARTICLE NUMBER: Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-κB and restricts bacterial proliferation Sjoerd J. L. van Wijk 1, 2, 7, 8, *, Franziska Fricke 3, *, Lina Herhaus 1, Jalaj Gupta 4, Katharina Hötte 5, Francesco Pampaloni 5, Paolo Grumati 1, Manuel Kaulich 1, Yu-shin Sou 6, Masaaki Komatsu 6, Florian R. Greten 4,7,8, Simone Fulda 2, 7, 8, Mike Heilemann 3, # and Ivan Dikic 1,5, # 1 Institute of Biochemistry II, Goethe University Medical School, Theodor-Stern-Kai 7, Frankfurt am Main, Germany 2 Institute for Experimental Cancer Research in Pediatrics, Goethe University, Komturstrasse 3a, Frankfurt am Main, Germany 3 Institute of Physical and Theoretical Chemistry, Goethe University, Max-von-Laue-Strasse 7, Frankfurt am Main, Germany 4 Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Paul-Ehrlich- Strasse 42-44, Frankfurt am Main, Germany 5 Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University, Max-von- Laue-Strasse 15, Frankfurt am Main, Germany 6 Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata , Japan 7 German Cancer Consortium (DKTK), Heidelberg, Germany 8 German Cancer Research Centre (DKFZ), Heidelberg, Germany * These authors contributed equally to this work NATURE MICROBIOLOGY DOI: /nmicrobiol Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

2 Supplementary Figures and Figure Legends Supplementary Figure 1 a, Representative laser scanning confocal images of comparative LAMP-1/GFP/DAPI imaging on intracellular S. Typhimurium. HeLa cells infected with S. Typhimurium strain SFH4 at 2 hour post-infection (hpi) were subjected to immunofluorescence using chain-specific antibodies against LAMP-1 (red). Cytosolic S. Typhimurium (GFP; green), HeLa nuclei and total intracellular bacteria were counterstained with DAPI (blue). Scale bar: 5 μm. b, Quantificationn of DAPI/ GFP/LAMP1-positive bacteria

3 at 2 hpi. Data are biological replicates from ~ 20 independent microscopic views (> 6000 intracellular bacteria, presented as means. c, Super-resolution images of M1 and K63 stained cells and intensity profiles along the blue dotted lines. Data are presented as intensities against distance in µm. d k, dstorm imaging reveals distinct spatial localization patterns of endogenous LRSAM1 and p62/sqstm1 on cytosolic S. Typhimurium. HeLa cells were infected with S. Typhimurium strain SFH4, fixed and subjected to immunostaining using primary antibodies recognizing endogenous LRSAM1 d-g and endogenous p62/sqstm1 h-k and AF647-conjugated secondary antibodies at 2 hpi. Representative conventional fluorescence: d, f, h, j, dstorm images: e, g, i, k. Scale bars: 1 μm. Results shown are representative of three biological replicates.

4 Supplementary Figure 2 a, OTULIN CRISPR HeLa cells were subjected d to S. Typhimurium infection and at 2 hpi washed with PBS and fixed with 4% paraformaldehyde (4% PFA) or 0.2% glutaraldehyde in 4% PFA (4% PFA / 0.2 % GA) for 200 minutes att room temperature. Cells were immunostained for linear Ubb (M1-AF647; magenta). Cytosolic S. Typhimurium (green), DNA was counterstained with DAPI (blue). Scale bar: : 5 μm. b, Representative laser scanning confocal images of M1/K633 ubiquitin chains co-localizingc g on cytosolic S. Typhimurium. HeLa cells infected with SFH4 at 2 hpi were subjected to immunofluorescence using chain-specific antibodies against linear Ub (M1-AF647; magenta) and K63 Ub (K63-

5 AF555; red). Shown are cytosolic S. Typhimurium (green) and DAPI (blue). Scale bar: 5 μm. c, Representative dual-colour dstorm imaging using chain-specific antibodies against M1- AF647 (orange) and K63-AF532 (blue) in HeLa cells subjected to SFH4 infection at 2 hpi. Boxed areas indicate co-localization of M1/K63 chains on cytosolic S. Typhimurium with a Mander s coefficient of 85-95% for both channels and regions. Scale bar: 1µm. Results shown are representative of three biological replicates.

6 Supplementary Figure 3 a, Representative confocal laser scanning images of HeLa cells transfected with indicated sirnas and subjected to S. Typhimurium infection and immunofluorescence using antibodies against total Ub (FK2; magenta; upper panels) and linear Ub (M1; magenta, lower panels). Cytosolic S. Typhimurium (green). Scale bar: 5 μm. b, Quantification of colocalization of FK2/GFP-positive S. Typhimurium (top) and M1/GFP- positive S. Typhimurium (bottom) using Pearson correlationn coefficients as quantitative measuree for signal accumulation at GFP-positive the indicated sirnas. ns: non-significant, * p < 0.05, unpaired, two-tailed Student s t-test. Data are biological replicates fromm 20 independent microscopic views (> 200 cells per condition), presented as means, error bars b indicatee s.d. c, Expression bacteria, ranging from 1 (anti correlated) to + 1 (perfect correlation) forr

7 analysis of (non-)induced OTULIN wild-type (WT) and C129S N-terminally 2 x Strep/HA (SSH)-tagged stable doxycycline (Dox)-inducible cell lines probed with antibodies against OTULIN and VINCULIN. Results shown are representative of two biological replicates. d, Representative confocal laser scanning images of S. Typhimurium-infected (non-) induced OTULIN wild-type (WT) and C129S stable doxycycline (Dox)-inducible cell lines at 2 hpi, immunostained with antibodies against linear Ub (M1, magenta), cytosolic S. Typhimurium (green) and DNA was counterstained with DAPI (blue). Scale bar: 10 μm. e, Quantification of the fraction M1/GFP-positive S. Typhimurium in S. Typhimurium-infected (non-)induced OTULIN wild-type (WT) and C129S stable doxycycline (Dox)-inducible cell lines at 2 hpi from Figure 2D. n.s.: non-significant, ** p < 0.01, *** p < 0.001, unpaired, two-tailed Student s t-test. Data are biological triplicates from > 175 cells per condition, presented as means, error bars indicate s.e.m.

$of the fraction M1/$ Typhimurium in S.

8 Supplementary Figure 4 a, Quantificati ion of the fraction M1/ /GFP-positive S. Typhimurium in S. Typhimurium-infected HeLa cells transfected with sicontrol or siotulin #2 and the indicated sirna-resistant OTULIN plasmids at 2 hpi. ns: non-significant, *** p < 0.001, unpaired, two-tailed Student s t-test. Data are biological duplicates from > 300 cells per condition, presented as means, error bars indicate s.e.m. b, Expression analysis of total t cell lysates from a, probed with antibodies against OTULIN and VINCULIN. V. Results shown are representative of two biological replicates.

$of the fraction$ HOIP/

9 Supplementary Figure 5 a,, Representative confocal laser scanning s images of OTULIN CRISPRR HeLa or wild-type ( WT) cells infected with S. Typhimurium strain SFH4 at 2 hpi, subjected to immunostaining with antibodies against endogenous HOIP (red) and linear Ub (M1; magenta). Cytosolic S. Typhimurium (green), DNA was w counterstained with DAPI (blue). Scale bar: 5 μm. b, Quantificationn of the fraction HOIP/ /GFP-positive S. Typhimurium in S. Typhimurium-infected OTULIN CRISPR HeLa or wild-type two-tailed Student s t-test. Data are (WT) cells at 2 hpi from Supplementary Figure 5A. ns: non-significant, unpaired, biological triplicates from > 150 cells per condition, presentedd as means,, error bars indicate s.e.m. c, Representative dual-colour dstorm imaging of endogenous linear Ub (M1-AF647;

10 orange) and HOIP (red) in S. Typhimurium-infected OTULIN CRISPR HeLa or wild-type (WT) cells at 2 hpi. Scale bar: 1µm. Results shown are representative of three biological replicates.

11 Supplementary Figure 6 a, Representative dual-colour dstorm imaging of endogenous linear Ub (M1-AF647; orange) and NEMO (AF532; blue) in S. S Typhimurium-infected HeLa cells transfected with the indicated sirnas at 2 hpi. Scale bar: 1 µm. b, Quantification of colocalization using Mander ss M1 coefficient between endogenous M1 Ubb and NEMO at the bacterial Ub coat in S. Typhimurium-infected HeLa cells transfected with the indicated sirnass at 2 hpi. Individual data points represent single bacteriaa in biological replicates. *** p < 0.001, Mann-Whitney-test. c, Representative dual-colour dstorm imaging of endogenous linear Ub (M1-AF647; orange) andd OPTINEURIN (OPTN) (AF532; blue) ) in S. Typhimurium-infected HeLa cells transfected with the indicatedd sirnas at 2 hpi. Scale bar: 1 µm. d, Quantification of colocalization using Mander s M1 coefficient between endogenous M1 Ub and OPTN at the bacterial Ub coat in S. Typhimurium-infected HeLa cells transfected

12 with the indicated sirnas at 2 hpi. Individual data points represent single bacteria in biological replicates. ** p < 0.01, *** p < 0.001, Mann-Whitney-test.

13 Supplementary Figure 7 a, Representative laser scanning confocal images of HeLa cells subjected to Salmonella infection and immunofluorescence using anti-ikkα/β-alexafluor555 (red) antibodies. Cytosolic S. Typhimurium (green), DNA was w counterstained with DAPI (blue). Scale bar: 5 μm. Results shown are representative of three biological replicates. b, Representative laser scanning confocal images of HeLa cells transiently expressing HA- infection and immunofluorescence using anti-ha/ /AlexaFluor555 antibodies. Cytosolic S. tagged IKKα (red; upper panels) and β (red; lower panels) were subjected d to S. Typhimurium Typhimurium (green), DNA was counterstained with DAPI (blue). Scale bar: 5 μm.. Results shown are representative of three biological replicates. c, c Representative dual-colour

14 dstorm imaging of endogenous linear Ub (M1-AF647; orange) and phosphorylated IKKα/β pser176/pser180 (AF532; blue) in S. Typhimurium-infected HeLa cells transfected with the indicated sirnas at 2 hpi. Scale bar: 1 μm. d, Quantification of colocalization using Mander s M1 coefficient between endogenous M1 Ub and phosphorylated IKKα/β pser176/pser180 at the bacterial Ub coat in S. Typhimurium-infected HeLa cells transfected with the indicated sirnas at 2 hpi. Individual data points represent single bacteria in biological replicates. * p < 0.05, ** p < 0.01, Mann-Whitney-test.

15 Supplementary Figure 8 a, Representative dual-colour dstorm imaging of endogenous K63-linked Ub (K63-AF647; orange) and NEMO (AF532; blue) in S. Typhimurium-infected b, Representative dual-colour dstorm imaging of endogenous K63-linked Ub U (K63-AF647; orange) and HeLa cells transfected with the indicatedd sirnas at 2 hpi. Scale bar: 1 µm. OPTN (AF532; blue) in S. Typhimurium-infected HeLa cells transfectedd with the indicated sirnass at 2 hpi. Scale bar: 1 µm. c,, Representative dual-colouorange) and phosphorylated IKKα/β dstorm imaging of endogenous K63-linked Ub (K63-AF647; pser176/pser180 (AF532; blue) in S. Typhimurium-infected HeLa H cells transfected with the

indicated sirnas at 2 hpi. Scale bar: 1 µm.

Typhimurium (SFH4) proliferation in indicated cell types

Equal numbers of mammalian cells subjected s too infection

1 % Triton X-100 in PBS and colonies were counted overnight

Data are biologicall triplicates,, presented as mean colony

wild-type sicontrol cells b, Quantification of intracellular

OTULIN CRISPR cells treatedd with control and OTULIN sirna

16 indicated sirnas at 2 hpi. Scale bar: 1 µm. Results shown are representative of three biological replicates. Supplementary Figure 9 a, Quantification of intracellular S. Typhimurium (SFH4) proliferation in indicated cell types treated with indicated sirna at 0.5 hpi and 6 hpi. Equal numbers of mammalian cells subjected s too infection were lysed at 2 hpi in 0.1 % Triton X-100 in PBS and colonies were counted overnight on plated dilutions on LB plates in duplicate. Data are biologicall triplicates,, presented as mean colony forming units (CFUs) per well, error bars indicate s.e.m, * p < 0.05, ** p < 0.01, unpaired, two-tailed Student ss t-test, compared to wild-type sicontrol cells b, Quantification of intracellular S. Typhimurium (SFH4) proliferation in indicated cell types treated with indicated sirna at 0.5 hpi and 6 hpi. Equal numbers of mammalian cells subjected s too infection were lysed at 2 hpi in 0.1 % Triton X-100 in PBS and colonies were counted overnight on plated dilutions on LB plates in duplicate. Data are biologicall triplicates,, presented as mean colony forming units (CFUs) per well, error bars indicate s.e.m, * p < 0.05, ** p < 0,01, unpaired, two-tailed Student ss t-test, compared to wild-type sicontrol cells or between OTULIN CRISPR cells treatedd with control and OTULIN sirna (indicated by black line) ).

17 Supplementary Figure 10 a, Schematic representation of the targeting vector and the targeted allele of the Fam105b/Otulin gene. The coding exons numbered in accordance with the initiation site as exon 1 are depicted by boxes with oblique lines. l LoxP and FRT sequences are indicated. Neo, neomycinn resistance gene cassette; DT-A,, Diphtheriaa toxin fragment A gene cassette. b, Genotyping of wild-type, heterozygotic and homozygotic Otulin mice. c, Expression analysis of intestinal crypt organoids derived from Otulin O fl/fl and Otulin fl/ /fl; CAG- Cre-ER+/- mice were treated with 4-hydroxytamoxifen (4-OHT; 1 mm) for 36 hours. Total protein lysates were resolved on SDS-PAGE and immunoblotted using g antibodiess against

18 Otulin and β-actin. d, Representative bright-field microscopic images of intestinal crypt organoids derived from Otulin fl/fl and Otulin fl/fl; CAG-Cre-ER+/- mice treated with 4- hydroxytamoxifen (4-OHT; 1 mm) or vehicle control (ethanol; EtOH) for 36 hours. Results shown are representative of at least two biological replicates.

Supplementary Figure 11 a, Quantification of mean cytosolic S.

Data are biological triplicatess from in total 30 independent microscopic

s.e.m, and OTULIN CRISPR HeLa cells subjected to S.

subjected to S. Typhimurium infection at 2 hpi.

1 % Triton X-100 in PBS and colonies weree counted overnight onn plated

19 Supplementary Figure 11 a, Quantification of mean cytosolic S. Typhimuriumm (GFP intensity) per cell in wild-type at 2 hpi. Data are biological triplicatess from in total 30 independent microscopic views (> 200 cells per condition), presented as means, error bars indicate s.e.m, and OTULIN CRISPR HeLa cells subjected to S. Typhimurium infection *** p < 0.001, unpaired, two-tailed Student s t-test. b, Quantification of intracellular S. Typhimurium proliferation in wild-type and OTULIN CRISPR HeLa cells subjected to S. Typhimurium infection at 2 hpi. Equal numbers of cells subjected to infection were lysed at 2 hpi in 0.1 % Triton X-100 in PBS and colonies weree counted overnight onn plated dilutions on LB plates in duplicate. Data are biological triplicates, presentedd as mean colony forming units (CFUs) per well, error bars indicate s.e.m, *** p < 0.001, unpaired, two-tailed Student s t-test.

20 c, Relative surface area of cytosolic (GFP-positive) Salmonella upon infection in EtOH and 4- OHT (Tam)-treated intestinal crypt organoids. Data are biological replicates (> 5 organoids), presented in boxplots (box contains 50 % of data points, middle line is median, whiskers and outliers represent the upper and lower 25 % of data, outliers are outside the 1.5 x interquartile range), * p < 0.05, Wilcoxon-Mann-Whitney test.

21 Supplementary Figure 12 Uncropped Western blots, brackets indicate lanes that were used in figures.

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:10.1038/nature11419 Supplementary Figure 1 Schematic representation of innate immune signaling pathways induced by intracellular Salmonella in cultured macrophages. a, During the infection Salmonella