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1 ARTICLE NUMBER: DOI: /NMICROBIOL Intermediate filaments enable pathogen docking to trigger type 3 effector translocation Brian C. Russo, Luisa M. Stamm, Matthijs Raaben, Caleb M. Kim, Emily Kahoud, Lindsey R. Robinson, Sayantan Bose, Ana L. Queiroz, Bobby Brooke Herrera, Leigh A. Baxt, Nirit Mor-Vaknin, Yang Fu, Gabriel Molina, David M. Markovitz, Sean P. Whelan, Marcia B. Goldberg NATURE MICROBIOLOGY 1

2 DOI: /NMICROBIOL Supplementary Tables Supplementary Table 2: Strains and plasmids. Reference Genotype or description or source Strains S. flexneri WT Serotype 2a strain 2457T 1 BS T cured of its virulence 2 plasmid S. flexneri ipac 2457T ipac::kan This Study S. Typhimurium WT SL1344 S.T. invg SL1344 invg 3 Y. psuedotuberculosis WT IP32953 Gift of J. Mecsas Y. pseudotuberculosis yopb IP32953 yopb Gift of J. Mecsas Y. pseudotuberculosis effectors IP2666 yophemojn 4 Y. pseudotuberculosis effectors IP2666 yophemojn ΔyopD 4 yopd Saccharomyces cerevisiae S288C Strain used for yeast cloning Gift of C. Lesser DH10B Invitrogen DH10B psft3ss pmt3ss, png162-virb, Spec r, Tet r, Kan r 5 Bacterial plasmids psft3ss Spec r, Tet r 5 pbad33 Cm r ATCC pipac pbad33 ipac, Cm r This Study pipac R362E pbad33 ipac R362E, Cm r This Study pipac R362L pbad33 ipac R362L, Cm r This Study pipac R362W pbad33 ipac R362W, Cm r This Study pipac S349A pbad33 ipac S349A, Cm r This Study pospb-tem pdsw206 ospb-tem β- lactamase, Amp r Gift of C. Lesser pospb-flag pdsw206 ospb-flag, Amp r Gift of C. Lesser pyope pyope TEM β-lactamase, Kan r Gift of J. Mecsas psope pdsw206-sope1-tem β- lactamase, Amp r Gift of C. Lesser pafa-1 pbr322-afa-1, Amp r 6 Yeast plasmids Vimentin pag416-vimentin This Study Keratin 8 pag415-keratin 8 This Study Keratin 18 pag415-keratin18 This Study IpaC WT pag413-ipac WT This Study IpaC R362E pag413-ipac R362E This Study IpaC R326L pag413-ipac R362L This Study IpaC R362W pag413-ipac R362W This Study IpaC S349A pag413-ipac S349A This Study SipC pag413-sipc This Study YopD pag413-yopd This Study IpaB pag415-ipab This Study ipgc pag415-ipgc This Study 2 NATURE MICROBIOLOGY

3 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figures Figure S1: Genetic selection of human-derived haploid cells identifies vimentin. (a) Hap-1 cell viability at 24 hours of infection with WT, WT pafa-1, or non-invasive NATURE MICROBIOLOGY 3

4 DOI: /NMICROBIOL (BS103) S. flexneri. Data depicted are the mean ± SEM of independent experiments. Uninfected, n=7; S.f. WT, n=6; S.f. WT pafa-1, n=2; S.f. non-invasive, n=3, where n represents the number of independent experimental assays. (b) Hap-1 cell viability following serial infections with S. flexneri producing the Afa-1 adhesin, which was used for the genome-wide selection. In the experiment depicted here, infections were performed on days 0, 1, 2, 3, and 4, whereas for the selection, infections were performed on days 0, 1, and 2 only. Data depicted are the mean ± SEM of three independent experiments for days 0-2, and mean ± SEM of two independent experiments for days 3-5. (c) Interactome analysis of top 81 genes enriched by the selection. Red circles (arrows) are proteins encoded by genes enriched by the selection; lines indicate protein-protein interactions; black circles indicate interacting proteins. 4 NATURE MICROBIOLOGY

$OspB-FLAG translocated into Vim +/+ or Vim -/- MEFs upon infection with S. flexneri. (a) Cytosolic fraction probed for FLAG, actin, and DnaK, a cytosolic bacterial protein, by western blot.$

5 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Figure S2: Efficient type 3 translocation by S. flexneri depends on intermediate filaments. OspB-FLAG translocated into Vim +/+ or Vim -/- MEFs upon infection with S. flexneri. (a) Cytosolic fraction probed for FLAG, actin, and DnaK, a cytosolic bacterial protein, by western blot. The two DnaK panels are from the same exposure of the same western blot, and all panels are from a single experiment, which is representative of three independent experiments. (b) Densitometry analysis of OspB-FLAG recovered from the cytosol in experiments depicted in a. The data are mean ± SEM of three independent experiments. **, P<0.01, Student s t-test. NATURE MICROBIOLOGY 5

DOI: 10.1038/NMICROBIOL.2016.25 Figure S3: S. flexneri requires intermediate filaments for efficient invasion. (a-b) Vimentin is required for efficient infection of MEFs by S. flexneri. Infection of Vim +/+ and Vim -/- MEFs.

6 DOI: /NMICROBIOL Figure S3: S. flexneri requires intermediate filaments for efficient invasion. (a-b) Vimentin is required for efficient infection of MEFs by S. flexneri. Infection of Vim +/+ and Vim -/- MEFs. (a) Representative images. Extracellular bacteria were labeled with anti-s. flexneri prior to permeabilization. All bacteria and cell nuclei were stained with DAPI and actin with phalloidin, following permeabilization. Merge, extracellular bacteria (green), DNA (blue), and actin (red). Scale bar, 20 µm. Arrows, internalized bacteria. Arrowhead, extracellular bacteria. (b) Quantification of infected cells from a. Data are mean ± SEM of three independent experiments. *, P<0.05; Student s t-test. (c) Intracellular bacteria upon S. flexneri infection of Vim +/+ and Vim -/- MEFs. Data are the mean ± SEM of six independent experiments. ***, P<0.001; Student s t-test. (d-e) Keratin 8 and keratin 18 are required for efficient infection of polarized Caco2 cells by S. flexneri. Keratin 8 or 18 was knocked down by shrna. (d) Intracellular bacteria upon S. flexneri infection of 6 NATURE MICROBIOLOGY

7 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION polarized Caco-2 cells via the basolateral surface. Data are mean ± SEM of four independent experiments. *, P<0.05, **, P<0.01; one-way ANOVA with Dunnett s post hoc test. (e) Levels of keratin 8 and keratin 18 following knockdown in cells used for experiments in panel d. Of note, the absence of either keratin 8 or keratin 18 led to the instability of the other, presumably because keratins exist as heterodimers in these cells 7. NATURE MICROBIOLOGY 7

DOI: 10.1038/NMICROBIOL.2016.25 Figure S4: S. flexneri infection does not cause visually detectable rearrangements of intermediate filaments. HeLa cells were infected with S. flexneri. Vimentin, keratin 8, or keratin 18 (green), and bacteria and cell nuclei (blue).

8 DOI: /NMICROBIOL Figure S4: S. flexneri infection does not cause visually detectable rearrangements of intermediate filaments. HeLa cells were infected with S. flexneri. Vimentin, keratin 8, or keratin 18 (green), and bacteria and cell nuclei (blue). Arrows, intracellular bacteria. Images are from one experiment that is representative of three independent experiments. Scale bar, 20µm. 8 NATURE MICROBIOLOGY

DOI: 10.1038/NMICROBIOL.2016.25 SUPPLEMENTARY INFORMATION Figure S5: Actin-rich entry foci form in the absence of intermediate filaments.

9 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Figure S5: Actin-rich entry foci form in the absence of intermediate filaments. Assessment of actin-rich entry foci upon infection of HeLa cells, Vim +/+ MEFs, or Vim -/- MEFs with S. flexneri. (a) Fluorescent images representative of four independent experiments. (b-c) The data are the mean ± SEM of four independent experiments. NS, not significantly different; *, P<0.05, Student s t-test. (b) Quantification of the percent of NATURE MICROBIOLOGY 9

10 DOI: /NMICROBIOL infected Vim +/+ and Vim -/- MEFs displaying actin-rich entry foci. (c) Quantification of the percent of total Vim +/+ and Vim -/- MEFs displaying actin-rich entry foci. (d) Quantification of the percent of Vim +/+ and Vim -/- MEFs infected. 10 NATURE MICROBIOLOGY

DOI: 10.1038/NMICROBIOL.2016.25 SUPPLEMENTARY INFORMATION Figure S6: IpaC topology and alignment with SipC and YopD.

11 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Figure S6: IpaC topology and alignment with SipC and YopD. (a) Topology analysis prediction of the orientation of IpaC within the plasma membrane using Spoctopus, which predicts a single transmembrane segment. Dotted line, extracellular domain; cross-hatched bar (TM), transmembrane segment; broken dashed line, intracellular domain. Bracket, region of the IpaC C-terminus required for invasion 8. (b) Alignment of the region of IpaC C-terminus required for invasion with YopD and SipC using ClustalΩ. *, conserved amino acids. Arrowheads, IpaC R362 and IpaC S349. NATURE MICROBIOLOGY 11

DOI: 10.1038/NMICROBIOL.2016.25 Figure S7: Variants of IpaC are produced in yeast.

12 DOI: /NMICROBIOL Figure S7: Variants of IpaC are produced in yeast. Representative images from protein interaction assays of vimentin with wild type IpaC or IpaC mutants. Experimental replicates are described in methods. Scale bar, 5 µm. 12 NATURE MICROBIOLOGY

13 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Figure S8: Lack of contribution of IpaB to IpaC-vimentin interaction in yeast protein-protein interaction assay. Yeast based protein-protein interaction assay. Data are the mean ± SEM; Empty-IpaC WT-Empty, n=9; Empty-IpaC WT-IpaB, n=8; Vimentin-IpaC WT-Empty, n=9; Vimentin-IpaC WT-IpaB, n=12; Vimentin-IpaC R326E- IpaB, n=6; Vimentin-IpaC R326L-IpaB, n=6; Vimentin-IpaC R326W-IpaB, n=9. The n for a condition represents the number of independent experimental assays. NS, nonsignificant; *, P<0.05, one-way ANOVA comparing each to either Vimentin-IpaC WT- Empty or Vimentin-IpaC WT-IpaB. NATURE MICROBIOLOGY 13

$OspB-FLAG translocated into HeLa cells upon infection with S. flexneri. Cytosolic fraction probed for OspB-FLAG and DnaK, a cytosolic bacterial protein, by western blot.$

14 DOI: /NMICROBIOL Figure S9: Type 3 translocation into HeLa cells is reduced for IpaC mutants that are defective for interactions with intermediate filaments. OspB-FLAG translocated into HeLa cells upon infection with S. flexneri. Cytosolic fraction probed for OspB-FLAG and DnaK, a cytosolic bacterial protein, by western blot. The two DnaK panels are from the same exposure of the same western blot. All panels are from a single experiment that is representative of two independent experiments. 14 NATURE MICROBIOLOGY

15 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Figure S10: IpaC mutants that are defective in interaction with intermediate filaments are secreted through the T3SS and form pores in erythrocyte membranes similarly to WT IpaC. (a-b) Induction of secretion of type 3 substrates from indicated S. flexneri strains by Congo red. (a) Western blot, representative of three independent experiments. (b) Densitometry analysis of IpaC secretion for the experiments depicted in panel a. Data are the mean ± SEM of three independent experiments. Means not significantly different by one-way ANOVA. (c-d) Efficiency of pore formation, as determined by lysis of erythrocytes by indicated S. flexneri strains. (c) Representative image of supernatants collected following erythrocyte lysis assay; red is released hemoglobin, and darker red indicates increased erythrocyte lysis, a NATURE MICROBIOLOGY 15

16 DOI: /NMICROBIOL marker of translocon pore formation in the erythrocyte membrane. (d) Quantification of data from experiments depicted in panel c. Data are the mean ± SEM of two to five independent experiments for each condition (n=5 for Media, WT, ΔipaC, ΔipaC vector, ΔipaC pipac WT, ΔipaC pipac R362W, and SDS; n=3 for ΔipaC pipac R362E and ΔipaC pipac R362L, and n=2 for ΔipaC pipac S349A). ***, P<0.001; one-way ANOVA with Dunnett s post hoc test. 16 NATURE MICROBIOLOGY

17 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Figure S11: E. coli psft3ss, but not S. flexneri, causes significant BCECF release from HEK293T cells. BCECF release from HEK293T cells infected with either S. flexneri or E. coli psft3ss. Data presented are the mean ± SD of four wells from one experiment and are representative of three independent experiments. *, P<0.05; oneway ANOVA with Dunnett s post hoc test. NS, not significant. NATURE MICROBIOLOGY 17

DOI: 10.1038/NMICROBIOL.2016.25 Figure S12: BCECF dye release upon infection with S. flexneri. BCECF release from Vim +/+ and Vim -/- macrophages infected with S.

18 DOI: /NMICROBIOL Figure S12: BCECF dye release upon infection with S. flexneri. BCECF release from Vim +/+ and Vim -/- macrophages infected with S. flexneri. Data presented are the mean ± SEM of three independent experiments. NS, not significant; Student s t-test. 18 NATURE MICROBIOLOGY

19 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Figure S13: Intermediate filaments are required for efficient bacterial docking independently of invasion. (a). Cytochalasin D blocks bacterial entry. Intracellular bacteria upon infection of Vim +/+ and Vim -/- MEFs with WT S. flexneri, as determined by differential fluorescent microscopy. The data are the mean ± SEM of two independent experiments. CytoD, cytochalasin D, which inhibits actin polymerization. *, P<0.05; Student s t-test. (b). Docking of WT S. flexneri to Vim +/+ and Vim -/- MEFs in the presence or absence of cytochalasin D, as determined by differential fluorescent microscopy. Data are the mean ± SEM of two independent experiments. *, P<0.05, **, P<0.01; Student s t-test. NATURE MICROBIOLOGY 19

DOI: 10.1038/NMICROBIOL.2016.25 Figure S14: Model of intermediate filament contribution to docking and bacterial effector translocation by the S. flexneri type 3 secretion system.

20 DOI: /NMICROBIOL Figure S14: Model of intermediate filament contribution to docking and bacterial effector translocation by the S. flexneri type 3 secretion system. (a) The type 3 secretion system is expressed in S. flexneri. (b) S. flexneri makes contact with host cell plasma membrane. (c) The translocon pore proteins are secreted through the type 3 secretion system and form the translocon pore in the plasma membrane. (d) In the presence of intermediate filaments, the type 3 secretion needle docks on the extracellular face of the translocon pore. (e) In the presence of intermediate filaments, there is efficient translocation of bacterial effector proteins into the cytosol of the host cell. 20 NATURE MICROBIOLOGY

21 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Figure S15: Raw data (western blots). NATURE MICROBIOLOGY 21

22 DOI: /NMICROBIOL Supplementary References 1. Labrec EH, Schneider H, Magnani TJ, Formal SB. Epithelial Cell Penetration as an Essential Step in the Pathogenesis of Bacillary Dysentery. J Bacteriol 88, (1964). 2. Maurelli AT, Baudry B, d'hauteville H, Hale TL, Sansonetti PJ. Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri. Infect Immun 49, (1985). 3. Day JB, Lee CA. Secretion of the orgc gene product by Salmonella enterica serovar Typhimurium. Infect Immun 71, (2003). 4. Auerbuch V, Golenbock DT, Isberg RR. Innate immune recognition of Yersinia pseudotuberculosis type III secretion. PLoS Pathog 5, e (2009). 5. Reeves AZ, Spears WE, Du J, Tan KY, Wagers AJ, Lesser CF. Engineering Escherichia coli into a Protein Delivery System for Mammalian Cells. ACS Synth Biol 4, (2015). 6. Labigne-Roussel AF, Lark D, Schoolnik G, Falkow S. Cloning and expression of an afimbrial adhesin (AFA-I) responsible for P blood group-independent, mannose-resistant hemagglutination from a pyelonephritic Escherichia coli strain. Infect Immun 46, (1984). 7. Eriksson JE, et al. Introducing intermediate filaments: from discovery to disease. J Clin Invest 119, (2009). 8. Terry CM, et al. The C-terminus of IpaC is required for effector activities related to Shigella invasion of host cells. Microb Pathog 45, (2008). 22 NATURE MICROBIOLOGY

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