depends on the translocation assembly module nanomachine

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1 ARTICLE NUMBER: DOI: /NMICROBIOL Effective assembly of fimbriae in Escherichia coli depends on the translocation assembly module nanomachine Christopher Stubenrauch 1, Matthew J. Belousoff 1, Iain D. Hay 1, Hsin-Hui Shen 1,2, James Lillington 3, Kellie L. Tuck 4, Kate M. Peters 5, Minh-Duy Phan 5, Alvin W. Lo 5, Mark A. Schembri 5, Richard A. Strugnell 6, Gabriel Waksman 3 & Trevor Lithgow 1,* 1 - Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton 3800, Australia 2 - Department of Materials Engineering, Monash University, Clayton 3800, Australia 3 - Institute of Structural and Molecular Biology (ISMB), University College London and Birkbeck College, Malet Street, London WC1E 7HX, UK. 4 - School of Chemistry, Monash University, Clayton 3800, Australia 5 - School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia 4072, Australia 6 - Department of Microbiology & Immunology, University of Melbourne, Parkville 3052, Australia * - Correspondence: trevor.lithgow@monash.edu NATURE MICROBIOLOGY 1

2 DOI: /NMICROBIOL Supplementary Figure S1. The architecture of usher proteins, exemplified by FimD. a, Structural map of FimD shows the domain boundaries according to the crystal structure (pdb 3RFZ). Trans-membrane β-strands shown as arrows within the three segments of the β-barrel domain. The plug domain, shown in green, is incorporated between β-strands 6 and 7. A protease-sensitive extracellular loop (Loop 7), between β-strands 13 and 14, is designated with a yellow asterisk. The N-terminal domain (NTD) and two C-terminal domains (CTD1 and CTD2) are displayed in the periplasm. b, Schematic of the chaperone-usher system for fimbrial manufacture. Multiple copies of the major fimbrial subunit FimA are built into the growing filament by the action of the usher (FimD). The tip subunit (FimH) is a lectin with mannose-binding activity. The domains of FimD have been color-coded as shown in panel a, and the β-barrel pore domain is represented as a wireframe to show the β-strands within the outer membrane. 2 NATURE MICROBIOLOGY

3 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION a Signal Sequence NTD TM 1-6 Plug TM 7-13 Loop7 TM CTD1 CTD FimD * b FimH FimG FimF FimA FimD Pore NTD Plug CTD1 CTD2 FimC NATURE MICROBIOLOGY 3

4 DOI: /NMICROBIOL Supplementary Figure S2. A rifampicin/t7-based assay to measure outer membrane protein assembly rates in E. coli. Cartoon depiction of the rifampicin blocking technique and 35 S pulse-labelling, and subsequent detection techniques of correctly folded outer-membrane proteins. In the case of FimD, correct folding and assembly in the outer membrane results in the a protease-sensitive loop between β-strands 13 and 14 being displayed on the extracellular surface of the outer membrane, and Proteinase K treatment converts the ~90 kda FimD to two fragments that migrate on SDS-PAGE at ~ kda and ~40 kda. Inset panel shows a comparison of total cell extracts prepared from E. coli harbouring pks02 (the plasmid that expresses fimd) were assayed as in Figure 1b, except that the radioactivity was not added, and that cells were induced or not induced for 5 minutes with IPTG, before a 60-minute "chase". The samples, corresponding to 5-times the amount of total cells normally analysed during radiolabelling experiments, were analysed by SDS-PAGE and phosphor-imaging. The upper panel shows a Coomassie blue stain of the cell extracts, with a red asterisk positioned in the approximate migration position for FimD: there is no over-expression of protein in this region of the gel. The lower panels show immunoblot analysis using antibodies raised to FimD and a control outer membrane protein: the expression level of FimD driven off the plasmid is moderate, and would not be in excess of the capacity of the β-barrel assembly machinery. 4 NATURE MICROBIOLOGY

5 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION A A A Proteinase K A A A BL21-DE3* T7 -S Rifampicin 35 IPTG S starve pulse 32 S chase BL21-DE3* T7 BL21-DE3* T7 Gene A 2 1 IPTG - + * time Native Gel Electrophoresis time Gel Electrophoresis kda Coomassie stain FimD OmpC Immunoblot NATURE MICROBIOLOGY 5

6 DOI: /NMICROBIOL Supplementary Figure S3. Peptide mapping of FimD fragments generated by Proteinase K digestion. a, MS1 chromatographic peak area-based quantitation where peptide coverage is plotted as a heat-map: red shows high abundance in peptide recovery, pink shows minimal detection of peptides, white shows complete absence of peptides. Note, sequence numbering is from the first translated methionine residue, no peptides were recovered corresponding to the signal sequence (residues 1-45) as expected. b, Peak areas of peptides shown in (a) were extracted by Skyline: SLNESGTNIQLVGYR (residues ), YSTSGYFNFADTTYSR (i, residues ), DGVIQVKPK (residues 6-514), FTDYYNLAYNK (residues 5-5) and LQLTVTQQLGR (residues ). Note that the cleavage N-terminal to DGVIQVKPK is non-tryptic, identifying one end of the Proteinase K cleavage site. This peptide (DGVIQVKPK) was only identified in the ~40 kda (C-terminal) fragment of FimD. For the ~45 kda B-fragment, the pink line beyond this cleavage denotes the presence of the peptides FTDYYNLAYNK (residues 5-5) and FTDYYNLAYNKR (residues 5-526), assisting designation of the cleavage regions that define the B-fragment. 6 NATURE MICROBIOLOGY

7 a DOI: /NMICROBIOL FimD N- fragment FimD Sequence 1 45 SUPPLEMENTARY INFORMATION B- fragment C- fragment b SLNESGTNIQLVGYR YSTSGYFNFADTTYSR DGVIQVKPK FTDYYNLAYNK LQLTVTQQLGR Normalized Peak Area Full N B C Normalized Peak Area Full N B C Normalized Peak Area Full N B C Normalized Peak Area Full N B C Normalized Peak Area Full N B C precursor [M+3] precursor [M+2] precursor [M+1] precursor precursor [M+4] precursor [M+3] precursor [M+2] precursor [M+1] precursor precursor [M+2] precursor [M+1] precursor precursor [M+3] precursor [M+2] precursor [M+1] precursor precursor [M+3] precursor [M+2] precursor [M+1] precursor NATURE MICROBIOLOGY 7

8 DOI: /NMICROBIOL Supplementary Figure S4. Statistics on FimD assembly (in bam and tam mutants). a, Histogram of the fitted first order observed rate constants for the appearance of the ~ kda N- terminal FimD fragment (N), the ~40 kda C-terminal fragment or the single (~45 kda) FimD fragment found in the tam mutants ( B ). Error bars correspond to the standard error of fit (n=4). b, Table of k obs for the appearance of FimD fragments for all strains in the study. c, Plots of relative density of each of the bands (N), (C) and (B) versus time (seconds) for each of the E. coli strains in the study. Highlighted as solid red squares is the data for the ~45 kda ( B ) fragment. The ΔtamA and ΔtamB mutants each accumulate much more of the ~45 kda fragment of FimD than any of the bam mutants. Error bars indicated SEM (n = 4). 8 NATURE MICROBIOLOGY

9 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION k obs (s -1 ) SEM a k obs (s -1 ) wildtype (N) (C)(N) bamb bamc (C) (N)(C) bame (N) (C)(N) tama (B) (C)(N) tamb (B) (C) b wildtype (N) wildtype (C) bamb (N) bamb (C) bamc (N) bamc (C) bame (N) bame (C) tama (N) tama (B) tama (C) tamb (N) tamb (B) tamb (C) c Relative density Wildtype (N) (B) (C) Relative density bamb (N) (B) (C ) Time (sec) Time (sec) bamc bame Relative density (N) (B) (C) Relative density (N) (B) (C) Time (sec) Time (sec) tama tamb Relative density (N) (B) (C) Relative density (N) (B) (C) Time (sec) Time (sec) NATURE MICROBIOLOGY 9

10 DOI: /NMICROBIOL Supplementary Figure S5. Epitope mapping of the ~45 kda fragment of FimD. a, Diagrammatic representation of the strep-tag II constructs of FimD, encoded by plasmids pcjs51 and pcjs29. b, E. coli harbouring plasmids encoding either N term -Strep (pcjs51) or C term -Strep (pcjs29) were assayed as in Figure 1b, except radioactivity was omitted and the cells were induced for 32 minutes. Analysis was by SDS-PAGE and blotting using Streptactin-HRP (horse radish peroxidase) reagent (Biorad, catalog # ). The 45 kda fragment was not observed. c, Diagrammatic representation of the His 6 -tag constructs of FimD, encoded by plasmids pcjs65 and pcjs66. d, E. coli harbouring plasmids (pcjs65 or pcjs66) were assayed as in Figure 1b, except that the cell concentration during the assay was 5.2-times greater and cells were chased for 32 minutes. Analysis was by SDS-PAGE, phosphor-imaging and blotting using Mouse anti-his 6 antibodies (BD Pharmingen, catalog # ). Minor amounts of N- and C- fragments formed in the ΔtamA cells are evident. A minor fraction of the B fragment carries the pcjs65 tag, suggesting that Proteinase K degrades FimD (i) at several sites across and N-terminal to the region between betastrands # 4 and #5 (i.e. the site of the pcjs65 insertion, and (ii) at a point that is N-terminal to betastrand #18 (i.e. N-terminal to the pcjs66 insertion). 10 NATURE MICROBIOLOGY

11 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION a Strep NTD Plug CTD1 CTD2 NTD Plug CTD1 CTD2 N term Strep C term Strep Strep b WT tama kda N term Strep C term Strep N term Strep C term Strep PK FimD c Streptactin N-terminal C-terminal NTD Plug CTD1 CTD2 H1 NTD Plug CTD1 CTD2 H2 d FimD FimD-H1 FimD-H2 kda WT tama WT tama WT tama PK FimD N-terminal B C-terminal 35 S-autoradiogram FimD N-terminal B C-terminal α-his 6 immunoblot NATURE MICROBIOLOGY 11

12 DOI: /NMICROBIOL Supplementary Figure S6. Loss of the TAM does not have a major impact on the assembly of PhoE. a, E. coli strains containing pks02(fimd) were assayed as in Fig. 2, but the chase incubation was for 16 minutes, wherein the samples were treated with or without Proteinase K for 10 minutes and TCA precipitation. The samples were analysed by SDS-PAGE, phosphor-imaging (upper panel) and immunoblotting using an antibody specific for the periplasmic protein BamD (lower panel). b, Crystal structure of PhoE oligomer of β-barrels showing the arrangement of 12 anti-parallel β-strands from a single polypeptide in each of the three β-barrels. (pdb 1PHO). Graphic prepared using PyMOL. c, E. coli strains harbouring pks07(phoe) were starved of methionine and cysteine were pulse-labelled with 35 S-methionine and 35 S-cysteine. The shaded triangles represent the time increment where aliquots were taken at 10 seconds, 2, 4, 8, 16 and 32 minutes. Duplicate aliquots were removed to tubes containing Semi-Native Sample Buffer, followed by 5 minute incubation at either C or C prior to electrophoresis. Analysis was by semi-native PAGE and phosphor-imaging. o oligomeric forms of PhoE; m PhoE monomers. d, The normalized density of the monomer band ( ) is plotted versus time for a single experiment highlighting the exponential decay of the monomer relative to the exponential increase in oligomer formation. The densitometry across the range of oligomer species ( ) is also plotted. The monomer is better focussed, allowing for more accurate densitometry, and so this data was analysed (including error bars representing the SEM calculated from the 4 biological replicates) and is presented in graphs in Supplementary Fig. S6e. e, Densitometry analysis of PhoE assembly in the various mutants, corresponding to data shown in Supplementary Fig. S6c. The normalized density of the monomer band is plotted versus time, where the solid line represents the fitted exponential decay, and error bars are the SEM calculated from 4 biological replicates. f, Quantitation of the assembly of PhoE oligomers from monomers at C. The histogram depicts the fitted first order observed rate constants of PhoE oligomer assembly from the data in Supplementary Fig. S6d. Error bars represent the standard error of the mean (SEM) (n=4). 12 NATURE MICROBIOLOGY

13 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION a kda WT PK bamb bamc bame tama tamb FimD N-terminal B C-terminal b α -BamD c 32 d o m o m o m o m o m o m Wildtype bamb bamc bame tama tamb Normalized relative density Oligomer Monomer C C e Wildtype bamb bamc f 10 Fitted rate constant k obs (s -1 ) 05 bame tama tamb 00 Wildtype bamb bamc bame tama tamb NATURE MICROBIOLOGY 13

14 DOI: /NMICROBIOL Supplementary Figure S7. Loss of the TAM does not have a major impact on the assembly of TolC. a, Crystal structure of TolC (pdb 1EK9): the oligomer is assembled from three subunits, each of which contributes 4 β-strands to a 12-stranded β-barrel. The long helical extensions delve into the periplasm. b, E. coli strains harbouring pmb11(tolc) were used to measure the assembly of TolC trimers (t) from monomers (m) as in Supplementary Fig. S6c, except the samples in semi-native sample buffer were incubated for five minutes at C (non-denaturing; to distinguish monomers and trimers) or C (denaturing; total TolC present). c, The normalized density of the TolC monomer band ( ) and the TolC trimer ( ) is plotted versus time, where the solid line represents the fitted exponential decay. d, Plots of the normalized relative intensity of the monomer band versus time (seconds) from data shown in Supplementary Fig. S7b, solid line represents the fitted exponential decay, error bars are the SEM calculated from 4 biological replicates. e, Histogram of the fitted first order observed rate constants corresponding to the assembly of TolC in the different E. coli strains, error bars represent the standard error of fit (n = 4). 14 NATURE MICROBIOLOGY

15 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION a b 32 c t m Wildtype bamb bamc bame tama tamb Normalized relative density Wildtype Trimer Monomer C C d Wildtype bamb bamc e Fitted rate constant k obs (s -1 ) bame tama tamb Wildtype bamb bamc bame tama tamb NATURE MICROBIOLOGY

16 DOI: /NMICROBIOL Supplementary Figure S8. Model for usher assembly by the β-barrel assembly machinery, with or without the activity of the TAM. At a fixed timepoint where the BAM+TAM+ cells have completed FimD folding, Proteinase K (PK) generates kda and 40 kda fragments because of the correct exposure of Loop 7 at the cell surface. At this time, the BAM+Δtam cells have partially folded a central section of FimD that did not start from the C-terminus. Ultimately, a correctly folded FimD is generated in both conditions. Based on a current model for how BamA builds and then buds a β-barrel substrate, we speculate that the 45 kda fragment represents that part of the FimD barrel domain that was protected from Proteinase K digestion by the BAM complex, represented here as a shaded shape. 16 NATURE MICROBIOLOGY

17 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION FimD BAM + TAM + +PK OM Fast Fast Substrate N C N C N C kda 40 kda FimD BAM + tama +PK OM Fast Slow Substrate N 45 kda N C N C C NATURE MICROBIOLOGY 17

18 DOI: /NMICROBIOL Supplementary Figure S9. Raw image files. The raw image files of the cropped radiographs, Coomassie-stained gels and immunoblots displayed in the Figures or Supplementary Figures. Apparent sizes in kda are indicated on the left. A blue box is used to indicate the cropped portion of the raw image that was displayed in the indicated Figure or Supplementary Figure. A pink box is used to indicate additional cropping made to raw files that were used to explain the pulse chase assay in Supplementary Fig. S2. 18 NATURE MICROBIOLOGY

19 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION (blue) Figure 1b and 2c (wildtype panel) (pink) Figure S2 right panel Figure 1c 2 1 Figure 1e Figure 2b 2 1 Figure 2c (ΔbamB panel) Figure 2c (ΔbamC panel) 2 1 Figure 2c (ΔbamE panel) Figure 2c (ΔtamA panel) NATURE MICROBIOLOGY 19

20 DOI: /NMICROBIOL Figure 2c (ΔtamB panel) 2 1 Figure 2e 2 1 Figure 2f (ΔtamA panel) 2 1 Figure 2f (ΔtamB panel) Figure 4b (PapC panel) Figure 4b (HtrE panel) Figure 4b (YbgQ panel) Figure 4b (YfcU panel) NATURE MICROBIOLOGY

21 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Figure S2 (Coomassie inset) 2 1 Figure S2 (FimD Western) 2 1 Figure S2 (OmpC Western) 2 1 (pink) Figure S2 (Assembly gel panel) (blue) Figure S7b (ΔbamB panel) 2 1 Figure S5b 10 Figure S5d (radiograph) Figure S5d (western) Figure S6a (radiograph) 2 1 Figure S6a (western) NATURE MICROBIOLOGY 21

22 DOI: /NMICROBIOL Figure S6c (wildtype panel) 2 1 Figure S6c (ΔbamB panel) 10 Figure S6c (ΔbamC panel) Figure S6c (ΔbamE panel) Figure S6c (ΔtamA panel) Figure S6c (ΔtamB panel) NATURE MICROBIOLOGY

23 DOI: /NMICROBIOL Figure S7b (wildtype panel) SUPPLEMENTARY INFORMATION 2 1 Figure S7b (ΔbamC panel) 10 Figure S7b (ΔbamE panel) Figure S7b (ΔtamA panel) Figure S7b (ΔtamB panel) 1 10 NATURE MICROBIOLOGY 23