Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori
- University of Washington, United States
- Fred Hutchinson Cancer Research Center, United States
- Princeton University, United States
- University of Delaware, United States
- Harvard Medical School, United States
- Newcastle University, United Kingdom
- Indiana University, United States
Figures
Figure 1
Helical cell surfaces feature areas of distinct curvatures.
(A) 3D SIM images of individual H. pylori cells stained with fluorescent wheat germ agglutinin (WGA). Top-down view (left) and 90-degree rotation about the long axis (right). Scale bar = 0.5 µm; …
Figure 2
The distribution of surface Gaussian curvature for helical cells is distinct from that of curved- and straight-rod cells.
Smooth histograms of the distribution of surface Gaussian curvatures for a population of cells (wild-type helical, yellow; curved-rod Δcsd2, teal; straight-rod Δcsd6, indigo) with poles included (A) …
Figure 3 with 4 supplements
Three-dimensional shape properties of a wild-type helical population.
Analysis of the wild-type population in Figure 2 from the 231 wild-type cells for which the cell centerline was well-fit by a helix. (A) Schematic of helical-rod shape parameters (cell centerline …
Figure 3—figure supplement 1
Evaluation of the subset of the wild-type population used to generate synthetic cells.
(A) Example cell centerlines (gray dots) and calculated helical fits (red lines), arranged from good (left) to poor (right) fit. (B) Histogram of the relative helical fit error for each of the cells …
Figure 3—figure supplement 2
Change in the distribution of cell surface Gaussian curvatures based on modulating helical rod parameters.
Left column, distribution of population helical rod parameters from Figure 3C–F. Center column, Gaussian surface curvature distribution of the synthetic cells in (Figure 3—figure supplement 3) …
Figure 3—figure supplement 3
Simulated helical cells demonstrating how variation in helical parameters alters surface Gaussian curvature.
Cell centerline (paired cells, left) and cell surface Gaussian curvatures (paired cells, right) of synthetic, idealized cells with parameters taken from the distribution of wild-type shapes. The …
Figure 3—video 1
Rotation of example cell centerlines (gray dots) and calculated helical fits (red lines), arranged from good (left) to poor (right) fit from Figure 3—figure supplement 1A.
Figure 4 with 4 supplements
Validation of PG metabolic probes.
(A) 10-fold dilutions showing LSH108 (rdxA::catsacB) or HJH1 (rdxA::amgKmurU) treated with 50 µg/ml fosfomycin or untreated and with or without 4 mg/ml MurNAc supplementation, from one …
Figure 4—figure supplement 1
Schematic of PG synthesis and incorporation of PG metabolic probes.
(A) MurNAc-alk diffuses across the cell membrane and is converted into UDP-MurNAc-alk, which can then be used in the synthesis of PG precursors. (B) Fosfomycin inhibits the conversion of UDP-GlcNAc …
Figure 4—figure supplement 2
The MIC of fosfomycin in H. pylori is 25 µg/ml.
Optical density of wild-type H. pylori cultures grown in a 96-well plate with a 2-fold dilution series of fosfomycin. Optical density was measured at 1 (light gray), 6 (medium gray), and 12 (dark …
Figure 4—figure supplement 3
Detected MurNAc-alk labeled muropeptides.
Labeled (A) pentapeptide monomer and (B) tetra-pentapeptide dimer ions. Parentheses indicate that the MurNAc-alk could be on either the tetra or penta portion of the dimer; these two species are …
Figure 4—figure supplement 4
Detected D-Ala-alk labeled muropeptides.
Figure 5 with 1 supplement
New cell wall growth appears dispersed along the sidewall, excluded from poles, and present at septa.
3D SIM imaging of wild-type cells labeled with an 18 min pulse of MurNAc-alk (A–D, yellow) or 18 min pulse of D-Ala-alk (E–H, yellow) counterstained with fluorescent WGA (blue). Color merged maximum …
Figure 5—video 1
Volumetric rendering and z-slices of the example cells in Figure 5.
Figure 6 with 2 supplements
New cell wall growth is excluded from the poles and enriched at negative Gaussian curvature and the major axis area.
(A) The calculation of relative concentration for a specific probe involves two steps of normalization. First, the raw signal is summed up in bins defined by the Gaussian curvature at the surface. …
Figure 6—figure supplement 1
For eight different example distributions (rows with brief labels to the left), five pieces of data are shown.
The five columns are as follows (from left to right): two views of an example rendering of a helical rod cell colored by the intensity of the raw signal at each point on the surface; the raw signal …
Figure 6—figure supplement 2
Curvature enrichment analysis of biological replicates of MurNAc-alk-, D-Ala-alk-, and mock-labeling.
(A) Sidewall only surface Gaussian curvature enrichment of relative concentration of new cell wall growth (y-axis) vs. Gaussian curvature (x-axis) of the three biological replicates pooled in Figure …
Figure 7 with 4 supplements
MreB is essential in LSH100 and is present as small foci enriched at negative Gaussian curvature.
(A) Schematic of transformation experiment testing MreB essentiality in LSH100 (WT) and IM4 (2XmreB) (left) and corresponding transformation frequencies (right). *=two recombinant clones with mreB …
Figure 7—figure supplement 1
MreB is essential in the G27 derivative LSH100.
(A) (Top) Schematic of the LSH100 native mreB locus with DNA and protein sequence for a small region at the C-terminus. Anti-MreB epitopes are annotated in yellow. (Middle) Schematic of the McGee …
Figure 7—figure supplement 2
MreB enrichment decreases with increasing positive Gaussian curvature.
Whole surface (sidewall and poles) Gaussian curvature enrichment of relative MreB concentration (y-axis) vs. Gaussian curvature (x-axis) of computational cell surface reconstructions of a population …
Figure 7—figure supplement 3
Curvature enrichment analysis of biological replicates of MreB.
(A) Sidewall only surface Gaussian curvature enrichment of relative MreB concentration (y-axis) vs. Gaussian curvature (x-axis) of the three biological replicates pooled in Figure 7: anti-MreB …
Figure 7—video 1
Volumetric rendering and z-slices of the example cells in Figure 7.
Figure 8 with 2 supplements
Amino acid substitution mutations in CcmA cause altered polymerization in vitro and alter cell shape in vivo.
(A–D) Negatively stained TEM images of purified CcmA. Scale bars = 100 nm, with representative images from one of three biological replicates. Wild-type CcmA lattices (A) (blue arrows) and helical …
Figure 8—figure supplement 1
CcmA lattices and bundles.
Negatively stained TEM images of purified CcmA. Scale bars = 200 nm. Lower magnification view than in Figure 8 of (A) wild-type CcmA, displaying both lattices (blue arrows) and extended helical …
Figure 8—figure supplement 2
Fourier transform of CcmA lattices shows regular alignment and spacing.
(A) Lattices formed from purified WT CcmA in 25 mM Tris pH 8. Scale bars = 100 nm. (B) Fourier transform of the region inside each corresponding box in (A) performed using Fiji (Schindelin et al., …
Figure 9 with 2 supplements
Wild-type CcmA appears as short foci on the side of the cell, but CcmA mutants I55A and L110S appear as foci in the interior of the cell.
3D SIM imaging of CcmA-FLAG cells immunostained with M2 anti-FLAG (A, C, D, yellow) or wild-type or CcmA amino acid substitution mutant cells immunostained with anti-CcmA (B, E–J, yellow); cells …
Figure 9—figure supplement 1
There is low signal in the no-FLAG and preimmune serum controls.
(A) Wild-type (no-FLAG) cells immunostained with M2 anti-FLAG (yellow) and counterstained with fluorescent WGA (blue). (B) Wild-type, (C) I55A, or (D) L110S CcmA cells immunostained with CcmA …
Figure 9—video 1
Volumetric rendering and z-slices of the example cells in Figure 9 and three example WT cells immunostained with anti-CcmA and counterstained with fluorescent WGA.
Figure 10 with 3 supplements
CcmA curvature preference correlates with the peak of new PG incorporation at the major axis area and MreB curvature preference correlates with new PG enrichment at negative Gaussian curvature.
Overlay of sidewall only surface Gaussian curvature enrichment of relative concentration (y-axis) vs. Gaussian curvature (x-axis) from a population of computational cell surface reconstructions with …
Figure 10—figure supplement 1
CcmA is excluded from the poles.
Whole surface (sidewall and poles) Gaussian curvature enrichment of relative signal abundance (y-axis) vs. Gaussian curvature (x-axis) derived from a population of computational cell surface …
Figure 10—figure supplement 2
Curvature enrichment analysis of biological replicates of CcmA-FLAG.
(A) Sidewall only surface Gaussian curvature enrichment of relative signal abundance (y-axis) vs. Gaussian curvature (x-axis) of the three biological replicates pooled in Figure 10: CcmA-FLAG …
Figure 10—figure supplement 3
CcmA mutants are not enriched at positive Gaussian curvature.
(A) Sidewall Gaussian curvature enrichment of relative signal abundance (y-axis) vs. Gaussian curvature (x-axis) for a population of computational cell surface reconstructions with poles excluded of …
Figure 11 with 4 supplements
MreB and CcmA contribute to cell wall synthesis patterning.
(A, B) Sidewall only Gaussian curvature enrichment of relative concentration (y-axis) vs. Gaussian curvature (x-axis) from a population of computational cell surface reconstructions of HJH1 (amgK …
Figure 11—figure supplement 1
Cell wall synthesis patterning but not MreB curvature preference is altered by loss of CcmA.
(A, B) Whole surface (sidewall and poles) Gaussian curvature enrichment of relative concentration (y-axis) vs. Gaussian curvature (x-axis) from a population of computational cell surface …
Figure 11—figure supplement 2
MreB is present as small foci along the sidewall in ΔccmA.
3D SIM imaging of ΔccmA cells immunostained with anti-MreB (A, C, D, yellow) or preimmune serum (B) and counterstained with fluorescent WGA (blue). Color merged maximum projection of anti-MreB (A) …
Figure 11—figure supplement 3
New cell wall growth appears as diffuse labeling and circumferential bands dispersed along the sidewall, excluded from poles, and present at septa in ΔccmA.
3D SIM imaging of ΔccmA cells labeled with an 18 min pulse of MurNAC-alk (A, C, D, yellow) or D-Ala-alk (E, G, H, yellow) or mock labeled (B, F) and counterstained with fluorescent WGA (blue). Color …
Figure 11—video 1
Volumetric rendering and z-slices of the example cells in Figure 11—figure supplements 2 and 3.
Author response image 1
Tables
Table 1
MurNAc-alk incorporation into PG
| Muropeptide (non-reduced) | Theoretical neutral mass | MurNAc-alk labeled H. pylori | Control H. pylori | ||||
|---|---|---|---|---|---|---|---|
| Observed ion (charge) | Rt* (min) | Calculated neutral mass | Observed ion (charge) | Rt* (min) | Calculated neutral mass | ||
| Di | 696.270 | 697.289 (1+) | 20.3 | 696.282 | 697.290 (1+) | 20.4 | 696.283 |
| Alk-Di | 734.286 | 735.307 (1+) | 30.5 | 734.300 | -† | - | - |
| Tri | 868.355 | 869.375 (1+) | 15.8 | 868.368 | 869.374 (1+) | 15.8 | 868.367 |
| Alk-Tri | 906.371 | 907.392 (1+) | 25.8 | 906.385 | - | - | - |
| Tetra | 939.392 | 940.411 (1+) | 20.4 | 939.404 | 940.412 (1+) | 20.4 | 939.405 |
| Alk-Tetra | 977.408 | 978.428 (1+) | 30.4 | 977.421 | - | - | - |
| Penta | 1010.429 | 1011.449 (1+) | 22.9 | 1010.442 | 1011.449 (1+) | 22.8 | 1010.442 |
| Alk-Penta | 1048.445 | 1049.464 (1+) | 32.9 | 1048.457 | - | - | - |
| TetraTri | 1789.736 | 895.889 (2+) | 33.4 | 1789.762 | 895.888 (2+) | 33.3 | 1789.761 |
| Alk-TetraTri | 1827.752 | 914.898 (2+) | 39.2 | 1827.781 | - | - | - |
| TetraTetra | 1860.774 | 931.407 (2+) | 35.0 | 1860.799 | 931.407 (2+) | 34.9 | 1860.799 |
| Alk-TetraTetra | 1898.789 | 950.416 (2+) | 39.7 | 1898.817 | - | - | - |
| TetraPenta | 1931.811 | 966.926 (2+) | 35.8 | 1931.837 | 966.925 (2+) | 35.7 | 1931.835 |
| Alk-TetraPenta | 1969.826 | 985.934 (2+) | 39.9 | 1969.853 | - | - | - |
-
* Rt, retention time.
†-, not detected. Muropeptides detected (confirming incorporation) via LC-MS analysis of MurNAc-alk labeled versus control PG digests. The control cells displayed no evidence of any MurNAc-alk incorporation.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Monoclonal ANTI-FLAG M2 antibody produced in mouse | Sigma | Cat# F1804, RRID:AB_262044 | IF(1:200) |
| Antibody | Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | Cat# A-11029, RRID:AB_2534088 | IF(1:200) |
| Antibody | Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | Cat#: A-11008; RRID: AB_143165 | IF(1:200) |
| Antibody | Polyclonal rabbit αCcmA | (Blair et al., 2018) | IF (1:200); WB (1:10,000) | |
| Antibody | Polyclonal rabbit αMreB (H. pylori) | (Nakano et al., 2012) | IF (1:500); WB (1:25,000) | |
| Commercial assay, kit | Click-iT Cell Reaction Buffer Kit | Invitrogen | Cat# C10269 | |
| Chemical compound, drug | Alexa Fluor 555 Azide, Triethylammonium Salt | Invitrogen | Cat# A20012 | |
| Chemical compound, drug | D-Ala-alk ((R)−2-Amino-4-pentynoic acid) | Boaopharma | Cat# B60090 | |
| Chemical compound, drug | MurNAc-alk | (Liang et al., 2017) | ||
| Chemical compound, drug | MurNAc | Sigma | Cat# A3007 | |
| Chemical compound, drug | Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate | Invitrogen | Cat# W11261 | |
| Chemical compound, drug | Wheat Germ Agglutinin, Alexa Fluor 555 Conjugate | Invitrogen | Cat# W32464 | |
| Other | ProLong Diamond Antifade Mountant | Invitrogen | P36961 |
Table 2
Strains used in this study.
| Strain | Genotype/description | Construction | Reference |
|---|---|---|---|
| LSH100 | Wild-type: mouse-adapted G27 derivative | - | Lowenthal et al., 2009 |
| LSH141 (Δcsd2) | LSH100 csd2::cat | - | Sycuro et al., 2010 |
| TSH17 (Δcsd6) | LSH100 csd6::cat | - | Sycuro et al., 2013 |
| LSH108 | LSH100 rdxA::aphA3sacB | - | Sycuro et al., 2010 |
| HMJ_Ec_pLC292-KU | E. coli TOP10 pLC292-KU | Transformation of TOP10 with pLC292-KU | This study |
| HJH1 | LSH100 rdxA::amgKmurU | Integration of pLC292-KU into LSH108 | This study |
| IM4 | LSH100 mcGee:mreB | Integration of pIM04into LSH100 | This study |
| JTH3 | LSH100 ccmA:2X-FLAG:aphA3 | - | Blair et al., 2018 |
| JTH5 | LSH100 ccmA:2X-FLAG:aphA3 rdxA::amgKmurU | Natural transformation of HJH1 with JTH3 genomic DNA | This study |
| KGH10 | NSH57 ccmA::catsacB | - | Sycuro et al., 2010 |
| LSH117 | LSH100 ccmA::catsacB | Natural transformation of LSH100 with KGH10 genomic DNA | This study |
| SSH1 | LSH100 ccmAI55A | Natural transformation with ccmA I55A PCR product | This study |
| SSH2 | LSH100 ccmAL110S | Natural transformation with ccmA L110S PCR product | This study |
| LSH142 (ΔccmA) | LSH100 ccmA::cat | - | Sycuro et al., 2010 |
| JTH6 | LSH100 rdxA::amgKmurU ccmA::cat | Natural transformation of HJH1 with LSH142 genomic DNA | This study |
Table 3
Primers used in this study.
| Primer name | Sequence (5’ to 3’) |
|---|---|
| AmgK_BamHI_F | GATAGGATCCTGACCCGCTTGACGGCTA |
| MurU_HindIII_R | GTATAAGCTTTCAGGCGCGCTCGC |
| RdxA_F1P1 | CAATTGCGTTATCCCAGC |
| RdxA_dnstm_RP2 | AAGGTCGCTTGCTCAATC |
| O#9 ProMreB (KpnI_5’) | TATTGGTACCCGCTTGATGTATTCATCAAAG |
| O#10 ProMreB_R | GATTAATTTGCTAAAAATCATAAAATAAACTCCTTGTTTTG |
| O#11 ProMreB_F | CAAAACAAGGAGTTTATTTTATGATTTTTAGCAAATTAATC |
| O#12 ProMreB (XhoI_3’) | TATTCTCGAGTTATTCACTAAAACCCACAC |
| O#36 pMcGee-Insert-F | CTGCCTCCTCATCCTCTTCATCCTC |
| O#45 MreBC-seq-F2 | GCACCTATTTTGGGGTTTGAAACC |
| O#47 MreB-seq-F2 | CATTGAGCGCTGGTTTTAAGGCGGTC |
| O#28 MreBseq-F3 | CGATCGTGTTAGTCAAAGGGCAGGGC |
| O#37 pMcGee-Insert-R | GGTGTACAAACATTTAAAGGTAGAG |
| O#68 McGee-1F | CATTTCCCCGAAAAGTGCCACGAGCTCGAAGGAGTATTGATGAAAAAGG |
| O#69 McGee-1R | CTAGAGCGGCCCCACCGCGGCCATCATTAACATCATTATCG |
| O#70 MCS-kan-F | CTCGAGGGGGGGCCCGGTACCCACAGAATTACTCTATGAAGC |
| O#71 MCS-kan-R | CCATTCTAGGCACTTATCCCCTAAAACAATTCATCCAGTAA |
| O#72 McGee-2F | TTACTGGATGAATTGTTTTAGGGGATAAGTGCCTAGAATGG |
| O#73 McGee-2R | CGGATATTATCGTGAGATCGCTGCAGACTGGGGGGAAACTCATGGG |
| O#74 McGee-R6K-F | CCCATGAGTTTCCCCCCAGTCTGCAGCGATCTCACGATAATATCCG |
| O#75 McGee-R6K-R | GTAACTGTCAGACCAAGTTTACTGCGGCCGCGCAAGATCCGGCCACGATGCG |
| O#76 R6K-amp-F | CGCATCGTGGCCGGATCTTGCGCGGCCGCAGTAAACTTGGTCTGACAGTTAC |
| O#77 R6K-amp-R | CCTTTTTCATCAATACTCCTTCGAGCTCGTGGCACTTTTCGGGGAAATG |
| O#78 MCS fragment | CCGCGGTGGGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCGGAATTCGCTTATCG |
| O#79 McGee-MCS-F | CGATAATGATGTTAATGATGGCCGCGGTGGGGCCGCTCTAG |
| O#80 McGee-MCS-R | GCTTCATAGAGTAATTCTGTGGGTACCGGGCCCCCCCTCGAG |
| Csd1F | GAGTCGTTACATTAATGTGCATATCT |
| G1480_DnStrmP2 | AAGGGTGCAATAACGCGCTAA |
| MreB_start_F | ATGATTTTTAGCAAATTAATCGG |
| MreB_cat_up_R | CACTTTTCAATCTATATCCGTGCCTCCGCCAATATC |
| C1 | GATATAGATTGAAAAGTGGAT |
| C2 | TTATCAGTGCGACAAACTGGG |
| Cat_mreB_dn_F | AGTTTGTCGCACTGATAAACTGAAATTGGCG |
| MreB_end_R | TTATTCACTAAAACCCACACGGCTGA |
| FabZ_up_F | GCTATCCCATGCTATTGATAGAC |
| Cat_mid_R | GTCGATTGATGATCGTTGTAACTCC |
| MreB_mid_dn_F | GATCAAAGCATCGTGGAATACATCC |
| Supp2_junc1_R_mid | AATTTGCTAAAAATCACTAA |
| MreB_up | AATACCAGCAACTTTTCAAAA |
| Supp1_Junction1_R | ATTTGCTAAAAACACACGGC |
| Catout | CCTCCGTAAATTCCGATTTGT |
| McGee_187 | GCGAGTATTACCACAAGTTTTC |
| CcmA SDM mi R | AGACTAGATTGGATCATTCCCTATTTATTTTCAATTTTCT |
| CcmA SDM mi F | ATAAAGAAAGGAGCATCAGATGGCAATCTTTGATAACAAT |
| CcmA SDM up R | ATTGTTATCAAAGATTGCCATCTGATGCTCCTTTCTTTAT |
| CcmA SDM dn F | AGAAAATTGAAAATAAATAGGGAATGATCCAATCTAGTCT |
| CcmA SDM dn R | GCTCATTTGAGTGGTGGGAT |
| SDM 155A F | ATTCTAAAAGCACGGTGGTGgcCGGACAAACCGGCTCGGTAG |
| SDM 155A R | CTACCGAGCCGGTTTGTCCGgcCACCACCGTGCTTTTAGAAT |
| SDM L110S F | TGGTGGAAAGGAAGGGGATTtcGATTGGGGAAACTCGCCCTA |
| SDM L110S R | TAGGGCGAGTTTCCCCAATCgaAATCCCCTTCCTTTCCACCA |
