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. 2022 Sep 8;11:e80111. doi: 10.7554/eLife.80111

Figure 7. Csd5 is required for CcmA-HaloTag to localize to the cell envelope at the major helical axis.

(A) Maximum intensity projections (MIPs) and frames from Z-stacks of H. pylori cells labeled with wheat germ agglutinin (WGA) to label the cell wall (magenta) and JF549 ligand to label CcmA-HaloTag (green), pixels are white where the two signals colocalize. Green arrows indicate cytoplasmic CcmA-HaloTag. White arrows indicate CcmA-HaloTag signal that colocalizes with the cell envelope. Scale bars = 1 µm. (B) Representative plots displaying the relative concentration of CcmA-HaloTag and WGA signal from one WT and one ∆csd5 cell at the cell surface (offset from surface = 0) and at computationally generated cell surfaces that are inside (offset from surface < 0) and outside (offset from surface > 0) of the cell surface. (C) Representative H. pylori cells from each population with Gaussian curvature values mapped on the cell surface. (D, E) Top: histogram of Gaussian curvature of cell surfaces of each population. Bottom: surface Gaussian curvature enrichment of relative concentration of CcmA signal in each population where 1 = uniform concentration of CcmA, shaded regions indicate SEM. In panels (C–F), WT n = 292, ∆csd6 n = 264 cells, ∆csd7 n = 355, ∆csd5 n = 326, ∆csd5/7 n = 523. Strains used: SSH39B, SSH49A, SSH50A, JS09, SSH70A, and LSH117.

Figure 7.

Figure 7—figure supplement 1. Cell shape of CcmA-HaloTag strains and analysis of CcmA-HaloTag surface signal in populations.

Figure 7—figure supplement 1.

(A) Scatterplot of outlines of H. pylori cells derived from phase-contrast images. (B) Histogram displaying side curvature of each population. WT (LSH100) n = 116 cells, ccmA::ccmA-Halotag (SSH38A) n = 133 cells, ccmA::ccmA-HaloTag, rdxA::ccmA (SSH39B) n = 190 cells. (C, D) Plots displaying the relative concentration of CcmA-HaloTag and WGA signal at the cell surface (offset from surface = 0 nm) and at computationally generated cell surfaces that are inside (offset from surface < 0 nm) and outside (offset from surface > 0 nm) of the cell surface. Crosses indicate SEM. Populations of WT (C) and ∆csd5 (D) cells from three independent experiments were pooled.
Figure 7—figure supplement 2. Cell shape and Csd1 levels of ccmA ∆NT ∆csd5 mutants.

Figure 7—figure supplement 2.

(A) Scatterplot of outlines of H. pylori cells derived from phase-contrast images. (B) Histogram displaying side curvature of each population. ccmA ∆NT ∆csd5 (SSH109A) n = 246 cells, ccmA ∆NT (SSH55A) n = 250 cells, ∆csd5 (LSH123) n = 215 cells. (C) Western blot of whole-cell extracts probed with ɑ-Csd1 polyclonal antibody showing levels of Csd1; ɑ-Cag3 polyclonal antibody was used as a loading control. Strains used: LSH100, LSH113, LSH123, SSH55A, and SSH109A.
Figure 7—figure supplement 2—source data 1. Raw, unedited Western blots probed with ɑ-Cag3 and ɑ-Csd1 antibodies in whole-cell lysates of H. pylori strains.