Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: ( a ) Growth curves of E. faecium WT, ΔsagA, and complementation strains with functional and nonfunctional sagA genes (n=3). Data are presented as mean value ± standard deviation. ( b ) ɑ-SagA western blot of E. faecium WT, ΔsagA, and complementation strains with functional and nonfunctional sagA genes, on both whole cell lysate (pellets) and total secreted proteins (secreted). Bottom panel shows total protein loading visualized by Stain-free imaging and serves as protein loading control. ( c ) Differential interference contrast (DIC) microscopy of E. faecium WT, ΔsagA, and ΔsagA/psagA complemented strains. White arrows point to cell clusters. Scale bar = 5 µm. ( d ) Transmission electron microscopy (TEM) of E. faecium WT, ΔsagA, and ΔsagA/psagA complemented strains. White arrows point to failed cell separation in ΔsagA. Green arrow points to undegraded peptidoglycan in ΔsagA. Scale bar = 0.2 µm. Figure 1—source data 1. Excel file containing numeric data used to generate . Figure 1—source data 2. Uncropped western blots and gels (total protein) used to generate . Figure 1—source data 3. Uncropped images used to generate . Figure 1—source data 4. Uncropped images used to generate . Figure 1—source data 5. Validation of sagA deletion and image of bacterial strain growth. Uncropped images used to generate .

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques: Functional Assay, Standard Deviation, Western Blot, Staining, Imaging, Microscopy, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: ( a ) PCR validation of Δ sagA deletion. ( b ) Δ sagA cells are sedimented at the bottom of the tube compared to E. faecium wild-type (WT) and Δ sagA / p sagA . Figure 1—figure supplement 1—source data 1. Uncropped gel used to generate and uncropped photo used to generate .

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques:

Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: ( a ) Approximate minimum inhibitory concentration (MIC) determinations via antibiotic test strips for E. faecium wild-type (WT), Δ sagA and Δ sagA / p sagA . White arrows point to MIC values (summary of MIC determination is in ). ( b ) Liquid culture MIC determination for ampicillin. Growth curves of E. faecium WT, Δ sagA , and Δ sagA / p sagA in the presence of serially diluted ampicillin (n=3). Data are presented as mean value ± standard deviation. MIC is defined as minimum tested concentration at which no bacterial growth is observed. Figure 1—figure supplement 2—source data 1. Uncropped images used to generate . Figure 1—figure supplement 2—source data 2. Excel file containing numeric data used to generate .

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques: Concentration Assay, Standard Deviation

Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: ( a ) Representative cryo-electron tomography (cryo-ET) images of E. faecium wild-type (WT), Δ sagA , and Δ sagA /p sagA are shown in the top row. The cell wall, septum, divisome, and ribosome are indicated by white arrows. 3D segmentations are shown in the bottom row. The cell wall is annotated in blue, the membrane in green, and the divisome in magenta. Scale bar = 100 nm. ( b ) The upper panel shows a diagram illustrating how measurements are collected. In the lower panel, a representative cryo-ET image of the septum is shown. Scale bar = 50 nm. ( c ), Comparison of cell wall thickness. The violin plot displays the distribution of cell wall thickness, with E. faecium WT (n=66) shown in blue, Δ sagA (n=96) shown in red, and Δ sagA /p sagA (n=69) shown in magenta. Black dotted lines represent median ( E. faecium WT: 47.48 nm, Δ sagA : 50.11 nm, Δ sagA / p sagA : 46.42 nm) while the colored dotted lines represent quartiles. Welch’s t -est was used to calculate statistical significance. ns, p≥ 0.05. ( d ) Comparison of septum thickness. The violin plot displays the distribution of septum thickness, with E. faecium WT (n=26) shown in blue, Δ sagA (n=62) shown in red, and Δ sagA /p sagA (n=16) shown in magenta. Black dotted lines represent median ( E. faecium WT: 54.33 nm, Δ sagA : 54.86 nm, ΔsagA /p sagA : 57.50 nm) while the colored dotted lines represent quartiles. Welch’s t- test was used to calculate statistical significance. ns, p≥ 0.05. ( e ) Comparison of divisome architecture. To assess potential alterations in the divisome architecture, the distance between the apical end of the septum and the membrane-proximal ring was divided by the distance between the apical end of the septum and the membrane-distal ring. The violin plot displays the distribution of the ratios, with E. faecium WT (n=31) shown in blue, Δ sagA (n=40) shown in red, and Δ sagA /p sagA (n=42) shown in magenta. Black dotted lines represent median ( E. faecium WT: 0.5, Δ sagA : 0.5, ΔsagA /p sagA : 0.5) while the colored dotted lines represent quartiles. Welch’s t -test was used to calculate statistical significance. ns, p≥0.05. Figure 2—figure supplement 1—source data 1. Excel file containing numeric data to generate .

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques: Tomography, Membrane, Comparison

Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: ( a ) Cartoon model depicting cell division site in the xy coordinate plane (left) and xz coordinate plane (right). The two boxes shown in right represent the cryo-focused ion beam (cryo-FIB) milling patterns used to generate thin sections of E. faecium for subsequent cryo-electron tomography (cryo-ET) imaging. ( b–d ) Representative tomographic slices (n=2) of E. faecium strains: E. faecium wild-type (WT) ( b ), Δ sagA ( c ), and Δ sagA / p sagA . ( d ). The upper panels show the view of dividing cells in the xy coordinate plane and, while the corresponding bottom panels show the divisome complex (annotated with white arrows) at the highlighted areas in the corresponding upper panels in the xz coordinate plane, obtained by rotating the cell 90° along the highlighted axis. Scale bar = 100 nm. ( e ) A diagram indicating how measurements were collected. ( f ) Comparison of membrane to proximal ring distance. The violin plot displays the distribution of distance between the apical end of septum membrane to the proximal ring, with E. faecium WT (n=31) shown in blue, Δ sagA (n=40) shown in red, and Δ sagA /p sagA (n=42) shown in magenta. Black dotted lines represent median ( E. faecium WT: 7.39 nm, Δ sagA : 7.39 nm, Δ sagA /p sagA : 7.39 nm) while the colored dotted lines represent quartiles. Welch’s t -est was used to calculate statistical significance. ns, p≥0.05. ( g ) Comparison of membrane to distal ring distance. The violin plot displays the distribution of distance between the apical end of septum membrane to the distal ring, with E. faecium WT (n=31) shown in blue, Δ sagA (n=40) shown in red, and Δ sagA /p sagA (n=42) shown in magenta. Black dotted lines represent median ( E. faecium WT: 14.77 nm, Δ sagA : 15.83 nm, Δ sagA /p sagA : 15.83 nm) while the colored dotted lines represent quartiles. Welch’s t- test was used to calculate statistical significance. *p< 0.05. Figure 2—figure supplement 2—source data 1. Excel file containing numeric data used to generate .

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques: Tomography, Imaging, Comparison, Membrane

Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: ( a-c ) Representative tomographic slices (n=3) of E. faecium strains: E. faecium wild-type (WT) ( a ), Δ sagA ( b ), and Δ sagA /p sagA ( c ). Cell division septa are indicated by white arrows. Scale bar = 100 nm. ( d ) A diagram indicating how septum angle measurements were collected. Acute angles are recorded for further analysis. ( e ) Comparison of septum angle. The violin plot displays the distribution of septum angle, with E. faecium WT (n=40) shown in blue, Δ sagA (n=49) shown in red, and Δ sagA /p sagA (n=37) shown in magenta. Black dotted lines represent median ( E. faecium WT: 88°, Δ sagA : 79°, Δ sagA / p sagA : 87°) while the colored dotted lines represent quartiles. Welch’s t -test was used to calculate statistical significance. *p< 0.05; ****p< 0.0001. ( f ), Pairwise comparison of septum angle in opposing septa. The paired plot displays the distribution of septum angle, with E. faecium WT (n=18) shown in blue, Δ sagA (n=16) shown in red, and Δ sagA / p sagA (n=16) shown in magenta. Two septum angles from opposing septa are linked with straight lines. Paired t- test was used to calculate statistical significance. ns, p≥0.05. Figure 2—source data 1. Excel file containing numeric data to generate .

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques: Comparison

Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: ( a ) Representative LC-MS chromatograms of mutanolysin-digested peptidoglycan isolated from sacculi of E. faecium wild-type (W)T (top), Δ sagA (middle), and Δ sagA / p sagA (bottom). Numbers correspond to each muropeptide annotated in the table ( b ) b Composition of mutanolysin-digested peptidoglycan isolated from E. faecium sacculi. a Peak numbers refer to ( a ). b GM, disaccharide (GlcNAc-MurNAc); 2 GM, disaccharide-disaccharide (GlcNAc-MurNAc-GlcNAc-MurNAc); 3 GM, disaccharide-disaccharide-disaccharide (GlcNAc-MurNAc-GlcNAc-MurNAc-GlcNAc-MurNAc); GM-Tri, disaccharide tripeptide (L-Ala-D-iGln-L-Lys); GM-Tetra, disaccharide tetrapeptide (L-Ala-D-iGln-L-Lys-D-Ala); GM-Penta, disaccharide pentapeptide (L-Ala-D-iGln-L-Lys-D-Ala -D-Ala). c The assignment of the amide and the hydroxyl functions to either peptide stem is arbitrary. Figure 3—figure supplement 1—source data 1. Excel file containing numeric data used to generate .

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation

Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: ( a ) Relative abundance of muropeptides isolated from mutanolysin-digested sacculi of E. faecium strains and analyzed by LC-MS (n=6). Green asterisks highlight changes in abundance of small muropeptides, purple asterisks highlight changes in abundance of crosslinked peptidoglycan fragments. Numbers correspond to different muropeptides from LC-MS analysis listed in legend. ( b ) Composition of muropeptides from E. faecium sacculi. a Peak numbers refer to ( a ). b GM, disaccharide (GlcNAc-MurNAc); 2 GM, disaccharide-disaccharide (GlcNAc-MurNAc-GlcNAc-MurNAc); 3 GM, disaccharide-disaccharide-disaccharide (GlcNAc-MurNAc-GlcNAc-MurNAc-GlcNAc-MurNAc); GM-Tri, disaccharide tripeptide (L-Ala-D-iGln-L-Lys); GM-Tetra, disaccharide tetrapeptide (L-Ala-D-iGln-L-Lys-D-Ala); GM-Penta, disaccharide pentapeptide (L-Ala-D-iGln-L-Lys-D-Ala -D-Ala). c The assignment of the amide and the hydroxyl functions to either peptide stem is arbitrary. ( c ) Relative abundance of GMDP relative to wild-type (WT) from LC-MS chromatograms (n=6). ( d ), NF-κB responses of HEK-Blue hNOD2 cells to live E. faecium strains (MOI = 1, n=6). NOD2 activation is expressed as fold change relative to untreated control. ( e ) Colony forming units (CFU) of E. faecium strains (MOI = 1) internalized in HEK-Blue hNOD2 cells (n=6). Dashed line indicates Limit of Detection (LOD). Data for ( a, c–e ) represent mean value ± standard deviation and analyzed with one-way ANOVA and Tukey’s multiple comparison post hoc test. *p≤0.05; **p≤0.01; ***p≤0.005; ****p≤0.001; ns, not significant. Figure 3—source data 1. Excel file containing numeric data used to generate .

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Activation Assay, Standard Deviation, Comparison

Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: ( a ) Schematic of tumor growth experiment: mice were provided water containing antibiotics for one week and started drinking bacteria three days before tumor implantation. Once the tumor reaches ~100 mm 3 , the measurement starts, and two days after treated with anti-PD-1 (MC38) or anti-PD-L1 (B16F10) every other day. ( b ) MC-38 tumor growth in Nod2 +/- or Nod2 -/- mice that were colonized with E. faecium WT and treated with anti-PD-1 starting at day 7. +/-=10 for Nod2 +/- +/-, n=6 for Nod2 -/- mice. ( c ) B16F10 tumor growth in C57BL/6 mice that were colonized with E. faecium wild-type (WT) or Δ sagA and treated with anti-PD-L1 starting at day 6. No bacterial colonization group as a control (black). n=7–8 mice per group. ( d ) Fecal colony forming units (CFU) analysis of E. faecium on HiCrome Enterococcus faecium agar plates from ( c ) at day 6. n=6 per group. Each dot represents one mouse. The line indicates the limit of detection (LOD, 4000 CFU g –1 ). Nd, not detected. Data represent means ± 95% confidence interval. ( e–j ) Quantification of tumor infiltrating CD45 + cells ( e ), FoxP3 + cells ( f ), CD3 + CD8 + cells ( g ), GranzymeB + CD8 + T cells ( h ), Ki67 + CD8 + T cells ( i ), and PD-1 + CD8 + T cells ( j ). For ( f, h-j ), fluorescence minus one (FMO) control was used to define gates. n=7 mice per group. Data for ( b ) and ( c ) represent mean ± SEM and were analyzed using a mixed effects model with Tukey’s multiple comparisons post hoc test. Data for ( e–j ) represent mean ± SEM and were analyzed by the Mann-Whitney U (one-tail) test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant. Figure 4—source data 1. Excel file containing numeric data used to generate .

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques: Bacteria, Tumor Implantation, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy

doi: 10.7554/eLife.95297

Figure Lengend Snippet: Deletion of sagA impairs peptidoglycan remodeling and cell separation in E. faecium and limits the activation of NOD2 in mammalian cells to promote immune checkpoint inhibitor cancer therapy in mouse models. Created with BioRender.com.

Article Snippet: Fecal samples were weighed, resuspended in sterile PBS, homogenized by douncing with sterile pestles, serially diluted in sterile PBS, and then plated by drip assay onto selective HiCrome Enterococcus faecium agar plates (HIMEDIA 1580) with Enterococcus faecium selective supplement (FD226, HIMEDIA).

Techniques: Activation Assay