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

  1. Steven Klupt
  2. Kyong Tkhe Fam
  3. Xing Zhang
  4. Pavan Kumar Chodisetti
  5. Abeera Mehmood
  6. Tumara Boyd
  7. Danielle Grotjahn
  8. Donghyun Park
  9. Howard C Hang  Is a corresponding author
  1. Department of Immunology and Microbiology, Scripps Research, United States
  2. Department of Integrative Structural & Computational Biology, Scripps Research, United States
  3. Department of Chemistry, Scripps Research, United States
5 figures and 8 additional files

Figures

Figure 1 with 2 supplements
Growth and morphology phenotypes of wild-type, ΔsagA, and psagA complemented E. faecium Com15 strains.

(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 …

Figure 1—source data 1

Excel file containing numeric data used to generate Figure 1a.

https://cdn.elifesciences.org/articles/95297/elife-95297-fig1-data1-v1.xlsx
Figure 1—source data 2

Uncropped western blots and gels (total protein) used to generate Figure 1b.

https://cdn.elifesciences.org/articles/95297/elife-95297-fig1-data2-v1.zip
Figure 1—source data 3

Uncropped images used to generate Figure 1c.

https://cdn.elifesciences.org/articles/95297/elife-95297-fig1-data3-v1.zip
Figure 1—source data 4

Uncropped images used to generate Figure 1d.

https://cdn.elifesciences.org/articles/95297/elife-95297-fig1-data4-v1.zip
Figure 1—source data 5

Validation of sagA deletion and image of bacterial strain growth.

Uncropped images used to generate Figure 1d.

https://cdn.elifesciences.org/articles/95297/elife-95297-fig1-data5-v1.zip
Figure 1—figure supplement 1
Analysis of E.faecium ΔsagA deletion and complementation studies.

(a) PCR validation of ΔsagA deletion. (b) ΔsagA cells are sedimented at the bottom of the tube compared to E. faecium wild-type (WT) and ΔsagA / psagA.

Figure 1—figure supplement 2
Antibiotic sensitivity of ΔsagA.

(a) Approximate minimum inhibitory concentration (MIC) determinations via antibiotic test strips for E. faecium wild-type (WT), ΔsagA and ΔsagA/ psagA. White arrows point to MIC values (summary of …

Figure 2 with 2 supplements
Deletion of sagA changes the angle of growing septum.

(a-c) Representative tomographic slices (n=3) of E. faecium strains: E. faecium wild-type (WT) (a), ΔsagA (b), and ΔsagA/psagA (c). Cell division septa are indicated by white arrows. Scale bar = 100 …

Figure 2—figure supplement 1
E.faecium ΔsagA exhibits no defects in cell wall and septum thickness.

(a) Representative cryo-electron tomography (cryo-ET) images of E. faecium wild-type (WT), ΔsagA, and ΔsagA/psagA are shown in the top row. The cell wall, septum, divisome, and ribosome are …

Figure 2—figure supplement 2
Deletion of sagA alters the position of cell division.

(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 …

Figure 3 with 1 supplement
Peptidoglycan profile and NOD2 activation of E.faecium ΔsagA.

(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 …

Figure 3—figure supplement 1
Peptidoglycan profile of E. faecium ΔsagA by LC-MS.

(a) Representative LC-MS chromatograms of mutanolysin-digested peptidoglycan isolated from sacculi of E. faecium wild-type (W)T (top), ΔsagA (middle), and ΔsagA/ psagA (bottom). Numbers correspond …

Figure 4 with 1 supplement
Immune checkpoint inhibitor antitumor activity and tumor immune profile of E.faecium ΔsagA colonized mice.

(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 …

Figure 4—figure supplement 1
Gating strategy used for flow cytometry analysis of tumor infiltrating lymphocytes (TILs).

We first identified total tumor infiltrating cells by forward and side scatter gating. We then selected single cells using forward scatter area (FSC-A) versus forward scatter height (FSC-H) …

Summary of SagA function in E. faecium and impact on host immunity during ICI cancer therapy.

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 …

Additional files

Supplementary file 1

Mutations detected in ΔsagA E. faecium Com15 strain.

https://cdn.elifesciences.org/articles/95297/elife-95297-supp1-v1.docx
Supplementary file 2

Summary of MIC determinations via antibiotic test strips for E. faecium wild-type (WT), ΔsagA, and ΔsagA/psagA (Figure 1—figure supplement 2a).

https://cdn.elifesciences.org/articles/95297/elife-95297-supp2-v1.docx
Supplementary file 3

E. faecium strains used in this study.

https://cdn.elifesciences.org/articles/95297/elife-95297-supp3-v1.docx
Supplementary file 4

Plasmids used in this study.

https://cdn.elifesciences.org/articles/95297/elife-95297-supp4-v1.docx
Supplementary file 5

Primers used in this study for generating complementation plasmids and empty vector.

https://cdn.elifesciences.org/articles/95297/elife-95297-supp5-v1.docx
Supplementary file 6

Primers used in this study for SagA mutagenesis.

https://cdn.elifesciences.org/articles/95297/elife-95297-supp6-v1.docx
Supplementary file 7

Masses of peptidoglycan fragments were detected with MSD API-ES.

https://cdn.elifesciences.org/articles/95297/elife-95297-supp7-v1.docx
MDAR checklist
https://cdn.elifesciences.org/articles/95297/elife-95297-mdarchecklist1-v1.docx

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