A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection
- Department of Microbiology and Immunology, University of Maryland School of Medicine, United States
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, United States
Figures
Figure 1
PGLYRP1 has a dual role in host defense against Bordetella pertussis.
Lung bacterial burdens in wild-type BALB/c (WT, black) and PGLYRP1-knockout (PGLYRP1 KO, red) at 4 (A) and 7 (B) days post-infection. Violin-plot visualizing single-cell expression levels of Pglyrp1 across cell types in the lungs of C57/B6 at 4DPI for both infected and sham-challenged mice, assessed via analysis of single-cell RNA sequencing data (C). RNAscope in situ hybridization visualization of Pglyrp1 transcripts in FFPE sections of B. pertussis-infected mouse lung tissue (D). Each dot represents a single Pglyrp1 mRNA molecule. Images are presented at ×20 and ×10 magnification of ex vivo bone marrow-derived neutrophils (E). Neutrophils were isolated from BALB/c (WT) or PGLYRP1 KO mice and incubated with B. pertussis for 2 (circles) or 24 (square) hr, and CFU enumerated by serial dilution on Bordet Gengou agar. In vitro bacterial killing assay assessing recombinant murine PGLYRP1 (mPGLYRP1, red) bactericidal activity at 6- and 24-hr post-incubation compared to BSA control against WT (G) or a TCT non-releasing strain (TCT−) at 6 hr (H). Enumeration of CFU following incubation of mPGLYRP1 with wild-type B. pertussis (parental) or mutant lacking the BpsB gene of the Bordetella polysaccharide (Bps) operon for 6 hr (I). Data represent CFU as a percentage of starting inoculum recovered at indicated time point as assessed by serial dilution of culture on BG agar. Data are presented as mean ± SEM; significance determined by unpaired t-test (A, B, F, G) or ANOVA (H, I). **p-value <0.01, ***p-value <0.005, ****p-value <0.001.
Figure 2
Assessment of PGLYRP1’s role in pulmonary inflammation and transcriptional responses during Bordetella pertussis infection.
Semi-quantitative scoring of hematoxylin and eosin (H&E) stained lung sections from wild-type BALB/c (black dots) and PGLYRP1 knockout (red dots) mice at 4 and 7 days post-infection with B. pertussis (A). Data represents average scores of three blinded investigators scoring degree and percentage of peribronchial infiltration and alveolar consolidation. Bulk RNA sequencing was performed on lung homogenates collected from WT and PGLYRP1 KO mice at 4DPI to assess differential gene expression. Volcano plots (B, D) and heat-maps (C, E) showing total (B, D) and immune-related (C, E) differentially expressed genes between B. pertussis challenged BALB/c (WT) and PGLYRP1 KO mice (B, C) or PGLYRP1 KO mice challenged with parental wild-type B. pertussis (WT) or TCT-under releasing strain TCT(−). (F) KEGG enrichment pathway analysis was performed on differentially expressed genes from lung tissue isolated from mice challenged with WT or TCT(−) B. pertussis. Data represent the mean of biological replicates; statistical analyses were performed by Mann–Whitney U test. *p-value = 0.003, **p-value <0.01.
Figure 3 with 1 supplement
PGLYRP1 selectively modulates NOD1 and NOD2 signaling in response to Bordetella pertussis peptidoglycan.
HEK293 reporter cells expressing either mouse (A, D) or human (B) NOD1 or (C) NOD2 were stimulated with recombinant mouse (A) or murine or human (B–D) PGLYRP1 (12.5 µg/ml), purified tracheal cytotoxin (TCT, 1 ng/ml) (A, B), muramyl dipeptide (MDP, NOD2 agonist) (C), or conditioned bacterial growth media (OD = 0.8) (D). NF-κB-driven SEAP reporter activity was measured to assess pathway activation. Reporter activation was quantified after 18–24 hr and normalized to PBS-treated cell controls as a zero baseline. Single-cell RNA sequencing was performed on lung tissue from BALB/c mice infected with B. pertussis at 4 days post-infection. Neutrophils were subjected to further sub-clustering and visualized as a UMAP (E). Violin plots visualizing expression of Nod1, Nod2, IL1a, and Pglyrp1 across neutrophil sub-clusters (F). Volcano plot demonstrating differentially expressed genes in Nod1 (blue) or Nod2 (red) expressing neutrophils (G) Single-cell analyses represent combined data from two mice per group. Statistical analyses were performed using Student’s t-test or adjusted p-values as appropriate. **p-value <0.01, ***p-value <0.005, ****p-value <0.001.
Figure 3—figure supplement 1
Transcriptional heterogeneity of neutrophil populations in the B. pertussis infected mouse lung.
Figure 4
TCT inhibits PGLYRP1 signaling via TREM-1.
Violin plot generated from single-cell RNA sequencing (scRNA-seq) displaying TREM-1 expression on neutrophils (green), alveolar macrophages (AM, gold), and inflammatory monocytes (IM) at 4DP1 (A). TREM-1 activation measured via NFAT-driven luminescence (relative luminescence units, RLU) in TREM-1 reporter cells treated with recombinant human PGLYRP1 with or without 5 µg/ml of Lys-type peptidoglycan derived from Staphylococcus aureus (5 µg/ml), TCT (5 µg/ml), or a DAP-type PGN from Bacillus subtilis (5 µg/ml) (B). Diagram depicting proposed model (C). All TREM-1 activation assays were performed with biological triplicates (n = 3). Statistical analyses were performed using unpaired two-tailed Student’s t-test. *p-value <0.05, **p-value <0.01, ***p-value <0.005, ****p-value <0.001.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (HEK-293) | HEK-Blue mouse NOD1 reporter cells | Invivogen | hkb-mnod1 | HEK-293, NF-κB-based SEAP reporter |
| Cell line (HEK-293) | HEK-Blue human NOD1 reporter cells | Invivogen | hkb-hnod1 | HEK-293, NF-κB-based SEAP reporter |
| Cell line (HEK-293) | HEK-Blue mouse NOD2 reporter cells | Invivogen | hkb-mnod2 | HEK-293, NF-κB-based SEAP reporter |
| Cell line (HEK-293) | HEK-Blue human NOD2 reporter cells | Invivogen | hkb-hnod2v2 | HEK-293, NF-κB-based SEAP reporter |
| Cell line (Jurkat) | TREM-1 reporter cells | Eurofins/DiscoverX | 793-1167C19 | EFC-based TREM-1 activation reporter |
| Genetic reagent (Mus musculus) | PGLYRP1 KO | PMID:20709292 | Provided generously by Dr. Roman Dziarski (Indiana University SOM) | |
| Strain, strain background (Bordetella pertussis) | TCT(+) | Goldman Lab, UNC (Lyon, 2001) | ||
| Strain, strain background (B. pertussis) | TCT(−) | Goldman Lab, UNC (Lyon, 2001) | ||
| Strain, strain background (B. pertussis) | ΔbpsB | Deora Lab, OSU | Provided generously by Dr. Rajendar Deora (OSU) | |
| Peptide, recombinant protein | Murine recombinant PGLYRP1 | R&D Systems (Bio-Techne) | Cat#: 2696-PGB | |
| Peptide, recombinant protein | Human recombinant PGLYRP1 | R&D Systems (Bio-Techne) | Cat#: 2590-PGB | |
| Biological sample (Bordetella pertussis) | Purified TCT | Goldman Lab, UNC | N/A | Isolated from bacterial culture in the Goldman lab |
| Chemical compound, drug | Purified Staphylococcus aureus peptidoglycan | Sigma-Aldrich | Cat#: SIG-77140-10MG | |
| Chemical compound, drug | Purified Bacillus subtilis peptidoglycan | Sigma-Aldrich | Cat#: SIG-69554-10MG |
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A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection
eLife 15:RP108947.
https://doi.org/10.7554/eLife.108947.3
