Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: (A) Schematic of yeast-display screen- A library of yeast cells, each displaying a single human protein encoded by a uniquely bar-coded plasmid was pooled and mixed with surface biotinylated Borrelia . The BASEHIT library was scaled to 96-well magnetic separation format for this screen. Magnetic separation using streptavidin microbeads, followed by next-generation sequencing was used to identify yeast displaying proteins that bind to the bacterial cells. Plasmid DNA was isolated and sequenced to identify the proteins (B) Host interactions with B . burgdorferi N40- spirochete from the cultures grown at 33°C or 37°C were surface biotinylated and used for yeast selections, as described above. The data shows average scores of four independent runs of the selection on the 33°C grown sample and one run on the 37°C grown sample. The scores were normalized for diverse microbes (i.e. Escherichia coli , Staphylococcus aureus , Bacillus subtilis , Shigella flexneri , and Bifidobacterium adolescentis ) as background correction for non-specific binding activity. (C) PGLYRP1 interaction with Borrelia species- Samples from 53 Borrelia isolates, grown at two different temperatures (when possible), were screened against the host protein library as described above for B . burgdorferi , as were 370 additional bacterial samples. The score for each gene is defined as the overall enrichment for that gene (relative to the unselected library) multiplied by the percentage of barcodes associated with the gene that enriched (defined as logFC >0). The calculated scores of Borrelia species were compared with those of the other bacteria. The bars represent mean ± SD and p-values were determined using a Mann-Whitney U-test.

Article Snippet: After washing, recombinant human PGLYRP1 (R&D Systems, #2590-PGB) was added to each well at a concentration of 500 ng/mL in PBS containing 1% BSA.

Techniques: Plasmid Preparation, Next-Generation Sequencing, Isolation, Selection, Binding Assay, Activity Assay, Bacteria, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: (A) ELISA results show the interaction of human PGLYRP1 with lysate of B . burgdorferi . The lysate was immobilized on microtiter wells and probed with increasing concentrations of either recombinant human PGLYRP1-Fc protein (1–100 ng, red) or Fc control protein (gray). The values plotted represent the mean ± SEM of three replicates from a single experiment. p-value is displayed in the graph and determined using a student t-test. (B and C) Binding of human (B) and mouse (C) PGLYRP1 to B . burgdorferi . The culture was grown to a density of 10 6 CFU/mL and incubated with varying concentrations of recombinant PGLYRP1-His 8 (10 μg/mL in green, 40 μg/ml in red). B . burgdorferi bound to recombinant PGLYRP1-His 8 was measured using a secondary AF488-His 6 monoclonal antibody by flow cytometry. Overlay histograms show protein binding to B . burgdorferi identified by Alexa Fluor 488-His 6 monoclonal antibody. Binding of recombinant human/murine CD55-His 8 (40 μg/ml in blue) to B . burgdorferi was used as control. The background binding of AF488-His 6 antibody alone with B . burgdorferi is shown in gray shaded region. Results from one independent experiment are shown here. For (B) and (C), the Y-axis represents relative cell counts calculated as a percentage of the maximum events ( Borrelia ).

Article Snippet: After washing, recombinant human PGLYRP1 (R&D Systems, #2590-PGB) was added to each well at a concentration of 500 ng/mL in PBS containing 1% BSA.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Control, Binding Assay, Incubation, Flow Cytometry, Protein Binding

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: (A) ELISA shows the binding of recombinant human PGLYRP1 at two concentrations, 100 and 500 ng/ml to peptidoglycan (PG) sacculi isolated from B . burgdorferi (Bb) as compared to a PBS negative control. The values plotted represent the mean ± SEM of two technical replicates from a single experiment. (B and C) Flow cytometry-based experiment showing binding of human (B) and mouse (C) PGLYRP1 to B . burgdorferi , after pre-incubating the protein in the absence (red) and presence (blue) of B . burgdorferi PG. Recombinant PGLYRP1-His 6 (1 μg/mL) was pre-incubated with Borrelia PG (10 μg/mL) and then added to live B . burgdorferi overlay histograms show PGLYRP1 binding to B . burgdorferi identified by Alexa Fluor 488, His 6 monoclonal antibody. Results from one independent experiment are shown here. For (B) and (C), the Y-axis represents relative cell counts calculated as a percentage of the maximum events ( Borrelia ).

Article Snippet: After washing, recombinant human PGLYRP1 (R&D Systems, #2590-PGB) was added to each well at a concentration of 500 ng/mL in PBS containing 1% BSA.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Isolation, Negative Control, Flow Cytometry, Incubation

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: Wild-type BALB/c and PGLYRP1 -/- mice (at least n = 7 in each group) were infected with 1x10 6 spirochetes by subcutaneous injection. (A and B) Skin B . burgdorferi burden was assessed by ear punch biopsies at 14 d (A) and 25 d (B) post infection, by qPCR for Borrelia specific gene ( flaB ) normalized to mouse β-actin . (C and D) B . burgdorferi burden was assessed in hearts (C) and joints (D) at day 25, by qPCR as above. Results from two independent experiments are shown. Each data point represents the value of an individual animal. (E) The extent of splenomegaly was expressed as spleen weights in WT and PGLYRP1 -/- mice infected with B . burgdorferi at day 25. Results from one independent experiment are shown here. Each data point represents the value of an individual animal. The bars represent mean ± SEM and p-values are calculated by student t-test.

Article Snippet: After washing, recombinant human PGLYRP1 (R&D Systems, #2590-PGB) was added to each well at a concentration of 500 ng/mL in PBS containing 1% BSA.

Techniques: Infection, Injection

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: (A) Histopathology scores from tibiotarsi for individual mice in infected wild-type mice (WT Inf) and infected PGLYRP1 -/- mice (PGLYRP1 -/- Inf) at 25 days post infection. Tibiotarsi were scored by blinded examination for tenosynovitis on a scale of 0 (negative) to 3 (severe). (B) The severity of cardiac inflammation in the heart of infected WT and PGLYRP1 -/- mice 25 d post infection. Hearts were scored in a blinded fashion for carditis on a scale of 0 (negative) to 5 (severe). Similar to tenosynovitis scores, PGLYRP1 -/- infected mice demonstrated no significant difference in carditis scores compared to WT mice. Results from at least two independent experiments (at least n = 7 in each group) are pooled and shown here. The bars represent mean ± SEM and p-values were calculated by Mann-Whitney U-test.

Article Snippet: After washing, recombinant human PGLYRP1 (R&D Systems, #2590-PGB) was added to each well at a concentration of 500 ng/mL in PBS containing 1% BSA.

Techniques: Histopathology, Infection, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: Antibody levels in uninfected wild type BALB/c (WT) and PGLYRP1 -/- mice were compared with those in the infected ones (at least n = 7 in each group). Representative results from one independent experiment are shown. (A) Whole cell lysate of B . burgdorferi was coated on microtiter plate and serum from either uninfected WT, infected WT, uninfected PGLYRP1 -/- or infected PGLYRP1 -/- mice was used at varying dilutions. The binding was measured by secondary goat anti-mouse IgG HRP-conjugated antibody. Statistically significant increase in binding was observed at IgG titers 1:200, 1:2000, 1:20000 in infected WT compared to infected knockout mice. (B-E) Levels of different IgG isotypes (IgG1, B; IgG2a, C; IgG2b, D; IgG3, E) were measured against B . burgdorferi lysate, using mouse serum at varying dilutions. The binding was measured by secondary goat anti-mouse IgG1, IgG2a, IgG2b or IgG3 HRP-conjugated antibody. Statistically significant increase was observed in IgG1, IgG2a, IgG3, and IgG2b at 1:200 and 1:2000 dilution in infected WT compared to infected PGLYRP1 -/- mice. Representative results from one independent experiment are shown. Each data point represents an individual animal in the corresponding group. The bars represent mean ± SEM and p values determined using student t test.

Article Snippet: After washing, recombinant human PGLYRP1 (R&D Systems, #2590-PGB) was added to each well at a concentration of 500 ng/mL in PBS containing 1% BSA.

Techniques: Infection, Binding Assay, Knock-Out

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: Serum cytokine profile was assessed in both wild-type and PGLYRP1 -/- mice 25 d post-infection ((at least n = 7 in each group) using a mouse cytokine/chemokine 31-plex (MD-31) array. An increase in pro-inflammatory cytokines IFN-γ (A) and related cytokines- CXCL-9 (B) and CXCL-10 (C) was observed in infected PGLYRP1 -/- mice as compared to parent BALB/c mice. Representative results from one independent experiment are shown. Each data point represents an individual animal in the corresponding group. The bars represent mean ± SEM and p-values determined using student t test.

Article Snippet: After washing, recombinant human PGLYRP1 (R&D Systems, #2590-PGB) was added to each well at a concentration of 500 ng/mL in PBS containing 1% BSA.

Techniques: Infection

Journal: PLoS Pathogens

Article Title: A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis

doi: 10.1371/journal.ppat.1009030

Figure Lengend Snippet: B . burgdorferi is a diderm bacteria where the outer membrane surrounds the peptidoglycan layer and protects it from the external environment. The peptidoglycan meshwork, in turn, surrounds the cytoplasm. Our results suggest PGLYRP1 binds to Borrelia peptidoglycan.

Article Snippet: After washing, recombinant human PGLYRP1 (R&D Systems, #2590-PGB) was added to each well at a concentration of 500 ng/mL in PBS containing 1% BSA.

Techniques: Bacteria, Membrane

Journal: bioRxiv

Article Title: A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection

doi: 10.1101/2025.09.26.678899

Figure Lengend Snippet: 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 or 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 20X and 10X magnification Ex vivo bone marrow derived neutrophil killing assay (E). Neutrophils were isolated from BALB/c (WT) or PGLYRP1 KO mice and incubated with B. pertussis for 2 (circles) or 24 (square) hours 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-hours post-incubation compared to BSA control against WT (F) or a TCT non-releasing strain (TCT-) (G). 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 (H). Data represents CFU as a percentage of starting inoculum recovered at indicated timepoint as assessed by serial dilution of culture on BG agar. Data are presented as mean ± SEM; significance determined by unpaired t-test or ANOVA as appropriate. **, p-value < 0.01, *** p-value < 0.005, **** p-value < 0.001

Article Snippet: HEK293-derived reporter cells expressing murine or human NOD1 and NOD2 with an, or TREM-1 and an NFkB–dependent luciferase or NF-kB inducible SEAP reporter (InvivoGen) and TREM-1-DAP12 reporter cells (Eurofins Discover X)in the Jurkat T cell background with an NFAT-dependent luciferase were were stimulated with B. pertussis tracheal cytotoxin TCT (generously provided by William Goldman, UNC), PGN fragments (TCT, S. aureus or B. subtilis PGN (Invivogen)) in the presence or absence of recombinant human PGLYRP1 (R&D systems) at a concentration of 5ug per well for 24 hours.

Techniques: Knock-Out, Infection, Expressing, RNA Sequencing, RNAscope, In Situ Hybridization, Ex Vivo, Derivative Assay, Isolation, Incubation, Serial Dilution, In Vitro, Recombinant, Activity Assay, Control, Mutagenesis

Journal: bioRxiv

Article Title: A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection

doi: 10.1101/2025.09.26.678899

Figure Lengend Snippet: 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 3 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 using adjusted p-values as indicated in the main text. *, p-value < 0.05, **, p-value < 0.01

Article Snippet: HEK293-derived reporter cells expressing murine or human NOD1 and NOD2 with an, or TREM-1 and an NFkB–dependent luciferase or NF-kB inducible SEAP reporter (InvivoGen) and TREM-1-DAP12 reporter cells (Eurofins Discover X)in the Jurkat T cell background with an NFAT-dependent luciferase were were stimulated with B. pertussis tracheal cytotoxin TCT (generously provided by William Goldman, UNC), PGN fragments (TCT, S. aureus or B. subtilis PGN (Invivogen)) in the presence or absence of recombinant human PGLYRP1 (R&D systems) at a concentration of 5ug per well for 24 hours.

Techniques: Staining, Knock-Out, Infection, RNA Sequencing, Gene Expression, Isolation

Journal: bioRxiv

Article Title: A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection

doi: 10.1101/2025.09.26.678899

Figure Lengend Snippet: (A–B) HEK293 reporter cells expressing either mouse (A) or human (B-D) NOD1 or NOD2 were stimulated with recombinant mouse (A) or human (B-D) PGLYRP1 (12.5 ug/mL), purified tracheal cytotoxin (TCT) (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 hours. 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

Article Snippet: HEK293-derived reporter cells expressing murine or human NOD1 and NOD2 with an, or TREM-1 and an NFkB–dependent luciferase or NF-kB inducible SEAP reporter (InvivoGen) and TREM-1-DAP12 reporter cells (Eurofins Discover X)in the Jurkat T cell background with an NFAT-dependent luciferase were were stimulated with B. pertussis tracheal cytotoxin TCT (generously provided by William Goldman, UNC), PGN fragments (TCT, S. aureus or B. subtilis PGN (Invivogen)) in the presence or absence of recombinant human PGLYRP1 (R&D systems) at a concentration of 5ug per well for 24 hours.

Techniques: Expressing, Recombinant, Purification, Activity Assay, Activation Assay, RNA Sequencing, Infection

Journal: bioRxiv

Article Title: A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection

doi: 10.1101/2025.09.26.678899

Figure Lengend Snippet: 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 5μg/mL of Lys-type peptidoglycan derived from Staphylococcus aureus , tracheal cytotoxin (TCT) or a DAP-type PGN from Bacillus subtilis (5 μg/mL) (B). Diagram depicting proposed model of exogenous PGLYRP1 boosting NOD1 and TREM-1 signaling, but dampening NOD2 signaling. Diagram further depicts B. pertussis co-opting these pathways via release of TCT to block TREM-1 signaling and bias NOD signaling (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

Article Snippet: HEK293-derived reporter cells expressing murine or human NOD1 and NOD2 with an, or TREM-1 and an NFkB–dependent luciferase or NF-kB inducible SEAP reporter (InvivoGen) and TREM-1-DAP12 reporter cells (Eurofins Discover X)in the Jurkat T cell background with an NFAT-dependent luciferase were were stimulated with B. pertussis tracheal cytotoxin TCT (generously provided by William Goldman, UNC), PGN fragments (TCT, S. aureus or B. subtilis PGN (Invivogen)) in the presence or absence of recombinant human PGLYRP1 (R&D systems) at a concentration of 5ug per well for 24 hours.

Techniques: Generated, RNA Sequencing, Expressing, Activation Assay, Recombinant, Derivative Assay, Blocking Assay, Two Tailed Test