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

A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection View ORCID Profile David M Rickert , Sasha Cardozo , View ORCID Profile Nicholas H Carbonetti , William E Goldman , View ORCID Profile Karen M Scanlon , View ORCID Profile Ciaran Skerry doi: https://doi.org/10.1101/2025.09.26.678899 David M Rickert 1 Department of Microbiology and Immunology, University of Maryland School of Medicine , 685 W. Baltimore Street, Baltimore, Maryland, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for David M Rickert Sasha Cardozo 1 Department of Microbiology and Immunology, University of Maryland School of Medicine , 685 W. Baltimore Street, Baltimore, Maryland, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicholas H Carbonetti 1 Department of Microbiology and Immunology, University of Maryland School of Medicine , 685 W. Baltimore Street, Baltimore, Maryland, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nicholas H Carbonetti William E Goldman 2 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Karen M Scanlon 1 Department of Microbiology and Immunology, University of Maryland School of Medicine , 685 W. Baltimore Street, Baltimore, Maryland, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Karen M Scanlon Ciaran Skerry 1 Department of Microbiology and Immunology, University of Maryland School of Medicine , 685 W. Baltimore Street, Baltimore, Maryland, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ciaran Skerry For correspondence: cskerry{at}som.umaryland.edu Abstract Full Text Info/History Metrics Data/Code Preview PDF ABSTRACT Bordetella pertussis , the etiologic agent of whooping cough, remains a serious public health concern despite widespread vaccination. Improved therapeutics and vaccines are urgently needed to treat and prevent pertussis disease. Host recognition of bacterial peptidoglycan (PGN), including B. pertussis extracellular PGN fragment tracheal cytotoxin (TCT), shapes the immune response to infection. Peptidoglycan recognition proteins (PGLYRPs) are a conserved family of innate immune molecules which bind bacterial PGN. While they function as immune signaling receptors in arthropods (termed PGRPs in arthropods), PGLYRPs in mammals have thus far been primarily recognized for their bactericidal activity. Previously thought to function only as antimicrobial peptides in mammals, the immune modulatory roles of this family of peptidoglycan recognition proteins are beginning to gain greater appreciation. Peptidoglycan recognition protein 1 (PGLYRP1) is a secreted antimicrobial protein. However, its role in mammalian host defenses and immune signaling during infection with Gram-negative pathogens, such as B. pertussis , remain largely unknown. Here, we identify a dual role for PGLYRP1 in modulating host immune responses to B. pertussis . Using knockout mice, single-cell and bulk transcriptomics and functional assays, we show that PGLYRP1 contributes to host antibacterial responses to B. pertussis . PGLYRP1 also dampens inflammatory responses and paradoxically inhibits bacterial killing later in infection. Mechanistically, PGLYRP1 enhances nucleotide oligomerization domain (NOD)-1 signaling in response to TCT while suppressing NOD2- and triggering receptor expressed on myeloid cells-1 (TREM-1)-mediated inflammatory pathways. TCT-bound PGLYRP1 selectively impairs TREM-1 activation compared to PGNs from other bacteria, revealing a novel bacterial immune evasion strategy. These findings demonstrate that B. pertussis co-opts PGLYRP1 to temper inflammation and alter immune signaling, revealing a novel immune evasion mechanism of manipulating the availability and structure of their exogenous peptidoglycan, revealing implications for host-pathogen evolution, vaccine design and host-directed therapeutics. INTRODUCTION Bordetella pertussis is a highly contagious respiratory pathogen and the causative agent of whooping cough, a disease characterized by severe, prolonged coughing and airway inflammation 1 - 3 . Despite widespread vaccination efforts, B. pertussis remains a public health concern due to waning vaccine-induced immunity and the pathogen’s capacity to evade host immune defenses 4 . Host detection of microbial components, including peptidoglycan fragments, is essential for initiating immune responses to infection. Understanding how these host-pathogen interactions shape disease outcomes is critical for developing improved strategies to fight infections. Peptidoglycan (PGN) is a fundamental component of the bacterial cell wall, composed of repeating disaccharide chains cross-linked by short peptides 5 . PGN structure is an important determinant of innate immune recognition and responses 6 , 7 . During cell wall turnover, some pathogenic bacterial species, including B. pertussis , release PGN fragments into the extracellular environment 8 - 10 . The biological roles of these fragments in infection and immunity are poorly defined. B. pertussis releases a disaccharide-tetrapeptide monomeric PGN fragment known as tracheal cytotoxin (TCT) 11 , 12 . TCT differs from B. pertussis cell wall PGN by having a 1,6-anhydro group on its MurNAc sugar due to incomplete PGN turnover 11 . The functional consequences of this 1,6-anhydro MurNAc modification are poorly understood 13 . TCT is recognized by the cytosolic pattern recognition receptor (PRR) NOD1 to activate NFkB-driven immune responses 14 , 15 . In vitro and ex vivo studies suggest that recognition of TCT by NOD1 in non-ciliated mucus-secreting cells leads to the accumulation of nitric oxide within neighboring ciliated airway epithelial cells and subsequent ciliostasis and extrusion of ciliated cells 16 , 17 . We recently demonstrated that TCT has broader immunomodulatory effects, skewing PGN-sensing toward NOD1 and away from NOD2, thereby dampening a stronger NOD2-dependent, pro-inflammatory cytokine production and generation of immune memory 18 . However, the broader immunological consequences of TCT release during infection, especially in the context of PGN-sensing by PRRs remain poorly defined. Peptidoglycan Recognition Proteins (PGLYRPs) are a conserved family of soluble extracellular innate immune receptors that bind PGN 19 . In arthropods, PGRPs act as extracellular pattern recognition receptors (PRR) that trigger downstream immune pathways 20 . In mammals, they have been characterized as bactericidal proteins that target PGN on Gram-positive bacterial surfaces 21 , 22 . However, recent studies have expanded their role beyond antimicrobial defense, suggesting function in regulating host immune responses. PGLYRP1 is a secreted protein stored in neutrophil granules, that is directly bactericidal against several bacterial species 23 . Recent work suggests that mammalian PGLYRPs also regulate adaptive immunity, including CD8+ T cell responses, in autoimmunity and cancer, and act as an accessory protein for the activation of inflammation amplifying receptor TREM-1 and NOD2-dependent signaling 24 - 26 . TREM-1 is a PRR which amplifies inflammatory responses during infection 27 . Activation of TREM-1 by its ligands, including PGLYRP1-PGN complexes, enhances production of proinflammatory cytokines and chemokines 28 . Excessive TREM-1 activation is associated with inflammatory pathology in sepsis and other diseases 29 . Despite growing interest in PGLYRPs, their roles in host-pathogen interactions, particularly in the context of Gram-negative bacteria and secreted PGN fragments, remains poorly defined. Here, we demonstrate that PGLYRP1 plays a dual role in host responses to B. pertussis infection, contributing to early bactericidal responses and fine-tuning inflammation in response to distinct PGN structures. We demonstrate that exogenous PGLYRP1 enhances NOD1 activation by TCT but failed to enhance muramyl dipeptide (MDP) activation of NOD2. Further, PGLYRP1 directly activates the pro-inflammatory receptor TREM-1, a response that is potentiated by sacculus derived PGN from Gram-positive bacteria Staphylococcus aureus or Bacillus subtilis . PGLYRP1-mediated activation of TREM-1 was not supported by B. pertussis extracellular PGN monomer TCT. These findings reveal that PGLYRP1 is not only contributes to anti-bacterial responses but suggests it may also be a context-specific immunomodulator whose function is shaped by the PGN it encounters. This work uncovers a previously unrecognized mechanism by which extracellular PGN and innate receptors together shape host inflammation during infection in a structure-specific manner. RESULTS Opposing roles of PGLYRP1 in early and late host defenses to B. pertussis PGLYRP1 has known antibacterial activity in vitro , particularly against Gram-positive bacteria 23 . To investigate whether PGLYRP1 contributes to host microbial defenses against B. pertussis , we intranasally infected adult BALB/c wild-type (WT) and PGLYRP1-knockout (PGLYRP1 KO) mice and assessed lung bacterial burden at 4- and 7-days post infection (DPI). Surprisingly, PGLYRP1 had opposing impacts on bacterial burden at these time points ( Fig. 1A&B ). At 4DPI, PGYLRP1 KO mice exhibited significantly higher bacterial burdens compared to WT controls (p < 0.01), indicating that PGLYRP1 contributes to early bacterial control. However, by 7DPI PGLYRP1 KO mice had significantly lower bacterial burden compared to WT mice (p < 0.001), suggesting that PGLYRP1 impairs bacterial clearance later in infection, potentially by regulating or modulating host immune responses. Download figure Open in new tab 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 20X and 10X 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) 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 (G) or a TCT non-releasing strain (TCT-) at 6 hours (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 hours (I). 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 To identify which cell types express PGLYRP1 following infection, we performed single cell RNA sequencing on lungs from uninfected and B. pertussis- infected C57BL/6 mice at 4DPI. In uninfected lungs PGLYRP1 expression was most prominent in neutrophils along with heterogenous expression in some lymphocyte and inflammatory monocyte (iMON) populations ( Fig. 1C ). Upon infection, the PGLYRP1-expressing cell-types expanded to include epithelial cells like ciliated cell, secretory cells, and AT1/AT2s in addition to granulocytes like basophils ( Fig. 1C ). RNAscope in situ hybridization confirmed expression in the airways of infected mice and in neutrophils ( Fig. 1D, E ). To determine whether PGLYRP1 contributes to antibacterial responses to B. pertussis , we performed in vitro and ex vivo killing assays. Based on transcriptomic data ( Fig. 1C ), we assessed the bactericidal potential of BMDNs from WT and PGLYRP1 KO mice incubated with B. pertussis for 2 or 24 hours. At 24 hours, PGLYRP1 KO neutrophils failed to control viable bacteria compared to WT neutrophils ( Fig. 1F ), indicating that PGLYRP1 contributes to neutrophil-mediated control of B. pertussis . In a cell-free assay, recombinant murine PGLYRP1 (mPGLYRP1) significantly reduced bacterial CFU after 24 hours of incubation compared to BSA control ( Figure 1G , p<0.001), validating its potential antibacterial role in neutrophil mediated control of B. pertussis growth. TCT release by B. pertussis is a product of inefficient recycling by the permease AmpG 11 . Replacing B. pertussis AmpG, with E. coli AmpG resulted in a strain (TCT-) which releases 99% less TCT (TCT(-)), while deletion of AmpG created a strain with 24-fold greater TCT release (TCT(+)) 30 . Since PGLYRP1 binds bacterial PGN and the addition of excess PGN has been shown to prevent PGLYRP bactericidal activity 31 ,,we hypothesized that B. pertussis TCT, an extracellular PGN fragment, may act as a decoy to divert PGLYRP1 activity. However, no increase in the bactericidal activity of recombinant mPGLYRP1 was noted between WT or TCT-deficient strains following incubation with PGLYRP1 ( Fig. 1H ), arguing against this hypothesis. Given prior work showing that Bordetella polysaccharides confer resistance to antimicrobial peptides 32 , we tested whether extracellular polysaccharides inhibit PGLYRP1 function. The Bps operon is required for effective production of B. pertussis surface polysaccharide 33 . Using a BpsB-deficient strain we observed significantly reduced resistance to mPGLYRP1 activity compared to WT bacteria ( Fig. 1I , p< 0.01), suggesting that endogenous polysaccharides can shield B. pertussis from PGLYRP1 activity. Together, these results indicate that PGLYRP1 plays a dual role during B. pertussis infection. PGLYRP1 enhances early bacterial control via neutrophil-mediated killing and direct bacteriostatic activity but may also play a deleterious role in controlling pulmonary infection at later stages of infection ( Figure 1 ). While TCT does not appear to interfere with PGLYRP1 activity, Bordetella polysaccharides, previously associated with biofilm production 33 , do, highlighting a key bacterial evasion strategy against host innate defenses. Peptidoglycan fragment TCT enhances PGLYRP1-mediated suppression of host responses in pertussis Given the opposing effects of PGLYRP1 on bacterial burden at 4DPI and 7DPI, we hypothesized that PGLYRP1 exerts antibacterial activity at early time points but may suppress protective immune responses or enhance deleterious responses later in infection, impairing bacterial control by the host. Recently, PGLYRP1 has been associated with PGN recognition by innate immune receptors, including NOD2 and TREM-1 24 , 26 . However, B. pertussis releases a NOD1 activating PGN fragment, TCT 14 . To assess the role of PGLYRP1 in B, pertussis -driven inflammation, we performed histological analysis of lungs from WT and PGLYRP1 KO mice at 4 and 7DPI. We observed significantly reduced immunopathology, based on the percentage and severity of bronchovascular bundle formation and infiltration of immune cells into alveolar spaces, in PGLYRP1 KO mice at both timepoints ( Fig. 2A , p < 0.05 at 4DPI, p < 0.01 at 7DPI). These findings suggest that PGLYRP1 contributes to inflammatory lung damage during B. pertussis infection. Download figure Open in new tab 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 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 by Mann Whitney U test p-values; *=0 . 003 , **, p-value < 0.01 To investigate PGLYRP1s role in immune signaling during B. pertussis infection, we performed bulk RNA sequencing on lungs from infected BALB/c (WT) and PGLYRP1 KO mice. PGLYRP1 KO mice exhibited increased expression of pro-inflammatory cytokines (e.g., IL6, IL23A, IL1A, IL1B , and IL36G ) and chemokines (e.g., CCL2, CCL3, CCL12 , and CCL28 ) alongside reduced expression of anti-inflammatory cytokine IL10 ( Fig. 2B-C ). This transcriptomic profile suggests a model in which PGLYRP1 dampens key cytokine responses while paradoxically promoting immunopathology. PGLYRP1 is a soluble PGN-binding protein. B. pertussis releases an extracellular PGN fragment, tracheal cytotoxin (TCT). We have recently demonstrated that TCT inhibits immune responses to B . pertussis 18 .We hypothesized that interactions with TCT contribute to PGLYRP1 influence on host inflammatory responses to B. pertussis . To test this, we compared the transcriptional responses of PGLYRP1 KO mice challenged with TCT-producing (parental) or TCT-deficient B. pertussis strains. Both strains induced a similar pattern of cytokine upregulation in the absence of PGLYRP1, but this increased inflammation was more robust in the absence of TCT ( Fig. 2D&E ). Specifically, PGLYRP1 KO mice infected with TCT(-) bacteria exhibited greater expression of IL23A, IL6, IL1B, and IL36G compared to WT infection, suggesting that TCT may potentiate the immune-dampening activity of PGLYRP1. PGLYRP1 has previously been shown to stimulate inflammatory responses to PGN 26 . These findings indicate that the immunomodulatory function of PGLYRP1 is informed by the PGN it interacts with. TCT release may be a strategy employed by B. pertussis to co-opt PGLYRP1 function and suppress protective inflammation, thereby promoting pathogen persistence. Supporting this, KEGG pathway analysis on differential gene expression of 4DPI WT (parental) and TCT(-) lungs confirmed enrichment of cytokine-cytokine receptor interactions, NOD signaling, and IL-17/TNF pathways in the presence of TCT ( Fig. 2F ). TCT-PGLYRP1 interactions drive NOD1 signaling To understand how TCT modulates PGLYRP1-dependent immune signaling, we investigated known mechanisms by which PGLYRP1 influences host PRR signaling. Recent studies suggest that intracellular PGLYRP1 can act as a co-factor in NOD2 signaling 24 and TCT is a known NOD1 agonist. Hence, we hypothesized that PGLYRP1 may enhance NOD1 signaling in response to the muropeptide TCT, a NOD1 exclusive agonist. To test this, recombinant PGLYRP1 was incubated with TCT (1 ng/mL) before stimulation of NOD1 and NOD2 reporter cells. Consistent with previous reports, TCT robustly stimulated a NOD1 response ( Fig. 3A ). Notably, both murine ( Fig. 3A , p < 0.005) and human ( Fig. 3B , p < 0.001) PGLYRP1 significantly enhanced TCT-mediated NOD1 activation compared to TCT alone. Importantly, PGLYRP1 alone did not induce NOD1 activation ( Fig. 3A ), suggesting the enhanced NOD1 stimulation was not by PGLYRP1 directly activating NOD1 ( Fig. 3A ). Interestingly, this enhanced activation was specific to NOD1 as neither TCT nor TCT+PGLYRP1 activated NOD2 reporter cells (data not shown) since TCT is not a NOD2 agonist. On the other hand, mPGLYRP1 decreased NOD2 recognition of NOD2 agonist muramyl dipeptdide (MDP, p < 0.01) ( Fig. 3C ), suggesting exogenous PGLYRP1 boosts responses to NOD1 stimulating PGN structures like TCT but dampens responses to NOD2 stimulatory PGNs. Download figure Open in new tab Figure 3. PGLYRP1 selectively modulates NOD1 and NOD2 signaling in response to Bordetella pertussis peptidoglycan. (A–B) HEK293 reporter cells expressing either mouse (A and D) or human (B) NOD1 or (C) NOD2 were stimulated with recombinant mouse (A) or murine or human (B-D) PGLYRP1 (12.5 ug/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 hours 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 To determine if PGLYRP1 modulates NOD responses to other potential PGNs released by B. pertussis , we stimulated reporter cells with mPGLYRP1 incubated with conditioned media from WT or TCT(-), or TCT(+) cultures. Consistent with our results using purified TCT, mPGLYRP1 amplified NOD1 signaling in response to B. pertussis conditioned media, likely due to increased recognition of extracellular TCT ( Fig. 3D , p < 0.01). In contrast, NOD2 signaling was significantly suppressed by mPGLYRP1 ( Fig. 3D ), consistent with our results with purified PGN ( Fig. 3A-C ). Together, these findings suggest that exogenous PGLYRP1 acts as a selective modulator of NOD signaling, enhancing NOD1 activation while dampening NOD2 responses. To assess the functional consequence of skewed NOD1 versus NOD2 expression and signaling during infection, we analyzed single-cell RNA sequencing data from lungs of BALB/c mice at 4DPI. We examined NOD1 and NOD2 expression across neutrophil sub-clusters ( Fig. 3E&F ), as neutrophils are the primary expressors of PGLYRP1 during B. pertussis infection ( Fig. 1C ). NOD2-expressing neutrophils showed higher expression of pro-inflammatory genes including Il1a, Ccl3 and Ptgs2 (COX-2) ( Fig. 3F&G ). In contrast, NOD1 expression correlated with genes involved in anti-inflammatory immune responses: Pla2g7 (degrades platelet activating factor), Tmsb4x which inhibits neutrophil migration, promotes repair and inhibits NFkB and increased expression of PGLYRP1, which may further positively regulate these pathways 34 . This identifies a correlation between pro-inflammatory cytokine expression and NOD2 signaling in neutrophils. Interestingly, when we further analyzed the clusters with high Nod2 expression, like clusters 0 and 4, we observed gene expression patterns which correspond to previously identified inflammatory neutrophil populations. Cluster 0 hallmark genes were associated with interferon-stimulated programming ( Calhm6, Cxcl9, Tma16, Slamf7, Baft2 ) (Sup. Fig. 1), and B. pertussis infection has been shown to cause both deleterious and beneficial interferon responses 34 - 36 . Additionally, poor B. pertussis host-responses have been linked to interferon-stimulated neutrophils, providing interesting context to this identified neutrophil cluster 37 , 38 . Furthermore, cluster 4 marker ( Hilpda, Csf3, Myl4, Odc1, Spp1 ) neutrophils have been identified in tumor exhaustion environments, and related to hypoxia response, highlighting another inflammatory neutrophil population (Sup. Fig. 1) 39 . These results highlight the heightened inflammatory phenotype of NOD2+ neutrophils and suggest that TCT-mediated skewing of NOD responses towards NOD1, amplified by PGLYRP1, may be a mechanism to suppress inflammation and promote B. pertussis persistence. TCT-PGLYRP1 complexes dampen TREM-1 activation, limiting inflammation during B. pertussis infection In addition to their role in NOD signaling, PGLYRP1-PGN complexes have been shown to activate TREM-1, driving NFkB-mediated inflammatory responses 26 . This ability to activate TREM-1 is boosted by the presence of a PGN from the sacculi of S. aureus, B. subtilis or E. coli 26 . To determine if TREM-1 contributes to host responses to B. pertussis , we first assessed TREM-1 expressing cell-types following B. pertussis infection in mice using single cell RNA-sequencing. Following B. pertussis infection, TREM-1 was primarily expressed by neutrophils, with lesser expression noted in monocytes and macrophages ( Fig. 4A ). Download figure Open in new tab 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 5µg/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 To determine if TCT influences PGLYRP1-dependent TREM-1 activation, we assessed the ability of TCT, a DAP-type PGN monomer, to enhance PGLYRP1-mediated TREM-1 activation. As reported previously, preparations of peptidoglycans isolated from S. aureus and B. subtilis enhanced PGLYRP1-mediated activation of a TREM-1 reporter cell line ( Fig. 4B , p < 0.01) 40 , 41 . Given the anti-inflammatory role of TCT observed in our data ( 18 and Figs 2 and 3 ), and known role for TREM-1 in B. pertussis pathogenesis 42 we proposed that TCT may be a poorer enhancer of PGLYRP-1 mediated TREM-1 activation. Consistent with our hypothesis the addition of TCT significantly reduced TREM-1 activity ( Fig. 4C ). This suggests that TCT, unlike PGN from the sacculi, fails to support and possibly even antagonizes PGLYRP1’s ability to activate TREM-1 ( Fig. 4C ). Further studies are required to discern the contribution of PGN structure and solubility in this model.. Combined with our previous work 42 , these findings highlight the importance of PGN in bacterial regulation of TREM-1 and its role in B. pertussis pathogenesis and disease and the potential impact of skewing TREM-1 responses. DISCUSSION Our study reveals a previously unrecognized role for the PRR, PGLYRP1, in orchestrating both antibacterial defenses and inflammatory responses during Bordetella pertussis infection. While traditionally characterized as a bactericidal effector which mediates its effects by binding PGN on the surface of Gram-positive bacteria, emerging work suggests its role may extend to mammalian immune signaling 25 . The contribution of PGLYRP1 to PRR mediated inflammatory responses in mammals and in particular how PGN structure impacts the outcomes of these interactions, remains largely unexplored. Our findings demonstrate that PGLYRP1 can functions as a PGN structure-specific interpreter which translates structure into context and pathogen specific immune signaling. This dual role expands our understanding of how innate immune receptors fine-tune host-pathogen interactions in the airway based on the structure of PGN encountered. Prior studies have also shown that endogenous PGLYRP1 bolstered NOD2-driven macrophage activation in response to GMTriP-K, but not in response to related PGN fragments such as GMDiP or MDP, highlighting the structural specificity of PGLYRP1-mediated immune recognition 24 . We expand on these findings to suggest exogenous PGLYRP1 biases NOD1 responses and dampens NOD2 responses. Furthermore, we suggest pathogens like B. pertussis have evolved to release PGN structures, such as TCT, which dampen the ability of PGLYRP1 to signal via TREM-1 ( Fig. 4C ), suggesting an active area of ongoing host-pathogen evolution. Here, we propose PGLYRP1 as a PGN-structure specific regulator of inflammatory responses. This is comparable to SIGLECs, which can distinguish self from non-self glycans to ignore or elicit immune responses 43 . During B. pertussis infection, PGLYRP1, predominantly expressed by neutrophils, contributes to early bacterial clearance, as PGLYRP1 KO mice exhibit significantly higher bacterial loads at 4DPI. The bactericidal activity of PGLYRP1 is diminished by the presence of extracellular polysaccharide, highlighting the potential for Bordetella pertussis -derived extracellular decoys to prevent antibiotic activity. PGLYRP1 KO mice exhibit elevated inflammatory cytokine expression, suggesting PGLYRP1 also serves an anti-inflammatory function. Transcriptomic analysis of PGLYRP1 KO lungs infected with mutants which fail to release TCT or the parental strain support the hypothesis that PGLYRP1 limits inflammation in a PGN structure-dependent manner: specifically, the monomeric PGN fragment TCT potentiates the anti-inflammatory activity of PGLYRP1. An alternative explanation for the discrepancy observed in bacterial burden at early and later timepoints of infection is that PGLYRP1-directed lysis of B. pertussis releases more host-available muropeptides, which PGLYRP1 can then interact with to enhance less protective NOD1-driven immune responses at the expense of more potent NOD2-driven responses ( Fig 1 ) 18 . Mechanistically, PGLYRP1 enhances NOD1 signaling in response to TCT while suppressing NOD2 signaling in response to MDP directing the polarization of immune responses away from a more inflammatory NOD2-driven response. Single cell RNA sequencing revealed that PGLYRP1 expression is enriched in NOD1+ neutrophils, which have less inflammatory profiles than NOD2+ neutrophils. These data suggest B. pertussis may exploit PGLYRP1 to amplify a NOD1-dominant signaling environment that blunts host inflammation. Recent work from our group highlighted the ability of TCT to polarize NOD responses, towards NOD1 as an immune evasion mechanism 18 . This work expands on this finding, highlighting the role of PGLYRP1 and PGN structure in these findings, but further work should validate and expand on how PGLYRP1 modulates responses to muropeptide outside the cellular and murine models used in these studies. These findings shift our understanding of mammalian PGLYRP1 from a passive bactericidal protein to an immunomodulator, whose contribution to inflammation is determined by the structure of PGN it encounters. Additionally, they inform our views of PGN structure and how it impacts host responses, suggesting bacterial release of PGNs, as a mechanism to dampen host immunity. These results propose a novel paradigm in host-pathogen interactions; that bacteria may release structurally tailored PGN fragments to bias host immune sensors and signaling in their favor. Excitingly, we report a novel axis by which PGLYRP1 regulates TREM-1, a known amplifier of inflammation. TREM-1 activation has been reported to strengthen NF-kB signaling pathways which NLRs like NOD1 and NOD2 canonically signal through 27 . While PGLYRP1 synergized with PGNs from S. aureus and a B. subtilis to enhance TREM-1 activation, the presence of TCT antagonized this effect ( Fig. 4 ). There are several differences in the structures of these PGNs: unlike monomeric TCT, PGN tested from other bacteria are derived from the sacculus and are insoluble, additionally B. subtilis PGN is amidated. Further studies are required to discern if solubility, size or other factors are responsible for differences in the effect on PGLYRP1-mediated TREM-1 activation. Additionally, TCT contains a 1,6 anhydroMurNAc. Bifidobacterium have been predicted to produce abundant anhydro-PGNs which markedly reduce LPS-stimulated cytokine expression in RAW264.7 cells, underscoring a potential anti-inflammatory effect of 1,6-anhydro-MurNAc containing PGNs, such as TCT. Previous work from our group demonstrated that inhibition of TREM-1 prevented immunopathology and inhibited cytokine and chemokine responses to infection, highlighting the potential utility of disturbed TREM-1 signaling for B. pertussis 42 . TREM-1 inhibitor treated mice showed reduced inflammation and inflammatory cytokine responses following B. pertussis infection highlighting the importance of this pathway in pertussis pathogenesis and the potential utility of its manipulation by bacteria. Further, our findings suggest that selectively targeting TREM-1 or PGN structure sensing by PGLYRP1 could attenuate disease severity without compromising bacterial clearance, a strategy aligned with emerging host-directed therapeutics in TB and sepsis 44 - 46 . However, the role of these players in generating effective antibody responses still needs to be elucidated so that we can understand the implications on long-term immunity. We hypothesize that B. pertussis may attenuate host immune responses not merely by evading recognition, but by actively hijacking host regulatory circuits through the release of specific PGN structures. Taken together, our findings support a model in which PGLYRP1 serves dual and temporally distinct roles during infection, by promoting bacterial killing while restraining inflammation in a PGN-structure specific manner. This duality may reflect temporal or disease stage specific changes in the types of PGN available to host PRRs, as 1,6-anhydro MurNAc containing PGNs are usually internal, unless released following bacterial lysis. The ability of B. pertussis to exploit this shift by releasing TCT to suppress excessive inflammation represents a novel immune evasion strategy. More broadly, this work demonstrates that the structure of PGN fragments is a key determinant of host immune outcomes. Innate receptors like PGLYRP1 relay this structural information to shape both defense and pathology, providing an avenue for pathogen hijacking by alternating the availability of different PGN structures. Targeting the TCT–PGLYRP1–TREM-1 axis may offer a novel therapeutic or vaccine-mediated approach to mitigate immunopathology in pertussis and potentially other infections involving secreted PGN. These findings reframe PGN as both a pathogen-associated molecular pattern and an immunomodulatory cue that both host and pathogen exploit to regulate the immune-inflammatory environment. Future studies using defined synthetic muropeptides will be necessary to pinpoint structural determinants of the reduced immune activation noted toward TCT. Likewise, how these alterations in PGN structure impact PGLYRP1 interactions with host PRRs remains unclear. METHODS AND MATERIALS Mice and Ethics Statement All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Maryland School of Medicine (protocol 00000108). Adult (6–8-week-old) BALB/c (Charles River, 028) and PGLYRP1 knockout (Pglyrp1 tm1Rdz /Pglyrp1 tm1Rdz in BALB/c background, generously provided by Dr. Roman Dziarski 47 ), mice were bred and housed under specific pathogen-free conditions. To control for and assess the potential of sex-related differences in pertussis pathogenesis both male and female mice were used in experiments. For transcriptomic studies, RNA was isolated from the lungs of PBS- or B. pertussis challenged female mice. Bacterial Strains and Infections B. pertussis strains, including wild-type BC36, a streptomycin-resistant Tohama I derivative, or isogenic mutants (TCT-, TCT+, BpsB KO) were cultured on Bordet-Gengou agar supplemented with 10% defibrinated sheep blood for in vivo infections or in Stainer-Scholte (SS) broth for in vitro assays. Strains were grown in SS broth to mid-log stage (OD = 0.8), and the resulting media was centrifuged and filtered to remove any bacteria to make the conditioned media used for cell culture assays. TCT mutants were generously supplied by William Goldman (University of North Carolina at Chapel Hill). TCT-strain was generated by expression of the E. coli AmpG in BC36, resulting in a 50-fold decrease in TCT release. TCT+ was generated by replacing native B. pertussis AmpG with a kanamycin resistance cassette, this resulted in a 24-fold increase in TCT release 30 . Adult mice were anesthetized and inoculated intranasally with 2 × 10 6 CFU of B. pertussis in 50 µL PBS. Lungs were harvested at 4DPI and 7DPI for bacterial burden analysis by plating of serially-diluted lung homogenates. RNA isolation and processing Lung tissue was harvested at 2 or 7DPI for RNA isolation by the Trizol-choloroform method. Briefly, lung tissue was homogenized using the Omni Bead Beader (Omni, Inc), phase separated using chloroform, and precipitated using isopropanol. RNA was then quantified and converted to cDNA (iScript cDNA Synthesis Kit). Quantitative real-time PCR (qPCR) was performed using the BIORAD CFX96 real-time PCR instrument. Gene expression was calculated as fold change relative to PBS-inoculated control animals using the 2 ^ −ΔΔCT threshold cycle method and normalized to Hypoxanthine phosphoribosyltransferase (HPRT) (internal housekeeping gene). Bulk RNA Sequencing At 4DPI, lungs were harvested from 3 WT or PGLYRP1 KO BALB/c mice infected with WT or TCT-deficient B. pertussis . Total RNA was extracted from homogenized lungs using Trizol Reagent (Invitrogen) according to the manufacturer’s protocol. RNA integrity was confirmed using a Bioanalyzer (Agilent), alignments were generated by HISAT2 sequencing performed using a Illumina NextSeq 1000. FastQC and RSeQC were used to assess read and alignment quality. Samtools was used to generate alignment statistics. Reads were aligned with the Mus musculus GRCm39 reference genome. Differential gene expression was analyzed using DESeq2, with significance defined as adjusted p-value < 0.05. Single-Cell RNA Sequencing Single-cell suspensions were prepared from lung tissues of duplicate infected and uninfected control C57BL/6 mice at 4DPI, barcoded and sequenced as individual mice. Following red blood cell lysis, cell viability was assessed by trypan blue staining, viable cells were sorted and loaded onto the 10x Genomics Chromium Controller for barcoding and library construction. Sequencing was performed on an Illumina NovaSeq platform. Data were processed using Cell Ranger and analyzed using the Seurat package in R. Quality control cutoffs of between 1000 and 10000 features and <20% mitochondrial DNA. Data were normalized and scaled in Seurat v5, and groups were integrated via Harmony-based integration. Clusters were identified, determined, and visualized via Seurat library workflow (RunPCA, FindNeighbors, FindClusters, RunUMAP) and then annotated via hallmark markers determined by differential gene expression analysis across clusters using Wilcoxon rank-sum tests. This dataset is now deposited with the Gene Expression Omnibus (GSE324217) Neutrophil Isolation and Bactericidal Assays Bone marrow-derived neutrophils (BMDNs) were isolated by density gradient centrifugation from adult C57BL6 mice and cultured in DMEM supplemented with 10% FBS. For killing assays, BMDNs were incubated with B. pertussis at a multiplicity of infection (MOI) of 10:1 for 2 or 24 hours. Supernatants were serially diluted and plated to quantify surviving CFU. Recombinant murine PGLYRP1 (R&D Systems) or BSA control was incubated with live B. pertussis or purified PGN fragments (TCT gifted by Prof. William Goldman or S. aureus PGN, Invivogen) to assess direct bactericidal activity. RNAscope In Situ Hybridization RNA in situ hybridization was performed on 5um formalin fixed paraffin embedded lung sections with the RNAscope detection kit to confirm RNA sequencing identification of PGLYRP1 transcripts (Advanced Cell Diagnostics). Hybridization, amplification, and detection steps were carried out according to the manufacturer’s protocol. Briefly, slides were deparaffinized in xylene and rehydrated using an ethanol series. Sections were treated with hydrogen peroxide for 10 minutes at room temperature, followed by treatment with target retrieval reagent (Advanced Cell Diagnostics). Sections were hybridized with target-specific probes (Advanced Cell Diagnostics) followed by amplification and fluorescent detection (Opal 570 and Opal 520). Slides were counterstained with hematoxylin and eosin and imaged using an ECHO Revolve 4 and analyzed with ImageJ. Reporter Cell Assays for NOD1, NOD2, and TREM-1 Activation 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 stimulated with B. pertussis tracheal cytotoxin TCT (1 ng/mL), conditioned media (1:20 ratio of conditioned bacterial media to cell culture media), 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. Supernatants were analyzed for reporter activity using colorimetric (NOD1 and NOD2 reporter cells; Promega) or luminescent (TREM-1 reporter cells; Invivogen) detection kits (Promega). TCT was quantitated from its peak area elution profile from the final purification step, For each preparation of TCT this area was compared to the peak area of a purified standard that had been quantitated by amino acid analysis and generously provided by William Goldman, (UNC) Histopathology Lungs were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 µm thickness. Slides were stained with hematoxylin and eosin (H&E). Scores are given as the average score from 3 blinded investigators based on percentage and severity of bronchovascular bundle formation and infiltration of immune cells into alveolar spaces. Statistics Significant differences in pathology scores were calculated at each timepoint using a 2-tailed Mann-Whitney U test. Each timepoint was analyzed independently. A p-value < 0.05 was considered significant. A p-value < 0.05 is denoted by *, < 0.01 by ** and < 0.005 by *** ACKNOWLEDGEMENTS This work was supported by NIH award R01AI167947. Prof. Roman Dziarski (Indiana University School of Medicine) generously provided PGLYRP1 KO mice. Prof. Rajendar Deora (Ohio State College of Medicine) generously provided the BpsB deletion mutant strain of B. pertussis . Funder Information Declared NIH Common Fund, https://ror.org/001d55x84 , R01 AI167947 Footnotes Text updated following reviewer critiques. Language improved throughout manuscript. Figure 1 edited to show uninfected and infected datasets. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE324217 References 1. ↵ Johnston ID , Anderson HR , Lambert HP , Patel S. Respiratory morbidity and lung function after whooping-cough . Lancet . 1983 ; 2 ( 8359 ): 1104 – 8 . Epub 1983/11/12. doi: 10.1016/s0140-6736(83)90626-8 . PubMed PMID: 6138644 . OpenUrl CrossRef PubMed Web of Science 2. Paddock CD , Sanden GN , Cherry JD , Gal AA , Langston C , Tatti KM , Wu KH , Goldsmith CS , Greer PW , Montague JL , Eliason MT , Holman RC , Guarner J , Shieh WJ , Zaki SR . Pathology and pathogenesis of fatal Bordetella pertussis infection in infants . Clin Infect Dis . 2008 ; 47 ( 3 ): 328 – 38 . doi: 10.1086/589753 . PubMed PMID: 18558873 . OpenUrl CrossRef PubMed 3. ↵ Birkebaek NH , Kristiansen M , Seefeldt T , Degn J , Moller A , Heron I , Andersen PL , Moller JK , Ostergard L. Bordetella pertussis and chronic cough in adults . Clin Infect Dis . 1999 ; 29 ( 5 ): 1239 – 42 . doi: 10.1086/313448 . PubMed PMID: 10524969 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Wang S , Zhang S , Liu J. Resurgence of pertussis: Epidemiological trends, contributing factors, challenges, and recommendations for vaccination and surveillance . Hum Vaccin Immunother . 2025 ; 21 ( 1 ): 2513729 . Epub 20250609. doi: 10.1080/21645515.2025.2513729 . PubMed PMID: 40491090 ; PMCID: PMC12153400 . OpenUrl CrossRef PubMed 5. ↵ Schleifer KH , Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications . Bacteriol Rev . 1972 ; 36 ( 4 ): 407 – 77 . doi: 10.1128/br.36.4.407-477.1972 . PubMed PMID: 4568761 ; PMCID: PMC408328 . OpenUrl FREE Full Text 6. ↵ Sanchez M , Kolar SL , Muller S , Reyes CN , Wolf AJ , Ogawa C , Singhania R , De Carvalho DD , Arditi M , Underhill DM , Martins GA , Liu GY . O-Acetylation of Peptidoglycan Limits Helper T Cell Priming and Permits Staphylococcus aureus Reinfection . Cell Host Microbe . 2017 ; 22 ( 4 ): 543 – 51 e4. Epub 20170921. doi: 10.1016/j.chom.2017.08.008 . PubMed PMID: 28943328 ; PMCID: PMC5679255 . OpenUrl CrossRef PubMed 7. ↵ Wolf AJ , Underhill DM . Peptidoglycan recognition by the innate immune system . Nat Rev Immunol . 2018 ; 18 ( 4 ): 243 – 54 . Epub 20180102. doi: 10.1038/nri.2017.136 . PubMed PMID: 29292393 . OpenUrl CrossRef PubMed 8. ↵ Rosenthal RS , Nogami W , Cookson BT , Goldman WE , Folkening WJ . Major fragment of soluble peptidoglycan released from growing Bordetella pertussis is tracheal cytotoxin . Infect Immun . 1987 ; 55 ( 9 ): 2117 – 20 . doi: 10.1128/iai.55.9.2117-2120.1987 . PubMed PMID: 2887513 ; PMCID: PMC260665 . OpenUrl Abstract / FREE Full Text 9. Melly MA , McGee ZA , Rosenthal RS . Ability of monomeric peptidoglycan fragments from Neisseria gonorrhoeae to damage human fallopian-tube mucosa . J Infect Dis . 1984 ; 149 ( 3 ): 378 – 86 . doi: 10.1093/infdis/149.3.378 . PubMed PMID: 6425421 . OpenUrl CrossRef PubMed 10. ↵ Jutras BL , Lochhead RB , Kloos ZA , Biboy J , Strle K , Booth CJ , Govers SK , Gray J , Schumann P , Vollmer W , Bockenstedt LK , Steere AC , Jacobs-Wagner C. Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis . Proc Natl Acad Sci U S A . 2019 ; 116 ( 27 ): 13498 – 507 . Epub 20190617. doi: 10.1073/pnas.1904170116 . PubMed PMID: 31209025 ; PMCID: PMC6613144 . OpenUrl Abstract / FREE Full Text 11. ↵ Cookson BT , Tyler AN , Goldman WE . Primary structure of the peptidoglycan-derived tracheal cytotoxin of Bordetella pertussis . Biochemistry . 1989 ; 28 ( 4 ): 1744 – 9 . doi: 10.1021/bi00430a048 . PubMed PMID: 2541765 . OpenUrl CrossRef PubMed 12. ↵ Goldman WE , Klapper DG , Baseman JB . Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells . Infect Immun . 1982 ; 36 ( 2 ): 782 – 94 . doi: 10.1128/iai.36.2.782-794.1982 . PubMed PMID: 6177637 ; PMCID: PMC351298 . OpenUrl Abstract / FREE Full Text 13. ↵ Torrens G , Cava F. Mechanisms conferring bacterial cell wall variability and adaptivity . Biochem Soc Trans . 2024 ; 52 ( 5 ): 1981 – 93 . doi: 10.1042/BST20230027 . PubMed PMID: 39324635 ; PMCID: PMC11555704 . OpenUrl CrossRef PubMed 14. ↵ Magalhaes JG , Philpott DJ , Nahori MA , Jéhanno M , Fritz J , Le Bourhis L , Viala J , Hugot JP , Giovannini M , Bertin J , Lepoivre M , Mengin-Lecreulx D , Sansonetti PJ , Girardin SE . Murine Nod1 but not its humanorthologue mediates innate immune detection of tracheal cytotoxin . EMBO Rep . 2005 ; 6 ( 12 ): 1201 – 7 . doi: 10.1038/sj.embor.7400552 . PubMed PMID: 16211083 ; PMCID: PMC1369207 . OpenUrl Abstract / FREE Full Text 15. ↵ Chamaillard M , Hashimoto M , Horie Y , Masumoto J , Qiu S , Saab L , Ogura Y , Kawasaki A , Fukase K , Kusumoto S , Valvano MA , Foster SJ , Mak TW , Nuñez G , Inohara N. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid . Nat Immunol . 2003 ; 4 ( 7 ): 702 – 7 . Epub 20030606. doi: 10.1038/ni945 . PubMed PMID: 12796777 . OpenUrl CrossRef PubMed Web of Science 16. ↵ Kessie DK , Lodes N , Oberwinkler H , Goldman WE , Walles T , Steinke M , Gross R. Activity of Tracheal Cytotoxin of Bordetella pertussis in a Human Tracheobronchial 3D Tissue Model . Front Cell Infect Microbiol . 2020 ; 10 : 614994 . Epub 20210119. doi: 10.3389/fcimb.2020.614994 . PubMed PMID: 33585281 ; PMCID: PMC7873972 . OpenUrl CrossRef PubMed 17. ↵ Flak TA , Goldman WE . Signalling and cellular specificity of airway nitric oxide production in pertussis . Cell Microbiol . 1999 ; 1 ( 1 ): 51 – 60 . doi: 10.1046/j.1462-5822.1999.00004.x . PubMed PMID: 11207540 . OpenUrl CrossRef PubMed Web of Science 18. ↵ Rickert DM , Hill EM , Himmelberger RE , Rajbanshi N , Cardozo S , Bergamin De Castro T , Bharadwaj R , Scott AJ , Silverman N , Goldman WE , Carbonetti NH , Scanlon KM , Skerry C. B. pertussis tracheal cytotoxin biases NOD signaling to suppress IL-1 mediated inflammation and evade adaptive immunity . bioRxiv . 2025 :2025.08.08.669383. doi: 10.1101/2025.08.08.669383 . OpenUrl Abstract / FREE Full Text 19. ↵ Liu C , Xu Z , Gupta D , Dziarski R. Peptidoglycan recognition proteins: a novel family of four human innate immunity pattern recognition molecules . J Biol Chem . 2001 ; 276 ( 37 ): 34686 – 94 . Epub 2001/07/20. doi: 10.1074/jbc.M105566200 . PubMed PMID: 11461926 . OpenUrl Abstract / FREE Full Text 20. ↵ Dziarski R , Gupta D. The peptidoglycan recognition proteins (PGRPs) . Genome Biol . 2006 ; 7 ( 8 ): 232 . doi: 10.1186/gb-2006-7-8-232 . PubMed PMID: 16930467 ; PMCID: PMC1779587 . OpenUrl CrossRef PubMed 21. ↵ Kashyap DR , Wang M , Liu LH , Boons GJ , Gupta D , Dziarski R. Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems . Nat Med . 2011 ; 17 ( 6 ): 676 – 83 . Epub 20110522. doi: 10.1038/nm.2357 . PubMed PMID: 21602801 ; PMCID: PMC3176504 . OpenUrl CrossRef PubMed 22. ↵ Dziarski R , Gupta D. Mammalian PGRPs: novel antibacterial proteins . Cell Microbiol . 2006 ; 8 ( 7 ): 1059 – 69 . doi: 10.1111/j.1462-5822.2006.00726.x . PubMed PMID: 16819960 . OpenUrl CrossRef PubMed Web of Science 23. ↵ Lu X , Wang M , Qi J , Wang H , Li X , Gupta D , Dziarski R. Peptidoglycan recognition proteins are a new class of human bactericidal proteins . J Biol Chem . 2006 ; 281 ( 9 ): 5895 – 907 . Epub 20051214. doi: 10.1074/jbc.M511631200 . PubMed PMID: 16354652 . OpenUrl Abstract / FREE Full Text 24. ↵ Chen S , Putnik R , Li X , Diwaker A , Vasconcelos M , Liu S , Gondi S , Zhou J , Guo L , Xu L , Temme S , Bersch K , Hyland S , Yin J , Burstein E , Bahnson BJ , Gildersleeve JC , Grimes CL , Reinecker HC . PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection . Nat Commun . 2025 ; 16 ( 1 ): 1864 . Epub 20250221. doi: 10.1038/s41467-025-57126-9 . PubMed PMID: 39984444 ; PMCID: PMC11845746 . OpenUrl CrossRef PubMed 25. ↵ Schnell A , Huang L , Regan BML , Singh V , Vonficht D , Bollhagen A , Wang M , Hou Y , Bod L , Sobel RA , Chihara N , Madi A , Anderson AC , Regev A , Kuchroo VK . Targeting PGLYRP1 promotes antitumor immunity while inhibiting autoimmune neuroinflammation . Nat Immunol . 2023 ; 24 ( 11 ): 1908 – 20 . Epub 20231012. doi: 10.1038/s41590-023-01645-4 . PubMed PMID: 37828379 ; PMCID: PMC10864036 . OpenUrl CrossRef PubMed 26. ↵ Read CB , Kuijper JL , Hjorth SA , Heipel MD , Tang X , Fleetwood AJ , Dantzler JL , Grell SN , Kastrup J , Wang C , Brandt CS , Hansen AJ , Wagtmann NR , Xu W , Stennicke VW . Cutting Edge: identification of neutrophil PGLYRP1 as a ligand for TREM-1 . J Immunol . 2015 ; 194 ( 4 ): 1417 – 21 . Epub 20150116. doi: 10.4049/jimmunol.1402303 . PubMed PMID: 25595774 ; PMCID: PMC4319313 . OpenUrl Abstract / FREE Full Text 27. ↵ Arts RJ , Joosten LA , van der Meer JW , Netea MG . TREM-1: intracellular signaling pathways and interaction with pattern recognition receptors . J Leukoc Biol . 2013 ; 93 ( 2 ): 209 – 15 . Epub 20121029. doi: 10.1189/jlb.0312145 . PubMed PMID: 23108097 . OpenUrl CrossRef PubMed Web of Science 28. ↵ Bouchon A , Dietrich J , Colonna M. Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes . J Immunol . 2000 ; 164 ( 10 ): 4991 – 5 . doi: 10.4049/jimmunol.164.10.4991 . PubMed PMID: 10799849 . OpenUrl Abstract / FREE Full Text 29. ↵ Bouchon A , Facchetti F , Weigand MA , Colonna M. TREM-1 amplifies inflammation and is a crucial mediator of septic shock . Nature . 2001 ; 410 ( 6832 ): 1103 – 7 . Epub 2001/04/27. doi: 10.1038/35074114 . PubMed PMID: 11323674 . OpenUrl CrossRef PubMed Web of Science 30. ↵ Lyon RS . Tracheal cytotoxin production by the Bordetellae : Washington University , St Louis ; 2001 . 31. ↵ Lu X , Wang M , Qi J , Wang H , Li X , Gupta D , Dziarski R. Peptidoglycan Recognition Proteins Are a New Class of Human Bactericidal Proteins * . Journal of Biological Chemistry . 2006 ; 281 ( 9 ): 5895 – 907 . doi: 10.1074/jbc.M511631200 . OpenUrl Abstract / FREE Full Text 32. ↵ Fullen AR , Gutierrez-Ferman JL , Yount KS , Love CF , Choi HG , Vargas MA , Raju D , Corps KN , Howell PL , Dubey P , Deora R. Bps polysaccharide of Bordetella pertussis resists antimicrobial peptides by functioning as a dual surface shield and decoy and converts Escherichia coli into a respiratory pathogen . PLoS Pathog . 2022 ; 18 ( 8 ): e1010764 . Epub 20220815. doi: 10.1371/journal.ppat.1010764 . PubMed PMID: 35969621 ; PMCID: PMC9410548 . OpenUrl CrossRef PubMed 33. ↵ Parise G , Mishra M , Itoh Y , Romeo T , Deora R. Role of a putative polysaccharide locus in Bordetella biofilm development . J Bacteriol . 2007 ; 189 ( 3 ): 750 – 60 . Epub 20061117. doi: 10.1128/JB.00953-06 . PubMed PMID: 17114249 ; PMCID: PMC1797297 . OpenUrl Abstract / FREE Full Text 34. ↵ Xie X , Shi Q , Wu P , Zhang X , Kambara H , Su J , Yu H , Park SY , Guo R , Ren Q , Zhang S , Xu Y , Silberstein LE , Cheng T , Ma F , Li C , Luo HR . Single-cell transcriptome profiling reveals neutrophil heterogeneity in homeostasis and infection . Nat Immunol . 2020 ; 21 ( 9 ): 1119 – 33 . Epub 20200727. doi: 10.1038/s41590-020-0736-z . PubMed PMID: 32719519 ; PMCID: PMC7442692 . OpenUrl CrossRef PubMed 35. Ardanuy J , Scanlon K , Skerry C , Fuchs SY , Carbonetti NH . Age-Dependent Emects of Type I and Type III IFNs in the Pathogenesis of Bordetella pertussis Infection and Disease . J Immunol . 2020 ; 204 ( 8 ): 2192 – 202 . Epub 2020/03/11. doi: 10.4049/jimmunol.1900912 . PubMed PMID: 32152071 ; PMCID: PMC7141952 . OpenUrl Abstract / FREE Full Text 36. ↵ Ardanuy J , Scanlon KM , Skerry C , Carbonetti NH . DNA-Dependent Interferon Induction and Lung Inflammation in Bordetella pertussis Infection . J Interferon Cytokine Res . 2023 ; 43 ( 10 ): 478 – 86 . Epub 20230831. doi: 10.1089/jir.2023.0066 . PubMed PMID: 37651198 ; PMCID: PMC10599430 . OpenUrl CrossRef PubMed 37. ↵ Kumar A , Johnson D , Bukowski A , Noto MJ , Carbonetti NH . Interferon lambda signaling in neutrophils enhances the pathogenesis of Bordetella pertussis infection . J Leukoc Biol . 2025 ; 117 ( 2 ). doi: 10.1093/jleuko/qiae202 . PubMed PMID: 39302155 ; PMCID: PMC11879762 . OpenUrl CrossRef PubMed 38. ↵ Kumar A , Bukowski A , Carbonetti NH . Myeloid cell-specific type I interferon signaling mediates age-dependent inflammation and protection in Bordetella pertussis infection . Infect Immun . 2025 ; 93 ( 10 ): e0030625 . Epub 20250922. doi: 10.1128/iai.00306-25 . PubMed PMID: 40980929 ; PMCID: PMC12519780 . OpenUrl CrossRef PubMed 39. ↵ Veglia F , Hashimoto A , Dweep H , Sanseviero E , De Leo A , Tcyganov E , Kossenkov A , Mulligan C , Nam B , Masters G , Patel J , Bhargava V , Wilkinson P , Smirnov D , Sepulveda MA , Singhal S , Eruslanov EB , Cristescu R , Loboda A , Nefedova Y , Gabrilovich DI . Analysis of classical neutrophils and polymorphonuclear myeloid-derived suppressor cells in cancer patients and tumor-bearing mice . J Exp Med . 2021 ; 218 ( 4 ). doi: 10.1084/jem.20201803 . PubMed PMID: 33566112 ; PMCID: PMC7879582 . OpenUrl CrossRef PubMed 40. ↵ Tocheva EI , López-Garrido J , Hughes HV , Fredlund J , Kuru E , Vannieuwenhze MS , Brun YV , Pogliano K , Jensen GJ . Peptidoglycan transformations during Bacillus subtilis sporulation . Mol Microbiol . 2013 ; 88 ( 4 ): 673 – 86 . Epub 20130327. doi: 10.1111/mmi.12201 . PubMed PMID: 23531131 ; PMCID: PMC3893088 . OpenUrl CrossRef PubMed 41. ↵ Sharif S , Singh M , Kim SJ , Schaefer J. Staphylococcus aureus peptidoglycan tertiary structure from carbon-13 spin dimusion . J Am Chem Soc . 2009 ; 131 ( 20 ): 7023 – 30 . doi: 10.1021/ja808971c . PubMed PMID: 19419167 ; PMCID: PMC2778264 . OpenUrl CrossRef PubMed 42. ↵ Gallop D , Scanlon K , Ardanuy J , Sigalov AB , Carbonetti NH , Skerry C. Triggering receptor expressed on myeloid cells-1 (TREM-1) contributes to Bordetella pertussis inflammatory pathology . Infect Immun . 2021 : IAI0012621 . Epub 2021/06/08. doi: 10.1128/IAI.00126-21 . PubMed PMID: 34097504 . OpenUrl CrossRef PubMed 43. ↵ Paulson JC , Macauley MS , Kawasaki N. Siglecs as sensors of self in innate and adaptive immune responses . Ann N Y Acad Sci . 2012 ; 1253 ( 1 ): 37 – 48 . Epub 20120130. doi: 10.1111/j.1749-6632.2011.06362.x . PubMed PMID: 22288608 ; PMCID: PMC3335958 . OpenUrl CrossRef PubMed Web of Science 44. ↵ Sigalov AB . A novel ligand-independent peptide inhibitor of TREM-1 suppresses tumor growth in human lung cancer xenografts and prolongs survival of mice with lipopolysaccharide-induced septic shock . Int Immunopharmacol . 2014 ; 21 ( 1 ): 208 – 19 . Epub 20140514. doi: 10.1016/j.intimp.2014.05.001 . PubMed PMID: 24836682 ; PMCID: PMC4088342 . OpenUrl CrossRef PubMed 45. Skerry C , Pinn ML , Bruiners N , Pine R , Gennaro ML , Karakousis PC . Simvastatin increases the in vivo activity of the first-line tuberculosis regimen . J Antimicrob Chemother . 2014 ; 69 ( 9 ): 2453 – 7 . Epub 20140522. doi: 10.1093/jac/dku166 . PubMed PMID: 24855121 ; PMCID: PMC4184365 . OpenUrl CrossRef PubMed Web of Science 46. ↵ Dey RJ , Dey B , Harrim M , Canfield ET , Lewinsohn DM , Bishai WR . Augmentation of the Riboflavin-Biosynthetic Pathway Enhances Mucosa-Associated Invariant T (MAIT) Cell Activation and Diminishes Mycobacterium tuberculosis Virulence . mBio . 2021 ; 13 ( 1 ): e0386521 . Epub 20220215. doi: 10.1128/mbio.03865-21 . PubMed PMID: 35164552 ; PMCID: PMC8844931 . OpenUrl CrossRef PubMed 47. ↵ Dziarski R , Platt KA , Gelius E , Steiner H , Gupta D. Defect in neutrophil killing and increased susceptibility to infection with nonpathogenic gram-positive bacteria in peptidoglycan recognition protein-S (PGRP-S)-deficient mice . Blood . 2003 ; 102 ( 2 ): 689 – 97 . Epub 2003/03/22. doi: 10.1182/blood-2002-12-3853 . PubMed PMID: 12649138 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted March 25, 2026. Download PDF Data/Code Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection David M Rickert , Sasha Cardozo , Nicholas H Carbonetti , William E Goldman , Karen M Scanlon , Ciaran Skerry bioRxiv 2025.09.26.678899; doi: https://doi.org/10.1101/2025.09.26.678899 Share This Article: Copy Citation Tools A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection David M Rickert , Sasha Cardozo , Nicholas H Carbonetti , William E Goldman , Karen M Scanlon , Ciaran Skerry bioRxiv 2025.09.26.678899; doi: https://doi.org/10.1101/2025.09.26.678899 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)