Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to next-generation β-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum β-lactam resistance, is classically mediated by PBP2a, a penicillin-binding protein encoded by mecA (or mecC ) in MRSA. Thus, presence of mec genes among S. aureus spp. serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA -deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as methicillin-resistant lacking mec (MRLM), are currently being identified in increasing numbers among natural resistant isolates of S. aureus . The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs, can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum β-lactam resistance in S. aureus . It thus suggests reconsideration of the current approach toward diagnosis and treatment of β-lactam-resistant S. aureus infections. IMPORTANCE: In Staphylococcus aureus , high-level, broad-spectrum resistance to β-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA . This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to β-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA , as seen by increased bacterial survival in the presence of β-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and G GDEF d omain p rotein containing p hosphodiesterase ( gdpP ) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of β-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

Bordetella pertussis , the causative agent of whooping cough, is a Gram-negative bacterium, which colonizes the respiratory tract and evades clearance through multiple virulence factors. One such factor, tracheal cytotoxin (TCT), is a monomeric peptidoglycan (PGN) fragment released extracellularly due to inefficient recycling by the permease AmpG. Although TCT is known to be a potent NOD1 agonist in vitro , its role in vivo remains poorly defined. Here, we demonstrate that TCT release during infection suppresses pulmonary inflammation. Mice infected with a TCT over-releasing strain, TCT(+), exhibited reduced lung immunopathology compared to wild-type or TCT-under releasing, TCT(−), strains. Reporter assays revealed that TCT(+) preferentially enhanced NOD1 activation, while TCT(−) strains skewed signaling towards NOD2. In vivo , NOD2 knockout mice displayed reduced lung inflammation, implicating NOD2-driven responses in pulmonary pathology. Transcriptomic analysis identified IL-1β as closely associated with expression of NOD2 but not NOD1. In support, TCT(+) infection led to reduced IL-1β protein levels than WT or TCT(−). CellChat analysis of single-cell RNA-sequencing data identified alveolar macrophages as the primary source of IL-1β and airway fibroblasts as key responders. Infected airway fibroblasts upregulated inflammatory chemokines such as CXCL13 and CCL19, promoting lymphocyte recruitment. Proteomic profiling and flow cytometry confirmed reduced chemokine levels and B cell recruitment in TCT(+) infections. Importantly, IL-1 receptor knockout mice showed significantly impaired recruitment of B and CD4+ T cells and higher bacterial burdens, despite unchanged innate cell infiltration. This underscores the role of IL-1 in coordinating adaptive immunity. Together, these findings support a model in which TCT release skews host signaling towards NOD1, suppresses NOD2 and IL-1 mediated fibroblast activation, blunts chemokine-driven lymphocyte recruitment, and impairs adaptive immune clearance. These results suggest that TCT release is an immune evasion strategy to dampen inflammation, prolong persistence and prevent the development of protective adaptive responses and highlights divergent outcomes following engagement of NOD1 or NOD2 during infection. Graphical Highlights: ➢ B. pertussis produces both NOD1 and NOD2 activating PGNs. ➢ Release of TCT polarizes toward NOD1 responses. ➢ NOD2 activation in myeloid cells drives IL-1 family cytokine production. ➢ IL-1 family cytokines skew fibroblasts towards an inflammatory phenotype, leading to chemokine release, extracellular remodeling, and recruitment of lymphocytes ➢ TCT release dampens the recruitment of adaptive lymphocytes to facilitate bacterial persistence.

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.

Bordetella pertussis , the etiologic agent of whooping cough, remains a serious public health concern despite widespread vaccination. Improved therapeutics and vaccines are urgently needed. 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 innate immune family that bind bacterial PGN and are primarily known for bactericidal activity in mammals; however, their immune modulatory roles are beginning to gain appreciation. The role of PGLYRPs in mammalian host defenses to Gram-negative pathogens, such as B. pertussis , remains largely unknown. Here, using knockout mice, single-cell and bulk transcriptomics, and functional assays, we identify a dual role for PGLYRP1 in modulating host immune responses to B. pertussis . PGLYRP1 contributes to antibacterial responses and paradoxically dampens inflammatory responses and inhibits bacterial killing later in infection. Mechanistically, PGLYRP1 enhances NOD1 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. These findings demonstrate that B. pertussis co-opts PGLYRP1 to alter immune signaling, revealing a novel immune evasion mechanism with implications for vaccine design and host-directed therapeutics.

Peptidoglycan (PGN) is a mesh like polymer that builds and protects the bacterial cell wall. Across and even within species, PGN varies considerably in structure and composition, and this structural diversity shapes how released fragments are detected by the host. The varied structural features of PGN can also dictate its ability to act as a toxin or danger signal. Traditionally, bacterial cell wall components have been classified as toxic, based on their ability to cause extensive host cell damage, but the specific immune responses triggered by distinct PGN structural motifs when they are released into the extracellular space, and their potential direct role as toxins are not yet fully understood. Emerging evidence suggests that PGN is recognized by the host not only through its canonical di-/tripeptides, but also through novel structural motifs that are sensed by host pattern recognition receptors (PRRs). However, the mechanisms underlying the recognition of these structurally diverse fragments and their role in promoting immune activation, bacterial pathogenesis, and immune evasion remain poorly understood. In this review, we describe how structurally distinct muropeptides are synthesized, processed, and selectively sensed by host PRRs, and how differential immune recognition shapes PRR activation, thereby influencing host–pathogen interactions and infection outcomes.