Findings uncover a previously unrecognized structural role of extracellular phospholipids in biofilm architecture and suggest that targeting Lys-PG and its biosynthetic pathway represents a promising strategy for biofilm control.
Abstract
Staphylococcus aureus biofilms contribute significantly to persistent infections and antibiotic resistance, supported by a complex extracellular matrix. While their proteins, polysaccharides, and extracellular DNA have been well studied, the role of phospholipids in biofilm architecture remains underexplored. Here, we identify extracellular phospholipids within the biofilm matrix, particularly lysyl-phosphatidylglycerol (Lys-PG), as critical structural elements in S. aureus biofilms. Bacterial phospholipase A1 (PLA1), which hydrolyzes phospholipid acyl ester bonds, effectively dispersed pre-formed biofilms and prevented biofilm formation by hydrolyzing extracellular phospholipids, without affecting bacterial growth or exhibiting cytotoxicity. Microscopic analyses revealed that PLA1 disrupts membranous nanostructures integral to biofilm stability. Lipidomic analysis demonstrated an enrichment of Lys-PG with specific fatty acid species within the biofilm matrix and confirmed their hydrolysis by PLA1. Mechanistically, Lys-PG promotes bacterial aggregation by acting as a molecular glue through electrostatic and hydrophobic interactions. Deletion of mprF, responsible for Lys-PG synthesis, markedly impaired biofilm formation. These findings uncover a previously unrecognized structural role of extracellular phospholipids in biofilm architecture and suggest that targeting Lys-PG and its biosynthetic pathway represents a promising strategy for biofilm control.
ABSTRACT Staphylococcus aureus is a leading cause of biofilm-associated infections, in which communities of bacterial cells are encased in an extracellular matrix composed of polysaccharides, proteins, and extracellular DNA (eDNA) that protect bacteria from host immune defense and antibiotics. Despite their importance, the mechanisms by which matrix components are released from bacterial cells and incorporated into the biofilm matrix remain poorly understood. Using a drip-flow biofilm system, we showed that membrane vesicles (MVs) were associated with the biofilm matrix formed by S. aureus clinical isolate MN8. Proteomic analysis of biofilm matrix proteins and purified MVs showed that biofilm-derived MVs carried cytoplasmic, membrane, and extracellular proteins that closely resembled the protein composition of the biofilm matrix, but differed significantly from MVs produced by planktonic cultures. Biofilm-derived MVs carried significantly higher levels of DNA than MVs from planktonic cultures, and MV-associated DNA was sensitive to DNase treatment, suggesting that eDNA is primarily associated with the MV surface. Although strain MN8 is known to form polysaccharide-dependent biofilms, exogenously added DNase or proteinase K significantly impaired biofilm formation and integrity. Importantly, supplementation with biofilm-derived MVs, but not MVs from planktonic cultures, significantly restored biofilm formation in enzyme-treated static cultures. Together, these findings provide evidence that S. aureus MVs are generated within biofilms, and that these MVs serve as an important resource of matrix components and contribute to biofilm formation. IMPORTANCE Extracellular membrane vesicles (MVs) are important mediators of intercellular communication and have been implicated in the bacterial physiology and pathogenesis. MVs in fungi and gram-negative bacteria mediate key biofilm processes, such as formation and structural maintenance. However, MV production and function in biofilm formation in gram-positive bacteria have remained largely unexplored. Here, we report for the first time the purification and characterization of MVs derived from Staphylococcus aureus biofilms. Our studies demonstrate that S. aureus MVs are important components of the biofilm matrix that contribute to biofilm formation by serving as carriers of key matrix components. This work advances our limited understanding of MVs in gram-positive bacteria and reveals a previously unrecognized mechanism contributing to S. aureus biofilm formation. Extracellular membrane vesicles (MVs) are important mediators of intercellular communication and have been implicated in the bacterial physiology and pathogenesis. MVs in fungi and gram-negative bacteria mediate key biofilm processes, such as formation and structural maintenance. However, MV production and function in biofilm formation in gram-positive bacteria have remained largely unexplored. Here, we report for the first time the purification and characterization of MVs derived from Staphylococcus aureus biofilms. Our studies demonstrate that S. aureus MVs are important components of the biofilm matrix that contribute to biofilm formation by serving as carriers of key matrix components. This work advances our limited understanding of MVs in gram-positive bacteria and reveals a previously unrecognized mechanism contributing to S. aureus biofilm formation.
Jinger Lei, Misaki Foster, Emery L. Ng et al.· Journal of Bacteriology· 0 citations
Biofilms represent the predominant microbial lifestyle in clinical and environmental settings, where the extracellular polymeric substance (EPS) matrix provides structural integrity, metabolic cooperation, and pronounced antimicrobial tolerance. This matrix forms a dynamic macromolecular network of polysaccharides, proteins, extracellular DNA (eDNA), lipids, and associated ions that collectively regulate hydration, adhesion, diffusion resistance, and persistence. Across ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens and Candida albicans, conserved matrix components shape biofilm architecture, enabling nutrient sequestration, immune evasion, and chronic infection. Mechanistic advances in biofilm eradication highlight the roles of enzymatic depolymerization, antibiotic penetration dynamics, nanoparticle-mediated disruption, bacteriophage-encoded depolymerases, and antibody-guided targeting. Parallel progress in biofilm inhibition emphasizes quorum-sensing interference, adhesion blockade, surface engineering, vaccines, and immunomodulatory strategies that prevent early community establishment. Emerging approaches-including peptide nucleic acids, aptamers, CRISPR-based antimicrobials, and biofilm-responsive delivery systems-enable precise targeting of genetic and structural vulnerabilities. Together, these developments provide a mechanistic foundation for next-generation antibiofilm interventions and support translational strategies aimed at recalcitrant, persistent infections.
John H. T. Luong, A. Gedanken· Biotechnology Advances· 0 citations
Bacterial extracellular vesicles (BEVs) are nano-sized lipid bilayer particles secreted by bacteria, capable of carrying various proteins, lipids, nucleic acids, and pathogen-associated molecular patterns (PAMPs). The biosynthetic pathway of BEVs determines their load components, physicochemical properties, and different biological activities. Increasing evidence indicates that BEVs play an important role in mediating host immune responses and the dynamic regulation of bacterial biofilms. Additionally, BEVs may serve as a molecular bridge between the two. BEVs derived from pathogens can trigger pro-inflammatory cascades, assist bacteria in immune evasion, and further accelerate the maturation of biofilms, forming a vicious cycle of persistent infection and inflammatory damage. In contrast, BEVs derived from probiotics can maintain host immune homeostasis and exert direct anti-biofilm and synergistic antibacterial effects, thereby breaking the pathological cycle. However, significant methodological research bottlenecks have greatly hindered the comparability and clinical translation of BEVs research. This article systematically summarizes the classification of BEVs and their biosynthetic mechanisms, compares the differential effects of BEVs from pathogenic bacteria and probiotic bacteria on immunity, clarifies the dual regulatory role of BEVs throughout the life cycle of biofilms, and highlights the bridging function of BEVs in the immune–biofilm interaction. Additionally, this article also discusses the current development of BEVs in clinical translation applications, such as vaccine development, antibiotic delivery, and mucosal inflammation intervention, and outlines the key industrial and clinical challenges faced in the future development of BEVs-based therapeutic approaches.
Qingyu Zhang, Bei-Lei Zhang, Mohd Shafiq Aazmi et al.· Biomolecules· 0 citations
Staphylococcus aureus is a major human pathogen that causes persistent infections characterised by the formation of aggregates such as surface-attached biofilm and staphylococcal abscess communities (SACs). Both consist of dense bacterial populations associated with self-produced matrices that impair immune cell and antibiotic access. Surface-attached biofilms have mostly protein- or polysaccharide-rich matrices, whereas SACs are spherical structures within tissue encased in a fibrin pseudocapsule and microcolony-associated meshwork. It remains unclear whether SAC are simply biofilms within tissue or if they display distinct features with unique genetic regulation. Here, we investigated genetic determinants of SAC and biofilm formation using four S. aureus deletion mutants targeting staphylokinase (Δsak), coagulase (Δcoa), the alternative sigma factor SigB (ΔrpoF), and sortase A (ΔsrtA). SACs were grown in collagen gels, whilst biofilms were grown on titanium discs, with bacterial viability, fibrin deposition, and biofilm biomass assessed by microscopy, image analysis, and crystal violet staining. Although deletion of coa or sak did not significantly impact either SAC or biofilm formation, biofilm surface coverage was markedly increased in ΔsrtA and biomass decreased in ΔrpoF; however, these mutations had no effect on SAC. These findings suggest that certain mutations can have different effects in the two experimental systems, and that the tested gene deletions were more important for surface-associated biofilm development than SAC formation.
Darine D'Adam, M. Chittò, Livia S. Kiener et al.· Biofilm· 0 citations
ABSTRACT Staphylococcus aureus forms biofilms on biotic and abiotic surfaces, enabling persistent infections that evade immune clearance. Although many S. aureus strains can produce the biofilm-associated exopolysaccharide, poly-β-1,6-N-acetyl-D-glucosamine (PNAG), they often form protein-dominated biofilm matrices in vitro, leaving a role for PNAG unclear. Using intravital imaging in a foreign-body infection model, we found that PNAG in biofilms hinders neutrophil access and delays bacterial clearance. Neutrophil elastase was crucial for eventual biofilm clearance. In vivo PNAG labeling revealed that the exopolysaccharide forms a physical barrier that prevents neutrophils from reaching bacterial clusters. In contrast, PNAG-deficient strains permitted greater neutrophil infiltration and were cleared more rapidly than wild-type bacteria. Enzymatic degradation of PNAG with the glycoside hydrolases PgaB or dispersin B (DspB) disrupted the biofilm, restored neutrophil access, and enhanced bacterial clearance. Together, these findings identify PNAG as a key structural barrier protecting S. aureus from innate immunity and suggest that targeting PNAG with glycoside hydrolases may offer a promising therapeutic strategy for biofilm-associated S. aureus infections. IMPORTANCE The biofilm-associated exopolysaccharide PNAG is frequently expressed in Staphylococcus aureus clinical isolates but is often reduced during laboratory passage, with expression highly dependent on growth conditions. While in vitro analyses have revealed that PNAG is not a dominant matrix component, our intravital imaging of community-acquired methicillin-resistant S. aureus (CA-MRSA) skin infections demonstrates that PNAG is robustly produced in vivo and plays a central role in immune evasion. These findings highlight how PNAG function in tissue environments may be non-obvious in vitro and underscore the need for in vivo models to understand biofilm pathogenesis. By revealing PNAG as a key barrier to neutrophil-mediated clearance, this work positions PNAG and PNAG-targeting glycoside hydrolases as compelling therapeutic candidates for treating antibiotic-resistant S. aureus biofilm infections, a major cause of morbidity in both healthcare and community settings. The biofilm-associated exopolysaccharide PNAG is frequently expressed in Staphylococcus aureus clinical isolates but is often reduced during laboratory passage, with expression highly dependent on growth conditions. While in vitro analyses have revealed that PNAG is not a dominant matrix component, our intravital imaging of community-acquired methicillin-resistant S. aureus (CA-MRSA) skin infections demonstrates that PNAG is robustly produced in vivo and plays a central role in immune evasion. These findings highlight how PNAG function in tissue environments may be non-obvious in vitro and underscore the need for in vivo models to understand biofilm pathogenesis. By revealing PNAG as a key barrier to neutrophil-mediated clearance, this work positions PNAG and PNAG-targeting glycoside hydrolases as compelling therapeutic candidates for treating antibiotic-resistant S. aureus biofilm infections, a major cause of morbidity in both healthcare and community settings.
Rachel M. Kratofil, R. Sejuty, Trevor E Randall et al.· mBio· 0 citations
An electron cryomicroscopy structure of the 60 kDa CdrA adhesive N-terminus is reported, which combined with electron cryotomography of focused-ion beam milled specimens, allows for a complete in situ model of the native adhesin and reveals a small adhesive domain (called ADEPT) at the distal tip of CdrA that is nearly perfectly conserved across the P. aeruginosa pangenome.
Olivia E. R. Smith, Camila M. Clemente, Antonina Andreeva et al.· bioRxiv· 0 citations