Sebastian, C. C. | Search bioRxiv

bioRxiv2026

The archaic CUP pilus SMF-1 uses an antiparallel bundling mechanism to initiate biofilm formation

Bacterial biofilms represent a survival strategy that enables microbial communities to withstand environmental stress. Stenotrophomonas maltophilia is an emerging, antibiotic-resistant, Gram-negative, opportunistic pathogen that frequently colonizes the lungs of individuals with cystic fibrosis. Its chaperone-usher pathway (CUP) pilus, SMF-1, is present in nearly all clinical isolates and is essential for biofilm development; however, its molecular architecture has remained unknown. Here, we present a 4.0 [A] cryo-EM structure revealing that SMF-1 is an archaic, rather than a classic, CUP pilus. SMF-1 forms thin, zigzag filaments that assemble into defined antiparallel "pili-couples," which further aggregate into thick bundles. These bundles act as robust intercellular tethers, facilitating the rapid cell-to-cell aggregation required for biofilm initiation. Despite high sequence and structural similarity to classic CUP systems, SMF-1 lacks the interfaces required to form a rod-like architecture, suggesting it may represent an evolutionary intermediate between the CUP classes. Finally, we demonstrate that SMF-1 producing bacteria initiate biofilm formation within 24 hours and that flagella can further accelerate this process. Together, these findings uncover a conserved bundling mechanism that promotes bacterial colonization and may contribute to pathogenicity.

microbiology↗

bioRxiv2025

Increased Low Molecular Weight Mucins in Muco-Obstructive Airway Disease Limit Staphylococcus aureus Growth

Muco-obstructive airway diseases result in an increase in mucus accumulation and a decrease in mucus clearance. MUC5B is the most abundant secreted mucin in the human airways, and MUC5B mucin strands dimerize to create the mucus mesh network in the healthy respiratory tract. In muco-obstructive airway diseases like cystic fibrosis (CF), immune cells and bacteria release enzymes that degrade MUC5B into smaller fragments that become entangled and compacted, contributing to pathogenesis. We utilized synthetic cystic fibrosis sputum media (SCFM) to examine how mucin polymers can impact Staphylococcus aureus, a common CF pathogen that persists despite highly effective modulator therapies to correct CF disease. We found low molecular weight (LMW) mucin negatively impacts S. aureus survival and biofilm biomass compared to high molecular weight (HMW) mucin. Adding extracellular DNA to SCFM with LMW mucin was not sufficient to restore growth. LMW mucin had a broad negative impact on S. aureus laboratory strains and CF clinical isolates. We next tested other CF pathogens, including Pseudomonas aeruginosa and nontypeable Haemophilus influenzae, and saw no significant differences in growth in HMW or LMW mucin. LMW mucin did not significantly impact Staphylococcus epidermidis growth, indicating there may be specific interactions with S. aureus. Overall, this work highlights how interactions with pathogenic mucins may limit S. aureus growth in the diseased airways while supporting low-level persistence, and its ability to thrive in the presence of longer mucin strands may help to explain why S. aureus is well adapted to survive in the healthy respiratory tract. IMPORTANCEStaphylococcus aureus is a common colonizer of the healthy human airways that is also known to establish persistent infections in the lungs of people with muco-obstructive airway diseases, including cystic fibrosis. Yet, few studies have investigated how S. aureus interactions with airway mucins in chronic airway disease may regulate bacterial persistence or pathogenesis. In this study, we report that low molecular weight mucins representative of short, degraded mucin polymers found at high abundance in muco-obstructive airway disease suppress S. aureus growth and limit bacterial biofilm formation and aggregation. Longer mucin polymers did not negatively affect S. aureus survival. Other common CF pathogens were not significantly affected by low molecular weight mucins, suggesting there are species-specific interactions between airway mucins and S. aureus in the chronically diseased lung. This work highlights how pathogenic mucins found in muco-obstructive disease can affect S. aureus growth and promote phenotypes associated with airway persistence.

microbiology↗