The structure reveals for the first time the interaction between a biofilm exopolysaccharide and matrix protein, as well as insights into conformational changes of exopolysaccharide induced by this binding, and provides a generalizable approach for studying the biophysical and biochemical properties of carbohydrate-dependent biofilm assembly.
Abstract
Biofilms serve as a protective mechanism for bacteria, including many pathogens. To form such communities, bacteria secrete macromolecules that form an extracellular matrix serving as a barrier against environmental threats, such as predation, antibiotics, and the host immune system. To be effective, a biofilm must anchor to foreign surfaces and retain sufficient stiffness, in the environment or in a host. However, how this matrix self-organizes to support biofilm formation remains a mystery at the molecular level. The human pathogen Vibrio cholerae produces biofilms primarily composed of an exopolysaccharide called VPS (Vibrio polysaccharide), consisting of an unusually-modified repeating tetrasaccharide core unit. VPS engages with two secreted adhesion proteins, Bap1 and RbmC, which adhere the biofilm to abiotic and biotic surfaces, and serve to strengthen the biofilm by interacting with VPS using a conserved β-propeller. To pinpoint the interaction between the adhesins and purified segments of VPS, we determined the ∼1.6 Å X-ray crystal structure of Bap1 bound to fragmented VPS and used the structure to carry out molecular dynamics simulations. The structure revealed a single binding site consisting of one tetrasaccharide unit involving an induced magnesium binding site. Unexpectedly, the tetrasaccharide adopted a bent state caused by a rotation of the glycosidic bond between the central two monosaccharide units. Using a combination of mutagenesis, light scattering, and in situ fluorescent microscopy, we demonstrate that Bap1 not only facilitates biofilm adhesion, but is also required for proper VPS organization, through the identified binding pocket. Our structure reveals for the first time the interaction between a biofilm exopolysaccharide and matrix protein, as well as insights into conformational changes of exopolysaccharide induced by this binding. Our findings provide a generalizable approach for studying the biophysical and biochemical properties of carbohydrate-dependent biofilm assembly, which may lead to new ways to treat disease caused by biofilm-forming bacterial pathogens by disrupting the exopolysaccharide-protein interactions.
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
Polysaccharides remain the least understood biomacromolecules, particularly in terms of the relationship between their chemical structure and physical properties. On the other hand, polysaccharides often serve as the main structural components in biofilms: surface-attached aggregates of bacterial cells encased within a mechanically resilient extracellular matrix. The large chemical space explored by bacteria within biofilms provides excellent opportunities to establish the structure-function relationship for polysaccharides. In this paper, we systematically characterize various polymer properties of Vibrio polysaccharide (VPS), the major exopolysaccharide in biofilms formed by Vibrio cholerae, the causative agent of pandemic cholera. Using a combination of shear rheology, dynamic and static light scattering, and small-angle X-ray scattering, we measure the viscosity, molecular weight, persistence length, radius of gyration, and hydrodynamic radius of this chemically unique biopolymer. Combining all-atom and coarse-grained simulations, we show how the conformational flexibility of a single glycosidic linkage within each VPS monomer can lead to dramatic compaction of the entire polymer chain and nonclassical entanglement behavior. Our comprehensive quantification represents a rare endeavor for bacterial biofilms, whose matrix composition and physical properties remain largely nebulous; it also represents a significant step towards a detailed understanding of the molecular origins of biofilm mechanics.
Kee-Myoung Nam, Nathan Fowler, Rajan Kandel et al.· bioRxiv· 0 citations
ABSTRACT Biofilm formation underlies the environmental persistence and transmission of Vibrio cholerae, the etiological agent of cholera. The Vibrio polysaccharide (VPS) is the principal structural component of the mature biofilm matrix, yet the enzymatic logic governing its assembly has remained incompletely defined. VPS is synthesized as two closely related polymers that share a tetrasaccharide repeat unit but differ at a single monosaccharide position. Here, we systematically define VPS assembly by integrating targeted gene deletions with liquid chromatography-mass spectrometry profiling of lipid-linked intermediates, and comparative structural modeling of biosynthetic enzymes. Our results establish that VPS is produced through an ordered Wzx/Wzy-dependent pathway. VpsL functions as the initiating phosphoglycosyltransferase, generating bactoprenyl diphosphate-linked glucose. A VpsA/VpsB/VpsK module analogous to the enterobacterial common antigen machinery synthesizes and transfers an N-acetyl-mannosaminuronic acid (ManNAcA)-derived residue, after which VpsJ, which we propose as a new class of epimerase, catalyzes C5 epimerization to generate the rare bacterial sugar L-N-acetyl-gulosaminuronic acid (L-GulNAcA). Additional tailoring reactions mediated by VpsH, a previously unidentified protein with few sequence or structural homologs, and VpsG introduce glycine and acetyl modifications that are dispensable for repeat-unit assembly but influence matrix properties. Subsequently, glycosyltransfer reactions by VpsI and VpsD complete the tetrasaccharide repeat unit, with VpsD exhibiting substrate flexibility that accounts for the formation of both major and minor VPS variants. Downstream, VpsE and VpsF act following repeat-unit assembly, consistent with flippase and polymerase functions, respectively. Together, our findings establish a molecular framework for VPS assembly and deepen our understanding of the mechanisms that drive biofilm formation in Vibrio cholerae. IMPORTANCE Biofilm formation is an integral part of Vibrio cholerae’s infection cycle, requiring production of the exopolysaccharide Vibrio polysaccharide (VPS). Together, these findings define the sequential enzymatic steps of VPS biosynthesis. This molecular map of VPS production identifies multiple enzymatic nodes as potential anti-biofilm targets and provides a mechanistic foundation for understanding how V. cholerae modulates biofilm architecture to enhance environmental survival and transmission. Biofilm formation is an integral part of Vibrio cholerae’s infection cycle, requiring production of the exopolysaccharide Vibrio polysaccharide (VPS). Together, these findings define the sequential enzymatic steps of VPS biosynthesis. This molecular map of VPS production identifies multiple enzymatic nodes as potential anti-biofilm targets and provides a mechanistic foundation for understanding how V. cholerae modulates biofilm architecture to enhance environmental survival and transmission.
Manoj K. Dooda, A. Potapova, S. Piepoli et al.· mBio· 0 citations
Results show that HsbA proteins in Mucor lusitanicus function as regulators that couple fungal surface remodeling with developmental transitions, thereby coordinating environmental adaptation and host–pathogen interactions.
Anna Molnár, Amanda Grace Vaz, M. Homa et al.· Frontiers in Cellular and In...· 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
Microbial organisms assemble a diverse array of surface structures to facilitate critical functions including motility, adhesion, and biofilm formation. As extracellular organelles, pili and related surface structures must be able to function in harsh environments and withstand various stressors. Microbes have evolved different strategies to assemble structures able to function under these challenging conditions. This review focuses on bacterial and archaeal systems that utilize donor-strand exchange (DSE) interactions between subunit proteins. DSE is a noncovalent assembly mechanism where one subunit contributes a β-strand to complete the structure of its neighboring subunit. This subunit-subunit interaction is one of the strongest noncovalent interactions known, with the resulting fiber being capable of withstanding extreme environmental stresses and shear forces. We summarize the structural biology and biogenesis of these surface structures, highlighting how DSE-mediated polymerization contributes to the assembly of extracellular structures in both the bacterial and archaeal domains.
Karla Cardenas Arevalo, D. Thanassi· Annual Review of Microbiolog...· 0 citations