Hinbest, A. J. | bioRxiv Science

bioRxiv2026

Structure of a key adhesin-exopolysaccharide interaction provides insights into matrix assembly in Vibrio cholerae biofilms

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 {beta}-propeller. To pinpoint the interaction between the adhesins and purified segments of VPS, we determined the [~]1.6 [A] 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.

microbiology↗

bioRxiv2025

Vibrio cholerae interaction with predatory bacteria on chitin suggests an alternative mode of biofilm formation in marine snow conditions

Vibrio cholerae is a ubiquitous marine microbe that solubilizes and consumes chitin in the marine water column. In both the marine environment and the intestinal track, V. cholerae forms biofilms; a key question regarding the lifestyle of V. cholerae is how do the diverse substrates that it encounters influence its biofilm formation and, in turn, shape its ecological interactions. Here, we use the predator-prey interaction between Bdellovibrio bacteriovorus and V. cholerae as a model to explore how the environmental chitin substrate alters V. cholerae biofilm formation and predator-prey interactions. We find that glass-bound biofilms provide strong protection for V. cholerae against predation while also allowing a population of predatory B. bacteriovorus to remain in place. In contrast, chitin-bound biofilms offer less protection against B. bacteriovorus predation and do not maintain a stable population of B. bacteriovorus. Using percolation and population dynamics models, we predict that these changes in predator-prey dynamics can be mostly explained by alterations in biofilm architecture between the two conditions, which changes the fraction of prey available to B. bacteriovorus. Performing targeted biofilm matrix deletions, we confirm this prediction by recapitulating key features of the chitin predator-prey interactions on glass surfaces. Following on this observation, we show that V. cholerae biofilms grown on chitin produce much less of the canonical biofilm matrix components and instead rely on other extracellular structures. Overall, our experiments detail how growth substrate can alter biofilm matrix composition and how these changes in biofilm architecture and cellular arrangement can impact higher-order ecological interactions.

microbiology↗