Periodic Reporting for period 3 - BioMatrix (Structural Biology of Exopolysaccharide Secretion in Bacterial Biofilms)
Reporting period: 2021-08-01 to 2023-01-31
For much of the bacterial life cycle, biofilm formation is a preferred mode of growth as it provides protection from noxious stimuli and environmental stress. Within a biofilm, bacteria secrete and become embedded in thick extracellular matrix, which serves as both a protective cushion and medium for intercellular communication. It secures strict temporal and spatial coordination of processes of functional differentiation, horizontal gene transfer and programmed cell death, that make the biofilm more akin to a multicellular organism rather than a passive mass of held-together cells. Importantly, biofilm formation has been associated with pathogen persistence, antimicrobials resistance development and roles in both chronic and acute bacterial infections, underscoring the medical relevance of this key developmental process.
Biofilm exopolysaccharides (EPS), which constitute a major structural and protective component of the biofilm matrix, are proposed to be secreted by dedicated protein nanomachineries in the cell envelope. In the 'Structural Biology of Biofilms' we integrate expertise in biofilm formation, membrane protein biology, and bacterial secretion with high-resolution structural biology approaches such as single-particle cryo-EM and X-ray crystallography to provide mechanistic insights into biofilm-promoting exopolysaccharide secretion in bacteria. The goal of this project is to undertake a holistic structure-function approach and make substantial progress towards understanding these key structural determinants of biofilm formation in Gram-negative bacteria, and in particular in opportunistic pathogens that represent important models for acute and chronic bacterial infections.
Despite strong conservation of the BcsAB tandem, in the economically relevant BC superproducer G. hansenii cellulose is secreted in a drastically different manner: a longitudinal nanoarray of synthase terminal complexes (TCs) assembles the EPS into a crystalline cellulose ribbon with implications in cell motility, flotation and substrate colonization. Crystalline BC secretion is dependent on two accessory subunits earlier proposed to interact in the periplasm, BcsD and BcsH. We recently provided the first atomic-resolution insights into BcsHD mediated BC crystallinity: BcsH drives BcsD oligomerization into a three-dimensional supramolecular scaffold. We showed that, in situ, the BcsHD assemblies share remarkable morphological similarities with the recently discovered cortical belt, namely an intracellular cytoskeleton that spatially correlates with the cellulose exit sites and the assembled crystalline cellulose ribbon. Finally, we detected specific protein-protein interactions between the BcsHD components and the regulatory BcsAPilZ module, further supporting that BcsHD features an unexpected intracellular localization for inside-out control of TC array formation and crystalline cellulose secretion.