Periodic Reporting for period 3 - BioMatrix (Structural Biology of Exopolysaccharide Secretion in Bacterial Biofilms)
Reporting period: 2021-08-01 to 2023-01-31
Over the past few decades, the notion of bacteria as unicellular, free-swimming organisms has been entirely overhauled. It has become increasingly evident that bacteria never exist as independent and self-sufficient entities but are rather in constant communication with their confrères and the environment in order to rapidly fine-tune their physiology and virulence potential. This is especially true for opportunistic pathogens such as Pseudomonas aeruginosa, Vibrio cholerae and Escherichia coli, which are causative agents for a variety of human diseases or disease-accompanying infections. Their ability to transition from free-living to indwelling pathogenic lifestyle largely depends on collaborative group behaviors such as quorum-sensing, swarming and biofilm formation, which in turn employ highly regulated signal transduction mechanisms whose intricate details are only recently beginning to emerge.
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.
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.
Cellulose is the most abundant biopolymer on Earth and while it is the predominant building constituent of plants, it is also a key extracellular matrix component in the biofilms of many bacteria. In our seminal 2017 study, we showed that in Escherichia coli, the BcsAB cellulose synthase tandem assembles into a stable megadalton-sized macrocomplex with four accessory subunits, which are either essential for (BcsR and BcsQ) or enhance (BcsE and BcsF) cellulose secretion. In our recent works, we presented the first cryo-EM structures of an assembled Bcs secretion macrocomplex and sub-assemblies, as well as multiple crystallographic structures of individual Bcs subunits and/or multi-component complexes between regulatory Bcs components. We demonstrated that enterobacterial BcsE packs a subtle but diverse toolkit to fine-tune cellulose production and provided structural and functional data that support multidomain evolution, fold conservation and synthase-activating, intercalated c-di-GMP complexation on one hand, together with co-transcriptional regulation of the essential for secretion bcsRQ genes, post-translational BcsRQ recruitment and facilitated membrane targeting through BcsF interactions on the other. We further demonstrated that the essential BcsRQ tandem shares many structural and functional properties with membrane protein-sorting NTPases suggesting a direct role in BcsA’s membrane insertion and structure-function integrity. We further showed unexpected subunit stoichiometry, which begs a rethinking of the generally accepted model for equimolar BcsAB synthase assemblies throughout the bacterial domain of life. Finally, we showed an unexpected mechanism for asymmetric secretion system assembly via superhelical periplasmic BcsB oligomerization, which is likely conserved not only among enterobacteria but might also explain synthase nanoarrays and secretion efficiency in a diverse range of bacterial species.
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.
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.
Over the next years, we will establish and/or continue several ambitious axes of research. First, we will continue our efforts to obtain a multiscale mechanistic model of bacterial cellulose secretion, not only in the model enterobacterial system that we have been successfully unraveling so far, but also in biotechnologically, agriculturally and medically important bacterial species. In particular, we will obtain an integrated understanding of the molecular and supramolecular determinants of crystalline versus amorphous cellulose secretion, as well as the tight interplay between cellulose secretion, its chemical modifications and the downstream interactions with other extracellular matrix components that appear to play synergistic roles in host-pathogen interactions. Second, we will expand our studies to other systems for biofilm exopolysaccharide secretion, as well as the ability to sense and respond to the intracellular second messenger c-di-GMP. Third, we will continue to investigate the multilevel control of exopolysaccharide secretion versus flagellar motility in Gram-negative species, and in particular the role of the second messenger c-di-GMP (Krasteva & Sondermann, Nature Chemical Biology 2017). Finally, as we are dedicated to training young scientists in the various stages of academic studies and scientific careers, we will recruit and train multiple interns and longer-term researchers to contribute experimentally to the various aspects of this ambitious project.