Periodic Reporting for period 2 - BioMatrix (Structural Biology of Exopolysaccharide Secretion in Bacterial Biofilms) Período documentado: 2020-02-01 hasta 2021-07-31 Resumen del contexto y de los objetivos generales del proyecto 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. To do this, we are working on the following specific aims:Specific Aim 1 : To determine the global architecture of exopolysaccharide secretion systems. Specific Aim 2 : To determine the structure of individual subunits, as well as the temporal sequence and interdependence among secretion system components.Specific Aim 3 : To determine the functional roles of identified structural motifs, protein-protein interfaces, ligand-binding sites, etc. in the native context of exopolysaccharide secretion in cells and biofilms.Specific Aim 4 : To identify activity-modifying compounds for the selective targeting of biofilm-promoting exopolysaccharide secretion systems Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha Secreted extracellular matrix components present important determinants for bacterial biofilm formation, survival and virulence. Many Gram-negative species rely on functionally homologous synthase-dependent systems for secretion of exopolysaccharides such as bacterial cellulose, alginate, Pel and poly-N-acetyl-glucosamine compounds. Although bacterial cellulose was first described at the end of the 19th century, it was not until a century later that biochemical studies on the Gluconacetobacter xylinus cellulose synthase led to the identification of c-di-GMP as its allosteric activator. With the revolution of DNA sequencing and genome assemblies in the beginning of the 21st century, c-di-GMP-metabolizing enzymes were discovered, often in multiple and diverse forms, in most characterized bacterial species and the dinucleotide revealed itself as a master regulator of exopolysaccharide secretion and biofilm formation. Over the last few years, I and my ‘Structural Biology of Biofilms’ team focused our efforts on the detailed structural and mechanistic studies of enterobacterial cellulose secretion: from the expression, assembly and global architecture of the multi-component secretion system to the specific regulatory mechanics of c-di-GMP sensing and secretion control. Overall our studies led to unprecedented insights into the structural organization and nucleotide-dependent regulation of the system and we prioritized communication of our findings in the form of few but truly comprehensive publications in open-access, high-impact journals (e.g. Zouhir S*, Abidi W* et al., mBio 2020; Abidi W*, Zouhir S* et al., Science Advances 2021).Bacterial cellulose is a widespread biofilm component that can modulate microbial fitness and virulence both in the environment and in infected hosts. Whereas its biosynthesis typically involves a c-di-GMP controlled BcsAB synthase tandem across the bacterial domain of life (Römling and Galperin, Trends Microbiol 2015), in many beta- and gamma-proteobacteria it is carried out by sophisticated E. coli-like Bcs secretion systems, where multiple additional subunits are either essential for secretion or contribute to the maximal production of the polysaccharide in vivo (Krasteva et al., Nat Comm 2017).In our seminal 2017 study, we showed that in enterobacteria most of the inner-membrane and cytosolic Bcs components (BcsRQABEF) interact stably to form a megadalton-sized Bcs macrocomplex with an unprecedented layered, multimeric and asymmetric architecture. Nevertheless, the low-resolution of these initial studies precluded us from deciphering how the system is assembled and regulated or the temporal sequence, stoichiometry and interdependence of Bcs subunit interactions. Importantly, homologs of the synthase BcsAB tandem have been studied repetitively and in great detail (e.g. Morgan et al. Nature 2013) and functional studies limited to individual subunits have assigned roles to some non-essential components (e.g. Thongsomboon et al., Science 2018), however the global architecture of the E. coli-type Bcs secretion systems, as well as the atomic-resolution structures and specific regulatory roles of most components had remained enigmatic. 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 protein-sorting SIMIBI NTPases and that the significant effects of both subunits on BcsA’s biochemical detection, as well as direct interactions with the synthase, point toward a direct role in BcsA’s membrane sorting 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. Importantly, our research tackles very dynamic and competitive areas of research in microbiology including bacterial secretion, biofilm formation, cyclic dinucleotide signaling, gene expression regulation and co-translational macrocomplex assembly. With bacterial cellulose rapidly finding many and diverse biotechnological applications, we are confident that our contribution to the field will be of great interest to a broad and multidisciplinary audience of readers. Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha) 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 (Krasteva et al., Nature Communications 2017; Zouhir et al., mBio 2020; Abidi et al., Science Advances 2021), but also in biotechnologically 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, in particular the synthase- (Alginate and Pel) and flippase- (Psl and Vps) systems of Pseudomonas aeruginosa and Vibrio cholerae, aim to unravel their assembled and functional states, as well as the ability to sense and respond to the intracellular second messenger c-di-GMP. It is important to note that although the Pseudomonas EPS secretion systems profit from decades of structural and microbiological studies on individual protein subunits, the global architecture and mechanistic interplay among these building blocks, as well as means to modulate secretion, remain to be discovered. 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. Structure of the Enterobacterial Cellulose Secretion System
