Periodic Reporting for period 4 - BAS-SBBT (Bacterial Amyloid Secretion: Structural Biology and Biotechnology.) Período documentado: 2019-12-01 hasta 2020-11-30 Resumen del contexto y de los objetivos generales del proyecto Many bacteria are found as multicellular organizations called biofilms. A major component of biofilms formed by many Gram-negative bacteria is the bacterial functional amyloid ‘curli’. Amyloids are best known from protein misfolding and aggregation events seen in pathological amyloid diseases such as Alzheimer’s and Parkinson’s disease. Curli, however, are the product of a dedicated protein secretion machinery, and form the native, functional form of the component proteins.In the BAS-SBBT research program we study the structural and molecular biology of E. coli curli biosynthesis and address the fundamental questions concerning curli formation: Q1: What is the mechanism employed by the Type VIII secretion transporter CsgG?Q2: How is premature curlin aggregation and cytotoxicity controlled prior to secretion?Q3: What is the structure of the curli fibers and how is assembly coordinated with secretion?Q4: Can we use the unique properties of the Type VIII secretion pathway for biotechnological applications? Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha A major question in functional amyloid assembly is how bacterial cells avoid the build-up or activity of possibly toxic intermediates known to accompany pathological amyloid formation in human wasting diseases. Contrary to pathological amyloids, the fiber confirmation in functional amyloids is not the result from protein misfolding, but represents the native, functional state of the protein. It is unknown whether the nucleation and elongation of the functional amyloid fibrils take a similar pathways than what has been described for pathological amyloids.We addressed these questions by means of high speed atomic force microscopy (AFM) imaging of the fibrillation pathway of curli subunit CsgA, starting from pure solution of CsgA monomers. Strikingly, curli follow a one-step nucleation process, where monomeric species contemporaneously fold and oligomerize into minimal fiber units that have growth characteristics identical to the mature fibrils. This is in contrast to most pathological amyloids where nucleation is a multistep process involving amorphous aggregates and distinct oligomeric intermediates, which are thought to form the main cytotoxic species involved in pathological amyloidogenesis. If we consider the biological context in which curli are formed, one-step direct nucleation can be considered as an adaptation to avoid the formation of potentially cytotoxic aggregation intermediates. Although more examples are needed, the absence of a lengthy induction time could be a defining trait of functional amyloids. A first paper reporting these new findings has been published: Sleutel M et al (2017) Nucleation and growth of a bacterial functional amyloid at single-fiber resolution. Nat Chem Biol. 13(8):902-908.In a follow-up on the curli dynamics study we determined the ultrastructural characteristics of this functional amyloid by nuclear magnetic resonance (NMR) and cryogenic electron microscopy (cryo-EM), and provide direct observation of the nucleating species of this amyloid assembly pathway. These confirm the direct nucleation pathway of this functional amyloid, and show that curli display a biphasic behavior, in which they are found as intrinsically unfolded monomers during the secretion process, and folded amyloid upon assembly. These data again highlight adaptations associated with safe and evolutionary optimized amyloid assembly. (Brajabandhu et al. 2021 – in preparation) In addition to curli, several bacterial functional amyloids are known to exist in different bacterial genera. Based on a literature review of available properties of known functional amyloid we find that these can be classified as intrinsic and facultative amyloid structures, where the amyloid fold forms the unique or an alternative native state of the component proteins, respectively. In this same study, we review the implication of bacterial amyloids as potential virulence factors. This study and opinion forms a comprehensive basis for the further study of biological and structural aspects of bacterial amyloids. (Van Gerven, N et al (2018) The role of functional amyloids in bacterial virulence, Journal of molecular biology 430 (20): 3657-3684.Another fundamental question in the curli assembly pathway relates to the subunit secretion process and the coordination with fiber assembly at the cell surface. On the bacterial cells, these processes are coordinated by the curli secretion channel CsgG and its extracellular accessory protein CsgF. To gain an atomic insight in these processes we solved the cryo-EM structure of the CsgG:CsgF complex (Van der Verren et al. (2020) A dual-constriction biological nanopore resolves homonucleotide sequences with high fidelity. Nature biotechnology 38 (12), 1415-1420). These structural insights form the basis for the molecular understanding of the secretion – assembly process. A second manuscript on the biological aspects of the CsgG:CsgF complex is in preparation (Van der Verren et al. 2021 – in preparation).A second aspect of the CsgG:CsgF structural study is the design of CsgG based nanopores for nanopore sensing applications. Our structural studies of the curli secretion channel CgsG revealed that the protein forms a constitutively open channel of 3.5 nm that is constricted in the center by a 0.9 nm wide and 1.5 nm high constriction formed by three concentrically stacked residues on a short constriction loop (CL). We found that reconstituted channels show stable single channel conductance levels, and that channel conductance properties and diameter could be altered by specific mutations in the CL loop. The channel architecture, stability and chemical properties in the constriction are such that it can be used for nanopore sensing application, in which the nature of a passing solute is measured based on the alteration in conductance levels. The use of mutant CsgG-like channels for nanopore sensing application has been protected under PCT application PCT/EP15201357.9 and has been licensed to Oxford Nanopore Technologies Ltd., who use a mutant CsgG pore for DNA sequencing applications using their handheld and tabletop sequencers MinION, GridION and PromethION. A collaboration agreement with Oxford Nanopore Technologies foresees in the further identification and development of CsgG-like pores for nanopore sensing applications. In 2017, two further patent applications were filed, concerning the use of CsgG double pores (PCT/GB1707122.6) and CsgG:CsgF complexes (PCT/EP17179099.1) for nanopore based sensing applications. The introduction of mutant CsgG pores into the nanopore sequence devices has provided a breakthrough shift in sequencing speed and accuracy compared to the previous baseline pore used by the company. 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) Published data from the first reporting period provide unprecedented insights in curli fibrillation at single fiber resolution. Hitherto, curli formation and inhibition had only been studied using bulk solvent techniques, where direct observation of rare and short-lived events such as nucleation are not possible. The unravelling of the transport mechanism employed by the curli translocation channel CsgG is ongoing. We anticipate that in the second half of the project, we will obtain atomic structures of CsgG bound to its various secretion partners and map out the interaction pathway of the curli subunits en route to the cell surface. Use of CsgG-based pores for nanopore sensing applications The role of functional amyloids in bacterial virulence Graphical abstract - nucleation and growth dynamics of the functional amyloid curli
