Biology subjects

Hansen, K. H.

Publications and source records attributed to Hansen, K. H..

5 recordsLinked to original sources

bioRxiv2026

Structural and Mechanistic Basis of FapB Accessory Functional Amyloid Assembly in Pseudomonas

Functional amyloid formation in Pseudomonas biofilms is coordinated by the six-gene Fap operon (fapA-fapF), which governs the production, secretion, and assembly of extracellular fibrils that reinforce the biofilm matrix. FapC, FapB, and FapE constitute the extracellular amyloid. FapC is the major fibrillar component, whereas FapB and FapE occur at lower abundance and other accessory Fap proteins regulate fibril composition, processing, and secretion. We recently determined the structure of FapC, a cross-b-solenoid major Pseudomonas extracellular fibril. The assembly mechanism and architecture of FapB, however, have remained undefined. Here, we elucidate the structural and mechanistic basis of FapB accessory amyloid, from an intrinsically disordered monomer to a cross-b-solenoid fibril. Using well-defined monomeric preparations, controlled fibrillation assays, NMR spectroscopy, cryo-EM, and structure prediction, we show that FapB efficiently self-assembles into structurally homogeneous helical fibrils. FapB fibrillation is highly sensitive to its initial oligomeric state and purity, as well as to environmental factors such as pH. Monomeric FapC does not measurably perturb the disordered conformational ensemble of FapB, whereas preformed FapB and FapC fibrils reciprocally cross-seed each other, revealing direct kinetic coupling between the two amyloid components. Finally, we present a 3.4A resolution cryo-EM structure of the FapB accessory fibril, revealing a compact cross-b-solenoid architecture closely related to that of FapC. Together, these findings establish FapB as an independently amyloidogenic, structurally defined component of the Fap system, provide structural basis for the first of accessory cross-b-solenoid fibril, and reveal a mechanistic framework for the post-secretion assembly of multicomponent extracellular functional amyloids in Pseudomonas biofilms.

microbiology↗

bioRxiv2025

Structural Basis of Pseudomonas FapC Biofilm-Forming Functional Amyloid Formation

Biofilm-protected Pseudomonas aeruginosa causes chronic infections that are difficult to treat. FapC, the major biofilm forming functional-amyloid in Pseudomonas, is essential for biofilm integrity, yet its structural details remain unresolved. Using an integrative structural biology approach, we combine solution NMR-based structural ensemble of unfolded monomeric FapC, a [~]3.3 [A] resolution CryoEM density map of FapC fibril, and all-atom MD simulations to capture transition from unfolded to folded monomer to fibrillar fold, providing a complete structural view of FapC biogenesis. CryoEM reveals a unique triple-layer {beta}-solenoid cross-{beta} fibril composed of a single protofilament. MD simulations initiated from monomeric and fibrillar FapC mapped structural transitions, offering mechanistic insights into amyloid assembly and disassembly. Understanding FapC reveals how Pseudomonas exploits functional amyloids for biofilm formation and establishes a structural and mechanistic foundation for developing therapeutics targeting biofilm-related infection and antimicrobial resistance.

microbiology↗

bioRxiv2024

High-Sensitivity Analysis of Native Bacterial Biofilms Using Dynamic Nuclear Polarization-Enhanced Solid-State NMR

Bacterial biofilms cause persistent infections that are difficult to treat and contribute greatly to antimicrobial resistance. However, high-resolution structural information on native bacterial biofilms remain very limited. This limitation is primarily due to methodological constraints associated with analyzing complex native samples. Although solid-state NMR (ssNMR) is a promising method in this regard, its conventional applications typically suffer from sensitivity limitations, particularly for unlabeled native samples. Through the use of Dynamic Nuclear Polarization (DNP), we applied sensitivity enhanced ssNMR to characterize native Pseudomonas fluorescens colony biofilms. The increased ssNMR sensitivity by DNP enabled ultrafast structural characterization of the biofilm samples without isotope-labelling, and chemical or physical modification. We collected 1D 13C and 15N, and 2D 1H-13C, 1H-15N and 13C-13C ssNMR spectra within seconds/minutes or hours, respectively which enabled us to identify biofilm components as polysaccharides, proteins, and eDNA effectively. This study represents the first application of ultrasensitive DNP ssNMR to characterize a native bacterial biofilm and expands the technical scope of ssNMR towards obtaining insights into the composition and structure of a wide array of in vitro and ex vivo biofilm applications. Such versatility should greatly boost efforts to develop structure-guided approaches for combating infections caused by biofilm-forming microbes.

microbiology↗

bioRxiv2024

High-resolution 2D Solid-State NMR provides insights into Nontuberculous Mycobacteria

We present a high-resolution magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize native nontuberculous mycobacteria (NTM). We studied two different NTM strains, Mycobacterium smegmatis, a model, non-pathogenic strain, and Mycobacterium abscessus, an emerging and important human pathogen. Native hydrated NTM samples were studied at natural abundance without isotope-labelling and any chemical or physical modification. We utilized 1D 13C and 2D 1H-13C ssNMR spectra and peak deconvolution to identify NTM cell-wall chemical sites. More than [~]100 distinct 13C signals were identified in the ssNMR spectra. The signals originating from both the flexible and rigid fractions of the native bacteria samples were selectively analyzed by utilizing either CP or INEPT based 13C ssNMR spectra. CP buildup curves provide insights into the dynamical similarity of the cell-wall components for NTM strains. Signals from peptidoglycan, arabinogalactan and mycolic acid were identified. We also provide tentative assignments for [~]30 polysaccharides by using well resolved 1H/13C chemical shifts from the 2D INEPT-based 1H-13C ssNMR spectrum. As an orthogonal way of characterizing the bacteria, electron microscopy (EM) was used to provide spatial characterization. ssNMR and EM data suggest that M. abscessus cell-wall is composed of a smaller peptidoglycan layer which is more flexible compared to M. smegmatis, which may be related to its higher pathogenicity. Here in this work, we used high-resolution 2D ssNMR first time to characterize native NTM strains and identified chemical sites. These results will aid the development of structure-based approaches to combat NTM infections.

microbiology↗

bioRxiv2023

Initial Steps of Chaperone-Aided Fibrillation of Pseudomonas aeruginosa Biofilm Forming Functional Amyloid FapC

Functional bacterial amyloids (FuBA) play a crucial role in the formation of biofilms, which are mediating chronic infections and contribute to antimicrobial resistance. This study focuses on the FapC protein from Pseudomonas, a major contributor to biofilm formation. We investigate the initial steps of FapC amyloid formation and the impact of the chaperone-like protein FapA on this process. Using solution NMR spectroscopy, we show that both FapC and FapA, which are part of the same biofilm-forming protein operon, are intrinsically disordered proteins (IDPs) in their soluble monomeric state. These SSPs were determined and compared to the Alphafold models. We further demonstrate that the IDP chaperone FapA interacts with FapC and significantly slows down the formation of FapC fibrils, while maintaining the fibril morphology unchanged. Our NMR titration experiments reveal that [~]18% of the resonances show FapA induced chemical shift perturbations (CPSs) which has not been previously observed, the largest being for A82, N201, C237, C240, A241 and G245 residues. These sites may suggest a specific interaction site and/or hotspots of fibrillation inhibition/control interface at the R1/L2 and L2/R3 transition areas and at the C-terminus of FapC. Remarkably, [~]90% of FapA NMR signals exhibit substantial CSPs upon titration with FapC. A temperature dependent effect of FapA was observed on FapC by ThT and NMR experiments. This study provides a detailed understanding of the interaction between the chaperone/chaperone-like FapA and the functional amyloid protein FapC, shedding light on the regulation and slowing down of amyloid formation. Our findings have important implications for the development of therapeutic strategies targeting biofilms and associated infections, leveraging these structural and mechanistic insights.

microbiology↗