Biology subjects

Pinkas, D.

Publications and source records attributed to Pinkas, D..

5 recordsLinked to original sources

bioRxiv2026

C-terminal domain of the filamentous hemagglutinin FhaB is crucial for interaction of Bordetella pertussis with ciliated epithelial cells

Bordetella pertussis, the causative agent of whooping cough, produces a [~]370 kDa filamentous hemagglutinin FhaB that serves as a major bacterial adhesin in airway infection. FhaB is secreted via a two-partner secretion pathway and under in vitro culture conditions it is proteolytically processed to the shed [~]230 kDa FHA antigen used currently in acellular pertussis vaccines. We show that FhaB remains largely unprocessed during B. pertussis adhesion to ciliated airway epithelial cells and that its C-terminal domain (CT) is essential for the adhesin function of FhaB. CT deletion did not affect FhaB folding, secretion, or surface exposure, but abolished B. pertussis adhesion to primary human nasal ciliated epithelial cells, thus preventing bacterial colonization of the nasal mucosa and shedding and transmission of the pathogen in a murine nasal infection model. In situ cryo-electron tomography revealed a structural reorganization of the FhaB filaments upon contact with the cilia, presumably due to export of the CT from bacterial periplasm and its subsequent delivery across the ciliary membrane. These findings establish the CT of FhaB as a critical determinant of upper airway colonization by B. pertussis and identify the unprocessed FhaB as the biologically relevant adhesin form involved in airway infection. The revised model of FhaB biogenesis underpins its unique mode of action in pertussis pathogenesis and makes the CT domain to a candidate antigen for future pertussis vaccines.

microbiology↗

bioRxiv2025

Cryo-FIB Lift-out and Electron Tomography Workflow for Bacteria-Nanopillar Interface Imaging Under Native Conditions: Investigating Dragonfly Inspired Bactericidal Titanium Surfaces

Dragonfly- and cicada-wing-inspired titanium nanostructured surfaces exhibit promising bactericidal properties. However, direct visualisation of the bacteria-material interface under hydrated conditions remains limited, restricting experimental interrogation how bacteria interact with nanostructured surfaces. This limitation reflects a long-standing methodological gap, as visualisation of bacteria-nanostructure interfaces has largely relied upon indirect or dehydrated imaging approaches. Cryo-electron tomography (cryo-ET) enables three-dimensional visualisation of cellular ultrastructure in a native hydrated environment, but imaging bacteria attached to metallic nanostructures by cryo-ET requires preparation of thin electron-transparent lamellae. Obtaining such lamellae from vitrified bacteria on titanium substrates is technically challenging because cryo-focused ion beam (cryo-FIB) milling must simultaneously section soft biological material and hard metal whilst bacteria embedded in vitreous ice are not directly visible. Here, we establish a correlative cryogenic workflow enabling targeted extraction and cryo-ET imaging of defined bacterium-nanopillar interfaces. Individual bacteria interacting with titanium nanopillars are identified by correlative cryo-fluorescence microscopy, followed by targeted cryo-FIB lift-out, transfer to a receiver grid, and thinning into electron-transparent lamellae. The data presented demonstrate that bacteria-nanostructure interfaces can be targeted, extracted, and structurally analysed in situ under fully hydrated conditions. This workflow provides a methodological framework for future cryo-ET studies of bacteria-nanotopography interactions.

microbiology↗

bioRxiv2025

In situ protein crystallography: a single-crystal electron diffraction pipeline for structure determination inside living cells.

Intracellular crystallization is an emerging approach in structural biology that bypasses the need for protein purification. An InCellCryst pipeline was recently established for structure determination by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of cells containing intracellular crystals, precluding high-resolution structural studies on proteins with low numbers of crystals. To overcome this limitation, we combined the InCellCryst approach with the advantages of 3D electron diffraction and established the new IncellED method. Using microcrystals of the HEX-1 protein from Magnaporthe grisea, grown inside High Five insect cells, we demonstrate that electron diffraction data collected from a single crystal yields a high-resolution structure at 1.9 [A] resolution, comparable to 1.8 [A] achieved by serial X-ray crystallography. The IncellED methodology opens structure determination for proteins that crystallize with low efficiency and use widely available electron cryo-microscope infrastructure, paving the way for intracellular macromolecular electron crystallography at high resolution.

biophysics↗

bioRxiv2023

Cellular Uptake and Fate of Cationic Polymer-Coated Nanodiamonds Delivering siRNA: A Mechanistic Study

Gene silencing using small interfering RNAs (siRNAs) is a selective and promising approach for treatment of numerous diseases. However, broad applications of siRNAs are compromised by their low stability in a biological environment and limited ability to penetrate cells. Nanodiamonds (NDs) coated with cationic polymers can enable cellular delivery of siRNAs. Recently, we developed a new type of ND coating based on a random copolymer consisting of (2-dimethylaminoethyl) methacrylate (DMAEMA) and N-(2-hydroxypropyl) methacrylamide (HPMA) monomers. These hybrid ND-polymer particles (Cop+-FND) provide near-infrared fluorescence, form stable complexes with siRNA in serum, show low toxicity, and effectively deliver siRNA into cells in vitro and in vivo. Here, we present data on the mechanism of cellular uptake and cell trafficking of Cop+-FND:siRNA complexes and their ability to selectively suppress mRNA levels, as well as their cytotoxicity, viability and colloidal stability. We identified clathrin-mediated endocytosis as the predominant entry mechanism for Cop+-FND:siRNA into U-2 OS human bone osteosarcoma cells, with a substantial fraction of Cop+-FND:siRNA following the lysosome pathway. Cop+-FND:siRNA potently inhibited the target GAPDH gene with negligible toxicity and sufficient colloidal stability. Based on our results, we suggest that Cop+-FND:siRNA can serve as a suitable in vivo delivery system for siRNA.

biophysics↗

bioRxiv2020

Alternative isoforms of KDM2A and KDM2B lysine demethylases negatively regulate canonical Wnt signaling

A precisely balanced activity of canonical Wnt signaling is essential for a number of biological processes and its perturbation leads to developmental defects or diseases. Here, we demonstrate that alternative isoforms of the KDM2A and KDM2B lysine demethylases have the ability to negatively regulate canonical Wnt signaling. These KDM2A and KDM2B isoforms (KDM2A-SF and KDM2B-SF) lack the N-terminal demethylase domain, but they are able to bind to activated promoters in order to repress them. We have observed that KDM2A-SF and KDM2B-SF bind to and repress the promoters of AXIN2 and CYCLIN D1, two canonical Wnt signaling target genes. Moreover, KDM2A-SF and KDM2B-SF can repress a Wnt-responsive luciferase reporter. The transcriptional repression mediated by KDM2A-SF and KDM2B-SF, but also by KDM2A-LF, is dependent on their DNA binding domain, while the N-terminal demethylase domain is dispensable for this process. Surprisingly, KDM2B-LF is unable to repress both the endogenous promoters and the luciferase reporter. Finally, we show that both KDM2A-SF and KDM2B-SF are able to interact with TCF7L1, one of the transcriptional mediators of canonical Wnt signaling. KDM2A-SF and KDM2B-SF are thus likely to affect the transcription of the TCF7L1 target genes also through this interaction.

molecular biology↗