Biology subjects

Fuzik, T.

Publications and source records attributed to Fuzik, T..

5 recordsLinked to original sources

bioRxiv2024

Dynamics of bacterial biofilm development imaged using light sheet fluorescence microscopy

Biofilm formation exacerbates bacterial infections and interferes with industrial processes. However, the dynamics of biofilm development is not entirely understood. Here, we present a microfluidic cultivation system that enables continuous imaging of biofilm growth using light sheet fluorescence microscopy (LSFM). We studied the development of biofilms of the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. Due to the low phototoxicity of LSFM, biofilms can be continuously imaged without adverse effects on their development. Whereas S. aureus forms 50-70-m-thick mushroom-like structures, a P. aeruginosa biofilm is 10-15 m thick with cell clusters 25 m in diameter. A combined biofilm, inoculated with an equal OD600 ratio of S. aureus and P. aeruginosa, resulted in the formation of large mushroom-like clusters of S. aureus cells that were subsequently dispersed by invading P. aeruginosa. A higher inoculation ratio favoring P. aeruginosa resulted in the formation of small and stable S. aureus clusters overgrown with P. aeruginosa cells. Applying conditioned media from S. aureus and P. aeruginosa coculture to a single-species S. aureus biofilm induced its dispersion. Integrating a microfluidic system into LSFM enables the visualization of biofilm formation dynamics and the effects of compounds on biofilm development.

microbiology↗

bioRxiv2023

The structure of immature tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV) is a medically important flavivirus that poses a significant health threat in Europe and Asia. However, the structure of the immature form of TBEV remains unknown. Here, we employed state-of-the-art cryogenic electron microscopy (cryoEM) to determine the structure of the immature TBEV particle. The immature TBEV particle has a diameter of 56 nm and its surface glycoproteins are organised into spikes characteristic of immature flaviviruses. The cryoEM reconstructions of the whole virus and of the individual spike enabled us to build atomic models of the major viral components, the E and prM proteins. The insights obtained from our study provide a foundation for understanding the early stages of TBEV assembly and maturation. The pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in metastable conformation. Destabilisation of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM cleavage, the collapse of E protein ectodomains onto the virion surface concurrent with significant movement of the membrane domains of both E and M, and release of the pr fragment from the particle render the virus mature and infectious. This knowledge contributes to our understanding of the flavivirus life cycle.

microbiology↗

bioRxiv2023

Cryo-electron tomography of enterovirus cell entry and endosome escape

Enveloped viruses deliver their genomes into the cell cytoplasm by membrane fusion; in contrast, membrane penetration by non-enveloped viruses is more diverse and less well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. To initiate infection, most enteroviruses enter cells by endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing rhinovirus 2, echovirus 18, echovirus 30, or enterovirus 71 deform, rupture, and release their content into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of enterovirus particles that released their genomes in endosomes, but did not prevent them from reaching the cytoplasm. Inhibiting N-WASP-mediated post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. We show that the rupture of endosomes also occurs in uninfected cells. In summary, our results indicate that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus genomes and particles into the cytoplasm. Since the studied enteroviruses employ different receptors for cell entry but are all delivered into the cytoplasm by cell-mediated endosome disruption, it is possible that many other enteroviruses utilize endosome rupture to infect cells.

microbiology↗

bioRxiv2023

Structure and replication cycle of a virus infecting climate-modulating alga Emiliania huxleyi

The globally distributed marine alga Emiliania huxleyi produces reflective calcite disks (coccoliths) that increase the albedo of ocean water and thus reduce the heat absorption in the ocean, which cools the Earths climate. The population density of E. huxleyi is restricted by nucleocytoplasmic large DNA viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane decorated with numerous transmembrane proteins. The virions are prone to deformation, and parts of their capsids deviate from the icosahedral arrangement. EhV-201 virions infect E. huxleyi by using their fivefold vertex to bind to a host cell and fuse the viruss inner membrane with the plasma membrane. Whereas the replication of EhV-201 probably occurs in the nucleus, virions assemble in the cytoplasm at the surface of endoplasmic reticulum-derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. Upon the completion of genome packaging, the capsids change conformation, which enables them to acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which allows the continuous release of virions by exocytosis. Our results provide insight into how EhVs bypass the surface protective layers of E. huxleyi and exploit the organelles of an infected cell for progeny assembly.

molecular biology↗

bioRxiv2018

Structure and genome ejection mechanism of Podoviridae phage P68 infecting Staphylococcus aureus

Phages infecting S. aureus have the potential to be used as therapeutics against antibiotic-resistant bacterial infections. However, there is limited information about the mechanism of genome delivery of phages that infect Gram-positive bacteria. Here we present the structures of S. aureus phage P68 in its native form, genome ejection intermediate, and empty particle. The P68 head contains seventy-two subunits of inner core protein, fifteen of which bind to and alter the structure of adjacent major capsid proteins and thus specify attachment sites for head fibers. Unlike in the previously studied phages, the head fibers of P68 enable its virion to position itself at the cell surface for genome delivery. P68 genome ejection is triggered by disruption of the interaction of one of the portal protein subunits with phage DNA. The inner core proteins are released together with the DNA and enable the translocation of phage genome across the bacterial membrane into the cytoplasm.

microbiology↗