Biology subjects

Franzkoch, R.

Publications and source records attributed to Franzkoch, R..

7 recordsLinked to original sources

bioRxiv2023

Structure and regulation of GSDMD pores at the plasma membrane of pyroptotic cells

Gasdermin D (GSDMD) executes inflammatory cell death pyroptosis by permeabilizing the plasma membrane (PM). We introduce polymer-supported PM (PSPM) to gain access to the cytoplasmic side of the PM with imaging techniques while preserving the native PM complexity and lipid microenvironment. By combining PSPM with DNA-PAINT super-resolution microscopy we visualized, for the first time, GSDMD nanostructures directly at the PM of pyroptotic cells. We resolved diverse macromolecular architectures with ring-and arc-shaped GSDMD oligomers that enable PM permeabilization. The pyroptotically-inactive mutant GSDMD-C192A (human C191A) still interacts with the PM however fails to form pores. GSDMD expression levels affect pore density but not permeabilization ability. Finally, we identified the local PI(3,4,5)P3 concentration as a key regulatory element of PM permeabilization. Increase in PI(3,4,5)P3 levels in the PM during pyroptosis facilitates growth into large ring-shaped pores. Using molecular dynamics (MD) simulations, we identified the mechanism by which PI(3,4,5)P3 stabilizes the GSDMD assembly.

biophysics↗

bioRxiv2023

Resolving exit strategies of mycobacteria by combining high-pressure freezing with 3D-correlative light and electron microscopy

The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of vacuolar and cytosolic mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium-containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system. Despite the availability of well-established light and advanced electron microscopy techniques in this system, a correlative approach that integrates both methodologies with almost native ultrastructural preservation is still lacking at the moment. This is most likely due to the low ability of D. discoideum to adhere to surfaces, which results in cell loss even after fixation. To address this problem, we improved the adhesion of cells and developed a straightforward and convenient workflow for 3D-correlative light and electron microscopy. This approach includes high-pressure freezing, which is an excellent technique for preserving membranes. Thus, our method allows to monitor the ultrastructural aspects of vacuole escape which is of central importance for the survival and dissemination of bacterial pathogens.

cell biology↗

bioRxiv2023

ER-dependent membrane repair of mycobacteria-induced vacuole damage

Several intracellular pathogens, such as Mycobacterium tuberculosis, damage endomembranes to access the cytosol and subvert innate immune responses. The host counteracts endomembrane damage by recruiting repair machineries that retain the pathogen inside the vacuole. Here, we show that the endoplasmic reticulum (ER)-Golgi protein oxysterol binding protein (OSBP) and its Dictyostelium discoideum homologue OSBP8 are recruited to the Mycobacterium-containing vacuole (MCV) after ESX-1-dependent membrane damage. Lack of OSBP8 causes a hyperaccumulation of phosphatidylinositol-4-phosphate (PI4P) on the MCV and decreased cell viability. OSBP8-depleted cells had reduced lysosomal and degradative capabilities of their vacuoles that favoured mycobacterial growth. In agreement with a function of OSBP8 in membrane repair, human macrophages infected with M. tuberculosis recruited OSBP in an ESX-1 dependent manner. These findings identified an ER-dependent repair mechanism for restoring MCVs in which OSBP8 functions to equilibrate PI4P levels on damaged membranes. ImportanceTuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the ER-Golgi lipid transfer protein OSBP8 in the D. discoideum/ M. marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defence mechanism against intracellular pathogens such as M. tuberculosis.

cell biology↗

bioRxiv2022

Early endosomes act as local exocytosis hubs to repair endothelial membrane damage

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of membrane required for wound repair are poorly understood. Here, we show that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, we demonstrate that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal SNARE VAMP2, which is crucial for efficient membrane repair. Thus, the here identified Ca2+-evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

cell biology↗

bioRxiv2022

From vacuole to cytosol: Disruptive invasion triggers cytosolic release of Salmonella Paratyphi A and subsequent cytosolic motility favors evasion of xenophagy

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Typhoidal S. enterica serovars like Paratyphi A (SPA) are human restricted and cause a severe systemic disease, while many S. enterica serovars like Typhimurium (STM) have broad host range, and in human hosts usually lead to self-limiting gastroenteritis. There are key differences between typhoidal and non-typhoidal Salmonella in pathogenesis, but underlying mechanisms remain largely unknown. Several genes encoding Salmonella pathogenicity island (SPI) effector proteins are absent or pseudogenes in SPA. Expression of virulence and metabolism genes show differential expression compared to STM. The intracellular transcriptomic architecture and phenotypes during presence in epithelial cells were recently described. Surprisingly, induction of motility, flagella and chemotaxis genes showed distinct expression patterns in intracellular SPA vs. STM and led to cytosolic motility of SPA. This study applies single cell microscopic analyses approaches to investigate the triggers and cellular consequences of cytosolic motility. Live cell imaging (LCI) revealed that SPA invades host cells in a highly cooperative manner. Extensive membrane ruffling at the invasion site leads to increased membrane damage in the nascent SCV with subsequent cytosolic release. After release into the cytosol, motile bacteria showed same velocity as under culture conditions used for infection. Reduced capture of SPA by autophagosomal membranes was observed by LCI and electron microscopy. Our results reveal flagella-mediated cytosolic motility as possible xenophagy evasion mechanism that could drive disease progression and contributes to dissemination of invasion-primed SPA during systemic infection. ImportanceIntracellular pathogens are commonly adapted to life in host cells either in a pathogen-containing vacuole, or free in host cell cytosol. However, transitions between these lifestyles are possible and demand specific adaptations, especially to avoid recognition and killing by host cell-autonomous immune defense. Salmonella enterica serovar Paratyphi A (SPA) belongs to typhoidal Salmonella able to cause live-threatening systemic infections in human hosts. We observed that SPA invades host cells in a way that often results in damage of the nascent vacuole and release of SPA in host cell cytosol. Here, SPA deploy flagella-mediated motility for rapid locomotion within infected cells. We demonstrate on single cell level that flagella-mediated motility enables evasion of xenophagic capture and control by the host cells. SPA uses a novel form of intracellular motility to successfully colonize human host cells.

microbiology↗

bioRxiv2022

Single molecule analyses of Salmonella translocated effector proteins reveal targeting to and dynamics in host cell endomembranes

Bacterial pathogens deliver proteins in temporal and spatial coordinated manner to manipulate mammalian host cells. The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. The pathogen resides in a membrane-bound compartment termed Salmonella-containing vacuole (SCV). By Salmonella- induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector molecules translocated by the SPI2-encoded type III secretion system (SPI2-T3SS) into host cells. A subset of these effectors is associated with, or integral in SCV and SIF membranes. It remained to be determined how SPI2-T3SS effectors reach their subcellular destination, and how these effectors interact with endomembranes remodeled by Salmonella. We deployed self-labeling enzyme (SLE) tags as novel approach to label translocated effector proteins in living host cells, and analyzed their dynamics on single molecule level. We found that SPI2-T3SS effector proteins diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differed between various effector proteins investigated and was dependent on membrane architecture of SIF. In the early infection, we observed host endosomal vesicles associated with Salmonella effector proteins. Effector-positive vesicles continuously fused with SCV and SIF membranes, providing a route of effector delivery by SPI2-T3SS translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This novel mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.

microbiology↗

bioRxiv2021

Single cell analyses reveal distinct adaptation of typhoidal and non-typhoidal Salmonella enterica serovars to intracellular lifestyle

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Human-restricted typhoidal S. enterica serovars Typhi (STY) or Paratyphi A (SPA) cause severe typhoid or paratyphoid fever, while S. enterica serovar Typhimurium (STM) has a broad host range and in human hosts usually lead to a self-limiting gastroenteritis. Due to restriction of STY and SPA to primate hosts, experimental systems for studying the pathogenesis of typhoid and paratyphoid fever are limited. Therefore, STM infection of susceptible mice is commonly considered as model system for studying these diseases. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2-T3SS) is a key factor for intracellular survival of Salmonella. Inside host cells, the pathogen resides within the Salmonella-containing vacuole (SCV) and induces tubular structures extending from the SCV, termed Salmonella-induced filaments (SIF). This study applies a set of single cell analyses approaches such as dual fluorescent protein reports, effector translocation, or correlative light and electron microscopy to investigate the fate and activities of intracellular STY and SPA. The SPI2-T3SS of STY and SPA is functional in translocation of effector proteins, SCV and SIF formation. However, only a low proportion of intracellular STY and SPA are actively deploying SPI2-T3SS and STY and SPA exhibited a rapid decline of protein biosynthesis upon experimental induction. A role of SPI2-T3SS for proliferation of STY and SPA in epithelial cells was observed, but not for survival or proliferation in phagocytic host cells. Our results indicate that reduced intracellular activities are factors of the stealth strategy of STY and SPA and facilitate systemic spread and persistence of the typhoidal Salmonella.

microbiology↗