Biology subjects

Greune, L.

Publications and source records attributed to Greune, L..

6 recordsLinked to original sources

bioRxiv2026

Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

Bacterial exit from host cells is essential for dissemination yet remains poorly understood. Here, we define a non-lytic egress pathway exploited by the enteric pathogen Yersinia pseudotuberculosis that co-opts the host exocytosis machinery in intestinal epithelial cells. The bacteria secrete a CNF-family toxin that activates the Cdc42-PLC {gamma}1-IP3-IP3R signaling cascade, triggering SNARE-dependent fusion of the Yersinia-containing vacuoles with the cell membrane via VAMP7, Stx4, and SNAP23. This controlled exocytotic release preserves epithelial barrier integrity and occurs infrequently, representing a key rate-limiting step in systemic spread. These findings establish the host exocytotic machinery as an active determinant of bacterial egress, uncover a conserved vesicle trafficking pathway hijacked by intracellular pathogens for dissemination, and assign a new role for bacterial toxins in regulating host cell exit. Authors SummaryMany bacterial pathogens penetrate and cross protective host cell barriers to spread within the body. While the mechanisms by which enteric bacteria enter host cells are well characterized, far less is known about how they exit host cells after invasion. In this study, we investigated how the enteric pathogen Yersinia pseudotuberculosis exits from human gut epithelial cells after crossing them. We found that they egress at the basolateral side of the intestinal cells through a controlled, non-destructive process that preserves the integrity of the epithelial barrier. To achieve this, the bacteria co-opt host signaling pathways involved in host cell vesicle release (exocytosis) to promote controlled egress. These exit events are rare, suggesting that bacterial escape from cells is a major bottleneck during infection. Importantly, we show that this process is enhanced by a secreted toxin of the Cytotoxic Necrotizing Factor (CNF) family (CNFY), which promotes the fusion of the bacteria-containing vacuole with the basolateral cell membrane. This finding uncovers a previously unrecognized role of bacterial toxins in facilitating bacterial cell egress.

microbiology↗

bioRxiv2024

Immune priming in the insect gut: a dynamic response revealed by ultrastructural and transcriptomic changes

Research on forms of memory in innate immune systems has recently gained momentum with the study of trained immunity in vertebrates and immune priming in invertebrates. Immune priming provides protection against previously encountered pathogens. However, causes and mechanisms of immune priming are still not well understood in most organisms. In this work, we combine RNA sequencing with transmission electron microscopy to investigate the dynamic processes during priming in the gut of a well-established model for oral immune priming, consisting of the host Tribolium castaneum and its entomopathogen Bacillus thuringiensis tenebrionis (Btt). We show that priming with specific, pathogen-derived virulence-relevant factors causes damage in the gut of T. castaneum larvae, which leads to an early physiological stress response as well as the upregulation of a specific set of immune genes. This response diminishes over time yet enables the gut to upregulate genes known to interfere with Btt virulence when T. castaneum larvae are later exposed to infectious Btt spores. The identification of these processes increases our understanding of immune priming as a dynamic process where cellular responses in concert with specific gene regulation prepare the gut tissue and thereby enable more efficient protection against infection. Such work can further help us understand the origin and mechanism of innate immune memory. Author summaryInvertebrate animals do not possess a classical adaptive immune system. And yet, many of these animals show forms of immune memory, collectively called immune priming. In this work, we investigate the causes of oral immune priming in the red flour beetle Tribolium castaneum in response to its bacterial entomopathogen Bacillus thuringiensis tenebrionis. Primary exposure to Btt spent culture media supernatants enables the larvae of T. castaneum to better withstand a subsequent infection with infectious Btt spores. We show that exposing T. castaneum larvae to those supernatants leads to damage in the gut resulting in a strong stress and immune response early after priming and a targeted up-regulation of beneficial genes upon the secondary re-encounter with Btt. These results suggest fundamental stress responses can be involved in innate immune memory phenomena, which has implications in translational research across species.

evolutionary 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↗

bioRxiv2021

Endocytic vacuole formation by WASH-mediated endocytosis

Endocytosis is a fundamental cellular process facilitated by diverse mechanisms. Remarkably, several distinct clathrin-independent endocytic processes were identified and characterized following virus uptake into cells. For some, however, mechanistic execution and biological function remain largely unclear. This includes an endocytic process exploited by human papillomavirus type 16 (HPV16). Using HPV16, we examined how vesicles are formed by combining systematic cellular perturbations with electron and video microscopy. Cargo uptake occurred by uncoated, inward-budding pits. Mechanistically, vesicle scission was facilitated by actin polymerization controlled through the actin nucleation promoting factor WASH. While WASH typically functions in conjunction with the retromer complex on endosomes during retrograde trafficking, endocytic vesicle formation was largely independent of retromer itself and the heterodimeric membrane-bending SNX-BAR retromer adaptor, thereby uncovering a role of WASH in endocytosis in addition to its canonical role in intracellular membrane trafficking.

cell biology↗

bioRxiv2021

A novel adhesive complex at the base of intestinal microcilli

Intestinal epithelial cells form dense arrays of microvilli at the apical membrane to enhance their functional capacity. Microvilli contain a protocadherin-based intermicrovillar adhesion complex localized at their tips which regulates microvillar length and packaging. Here, we identify a second adhesive complex in microvilli of intestinal epithelial cells. This complex is localized at the basal region of microvilli and consists of the adhesion molecule TMIGD1, the phosphoprotein EBP50 and the F-actin - plasma membrane cross-linking protein ezrin. Ternary complex formation requires unmasking of the EBP50 PDZ domains by ezrin binding and is strongly enhanced upon mutating Ser162 located in PDZ domain 2 of EBP50. Dephosphorylation of EBP50 at S162 is mediated by PP1, a serine/threonine phosphatase localized at the microvillar base and involved in ezrin phosphocycling. Importantly, the binding of EBP50 to TMIGD1 enhances the dynamic turnover of EBP50 at microvilli in a Ser162 phosphorylation-dependent manner. We identify an adhesive complex at the microvillar base and propose a potential mechanism that regulates microvillar dynamics in enterocytes.

cell biology↗

bioRxiv2018

Infectious Entry of Merkel Cell Polyomavirus

Merkel Cell Polyomavirus (MCPyV) is a small, non-enveloped tumor virus associated with an aggressive form of skin cancer, the Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses as it requires sulfated polysaccharides such as heparan sulfates and/or chondroitin sulfates for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co-receptor). To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis or glycosphingolipid-enriched carriers. The viruses internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry.\n\nImportanceMCPyV is the first polyomavirus directly implicated in the development of an aggressive human cancer, the Merkel Cell Carcinoma (MCC). Although MCPyV is constantly shed from healthy skin, MCC incidence increases among aging and immunocompromised individuals. To date, the events connecting initial MCPyV infection and subsequent transformation still remain elusive. MCPyV differs from other known polyomaviruses concerning its cell tropism, entry receptor requirements, and infection kinetics. In this study, we examined the cellular requirements for endocytic entry as well as the subcellular localization of incoming virus particles. A thorough understanding of the determinants of the infectious entry pathway and the specific biological niche will benefit prevention of virus-derived cancers such as MCC.

microbiology↗