Biology subjects

Werkmeister, E.

Publications and source records attributed to Werkmeister, E..

5 recordsLinked to original sources

bioRxiv2026

Galectin-3 recruitment at the Mycobacterium tuberculosis-containing phagosome is critical in macrophage but dispensable in epithelial cells

Mycobacterium tuberculosis (Mtb) virulence relies in part on its ability to induce phagosomal membrane rupture, enabling bacterial access to the host cell cytosol. This process is largely mediated by the ESX-1 secretion system, which is present in Mtb but absent from the vaccine strain BCG. Galectin-3 (Gal3), a {beta}-galactoside-binding lectin, is recruited to damaged endomembranes and functions as a cytosolic sensor of membrane disruption. However, the kinetics and quantitative features of Gal3 recruitment to Mtb-containing vacuoles have remained poorly characterized. Here, we performed a longitudinal quantitative imaging study of Gal3 recruitment in human macrophages over a five-day infection period. Gal3 was recruited to mycobacteria-containing vacuoles shortly after infection with both live and heat-killed Mtb. Differences between the two conditions emerged from day 1 post-infection and persisted until macrophage death. A similar kinetic profile was observed with recombinant BCG::ESX-1, whereas parental BCG failed to induce Gal3 recruitment, confirming the requirement for ESX-1-dependent membrane damage. Cytoplasmic Gal3 levels were higher in bystander macrophages than in infected cells and were comparable to non-infected controls, suggesting that diffuse cytoplasmic Gal3 is associated with cells lacking intracellular mycobacteria. Functional studies revealed that Gal3 silencing enhanced long-term intracellular Mtb replication, demonstrating a role for Gal3 in restricting bacterial growth. Importantly, Gal3 recruitment was not observed in alveolar epithelial cells. This cell-type specificity was confirmed in a microfluidic alveolus-on-chip model. Together, these findings identify sustained Gal3 recruitment to the mycobacteria-containing vacuole as a robust quantitative marker of ESX-1-dependent phagosomal rupture and Mtb virulence.

microbiology↗

bioRxiv2026

Development of a cost-effective Alveolus-on-Chip for studying Mycobacterium tuberculosis infection.

We developed a cost-effective human alveolus-on-chip based on 3D printing molds (3DP-Lung) to study early events of Mycobacterium tuberculosis (Mtb) infection in a physiologically human relevant microenvironment. This organ-on-chip platform is compatible with advanced imaging and recreates the alveolar-capillary interface by co-culturing primary human alveolar epithelial cells, endothelial cells and macrophages. We show that epithelial-only models display limited susceptibility to Mtb infection, whereas the integration of macrophages significantly enhances infection levels of the alveolar barrier and supports intracellular bacterial replication. Quantitative imaging reveals that macrophages act as a permissive niche, promoting Mtb infection of both epithelial and endothelial compartments. This accessible organ-on-chip platform enables robust modeling of early events of host-respiratory pathogen interactions and provides a valuable tool for studying tuberculosis pathogenesis in human-relevant conditions. More broadly, it lowers technical and economic barriers to accelerate the adoption of organ-on-chip technologies for studying human specific infection. SummaryA cost-effective human alveolus-on-chip enables physiologically relevant modeling of early host pathogen interaction and revealing a key role of macrophages in Mycobacterium tuberculosis infection

microbiology↗

bioRxiv2025

Oncogenic mutations convert MET from a pro-apoptotic tumor suppressor to an oncogenic driver

Dependence receptors can exert both oncogenic and tumor-suppressive activities. In cancers, downregulation of dependence receptors or overexpression of their ligands are well-established mechanisms that drive tumor progression. However, direct genetic alterations abolishing the pro-apoptotic function of dependence receptors have not been documented so far. MET, a receptor tyrosine kinase classically viewed as an oncogene, has also been proposed to act as a dependence receptor through its caspase-mediated cleavage, but whether this property impacts tumorigenesis remained unknown. In [~]3% of lung adenocarcinomas, MET mutations leading to exon 14 skipping (METex14Del) remove both the caspase site and the adjacent CBL-binding motif, thereby preventing generation of the pro-apoptotic p40MET fragment. METex14Del promotes sustained signaling, enhanced invasion, apoptosis resistance, and tumor growth in HGF-humanized mice. Genome editing revealed that combined --but not individual-- mutations of the caspase and CBL sites phenocopy METex14Del. Moreover, inducible re-expression of p40MET in METex14Del-expressing cells restored apoptosis and suppressed tumor formation. Altogether, our findings identify MET exon 14 skipping as the first oncogenic mutation that drives tumorigenesis by abolishing the tumor-suppressive pro-apoptotic function of a dependence receptor, thereby redefining the oncogenic potential of MET.

cancer biology↗

bioRxiv2021

The Endocytic Recycling Compartment Serves as a Viral Factory for Hepatitis E Virus

Background & AimsAlthough Hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet and it is currently unknown whether HEV infection leads to cellular membrane modelling as many positive-strand RNA viruses. HEV genome encodes three proteins, the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we aimed to probe infectious particles and viral factories in HEV-producing cells, using antibodies directed against the different ORF2 isoforms. MethodsWe generated monoclonal antibodies that specifically recognize the particle-associated ORF2i form, and antibodies that recognize the different ORF2 isoforms. We used them in confocal and electron microscopy approaches to probe viral factories in HEV-producing cells. We performed an extensive colocalization study of viral proteins with subcellular markers. We analyzed the impact of silencing Rab11, a central player of the endocytic recycling compartment (ERC). ResultsOne of the antibodies, named P1H1 and targeting the N-terminus of ORF2i, recognized delipidated HEV particles. Confocal and ultrastructural microscopy analyses of HEV-producing cells revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. These subcellular structures were enriched in ORF2 and ORF3 proteins, and were dependent on the ORF3 expression and ORF2i capsid protein assembly. Colocalization and silencing analyses revealed that these structures are derived from the ERC. ConclusionsOur study reveals that HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection. Lay summaryHepatitis E virus (HEV) is the leading cause of acute hepatitis worldwide but many steps of its lifecycle are still elusive. Thanks to the development of new antibodies that recognize the different forms of the HEV capsid protein, we were able to visualize vesicular and tubular structures that were established by the virus in the host cell. In addition, extensive efforts to identify these structures led us to conclude that HEV hijacks the endocytic recycling compartment of the cell to form this network of vesicles and tubules, which might be the hallmark of HEV infection.

microbiology↗

bioRxiv2019

Rab11A regulates the constitutive secretory pathway during Toxoplasma gondii invasion of host cells and parasite replication.

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of DG and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile parasites suggesting that a Rab11A-dependent apically polarized transport of cargo regulates parasite motility.

microbiology↗