Biology subjects

Wegner, A.

Publications and source records attributed to Wegner, A..

7 recordsLinked to original sources

bioRxiv2024

Bacterial membrane vesicles of Pseudomonas aeruginosa activate AMPK signaling through inhibition of mitochondrial complex III

Bacterial membrane vesicles (BMVs) are secreted by many pathogenic bacteria and known to stimulate various host responses upon infection, thereby contributing to the pathogenicity of bacterial pathogens like Pseudomonas aeruginosa. While the effects of BMVs on host immune responses are well studied, little is known about their impact on cell metabolism and mitochondrial respiration. Here, we show that P. aeruginosa BMVs (1) reprogram cell metabolism of human lung cells, (2) negatively affect mitochondrial respiration by (3) specifically inhibiting complex III of the electron transport chain leading to (4) the activation of AMP-activated protein kinase (AMPK) signaling which in turn results in (5) AMPK-dependent inhibition of global protein synthesis.

cell biology↗

bioRxiv2024

Cell morphology as a quantifier for functional states of resident tissue macrophages

Resident tissue macrophages (RTMs) are essential for tissue homeostasis. Their diverse functions, from monitoring interstitial fluids to clearing cellular debris, are accompanied by characteristic morphological changes that reflect their functional status. While current knowledge of macrophage behaviour comes primarily from in vitro studies, their dynamic behavior in vivo is fundamentally different, necessitating a more physiologically relevant approach to their understanding. In this study, we employed intravital imaging to generate dynamic data from peritoneal RTMs in mice under various conditions and developed a comprehensive image processing pipeline to quantify RTM morphodynamics over time, defining human-interpretable cell size and shape features. These features allowed for the quantitative and qualitative differentiation of cell populations in various functional states, including pro- and anti-inflammatory activation and endosomal dysfunction. The study revealed that under steady-state conditions, RTMs exhibit a wide range of morphodynamical phenotypes, constituting a naive morphospace of behavioral motifs. Upon challenge, morphodynamic patterns changed uniformly at the population level but predominantly within the constraints of this naive morphospace. Notably, aged animals displayed a markedly shifted naive morphospace, indicating drastically different behavioral patterns compared to their young counterparts. The developed method also proved valuable in optimizing explanted tissue setups, bringing RTM behavior closer to the physiological native state. Our versatile approach thus provides novel insights into the dynamic behavior of bona fide macrophages in vivo, enabling the distinction between physiological and pathological cell states and the assessment of functional tissue age on a population level. Author summaryIn this study, we combine state-of-the-art in vivo imaging with advanced computational analysis to reveal the dynamic behavior of peritoneal resident tissue macrophages (RTMs) in their natural environment. These sentinel cells, which are crucial for tissue homeostasis, constantly monitor their environment and, in the process, undergo dynamic morphological changes that have remained largely uninvestigated due to technical limitations. Using two-photon microscopy, we captured time-lapse images of RTMs in the peritoneal serosa under various experimental conditions. Our customized image processing pipeline allowed a comprehensive assessment of cell morphology and dynamics and provided unprecedented insights into the behavior of RTMs in vivo, enabling us to distinguish cell populations in different physiological and pathological states. Our work opens up new avenues for the dynamic in situ phenotyping of macrophage functionality in disease contexts without their extraction from tissues and provides a novel perspective on the behavior of RTMs in their natural microenvironment. This versatile tool promises to advance our understanding of tissue homeostasis and macrophage function in health and disease, with potential applications in both basic research and clinical settings.

immunology↗

bioRxiv2023

Plant pathogenic fungi hijack phosphate starvation signaling with conserved enzymatic effectors

Phosphate availability modulates plant immune function and regulates interactions with beneficial, phosphate-providing, microbes. Here, we describe the hijacking of plant phosphate sensing by a family of Nudix hydrolase effectors from pathogenic Magnaporthe oryzae and Colletotrichum fungi. Structural and enzymatic analyses of the Nudix effector family demonstrate that they selectively hydrolyze inositol pyrophosphates, a molecule used by plants to monitor phosphate status and regulate starvation responses. In M. oryzae, gene deletion and complementation experiments reveal that the enzymatic activity of a Nudix effector significantly contributes to pathogen virulence. Further, we show that this conserved effector family induces phosphate starvation signaling in plants. Our study elucidates a molecular mechanism, utilized by multiple phytopathogenic fungi, that manipulates the highly conserved plant phosphate sensing pathway to exacerbate disease. One-Sentence SummaryA family of conserved enzyme effectors from pathogenic fungi manipulate plant phosphate sensing to promote infection.

plant biology↗

bioRxiv2023

Isolation and quantification of bacterial membrane vesicles for quantitative metabolic studies using mammalian cell cultures

Bacterial membrane vesicles (BMVs) are produced by most bacteria and participate in various cellular processes, such as intercellular communication, nutrient exchange, and pathogenesis. Notably, these vesicles can contain virulence factors, including toxic proteins, DNA, and RNA. Such factors can contribute to the harmful effects of bacterial pathogens on host cells and tissues. Although the general effects of BMVs on host cellular physiology are well known, the underlying molecular mechanisms are less understood. In this study, we introduce a vesicle quantification method, leveraging the membrane dye FM4-64. We utilize a linear regression model to analyze the fluorescence emitted by stained vesicle membranes to ensure consistent and reproducible vesicle-host interaction studies using cultured cells. This method is particularly valuable for identifying host cellular processes impacted by vesicles and their specific cargo. Moreover, it outcompetes clearly unreliable protein concentration-based methods. We (1) show a linear correlation between the quantity of vesicles and the fluorescence signal emitted from the FM4-64 dye, (2) introduce the "vesicle load" as a new semi-quantitative unit, facilitating more reproducible vesicle-cell culture interaction experiments (3) show that a stable vesicle load yields consistent host responses when studying vesicles from Pseudomonas aeruginosa mutants (4) demonstrate that typical vesicle isolation contaminants, such as flagella, do not significantly skew the metabolic response of lung epithelial cells to P. aeruginosa vesicles, and (5) identify inositol-1-monophosphatase (SuhB) as a pivotal regulator in the vesicle-mediated pathogenesis of P. aeruginosa.

cell biology↗

bioRxiv2023

PGM3 inhibition Shows cooperative Effects With Erastin inducing Pancreatic cancer cell death via activation of the Unfolded Protein Response

1Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor types and is notorious for its resistance to all types of treatment. PDAC resistance is frequently associated with a wide metabolic rewiring and in particular of the glycolytic branch named Hexosamine Biosynthetic Pathway (HBP). Here we show the effect of the combined treatment between an HBPs Phosphoglucomutase 3 (PGM3) enzyme inhibitor, named FR054, and erastin (ERA), a recognized ferroptosis inducer, on PDAC cell growth and survival. Noteworthy, the combined treatment applied to PDAC cell lines induces a significant decrease in cell proliferation and a concurrent enhancement of cell death. Furthermore, we show that this combined treatment induces Unfolded Protein Response (UPR), NFE2 Like BZIP Transcription Factor 2 (NRF2) activation, a change in cellular redox state, a greater sensitivity to oxidative stress, a major dependence on glutamine metabolism, and finally ferroptosis cell death. Our study discloses that HBP inhibition enhances, through UPR activation, the ERA effect and therefore might be a novel anticancer mechanism to be exploited as PDAC therapy.

cancer biology↗

bioRxiv2021

METAMDB: METABOLIC ATOM MAPPING DATABASE

AO_SCPLOWBSTRACTC_SCPLOWMetAMDB (https://metamdb.tu-bs.de/) is an open source metabolic atom mapping database, providing atom mappings for around 75000 metabolic reactions. Each atom mapping can be inspected and downloaded either as a RXN file or as a graphic in SVG format. In addition, MetAMDB offers the possibility of automatically creating atom mapping models based on user-specified metabolic networks. These models can be of any size (small to genome scale) and can subsequently be used in standard 13C metabolic flux analysis software.

systems biology↗

bioRxiv2020

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with transiently stable telomeres containing vectors, which allowed temporary selection and convenient screening of marker-free editing. We demonstrate that this approach provides vastly superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. The high performance of telomere vector-mediated coediting was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to greatly facilitate molecular research in B. cinerea and other fungi.

genetics↗