@article{Kretschmer2023,
Title = {Isolation and Quantification of Bacterial Membrane Vesicles for Quantitative Metabolic Studies Using Mammalian Cell Cultures},
Author = {Kretschmer, Marcel and Müller, Julia and Henke, Petra and Otto, Viktoria and Rodriguez, Alejandro Arce and Müsken, Mathias and Jahn, Dieter and Borrero-de Acuña, José Manuel and Neumann-Schaal, Meina and Wegner, Andre},
Editor = {},
Journal = {Cells},
Year = {2023},
Pages = {2674},
Volume = {12},
Doi = {10.3390/cells12232674},
Abstract = {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 unreliable protein concentration-based methods. We (1) show a linear correlation between the number 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 monophosphatase 1 (SuhB) as a pivotal regulator in the vesicle-mediated pathogenesis of P. aeruginosa.},
}
TY - JOUR
AU - Kretschmer, Marcel
AU - Müller, Julia
AU - Henke, Petra
AU - Otto, Viktoria
AU - Rodriguez, Alejandro Arce
AU - Müsken, Mathias
AU - Jahn, Dieter
AU - Borrero-de Acuña, José Manuel
AU - Neumann-Schaal, Meina
AU - Wegner, Andre
TI - Isolation and Quantification of Bacterial Membrane Vesicles for Quantitative Metabolic Studies Using Mammalian Cell Cultures
T2 - Cells
PY - 2023
SP - 2674
VL - 12
DO - 10.3390/cells12232674
AB - 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 unreliable protein concentration-based methods. We (1) show a linear correlation between the number 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 monophosphatase 1 (SuhB) as a pivotal regulator in the vesicle-mediated pathogenesis of P. aeruginosa.
ER -
