Descripción de un nuevo tipo de vesícula de membrana externa en bacterias gramnegativasimplicaciones en el contenido de dna y rna

Resumen

A structural study performed by transmission electron microscopy (TEM) after high-pressure freezing followed by freeze substitution (HPF-FS) of bacterial samples and Cryo-TEM, have revealed that Gram-negative bacteria naturally releases a more complex type of vesicle, termed Outer–Inner Membrane Vesicle (O-IMVs). Its formation is characterized by the protrusion of both outer and inner membranes, dragging along cytoplasmic component into vesicles. The presence of DNA inside this new type of vesicle was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. The production of these O-IMVs explains the presence of components from cytoplasm in membrane vesicles (MVs) and opens up new areas of study their functionality. Recently, small RNA (sRNAs) have emerged as crucial regulators in bacterial physiology. Several studies have reported the presence of sRNAs inside vesicles, but their role is still unknown. To characterize the RNA content in MVs from Pseudomonas aeruginosa PAOA1 a transcriptomic analysis from RNA obtained from vesicles and cells was conducted. The study reveals that MVs are enriched in RNAs ranging from 40 to 80 nt, which corresponds mainly to tRNAs. Moreover, the RNA-seq from vesicles obtained at different points of PAO1 growth curve was performed, showing that that vesicles containing RNA are produced before starting the transition from exponential to stationary growth phase. Our findings suggest that sRNAs are selectively packaged into MVs and actively secreted. Finally, a structural study was performed to characterize a tolR mutant derived from the probiotic strain Escherichia coli Nissle to confirm the “hypervesiculating” phenotype. TEM observation after HPF-FS of bacterial samples, together with cryo-TEM observation of plunge-frozen hydrated isolated MVs showed considerable structural heterogeneity in the EcN tolR samples. Moreover, experiments of MV uptake in Caco-2 cells using rhodamine evidenced that EcN tolR MVs displayed reduced internalization levels compared to the EcN wild-type MVs. These findings indicate that heterogenicity of MVs from EcN tolR mutants may have a major impact on vesicle functionality.