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Pseudomonas aeruginosais an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown thatP. aeruginosasecretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA–containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation byP. aeruginosa. Specifically, human EVs deliver miRNA let-7b-5p toP. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC inP. aeruginosabut also the corresponding orthologs inBurkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited toP. aeruginosa. Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7–family miRNAs are in clinical trials to reduce inflammation and because chronicP. aeruginosalung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistantP. aeruginosainfections.
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Structure‐preserving fixation allows scanning electron microscopy to reveal biofilm microstructure and interactions with immune cells
Abstract Pseudomonas aeruginosais a pathogen that forms robust biofilms which are commonly associated with chronic infections and cannot be successfully cleared by the immune system. Neutrophils, the most common white blood cells, target infections with pathogen‐killing mechanisms that are rendered largely ineffective by the protective physicochemical structure of a biofilm. Visualisation of the complex interactions between immune cells and biofilms will advance understanding of how biofilms evade the immune system and could aid in developing treatment methods that promote immune clearance with minimal harm to the host. Scanning electron microscopy (SEM) distinguishes itself as a powerful, high‐resolution tool for obtaining strikingly clear and detailed topographical images. However, taking full advantage of SEM's potential for high‐resolution imaging requires that the fixation process simultaneously preserve both intricate biofilm architecture and the morphologies and structural signatures characterising neutrophils responses at an infection site. Standard aldehyde‐based fixation techniques result in significant loss of biofilm matrix material and morphologies of responding immune cells, thereby obscuring the details of immune interactions with the biofilm matrix. Here we show an improved fixation technique using the cationic dye alcian blue to preserve and visualise neutrophil interactions with the three‐dimensional architecture ofP. aeruginosabiofilms. We also demonstrate that this technique better preserves structures of biofilms grown from two other bacterial species,Klebsiella pneumoniaeandBurkholderia thailandensis.
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- Award ID(s):
- 2150878
- PAR ID:
- 10482519
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Journal of Microscopy
- Volume:
- 293
- Issue:
- 1
- ISSN:
- 0022-2720
- Format(s):
- Medium: X Size: p. 59-68
- Size(s):
- p. 59-68
- Sponsoring Org:
- National Science Foundation
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