Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown that P. aeruginosa secretes 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 by P. aeruginosa. Specifically, human EVs deliver miRNA let-7b-5p to P. 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 in P. aeruginosa but also the corresponding orthologs in Burkholderia 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 to P. 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 chronic P. aeruginosa lung 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-resistant P. aeruginosa infections.

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa

“…We previously described the transfer of miRNAs secreted in AEC EVs to P. aeruginosa (19). To assess the transfer of other sRNAs besides miRNAs to P. aeruginosa , we re-analyzed RNA-seq data of EV-exposed P. aeruginosa and unexposed controls (GSE174710).…”

“…1.5*10 7 CFUs of PA14 were exposed to 1.5*10 10 EV isolated from AEC from three donors or vehicle controls for a PA14:EV ratio of 1:1000 and incubated for 15 h at 37°C at 225 rpm. Samples were processed and TMT labeled as described previously (19). All six samples were run in duplicate, peak intensities from duplicate runs were averaged and filtered to retain 1619 proteins that were detected in all replicate samples.…”

“…Two recent studies provide a comprehensive characterization of the sRNA content of EVs secreted by AEC -one reporting changes in the sRNA content of EVs secreted by the human airway epithelial cell line A549 in response to viral infection (40), and another describing cigarette smoke induced changes in the RNA content of EVs secreted by primary small airway epithelial cells (41). Prior studies of EVs secreted by airway epithelial cells (AEC) have focused mainly on their miRNA cargo (19,(42)(43)(44), whereas the function of other sRNA species contained in AEC EVs has not yet been reported. While there is a growing body of literature on the regulatory effects of EV sRNAs within a given organism, little is known about the ability of sRNAs secreted in eukaryotic EVs to regulate gene expression in prokaryotes.…”

Extracellular vesicles: Their functions in plant–pathogen interactions

“…In addition, mammalian cells can deliver sRNA to bacterial cells via EVs. These sRNAs are able to decrease the formation of antibioticresistant biofilms by suppressing the expression of several genes essential for biofilm formation (Koeppen et al, 2021). The secretion of EVs in P. syringae-infected Arabidopsis was approximately double that in uninfected controls (Rutter & Innes, 2017), indicative of the functional involvement of plant EVs in resistance to bacterial pathogens.…”

Commensal and Pathogenic Bacterial-Derived Extracellular Vesicles in Host-Bacterial and Interbacterial Dialogues: Two Sides of the Same Coin

“…A complex interaction has been established between commensal or pathogenic bacterial communities and the released bacterial EVs, as an important contributor to different bacterial interactions or competition strategies [ 86 ]. The specific bacterial EV cargo components may be also responsible for a particular EV function in the bacterial community, such as antibiotic resistance, biofilm formation, quorum sensing, and virulence factors [ 17 , 87 , 88 ]. These functions of EVs have been reported in both commensal microbiota and pathogenic communities to meet specific goals and increase their survival [ 20 ].…”