Tobramycin reduces key virulence determinants in the proteome of Pseudomonas aeruginosa outer membrane vesicles

Abstract

Tobramycin is commonly used to treat Pseudomonas aeruginosa lung infections in patients with Cystic Fibrosis (CF). Tobramycin treatment leads to increased lung function and fewer clinical exacerbations in CF patients, and modestly reduces the density of P. aeruginosa in the lungs. P. aeruginosa resides primarily in the mucus overlying lung epithelial cells and secretes outer membrane vesicles (OMVs) that diffuse through the mucus and fuse with airway epithelial cells, thus delivering virulence factors into the cytoplasm that modify the innate immune response. The goal of this study was to test the hypothesis that Tobramycin reduces the abundance of virulence factors in OMVs secreted by P. aeruginosa. Characterization of the proteome of OMVs isolated from control or Tobramycin-exposed P. aeruginosa strain PAO1 revealed that Tobramycin reduced several OMV-associated virulence determinants, including AprA, an alkaline protease that enhances P. aeruginosa survival in the lung, and is predicted to contribute to the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion by primary human bronchial epithelial cells. Deletion of the gene encoding AprA reduced the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion. Moreover, as predicted by our proteomic analysis, OMVs isolated from Tobramycin treated P. aeruginosa had a diminished inhibitory effect on Phe508del-CFTR Cl- secretion compared to OMVs isolated from control P. aeruginosa. Taken together, our proteomic analysis of OMVs and biological validation suggest that Tobramycin may improve lung function in CF patients infected with P. aeruginosa by reducing several key virulence factors in OMVs that reduce CFTR Cl- secretion, which is essential for bacterial clearance from the lungs.

Outer Membrane Vesicles Derived from Klebsiella pneumoniae Influence the miRNA Expression Profile in Human Bronchial Epithelial BEAS-2B Cells

“…Thermolabile toxins and cytolysin have been identified in OMVs produced by Escherichia coli [ 24 , 25 , 26 ]. Haemolytic phospholipase C and alkaline phosphates have been detected in the OMVs of Pseudomonas aeruginosa [ 27 , 28 , 29 ]. Keenan et al have found vacuolating cytotoxin A in the OMVs produced by Helicobacter pylori [ 18 , 30 , 31 ].…”

The Contribution of Membrane Vesicles to Bacterial Pathogenicity in Cystic Fibrosis Infections and Healthcare Associated Pneumonia

“…On another note, P. aeruginosa infections treated with tobramycin led to a reduced secretion of MV-associated virulence factors, including AprA, which is an alkaline protease that reduces CFTR-mediated chloride secretion. AprA is essential for the survival of P. aeruginosa in the lungs as it inhibits the bacterial clearance (Koeppen et al, 2019).…”

The Role of Proteomics in Bacterial Response to Antibiotics

“…Finally, significantly lower expression levels were also reported for the virulence factors AlpA/D/E, after tobramycin treatment. All the findings together suggest that tobramycin could improve lung function in cystic fibrosis patients [ 42 ]. Adaptive resistance of P. aeruginosa to tobramycin under planktonic conditions has also been investigated through proteomics analysis [ 41 ].…”

“…Examples include investigating different protein levels in resistant [ 30 ] and drug-tolerant bacterial phenotypes [ 31 ] ( Table 2 ), as well as reports on the bacterial adaptation to different growing conditions and bacterial catabolism [ 32 ], biomarker discovery [ 33 ], and pathogen-host cell interactions [ 34 , 35 ]. In several other studies, different proteome analysis approaches have been employed to study the bacterial response to commonly used antibiotics such as ciprofloxacin [ 36 , 37 , 38 , 39 , 40 ], tobramycin [ 41 , 42 ], colistin [ 43 , 44 , 45 ], polymyxin B [ 46 ], daptomycin [ 47 , 48 ], and silver nanoparticles [ 49 ] ( Table 2 ). In addition, many biological processes are regulated by post-translational modifications (PTMs).…”