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.

Fig 1. Core proteome of OMVs isolated from control P. aeruginosa.

We identified a total of 757 proteins in OMVs secreted by planktonic P. aeruginosa (PAO1). Of these, 466 proteins (62%) were detected in OMVs secreted by planktonic P. aeruginosa in at least one other published study, and 291 proteins were unique to the present study. The core proteome, defined as proteins detected in OMVs secreted by planktonic P. aeruginosa in this and all four previous studies [–50], was composed of 66 proteins.

Fig 2. Mean log2 iBAQ of unique and core proteins.

PAO1 OMV core proteins are significantly more abundant than unique proteins. ****P<0.0001 versus unique.

Fig 3. Conserved OMV proteins.

120 proteins were detected in OMVs from P. aeruginosa strain PAO1 as well as PA14 and two CF clinical isolates [25].

Fig 4. Differential abundance analysis of integrated peak intensities for 761 proteins associated with Tobi OMVs and Ctl OMVs.

Volcano plot of log2 fold changes and FDR-corrected p-values obtained with QPROT. 182 proteins with FDR < 0.05 were considered significantly differentially abundant (red circles). 165 proteins were down-regulated in Tobi OMVs while 17 proteins were up-regulated. Horizontal red dotted line indicates FDR = 0.05. Low abundance proteins, defined as proteins detected in fewer than 2 replicate samples, as well as proteins exclusively detected in one of the groups, were not included in the analysis.

Fig 5. Phe508del-CFTR Cl^- secretion.

(A) CFBE cells were treated with VX-809 (3 μM for 48 h) and exposed to vehicle, Ctl OMVs or OMVs isolated from Tobramycin treated P. aeruginosa (Tobi OMV) for 1.5 h prior to measurements of Phe508del CFTR Cl^- secretion (presented as μA/cm²). Ctl OMVs reduced VX-809 Phe508del CFTR Cl^- secretion compared to VX-809 alone (P<0.001). Phe508del CFTR Cl^- secretion in the VX-809+Tobi OMV treated cells was significantly higher than in the VX-809+Ctl OMV treated cells (P<0.05), while OMVs isolated from P. aeruginosa treated with Tobramycin did not significantly reduce VX-809 Phe508del CFTR Cl^- currents compared to VX-809 alone. (B) CF-HBE cells were treated with vehicle (Control), VX-809 alone (3 μM for 48 h), or VX-809 and PA14 wild type (WT) or VX-809 and PA14-ΔaprA for 6 h prior to measurements of Phe508del CFTR Cl^- secretion. VX-809 increase Phe508del CFTR Cl^- secretion (P<0.01). PA14 eliminated the VX-809 stimulated Phe508del CFTR Cl^- secretion (VX-809 versus VX-809+ PA14, P<0.05). The PA14 ΔaprA deletion mutant did not reduce Phe508del CFTR Cl^- secretion as much as PA14 (P<0.05). *** P<0.001, **P<0.01, *P<0.05.