Identification of Pseudomonas aeruginosa released proteins: effects of oxygen limitation and azithromycin treatment in clinical and laboratory strains

Colonization by Pseudomonas aeruginosa (Pa) is a hallmark of lung disease in cystic fibrosis (CF), where microaerobic conditions develop as a consequence of disease progression. Conditioned medium (CM) obtained from Pa clinical strain AA2, unlike the CM from laboratory strain PAO1, induces in airway epithelial cells IL-8 mRNA in both aerobic and microaerobic conditions. The effect was impaired by protease digestion. Shotgun proteomic analysis (Multidimensional protein identification technology: MudPIT) of conditioned medium of PAO1 and AA2 identified 451 and 235 individual proteins. Various proteins were found differentially regulated between strains and culture conditions. Among these eleven different proteases were found released by the AA2 strain, while fewer peptides of only four of them were detected in the PAO1 strain. Ecotin, a protease inhibitor, was found to be highly represented in PAO1 in comparison with AA2 grown in microaerobiosis. These results were confirmed by functional assay (zymography) and western blotting. The pattern of expression of several proteases and their inhibitor ecotin correlates with pro-inflammatory activity in vitro better than other candidate virulence factors. Only 31% of the Pa strains isolates from chronically infected CF patients expressed detectable metalloprotease activity while all the isolates derived from sporadically infected individuals scored positive (individual strains analyzed: 42, p<0.002). These results suggest that high-throughput approaches are critical to unravel the complexity of the pro-inflammatory microenvironment associated to the presence of Pa and to facilitate the identification of key molecules involved in Pa biology/pathology. There is considerable interest in the use of azithromycin (AZM) for the treatment of lung disease in patients with cystic fibrosis. Although its mechanism of action as an inhibitor of bacterial protein synthesis has been well established, it is less clear how AZM ameliorates the lung disease associated with P. aeruginosa, which is considered to be resistant to the drug. Modulation of Pa virulence factors was suggested as mechanism for AZM beneficial effects in CF patients. We tested the effects of azithromycin on clinical isolate AA2 to establish how this drug might interfere with the production of bacterial virulence factors that are relevant to the pathogenesis of airway disease in CF patients. We demonstrated that the increase of IL-8 mRNA in CF epithelial cells induced by CM from AA2 was significantly reduced when the clinical strain was grown in the presence of AZM, suggesting that this macrolide reduces Pa pathogenicity. In the attempt to gain information on the identity of the molecules released by Pa clinical strain before and after treatment with AZM we applied MudPIT. We found 5 upregulated and 7 downregulated proteins in CM from AA2 incubated with AZM. Peptides from the alkaline metalloproteinase precursor (APR) were less represented in CM derived from AA2 strain grown in presence of AZM than in those from the same strain cultured in absence of this macrolide. AZM was observed also to decrease the metalloprotease activity and APR expression in CM of Pa isolates derived from sporadically infected individuals while any effect was detected in CM of Pa isolates from chronically infected CF patients. These results was validated by means of zymography assay and western blot technique. The MudPIT analysis of released proteins from Pa clinical isolate grown alone and in presence of AZM gives suggestion on the macrolide ability to decrease the expression of substances that contributes to Pa virulence, such as alkaline metalloproteinase. The effects of AZM on the expression and release of selected polypeptides by Pa strains may help to explain the clinical benefits associated with macrolide therapy.