Pseudomonas Quinolone Signal Induces Oxidative Stress and Inhibits Heme Oxygenase-1 Expression in Lung Epithelial Cells

Abstract

causes lung infections in patients with cystic fibrosis (CF). The quinolone signal (PQS) compound is a secreted virulence factor that contributes to the pathogenicity of We were able to detect PQS in sputum samples from CF patients infected with but not in samples from uninfected patients. We then tested the hypothesis that PQS induces oxidative stress in host cells by determining the ability of PQS to induce the production of reactive oxygen species (ROS) in lung epithelial cells (A549 and primary normal human bronchial epithelial [NHBE]) cells and macrophages (J774A.1 and THP-1). ROS production induced by PQS was detected with fluorescent probes (dichlorodihydrofluorescein diacetate, dihydroethidium, and MitoSOX Red) in conjunction with confocal microscopy and flow cytometry. PQS induced ROS production in lung epithelial (A549 and NHBE) cells and macrophages (J774A.1 and THP-1 cells). NHBE cells were sensitive to PQS concentrations as low as 500 ng/ml. PQS significantly induced early apoptosis ( < 0.05, = 6) in lung epithelial cells, as measured by annexin/propidium iodide detection by flow cytometry. However, no change in apoptosis upon PQS treatment was seen in J774A.1 cells. Heme oxygenase-1 (HO-1) protein is an antioxidant enzyme usually induced by oxidative stress. Interestingly, incubation with PQS significantly reduced HO-1 and NrF2 expression in A549 and NHBE cells but increased HO-1 expression in J774A.1 cells ( < 0.05, = 3), as determined by immunoblotting and densitometry. These PQS effects on host cells could play an important role in the pathogenicity of infections.

Pseudomonas Quinolone Signal molecule PQS behaves like a B Class inhibitor at the I Q site of mitochondrial complex I

“…In contrast, isolated mitochondria from A549 cells also showed a ROS increase due to Rotenone (but not PQS) treatment, confirming the frequent observation that the cellular ROS defense mechanism may be lost during mitochondrial isolation . As described earlier, the effect of PQS on cells is concentration dependent, very high PQS values (100 µmol/L) led to cell death as indicated by the penetration of DAPI into cells, and significantly increased cellular and nuclear ROS values. In summary, we conclude that PQS at concentrations as in the sputum range of CF patients is sufficient to inhibit complex I at the I Q site without stimulating ROS formation.…”

“…In these cells, the MitoSOX signal was also more diffuse with a strong signal in the nucleus indicating lysis of mitochondria and onset of apoptosis. In the experiments shown here, only intact cells were analyzed for superoxide determination, which might be different from the analysis performed in the Abdalla et al study …”

“…A549 cells were chosen as a cell model, as PQS is secreted into the lungs of patients. 18,29 The response of individual complexes to PQS was further analyzed via in vitro experiments using mitochondrial-enriched fractions from bovine liver and from A549 cells.…”

“…When we used a higher PQS concentration (100 µmol/L), the fluorescence of MitoSOX increased significantly after 2 hours, indicating oxidative stress, but only in HeLa cells. In the study by Abdalla et al (2017), increased cellular and mitochondrial ROS levels upon PQS treatment (40 µg/L = 154 µmol/L, 4 hours) were reported using HEt and MitoSOX. High levels of PQS (100 µmol/L), however, had also cytotoxic effects in A549 cells.…”

“…26 With reverse electron flow, the effects of the various inhibitors on ROS production are inverted. 27,28 A recent study reported that PQS increases ROS levels in lung epithelial cells, 29 but the specific target of PQS in epithelial cells was not identified. Here, we tested the hypothesis that PQS, like HQNO, affects mitochondrial respiration or the activity of respiratory complexes.…”