Table 1.
Characteristics of K. pneumoniae strains [23].
Table 2.
Primers used for target and housekeeping genes.
Fig 1.
Analysis of early EMT-like phenotypic changes activation in the EMT processes in A549 cells infected with K. pneumoniae strains.
(A) The morphologycal analysis by differential interference contrast microscopy of the K. pneumoniae infected cells shows an elongated morphology with reduction of intercellular contacts compatible with EMT-like phenotype, similar to that displays by monolayers exposed to TGF-β1. Conversely, uninfected A549 cells retain a typical epithelial morphology, with clear cell-cell adhesions. (B) The real time RT-PCR analysis performed in A549 Kp-infected cells at MOI 1:25 showed a downregulation of the genes encoding for epithelial adhesion molecules (ITGB4 and E-cad) and an upregulation of genes encoding for mesenchymal markers vimentin and MMP9. A459 TGF-β1-treated cells were used as positive control of EMT-induction. The mRNA levels were normalized respect to uninfected cells and results are expressed as mean ± SD. (Mann-Whitney test: *p<0.01 vs uninfected cells; **p<0.01 vs uninfected and Kp-infected cells, ^p<0.01 vs uninfected cells and not significant vs Kp-infected cells). (C) The immunofluorescence analysis reveals a reduction of CD326/EpCAM positive cells in K. pneumoniae infected cells as compared to uninfected cells with typical bordered staining (61% vs 94%: Mann-Whitney test: p<0.05 vs uninfected cells). Furthermore, the infected cells, in analogy of what observed in TGF-β1-treated cells, showed positivity rates of vimentin close to 100% respect to 31% of uninfected cells (Mann-Whitney test: p<0.05).
Fig 2.
Analysis of cytotoxic effects induced by K. pneumoniae infection in A549 cells.
(A) The cell viability assay by cytofluorimetric analysis of PI fluorescent signal shows that K. pneumoniae infection induces cellular damage mostly related to MOI (ANOVA post test for linear trend: *p<0.01; Dunnett’s test: **p>0.01 or ***p>0.001 vs uninfected cells). (B) The representative flow cytometry plots at MOI 25:1, at which we have observed the lowest cytotoxic effect, display an increased amount of cellular events in the right gate (necrotic cells) for the A549 infected by K.pneumoniae strains. Legend: PI: Propidium Iodide.
Fig 3.
Evaluation of the intracellular ROS generation and the expression of antioxidant genes due to K. pneumoniae infection of A549 cells at lower MOI.
(A) The assessment of intracellular ROS generation by cytofluorimetric analysis shows the modulation of intracellular ROS induced by K. pneumoniae infection. The ROS overproduction is related to MOI (ANOVA post test for linear trend: *p<0.05; Dunnett’s test: **p>0.05 or ***p>0.01 vs uninfected cells). The representative flow cytometry plots at MOI 25:1 show an enhancement of DCFH-DA signal in A549 Kp-infected (flat green) respect to uninfected cells (brilliant green). (B) The confocal quantitative analysis of intracellular DCFH-DA fluorescent signal confirms the overproduction of ROS species in A549 Kp-infected cells (Student T test: *p<0.0001). In the representative digital images, the differences in the levels of DCFH-DA signal were expresses in a pseudo-color scale of fluorescence intensity, in which white is the highest and brown-red is the lowest intensity value of gray scale levels. Legend: MFI: Mean Fluorescence Intensity; FIU: Fluorescence Intensity Units. (C) SOD-1, SOD-2 and catalase mRNA levels were evaluated by real time RT-PCR in Kp-infected cells at MOI 1:25 and normalized respect to uninfected A549 cells. Results are expressed as mean ± SD. (Mann-Whitney test: *p<0.01 or ^p = not significant vs A549 uninfected cells).
Fig 4.
Evaluation of HIF-1α expression due to K. pneumoniae infection of A549 cells at lower MOI.
(A) HIF-1α mRNA levels were evaluated by real-time RT-PCR in A549 cells infected with K. pneumoniae strains at MOI 1:25 and normalized respect to uninfected A549 cells. Results are expressed as mean ± SD. To observe a significant increase of HIF-1α mRNA expression in A549 infected monolayers (Mann-Withney test: *p<0.01 vs uninfected cells). (B) Quantitative immunofluorescence analysis shows a significant increase of the cytoplasmic HIF-1α signals in A549 exposed to the infection of the K. pneumoniae strains (Student T test: *p<0.001 vs uninfected cells). The fluorescence intensity for cytoplasmic area was performed by the analysis of at least 100 cells for each sample in 5 different fields randomly taken from three independent experiments. The results are expressed as mean ± CI 95%.
Fig 5.
Analysis of EMT transcription factors expression in cultured airway epithelial cells infected with K. pneumoniae strains.
Snail 1–2, Twist 1–2 and Zeb 1–2 mRNA levels were assessed by real-time RT-PCR in A549 Kp-infected cells at MOI 1:25 and normalized respect to uninfected A549 cells. A459 TGF-β1-treated cells were used as positive control of EMT-induction. Results are expressed as mean ± SD. All the K. pneumoniae strains were able to induce a significant increase of Twist 1–2, Snail 1–2 and Zeb 1–2 mRNA production (Mann-Whitney test: *p<0.01 vs uninfected). The amount of TFs in cells infected by Kp strains show levels of induction more similar to that obtained treating A549 cells with TGF-ß1 (Mann-Whintey test: **p<0.05 or ^p = not significant vs Kp-infected cells).
Fig 6.
Effects of ROS scavenging on the counteraction of EMT-like process due to K. pneumoniae infection of A549 cells.
The A549 cells were pretreated for 12h before the infection with the resveratrol 50 μM. (A) The pretreatment with antioxidant has been shown to significantly decrease the oxidative stress in cells infected by all strains of K. pneumoniae (Student T test: *p<0.0001 vs A549 Kp-infected cells without resveratrol). Legend: MFI: Mean Fluorescence Intensity; FIU: Fluorescence Intensity Units. (B) After resveratrol, has been observed a significant impairment of HIF-1α mRNA expression and of HIF-1α intracellular production in A549 infected monolayers (Student T test: *p<0.05 or **p<0.01 vs A549 Kp-infected cells without resveratrol). (C) The real time RT-PCR analyses show that the resveratrol was able also to counteract the up-modulation of TFs related to EMT-like process dependent to Kp infection. (Mann-Withney test: *p<0.05 or **p<0.01 vs A549 Kp-infected cells without resveratrol).