Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells

Abstract

BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

Fig. 1. : reactive oxygen species (ROS) generation by NADPH oxidase activity during infection of human primary endothelial cells (HUVECs) by Streptococcus agalactiae. Experiments were performed at different periods of incubation using the GBS90356 type III strain of S. agalactiae and HUVECs in the presence or absence of NADPH inhibitors (10 μΜ DPI, 10 μΜ apocynin) or a PI3K inhibitor (5 μΜ LY294002). Non-infected HUVEC monolayers with and without serum were used as negative controls. Non-infected HUVEC monolayers with serum and 10 ng/mL TNF-α were used as positive controls. Data are expressed as mean ± SD of three independent experiments; *p ≤ 0.05 vs non-infected HUVECs; #p ≤ 0.05 vs S. agalactiae-HUVEC infection.

Fig. 2. : immunofluorescence assays of NADPH oxidase p47phox subunit activity during infection of human primary endothelial cells (HUVECs) by Streptococcus agalactiae. (A, C, E) Uninfected HUVECs and (B, D, F) HUVECs infected by S. agalactiae in the presence or absence of NADPH inhibitors (10 μΜ DPI, 10 μΜ apocynin). Anti-p47phox was labelled with Alexa 546 (red), S. agalactiae with fluorescein-isothiocyanate (FITC) (green), and the nuclei were stained with DAPI (blue). Scale bars, 10 μM.

Fig. 3. : expression of NADPH oxidase p47phox subunit during infection of HUVECs with Streptococcus agalactiae. Data were analysed at different periods of time by (A) real time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR), (B) immunoblotting, and (C) densitometry assays. Actin was used as a control for protein loading. Data are expressed as mean ± SD of three experiments; *p < 0.05.

Fig. 4. : influence of NADPH oxidase p47phox subunit on Streptococcus agalactiae-human primary endothelial cells (HUVECs) interaction. (A) Adherence to and (B) intracellular viability of S. agalactiae in HUVECs pre-treated with DPI (10 µM) or apocynin (10 µM) to inhibit p47phox. Data are expressed as mean ± SD of three experiments; *p < 0.05.

Fig. 5. : influence of the PI3K/Akt pathway on the Streptococcus agalactiae-human primary endothelial cells (HUVECs) interaction. (A) Adherence to and (B) invasion of S. agalactiae in HUVECs pre-treated with LY294002, an inhibitor of PI3K. Micrographic images of immunofluorescence staining indicated: (C) non-infected HUVEC monolayers as a negative control; (D) generation of actin stress fibres in HUVECs infected with S. agalactiae; (E, G, I) HUVECs pre-treated with DPI (10 µM), apocynin (10 µM), and LY294002 (5 µM), respectively; (F, H, J) HUVECs pre-treated with inhibitors (DPI, apocynin and LY294002, respectively) and infected with S. agalactiae. (K) Immunoblotting assay of phosphorylated Akt in infected HUVECs pre-treated with LY294002. Data are expressed as mean ± SD of three experiments. *p < 0.05.