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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 p47 phox -Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47 phox  (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 p47 phox  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 p47 phox . 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.

memórias do instituto oswaldo cruz

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