Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells
| Autor: | Thereza Christina Barja-Fidalgo, Jessica Silva Santos de Oliveira, Gabriela da Silva Santos, João Alfredo Moraes, Alessandra Mattos Saliba, Prescilla Emy Nagao, Ana Luíza Mattos-Guaraldi |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Microbiology (medical) lcsh:Arctic medicine. Tropical medicine Time Factors lcsh:RC955-962 Blotting Western lcsh:QR1-502 medicine.disease_cause Real-Time Polymerase Chain Reaction lcsh:Microbiology Bacterial Adhesion Streptococcus agalactiae 03 medical and health sciences chemistry.chemical_compound Phosphatidylinositol 3-Kinases HUVEC Reference Values Streptococcal Infections medicine Human Umbilical Vein Endothelial Cells Humans Protein kinase B PI3K/AKT/mTOR pathway chemistry.chemical_classification reactive oxygen species Reactive oxygen species Analysis of Variance NADPH oxidase 030102 biochemistry & molecular biology biology Chemistry NADPH Oxidases p47phox Molecular biology Oxidative Stress 030104 developmental biology PI3K/Akt pathway Apocynin biology.protein Original Article Proto-Oncogene Proteins c-akt Oxidative stress Intracellular Signal Transduction |
| Zdroj: | Memórias do Instituto Oswaldo Cruz Memórias do Instituto Oswaldo Cruz, Volume: 113, Issue: 6, Article number: e140421, Published: 05 APR 2018 Memórias do Instituto Oswaldo Cruz., Vol 113, Iss 6 (2018) |
| ISSN: | 1678-8060 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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