Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells
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 i...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Fundação Oswaldo Cruz (FIOCRUZ)
2018-04-01
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| Series: | Memorias do Instituto Oswaldo Cruz |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762018000600300&lng=en&tlng=en |
| _version_ | 1827833176021532672 |
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| author | Jessica Silva Santos de Oliveira Gabriela da Silva Santos João Alfredo Moraes Alessandra Mattos Saliba Thereza Christina Barja-Fidalgo Ana Luíza Mattos-Guaraldi Prescilla Emy Nagao |
| author_facet | Jessica Silva Santos de Oliveira Gabriela da Silva Santos João Alfredo Moraes Alessandra Mattos Saliba Thereza Christina Barja-Fidalgo Ana Luíza Mattos-Guaraldi Prescilla Emy Nagao |
| author_sort | Jessica Silva Santos de Oliveira |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T05:26:03Z |
| format | Article |
| id | doaj.art-ae9b44a5089b4acdb683229fa8e9809d |
| institution | Directory Open Access Journal |
| issn | 1678-8060 |
| language | English |
| last_indexed | 2024-03-12T05:26:03Z |
| publishDate | 2018-04-01 |
| publisher | Fundação Oswaldo Cruz (FIOCRUZ) |
| record_format | Article |
| series | Memorias do Instituto Oswaldo Cruz |
| spelling | doaj.art-ae9b44a5089b4acdb683229fa8e9809d2023-09-03T07:24:28ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz1678-80602018-04-01113610.1590/0074-02760170421S0074-02762018000600300Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cellsJessica Silva Santos de OliveiraGabriela da Silva SantosJoão Alfredo MoraesAlessandra Mattos SalibaThereza Christina Barja-FidalgoAna Luíza Mattos-GuaraldiPrescilla Emy NagaoBACKGROUND 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.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762018000600300&lng=en&tlng=enStreptococcus agalactiaeHUVECreactive oxygen speciesNADPH oxidasep47phoxPI3K/Akt pathway |
| spellingShingle | Jessica Silva Santos de Oliveira Gabriela da Silva Santos João Alfredo Moraes Alessandra Mattos Saliba Thereza Christina Barja-Fidalgo Ana Luíza Mattos-Guaraldi Prescilla Emy Nagao Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells Memorias do Instituto Oswaldo Cruz Streptococcus agalactiae HUVEC reactive oxygen species NADPH oxidase p47phox PI3K/Akt pathway |
| title | Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells |
| title_full | Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells |
| title_fullStr | Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells |
| title_full_unstemmed | Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells |
| title_short | Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells |
| title_sort | reactive oxygen species generation mediated by nadph oxidase and pi3k akt pathways contribute to invasion of streptococcus agalactiae in human endothelial cells |
| topic | Streptococcus agalactiae HUVEC reactive oxygen species NADPH oxidase p47phox PI3K/Akt pathway |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762018000600300&lng=en&tlng=en |
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