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.

Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17

“…(13) GBS90356 isolate was cultured on blood agar base (BAB; Oxoid, Cambridge, UK) plates containing 5% sheep defibrinated blood for 24 h at 37ºC and then grown in Brain Heart Infusion broth (BHI; Difco Laboratories Inc, Detroit, MI, USA) at 37ºC until an optical density (OD) of 0.4 at ƛ = 540 nm (~10 8 CFU/mL) was reached. (11) HUVEC culture -Primary HUVEC were obtained by treating umbilical veins with 0.1% collagenase IV solution (Sigma Chemical Co., St. Louis, MO, USA) as previously described. (11) Cells were used during first or second passages only, and subcultures were obtained by treating the confluent cultures with 0.025 % trypsin/0.2 % EDTA solution in phosphate-buffered saline (PBS) (150 mM NaCl, 20 mM phosphate buffer, pH 7.2 -all from Sigma Chemical Co., St. Louis, MO, USA).…”

“…(11) HUVEC culture -Primary HUVEC were obtained by treating umbilical veins with 0.1% collagenase IV solution (Sigma Chemical Co., St. Louis, MO, USA) as previously described. (11) Cells were used during first or second passages only, and subcultures were obtained by treating the confluent cultures with 0.025 % trypsin/0.2 % EDTA solution in phosphate-buffered saline (PBS) (150 mM NaCl, 20 mM phosphate buffer, pH 7.2 -all from Sigma Chemical Co., St. Louis, MO, USA).…”

“…(9,10) The ability of S. agalactiae to invade a number of host-cell types has been clearly demonstrated. (1,11) However, the invasion process is not well understood. Subversion of the PI3K/Akt pathway by S. agalactiae resulted in coordination of actin rearrangement and internalisation of the microorganism.…”

“…Subversion of the PI3K/Akt pathway by S. agalactiae resulted in coordination of actin rearrangement and internalisation of the microorganism. (11) PI3K is the major activator of Akt, playing a central role in fundamental biological processes including cell growth, proliferation, migration and survival, through phosphorylation of a plethora of substrates. (12) Previous studies showed that the integrity of lipid rafts and PI3K activity are required for S. agalactiae invasion to Ishikawa cells.…”

Inhibition of the PI3K/AKT Signaling Pathway or Overexpression of Beclin1 Blocks Reinfection of Streptococcus pneumoniae After Infection of Influenza A Virus in Severe Community-Acquired Pneumonia

“…The phosphorylation of Beclin1 mediated by protein kinase B (AKT) functions in oncogenesis and autophagy inhibition [19]. Meanwhile, the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway mediated by a pathogen contributes to coordination of actin rearrangement, thereby leading to internalization of the organism [20]. A study has suggested that the activated PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway is implicated in lung tumorigenesis and it has been related to high-grade tumors and advanced diseases [21].…”