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

<div><p> 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.</p></div

Nutritional composition and comparison between experimental chows.

a<p>Standard chow for rodents; Nuvital, Nuvilab - Colombro Paraná - Brazil;</p>b<p>Shortening Primor: Gaspar, Santa Catarina - Brazil; c Condensed Milk Moça Nestlé, São Paulo – Brazil.</p>c<p>Condensed Milk Moça Nestlé, São Paulo – Brazil.</p

Leptin evaluation.

<p>Data are presented as mean±standard error of the mean HF =  High Fat Diet. HFSC =  High Fat Diet + Standard Chow. HFEX =  High Fat Diet + Exercise. HFSCEX =  High Fat Diet +Standard Chow/Exercise. *Significantly different from the HF group (p<0.01).</p

Assessments performed on experiment day.

<p>All values are presented as mean±standard error of the mean. HF =  High Fat diet. HFSC =  High Fat Diet + Standard Chow. HFEX =  High Fat Diet+Exercise. HFSCEX =  High Fat Diet +Standard Chow/Exercise (n = 10/per group). Fat (%)  =  Fat percentage; Uro depot =  Urogenital fat depot (g/100 g of body mass); Vis depot  =  Visceral fat depot (g/100 g of body mass); Ret depot  =  Retroperitoneal fat depot (g/100 g of body mass). MAP =  Mean arterial pressure. HR =  Heart rate.</p><p>*Significantly different from HF group (p<0.001).</p>+<p>Significantly different from the HFEX group (p<0.01).</p>#<p>Significantly different from the HFSC group (p<0.01).</p

Experimental design and Aerobic Exercise Training.

<p>(A) Experimental design with dietary modification and aerobic exercise training, after week 12. (B) Aerobic Exercise Training (AET) performed by hamsters, organized by weeks, time, speed and angulations. High fat fed hamsters were subjected to AET in the last two months associated or not to diet modification.</p

Macromolecular permeability at post capillary venules after 30 minutes ischemia of the cheek pouch of hamsters fed with high fat chow (HF and HFEX, n = 10 each) and with dietary modification associated or not to AET (HFSC and HFSCEX, n = 10 each) during 20 weeks.

<p>T5, 5 minutes after and T10, 10 minutes after tourniquet release. Data are expressed as median ± 10–90 percentile, represented by vertical bars. * Significantly different from the HF group (p<0.05). HF =  High Fat Diet. HFSC =  High Fat Diet + Standard Chow. HFEX =  High Fat Diet + Exercise. HFSCEX =  High Fat Diet +Standard Chow/Exercise.</p

Lipid profile and Glucose evaluation of high fat chow (HF), High fat+Standard chow (HFSC), High fat +Aerobic Exercise (HFEX) and High fat +Standard Chow/Exercise (HFSC/EX) groups.

<p>All values are presented as mean±S.E.M. No significant difference was found between groups. HF =  High Fat Diet. HFSC =  High Fat Diet +Standard Chow. HFEX =  High Fat Diet +Exercise. HFSCEX =  High Fat Diet +Standard Chow/Exercise (n = 10/per group). TC =  total cholesterol; HDL =  high density lipoprotein; LDL =  low density lipoprotein; VLDL =  very low density lipoprotein; TGL =  triglycerides.</p

Mean arteriolar diameters after topical application of three concentrations of acetylcholine (10⁻⁸, 10⁻⁶ and 10⁻⁴ M) to the cheek pouch of hamsters fed with high fat diet (HF and HFEX, n = 10 each) and that had dietary modification associated or not to AET (HFSC and HFSCEX, n = 10 each) during 20 weeks.

<p>Data are shown as changes relative to baseline considered as 100%. Data are expressed as median±10–90 percentile, represented by vertical bars. *Significantly different from sedentary obese control group (p<0.001 and p<0.01, respectively) in all concentrations of Ach.</p