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Involvement of lipid microdomains in human endothelial cells infected

by Streptococcus agalactiae type III belonging to the

hypervirulent ST-17


Bacterial strain and growth conditions - S.

agalactiae capsular type III [GBS90356 cerebrospinal fluid (CSF)

strain] belonging to the hypervirulent ST-17 lineage isolated in Brazil from a

3-day-old male baby with fatal acute meningitis was used in this study.

Microorganism was identified as group B streptococci and typing by methods

previously described.


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 (~108 CFU/mL) was



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.


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).

Bacterial binding and intracellular viability assays - Confluent

cultures of HUVEC cells were pre-treated or not with MβCD (2 mM, Sigma Chemical Co.,

St. Louis, MO, USA), a lipid raft disruptor for 1 h or with LY294002, PI3K inhibitor

(5 µM, Sigma Chemical Co., St. Louis, MO, USA), or with both MβCD and LY294002 for

15 min at 37ºC. Then, HUVEC were allowed to interact with S.

agalactiae (MOI, 1:100 HUVEC/bacteria) during different periods of

incubation (1, 2 and 4 h) in 5% CO2 at 37ºC. For the bacterial binding

assays, infected monolayers were rinsed three times with M199 and lysed in a 0.5 mL

solution of 25 mM Tris, 5 mM EDTA, 150 mM NaCl and 1% Igepal (all from Sigma

Chemical Co., St. Louis, MO, USA). The viability of total bacteria (intracellular

plus surface adherent) was estimated by plating endothelial lysates and counting the

resulting colonies emerging in BAB plates containing 5% sheep defibrinated blood. To

measure bacterial internalisation, the infected monolayers were rinsed three times

with M199 medium and incubated for an additional 2 h period in M199 containing

bactericidal amounts of gentamicin (100 μg/mL, Sigma Chemical Co., St. Louis, MO,

USA) and penicillin G (5 μg/mL, Sigma Chemical Co., St. Louis, MO, USA). We also

performed a count of cells that invaded and adhered shortly after the interaction

with S. agalactiae and 0.5 h after. The number of internalised

bacteria was determined as outlined above. Adherence rates were determined as: [CFU

of total cell-associated (intracellular viable plus surface adherent) S.

agalactiae - CFU intracellular S. agalactiae].


Untreated HUVEC were used as negative control.


All experiments were repeated three times.

Field emission scanning electron microscopy (FESEM) - HUVEC

monolayers were infected with S. agalactiae for 2 h, washed with

PBS and incubated overnight at 4ºC in a solution of 3% paraformaldehyde plus 2.5%

glutaraldehyde made in 0.1 M cacodylate buffer. The strains were washed and

post-fixed in a solution of 1% OsO4 plus 8 mM potassium

ferrocyanide and 10 mM CaCl2 in 0.1 M cacodylate buffer. After washing

eight times with PBS, infected cells were dehydrated in a graded series of ethanol,

and the surface of some infected monolayers were scraped with scotch tape in order

to expose the inner organisation of HUVEC. All cells were dried to a critical point

with CO2 and coated with a thin gold layer. The gold-coated strains were

then observed in a JEOL field emission scanner, operating at 10 kV.


Fluorescence confocal microscopy - HUVEC cells pretreated or not

with MβCD were infected with S. agalactiae for 1 h, rinsed with PBS

and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The cells

were permeabilised with 0.5% Triton-X 100 in PBS for 30 min and incubated with

primary antibodies [anti-S. agalactiae or anti-flotillin-1 antibody

(clone 29) or anti-caveolin-1 or anti-caveolin-2] for 1 h at 37ºC. After incubation,

cells were washed for 30 min and incubated with Alexa Fluor 488 or Alexa Fluor

546-conjugated secondary antibodies for 1 h at 37ºC. Nuclei were labeled with 0.5

μg/mL 4’-6-diamidino-2-phenylindole (DAPI). Cells were mounted in ProLong Gold

antifade reagent and examined using a Zeiss Axiovert 100M laser confocal microscope

(Carl Zeiss, Germany) by using filters sets that were selective for each

fluorochrome wavelength channel. Images were acquired with a C2400i integrated

charge-coupled device camera (Hamamatsu Photonics, Shizuoka, Japan) and an Argus 20

image processor (Hamamatsu). Control experiments with no primary antibodies showed

only faint background staining (data not shown). All reagents and antibodies were

obtained from Molecular Probes (USA). These experiments were repeated three


Immunoblot analysis - HUVEC monolayers were infected during

different times with S. agalactiae as described above. Following

infection, the plates were chilled, and all subsequent steps were carried out at

4ºC. The HUVEC were rinsed with PBS containing 0.4 mM Na3VO4

and 1 mM NaF per mL. Next, the infected cells were scraped from the plate,

ressuspended in 1.5 mL of the same buffered solution, collected by centrifugation

for 1 min at 12,000 g, and lysed for 30 min in 100 µL of 50 mM Tris-HCL (pH 7.6)

containing 0.4 mM Na3VO4, 1 mM NaF, 1% Triton X-100, 100 µM of

phenylethylsulphonylfluoride, 40 µM of leupeptin and 2 mM EDTA. The proteins were

quantified, and 30 µg of protein of each extract was subjected to electrophoresis in

12% polyacrylamide separating gel (SDS-PAGE). Proteins were transferred to

nitrocellulose membranes (Biorad), which were blocked and then incubated with

primary antibodies. The membranes were incubated with second antibody

peroxidase-conjugated and the immunoreactivity was detected using an ECL Plus

detection kit (Amersham Biosciences, Buckinghamshire, UK). Autoradiographs were

quantified by scanning densitometry, and the resulting absorbance curves were

integrated by using the Scion Image Master. Densitometric analyses were performed on

gels with different exposure times, and the ones giving linear absorbance curves

were used to obtain semi quantitative assessment.


These experiments were repeated three times.

Statistical analysis - The values of different treatments were

compared using Student’s t-test and analysis of variance (ANOVA),

followed by Bonferroni t test for unpaired values. All of the

statistical analyses were performed at the p < 0.05 level of significance.


Effect of cholesterol depletion and PI3K inhibitor during S.

agalactiae GBS90356-HUVEC interaction - Effects of

MβCD (cholesterol depletion agent) and LY294002 (PI3K inhibitor) treatments on

S. agalactiae GBS90356 adherence to and invasion of HUVEC are

displayed in Fig. 1. Cholesterol depletion

affected bacterial binding to HUVEC 1 h post-infection (6.3 x 104 CFU/mL,

p < 0.001). A higher number of adherent bacteria (3.8 x 106 CFU/mL, p

< 0.001) was observed in HUVEC pre-treated with LY294002 in 1h and mainly after 4

h incubation (1.2 x 107 CFU/mL, p < 0.001) (Fig. 1A). However, a significant reduction of S.

agalactiae cytoadhesion was observed in HUVEC treated with LY294002 +

MβCD at all chosen times of incubation (1.3 x 106 CFU/mL in 1 h; 1.5 x

106 CFU/mL in 2 h; 3.9 x 106 CFU/mL in 4 h, p < 0.01)

(Fig. 1A). S. agalactiae

strain exhibited a strong invasive phenotype to HUVEC after 2 h post-infection.

Moreover, pre-treatment of HUVEC with MβCD and/or LY294002 led to a decrease in

invasion (p < 0.01) (Fig. 1B). The FESEM was

used to demonstrate the presence of intracellular GBS90356 strain (Fig. 1C). Inhibition assays with MβCD and

LY294002 suggest the involvement of lipid rafts and PI3K/AKT pathway during

S. agalactiae GBS90356 internalisation process in human

endothelial cells.

Colocalisation of S. agalactiae GBS90356 with flotillin-1 and

caveolin-1 - Cellular localisation of S. agalactiae

GBS90356 strain, caveolin-1 (Fig. 2A, D),

caveolin-2 (Fig. 2B, E) and flotillin-1 (Fig. 2C, F) in HUVEC are demonstrated by

immunofluorescence microscopy. Staining for flotillin-1 revealed a colocalisation

with GBS90356 strain in untreated HUVEC cells (Fig.

2C), but no colocalisation was detected in cells treated with MβCD, a

cholesterol depleting agent (Fig. 2F). We also

found a colocalisation of GBS90356 strain and caveolin-1 after pretreatment of HUVEC

with MβCD (Fig. 2D), but not in untreated cells

(Fig. 2A). By contrast, no significant

colocalisation could be observed between GBS90356 strain and caveolin-2 in HUVEC

cells treated or not with MβCD (Fig. 2B, E).

These results suggest that caveolin-1 and flotillin-1 could be involved in the

invasion of S. agalactiae GBS90356 strain in HUVEC cells.

AKT activation in HUVEC cells during S. agalactiae GBS90356

infection - Fig. 3 shows the

activation of PI3K/Akt pathway during the interaction between S.

agalactiae GBS90356 and HUVEC cells pretreated with LY294002 (PI3K

inhibitor) and/or MβCD (cholesterol depletion agent). Immunoblotting analysis

revealed higher levels of phosphorylated Akt with a peak at 15 min post-infection of

HUVEC by S. agalactiae. Inhibition assays with MβCD completely

abolished the AKT phosphorylation (Fig. 3A). To

verify whether the phosphorylation of Akt in HUVEC cells, treated or not with MβCD,

was PI3K-dependent or -independent following infection with GBS90356 strain, the

specific PI3K inhibitor LY294002 was incubated prior to bacterial infection.

Activation of the PI3K pathway occurred at 5 min post-infection and peak at 30 min.

Both inhibitors reduced the PI3K phosphorylation (Fig.

3B). Results were confirmed by densitometry analysis (Fig. 3C, D). Overall, the results indicate the

involvement of lipid rafts and PI3K/AKT pathway activation during S.

agalactiae internalisation in human endothelial cells.


Lipid rafts often serve as an entry site for many microorganisms. Studies have shown

that extraction of membrane cholesterol inhibited bacterial infection in the early

stages of invasion.


The mechanisms that underlie this interaction are starting to be unraveled.

Several pathogenic bacteria have been associated with lipid rafts, such as

Francisella tularensis, Helicobacter pylori,

Pseudomonas gengivalis and Mycobacterium



Seveau et al.


demonstrated for the first time that the cell adhesion molecule E-cadherin

is required in host lipid rafts to mediate Listeria monocytogenes

entry. A previous study showed that S. agalactiae exploited lipid

rafts to invade human endometrial cells.


In this work, we evaluated the influence of host cell lipid rafts during

S. agalactiae internalisation in HUVEC by using

methyl-β-cyclodextrin (MβCD), a water-soluble cyclic oligosaccharide that depletes

membrane cholesterol and disrupt lipid rafts.


Cholesterol-enriched membrane microdomains may provide a platform to concentrate

receptors on the host cell membrane.


Our data support the notion that lipid rafts on the plasma membrane of HUVEC

cells facilitate entry of S. agalactiae GBS90356 strain. The

significant decrease in cytoadhesion of GBS90356 strain to human endothelial cells

treated with MβCD indicated that cholesterol depletion from the cell membrane

perturbed the attachment of bacteria and altered the GBS90356 entry at post-binding

steps. The reduction in the number of GBS90356 in cholesterol-depleted cells

probably occurred at initial steps of infection, since significant inhibitory effect

was reduced after 4h post-infection and became undetectable at 24 h post-infection

(data not shown). Interaction of GBS90356 strain with cholesterol-enriched

microdomains occurred probably with the participation of flotillin-1 and

caveolin-1-enriched membrane microdomains.

Flotillins are present at the plasma membrane and endosomal structures and have been

implicated in many cellular processes, such as lipid raft formation, cellular

migration and adhesion, cell polarity, signaling by receptor tyrosine kinases and

mitogen activated protein kinases (MAPK), as well as membrane trafficking.






Several cargo molecules, such as the GPI-anchored protein CD59, cholera

toxin B subunit, virus, proteoglycans and proteoglycan bound ligands have been

suggested to utilise an internalisation pathway that depends on flotillin.




Previous data suggested that the highly dynamic flotillin microdomains

become static just prior to their internalisation, which might be caused by

coalescence of flotillin oligomers into larger oligomeric structures, participating

in the formation of specific non-caveolar microdomains.


Currently, our results suggest that S. agalactiae GBS90356

induces flotillin-1 assembly to specific flotillin microdomains, which induce

membrane curvature and thus generate membrane buds to entry to the HUVEC.

Interestingly, the enrichment of flotillin-1 on post-LAMP endocytic organelles

during maturing phagosomes might be involved in actin filament remodeling and lipid

changes for phagosome-lysosomes fusion.


Further studies to verify if flotillin-1 colocalise with S.

agalactiae in early endosomes are in progress.

Interestingly, treatment with MβCD decreased the colocalisation of GBS90356 strain

with flotillin-1, favoring bacterial interaction with caveolin-1. Cholesterol levels

are important for maintaining membrane fluidity, and its removal can reduce lateral

diffusion within the cell membrane. The reduction in fluidity could perhaps affect

distribution of receptors within the plasma membrane, which may damage signal

transduction events, polarisation and F-actin polymerisation.


Our results suggest that cholesterol depletion by MβCD may have exposed

caveolin-1 molecules, favoring recognition by GBS90356 strain. Caveolin-1 is

critical for enhancing the innate immune response, which contributes to survival

during LPS-induced sepsis.


Also observed in intracellular parasites as the inhibition of lysosomal

fusion, a classical escape mechanism was observed after infection by

Mycobacterium, Chlamydia,

Toxoplasma and Trypanosoma cruzi, for example.

Another way that pathogens can prolong their survival inside the host is by

prevention of host-cell apoptosis and by the modulation of reactive oxygen and

nitrogen species generation.


Szczepanski et al.


revealed that canine respiratory coronavirus enters HRT-18G cells via the

caveolin-1 dependent pathway. Thus, these previous results support the notion that

caveolin-1 might play a role in the interaction of S. agalactiae

and HUVEC cells.

Serine/threonine kinase Akt is activated by G protein-coupled receptors that induce

the production of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by PI3K.


In this work, we demonstrated that the integrity of lipid rafts microdomains

and the activity of PI3K/Akt are required for invasion of GBS90356 strain to human

endothelial cells. Indeed, the phosphorylation of Akt and PI3K were suppressed by

cholesterol depletion using MβCD, suggesting that membrane microdomain integrity is

important for PI3K/Akt activation during S. agalactiae infection.

Peres et al.


showed that membrane microdomains were an essential site for PI3K activation

during lysophosphatidic acid stimulation in Vero cells. Lipid rafts also induced

platelet aggregation via PI3K-dependent Akt phosphorylation by stromal cell-derived

factor-1α signaling.


We cannot exclude the possibility that the results obtained in our studies could be

specific to S. agalactiae type III belonging to the hypervirulent

ST-17, and that other capsular type III strains could behave in different ways. More

studies are necessary to unravel this possibility.

Our results demonstrate that lipid microdomain affects S. agalactiae

recognition by HUVEC through PI3K/Akt signaling pathway. In addition, S.

agalactiae cytoadhesion using membrane microdomains suggests a

selective role of lipid raft molecules, such as flotillin-1 and caveolin-1. Hence,

an understanding of the role of host membrane rafts in S.

agalactiae invasion may shed light on the molecular mechanisms of