by Streptococcus agalactiae type III belonging to the
MATERIALS AND METHODS
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
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
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
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
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
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