National Centre for Streptococcus, Provincial Laboratory for Public Health (Microbiology), Edmonton, Alberta, CanadaT6G 2J21
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, CanadaT6G 2J22
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, CanadaT6G 2J23
Author for correspondence: Gregory J. Tyrrell. Tel: +1 780 407 8949. Fax: +1 780 407 3864. e-mail: g.tyrrell{at}provlab.ab.ca
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ABSTRACT |
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Keywords: actin, streptococci, phosphatidylinositol 3-kinase, scanning electron microscopy
Abbreviations: GBS, group B streptococci; NCS, National Centre for Streptococcus; SBA, sheep blood agar; SEM, scanning electron microscopy; TEM, transmission electron microscopy
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INTRODUCTION |
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The mechanism(s) used by GBS to cause invasive disease in the host are not completely understood, but it is clear that GBS are able to invade host cells. This has been demonstrated for HEp-2 cells, HeLa cells, A549 cells, MDCK cells, human brain microvascular endothelial cells, etc. (Gibson et al., 1993 ; Hulse et al., 1993
; Kallman & Kihlstrom, 1997
; Lalonde et al., 2000
; Nizet et al., 1997
; Rubens et al., 1991
, 1992
; Tamura & Rubens, 1994
; Valentin-Weigand et al., 1996
). While GBS have been shown to be invasive against a wide variety of host cells, the events that occur upon the adherence and internalization have not been fully elucidated.
A number of host-cell structures have been suggested as receptors for GBS. These include laminin, cytokeratins, fibronectin, fibrinogen, etc. (Cheng et al., 2002 ; Spellerberg et al., 1999
, 2002
; Tamura & Nittayajarn, 2000
; Tamura & Rubens, 1995
). Most recently, Beckmann et al. (2002)
and Cheng et al. (2002)
have shown that the C5a peptidase in GBS plays a role in binding to fibronectin and invasion of A549 and HEp-2 cells. Other recent work by Tamura et al. (2002)
demonstrated that the GBS gene, glnQ, is required for GBS adherence to immobilized fibronectin and also plays a role in binding and invasion of A549 cells. However, while these components (laminin, cytokeratin, fibronectin, etc.) have been shown to bind or be involved in the binding and invasion of host cells by GBS, whether they function as the main GBS receptor in the host cell or whether a main GBS receptor exists is not completely clear.
In addition, previous investigators have found that invasion requires actin microfilaments, since this invasion is disrupted by cytochalasin D, an inhibitor of polymerization of actin (Gibson et al., 1993 ; Nizet et al., 1997
; Rubens et al., 1992
; Valentin-Weigand et al., 1996
, 1997
). Further work has shown that exploitation of eukaryotic signal-transduction pathways seems to be occurring as GBS invasion is inhibited by protein kinase inhibitors staurosporine and genistein. This has suggested that GBS uptake occurs via a multicomponent pathway for which very few components have been identified.
In the following study, we describe experiments examining the physical interaction of GBS with HeLa and MRC-5 cell surfaces and begin an analysis of the mechanism of internalization with respect to the involvement of the actin cytoskeleton and phosphatidylinositol 3-kinase.
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METHODS |
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GBS invasion assay.
The assay was performed as previously described, with some changes (Rubens et al., 1992 ). Briefly, GBS were passaged once on sheep blood agar (SBA) and then passaged in ToddHewitt broth overnight. A 0·5 McFarland in PBS was made and diluted 1:100. One hundred microlitres of this suspension (approx. 1·5x105 bacteria) was applied to confluent cell monolayers in 1 ml OPTI-MEM plus 4% fetal bovine serum contained in 24-well Falcon tissue culture plates (Becton Dickinson). The cell monolayers and GBS were incubated at 37 °C for the period of time specified depending on the experiment. The monolayers were washed three times with PBS and then incubated with medium containing 5 µg penicillin ml-1 and 100 µg gentamicin ml-1 for 2 h. After this incubation, the monolayers were again washed three times with PBS. The tissue culture cells were then trypsinized and plated onto SBA (output) and incubated overnight at 37 °C. Also, a further 1:100 dilution was made of the original input, and 100 µl of this was plated onto SBA, incubated overnight at 37 °C and used as the input count. After overnight incubation, colonies were counted (both input and output) to determine the number of GBS that had invaded the monolayer in relation to the number of GBS added to the monolayer.
Fluorescent microscopy.
To visualize GBS bound to eukaryotic cells, shell vials containing HeLa or MRC-5 cells were grown to confluency. One hundred microlitres of a 1:10 dilution of a 0·5 McFarland of GBS (overnight culture) (approx. 1·5x106 bacteria) was added to the shell vial and then incubated at 37 °C for 2 h. The cells were washed three times with PBS and fixed with 3% formaldehyde for 30 min followed by another wash three times with PBS. Rabbit Streptococcus antiserum group B (Difco) was then added, followed by a secondary rhodamine-conjugated goat anti-rabbit IgG Fc antibody (Chemicon). The specimens were then examined by confocal microscopy using a Zeiss 510 NLO (Carl Zeiss) laser-scanning microscope. For fibronectin visualization, the HeLa and MRC-5 cells were grown to confluency on 12 mm coverslips, fixed with 3% formaldehyde and stained with rabbit anti-human fibronectin antibody (Sigma) diluted 1:400. This was then washed with PBS, and anti-rabbit FITC conjugate was added (1:80). The coverslips were washed with PBS, mounted and examined using fluorescent microscopy. For actin recruitment visualization, HeLa cells were permeabilized after the 3% formaldehyde step with 0·1% Triton X-100. The cells were then stained with FITC phalloidin (Sigma) as previously described (Knutton et al., 1989 ).
Comparison of levels of fibronectin on HeLa and MRC-5 cells using an EIA.
MRC-5 or HeLa cells were grown to confluency in the wells of a 96-well tissue culture plate (Corning Costar). The media were removed from the cells, and the cells were fixed with 3% formaldehyde. A dilution of 1:400 of anti-fibronectin polyclonal antibody (Sigma) was made in 50 mM Tris-buffered saline. Two hundred microlitres of antibody was added to the wells containing each cell line in triplicate. The plate was incubated at 37 °C for 1 h then washed with 10 mM PBS/Tween three times. Two hundred microlitres of 1:30000 anti-rabbit IgG conjugated with alkaline phosphatase (Sigma) was then added to the wells. The plate was incubated at 37 °C for 1 h and then washed with 10 mM PBS/Tween three times. Two hundred microlitres of pNPP (alkaline phosphatase yellow) was then added to the wells and the plate incubated for 1 h at room temperature. Absorbance was read at 405 nm blanked against triplicate wells containing substrate and cells only.
Cytochalasin D treatment.
For both pre- and post-GBS infection, cytochalasin D (Sigma-Aldrich Canada) was added to HeLa cell monolayers at a concentration of 1·0 µg ml-1. For pretreatment, the cytochalasin D was added 1 h prior to the addition of GBS. GBS was allowed to interact with the HeLa cell monolayer for 2 h, after which the monolayer was washed three times with PBS and either treated with trypsin and plated onto SBA or prepared for scanning electron microscopy (SEM). For post-treatment, GBS were allowed to infect HeLa cells for 1 h before treatment with cytochalasin D. After the 1 h GBS exposure, the HeLa cells were treated with cytochalasin D for a further 1 h. The monolayer was then washed three times with PBS and prepared for SEM.
SEM and transmission electron microscopy (TEM).
For SEM, identical pre- and post-cytochalasin D treatment of HeLa cells exposed to GBS was carried out as described in the previous section with the exception that the monolayers were grown on 13-mm-diameter sterile thermanox plastic cell-culture coverslips (Nalge Nunc). After exposure to GBS with or without cytochalasin D treatment, the coverslips were prepared for SEM or TEM. For SEM, the coverslips were fixed with freshly prepared 2% (v/v) glutaraldehyde, in phosphate buffer, pH 7·3, overnight at 4 °C. The samples were then washed three times in phosphate buffer and further fixed in 1% (w/v) osmium tetroxide for 3 h, washed in phosphate buffer, then dehydrated in a graded series of ethanol at room temperature and critical-point-dried (Balzers critical point dryer #CPD 030). The coverslips were mounted on standard Cambridge SEM stubs, sputter-coated lightly with gold and examined in a Hitachi S 4100 field emission scanning electron microscope.
For TEM, after fixation and dehydration in a graded series of alcohol, the coverslips were transferred through two changes of propylene for 30 min each time. Samples were then transferred to a 1:1 mixture of propylene oxide and LX 112 resin (Ladd Research Industries) and left uncapped for 24 h at room temperature. On the following day, the samples were transferred to pure LX 112 for 1 h. Fresh LX 112 was placed onto the coverslips, and micro-centrifuge tubes filled with LX 112 were inverted on top of the coverslips. The samples were then cured for 24 h at 60 °C. Sections were then cut using a Reichert-Jung Ultracut and placed on 3 mm, 200 mesh Formvar-coated copper grids. These were stained with 5% uranyl acetate for 10 min and lead citrate for 6 min. Images were recorded on a Kodak #4489 electron microscope film using a Hitachi model H7000 transmission electron microscope.
Analysis of GBSHeLa cell interactions over a 6 h period.
To analyse the effect of GBS on HeLa cells over a 6 h period, HeLa cell monolayers were grown on thermanox coverslips in a 24-well transwell plate containing 1 ml medium per well. After 24 h, the HeLa cells reached confluency, and a 1:10 dilution of a 0·5 McFarland suspension of an overnight culture of GBS grown in ToddHewitt broth was added to the monolayers. At 1, 2, 3, 4, 5 and 6 h time points, coverslips were removed and washed three times with PBS. The coverslips were then prepared for SEM observation.
To determine whether GBS physical contact of the HeLa cells was required to cause HeLa cell destruction, HeLa cells were prepared by the same process as described above to the point where GBS was to be added. At this point, a 0·4-µm-pore-size transwell membrane was placed over the top as needed (Corning Costar). A 0·5 McFarland of an overnight culture of GBS was made, and 100 µl of this was added directly to the HeLa cell monolayer or the top of the transwell membrane. After a 6 h incubation at 35 °C, the monolayer was washed three times with PBS and then fixed with formaldehyde for 30 min. Monolayers were then washed three times with PBS and stained for 20 s with crystal violet. The coverslips were then washed three times with PBS and examined.
Inhibition of GBS invasion by wortmannin.
HeLa cells were grown to 8095% confluency in a 24 tissue culture plate. Wortmannin was added to the HeLa cells in concentrations ranging from 2 nM to 2 µM in triplicate and incubated for 30 min at 37 °C. GBS were then added and the plate incubated for 2 h at 37 °C. After 2 h, the cells were washed with PBS, and medium containing the appropriate concentration of wortmannin, penicillin and gentamicin was added. After a further 2 h incubation at 37 °C, the cells were washed with PBS, and 0·25% trypsin was added to each well. The plate was incubated at 37 °C until the cells were detached. The contents of each well were plated onto SBA, incubated overnight and c.f.u. determined on the following day.
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RESULTS AND DISCUSSION |
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Binding and invasion of NCS13 against HeLa cells
To quantitate the invasive ability of NCS13 against HeLa cells, a standard invasion assay was performed. This assay involves the use of antibiotics to kill any noninvasive bacteria after a defined incubation period, and the invasive bacteria are quantified as c.f.u. The assay showed that the initial invasion level was low but rapidly changed, with over 2% of the initial inoculum becoming intracellular after 180 min (Fig. 1).
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A suggested mechanism for epithelial cell destruction is an increase in the number of GBS invading the HeLa cell, which would correlate with the increase in number of GBS bound. It is possible that the increase in intracellular GBS physically reaches a limit, and the HeLa cell bursts. An alternative possibility is the activity of the GBS surface associated ß-haemolysin (Marchlewicz & Duncan, 1980 ; Platt, 1995
). Nizet et al. (1996)
have reported that the GBS ß-haemolysin can cause injury to lung epithelial cells, so it is possible that this haemolysin is destroying the HeLa cells after an extended period of contact with NCS13.
GBS interactions with MRC-5 cells
Rubens et al. (1991) and Gibson et al. (1993)
had previously reported the presence of GBS inside membrane-bound vacuoles within fibroblasts, so we wanted to determine how invasive the collection of GBS strains in Table 1
would be against the fibroblastic cell line MRC-5. Table 1
shows the mean c.f.u. (±SD) for the invasion of MRC-5 cells by GBS strains. All GBS strains invaded MRC-5 cells to a similar low level, and no GBS strain invaded MRC-5 cells at the level NCS13 invaded HeLa cells. Since the majority of GBS strains invaded MRC-5 cells less than they invaded HeLa cells, this suggested that there are important differences between HeLa cells and MRC-5 cells that affect the ability of GBS to invade. To be consistent in our study and allow direct comparisons with HeLa cells, we chose NCS13 for a further analysis of GBSMRC-5 cell interactions. A quantitative invasion assay of NCS13 against MRC-5 cells showed that, after a 180 min exposure to NCS13, the invasion rate was very low in comparison to HeLa cells (Fig. 1
). To determine whether the preventative step in NCS13 invasion of MRC-5 cells was the initial binding of the bacteria to the fibroblast surface, NCS13 was allowed to bind to the surface of MRC-5 cells grown on glass coverslips for 2 h. Bacteria bound to the surface were visualized via fluorescent confocal microscopy using anti-GBS rabbit antibody followed by rhodamine-labelled goat anti-rabbit antibody. Confocal imaging showed low numbers of NCS13 bound to the MRC-5 cell surface, suggesting that the poor invasion was due to a lack of binding (Fig. 4a
). SEM was used to observe the GBSMRC-5 cell interaction at a higher magnification. Fig. 4(b)
shows minimal binding of NCS13 to the MRC-5 cell surface, suggesting that this fibroblastic cell line does not possess receptors for GBS. In comparison, the binding of NCS13 to HeLa cells, as visualized using confocal microscopy and SEM, found that the cocci bound in large numbers to the surface of the HeLa cell, suggesting that, in contrast to MRC-5 cells, HeLa cells do possess receptors for GBS (Figs 2b
and 5a
).
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The finding that NCS13 does not bind to a cell line containing large amounts of fibronectin, suggesting that fibronectin is not acting as a receptor in this cell line, is somewhat contradictory to the findings of others. Work by Cheng et al. (2002) demonstrated that the GBS strain 090R invaded HEp-2 and A549 cells, and when 090R was mutated in the scpB gene (a GBS gene that encodes the fibronectin-binding protein SCPB), levels of binding and invasion were reduced. Further work by Tamura et al. (2002)
showed that the GBS strain COH1, when mutated by Tn917 in the glnQ gene, failed to bind to immobilized fibronectin, in comparison to the wild-type COH1, and exhibited decreased adherence and invasion of A549 cells, a cell line also containing fibronectin on its surface.
Based on these reports and others that have shown that GBS binds to immobilized fibronectin, we expected NCS13 to bind to the fibronectin-containing cell line MRC-5 (Beckmann et al., 2002 ; Cheng et al., 2002
; Tamura & Rubens, 1995
; Tamura et al., 2002
). A possible reason why we did not observe significant binding of NCS13 to MRC-5 cells may be that the cells have a different isoform of fibronectin. Fibroblastic cells such as MRC-5 cells may contain a form of fibronectin that does not permit GBS receptorfibronectin interactions to occur, whereas the small amount of fibronectin on HeLa cells (epithelial cells) is an isoform that has a higher affinity for NCS13. Another possibility is that the previous investigators used different GBS strains to those used in this study. Tamura & Rubens (1995)
examined the ability of nine strains of GBS to bind to fibronectin immobilized on a polystyrene-coated surface. The percentage of GBS bound to fibronectin in this manner varied widely ranging from 4 to 60% of the input, depending on the strain used. It is therefore possible that strains used in other studies have a greater ability to bind fibronectin in comparison to the strains used in this study. However, this seems unlikely as we examined 18 different strains from different sources, and all did not invade MRC-5 cells very well. Recently, Spellerberg et al. (2002)
explored the binding of GBS to a variety of extracellular matrix proteins. It was observed that GBS did not adhere to fibronectin as well as fibrinogen, thrombospondin and vitronectin, and only bound fibronectin slightly better than laminin (Spellerberg et al., 2002
).
These observations suggest that GBS may be using fibronectin as a receptor in some cell lines, but the bacterium clearly has the ability to utilize other host-cell structures to allow it to bind.
Electron microscopic analysis of NCS13 binding to HeLa cells
The binding of NCS13 to HeLa cells was examined in greater detail using SEM. A close examination of Fig. 6(a, b
) shows numerous HeLa-cell-derived microvilli in contact with the chains. Microvilli can be seen wrapped around the chains in both the SEM (Fig. 6a
, b
) and TEM (Fig. 7a
, b
) electron micrographs.
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Involvement of HeLa cell actin during NCS13 binding and invasion
The observation that HeLa cell microvilli interact intimately with GBS, and the knowledge that one of the major components of microvilli is an actin cytoskeleton, prompted us to explore the role of the actin cytoskeleton more closely. HeLa cells exposed to NCS13 were stained with FITC-labelled phalloidin to determine whether cellular actin is recruited to the site of GBS binding. Fig. 8(a, b
) shows actin at the site of GBS cocci binding to the HeLa cell surface, suggesting that cellular actin is recruited to the site of GBS binding. This finding was not completely unexpected, as involvement of host-cell actin in bacterial invasion is a theme demonstrated by a number of bacterial pathogens, most recently group A streptococci (Ozeri et al., 2001
). Ozeri et al. (2001)
demonstrated the recruitment of actin to the site of group A streptococcal binding as well as various other host-cell cytoplasmic components. The image of host-cell actin recruitment to the site of binding by NCS13 is similar to that seen for the group A streptococci, suggesting that similar binding and invasion mechanisms may be used by GBS (Fig. 8
).
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To visually examine the effect of cytochalasin D on the NCS13 HeLa cell interaction, SEM was used. Fig. 9(a, b
) shows that the cytochalasin D-treated HeLa cells had lost the short microvilli seen on the surface of untreated HeLa cells and that these were replaced by a smooth cell surface and large protrusions of cellular blebs. Besides the cellular changes seen in the HeLa cells, the binding of NCS13 to cytochalasin D-pretreated cells was reduced in comparison to untreated HeLa cells (Fig. 9a
, b
). This was also seen in HeLa cells first exposed to NCS13 and then treated with cytochalasin D (Fig. 9c
, d
), suggesting that cytochalasin D reduces GBS binding to HeLa cells but does not completely inhibit binding. The finding that binding is not completely inhibited may suggest that the GBS receptor(s) are independent of the actin cytoskeleton during the initial binding of GBS to the host-cell surface.
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Concluding remarks
In summary, we have shown that GBS are able to destroy HeLa cells after a 6 h incubation and that this cell destruction can occur only by direct GBSHeLa cell contact. GBS do not bind and invade MRC-5 cells (a cell line containing large amounts of fibronectin on its surface) to the same level as HeLa cells (a cell line containing a small amount of fibronectin on its cell surface in comparison to MRC-5 cells), suggesting that other host-cell receptors besides fibronectin may also have a role in GBShost-cell interactions. Once GBS are bound to HeLa cells, HeLa cell microvilli entwine the bacteria, which enter the HeLa cell in a polar fashion. Host-cell cytoskeletal actin is involved as this process is disrupted by cytochalasin D, and recruitment of actin is visible at the site of adherent chains of GBS on the HeLa cell. Also, the host-cell signalling enzyme, PI 3-kinase, is involved in the GBS internalization event as the event can be inhibited by wortmannin.
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ACKNOWLEDGEMENTS |
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We thank Glen D. Armstrong for critical review of the manuscript.
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Received 20 May 2002;
revised 15 August 2002;
accepted 5 September 2002.