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Infection and Immunity, October 1998, p. 4932-4941, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Characterization of Group B Streptococcal Invasion of Human Chorion and Amnion Epithelial Cells In Vitro

Scott B. Winram,1 Mechthild Jonas,2 Emil Chi,2 and Craig E. Rubens1,*

Department of Pediatrics, Division of Infectious Diseases, Children's Hospital and Regional Medical Center, University of Washington, Seattle, Washington 98105,1 and Department of Pathology, School of Medicine, University of Washington, Seattle, Washington 981952

Received 22 May 1998/Returned for modification 23 June 1998/Accepted 24 July 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Group B streptococci (GBS) have been cultured from the chorioamnionic membrane of pregnant women, usually in association with chorioamnionitis and premature labor (K. A. Boggess, D. H. Watts, S. L. Hillier, M. A. Krohn, T. J. Benedetti, and D. A. Eschenbach, Obstet. Gynecol. 87:779-784, 1996). Colonization and infection of placental membranes can be a prelude to neonatal GBS infections even in the presence of intact membranes (R. L. Naeye and E. C. Peters, Pediatrics 61:171-177, 1978), suggesting that GBS cause chorioamnionitis or establish amniotic fluid infections by partial or complete penetration of the placental membranes. We have isolated and grown cultures of primary chorion and amnion cells from human cesarean-section placentas. This has provided a biologically relevant model for investigating GBS adherence to and invasion of the two epithelial barriers of the placental membrane. GBS adhered to chorion cell monolayers to a high degree. Pretreatment of GBS with trypsin reduced adherence up to 10-fold, which suggested that the bacterial ligand(s) was a protein. GBS invaded chorion cells at a high rate in vitro, and invasion was dependent on cellular actin polymerization. GBS could be seen within intracellular vacuoles of chorion cells by transmission electron microscopy. We also demonstrated that GBS were capable of transcytosing through intact chorion cell monolayers without disruption of intracellular junctions. GBS also adhered to amnion cells; in contrast, however, these bacteria failed to invade amnion cells under a variety of assay conditions. GBS interactions with the chorion epithelial cell layer shown here correlate well with epidemiological and pathological studies of GBS chorioamnionitis. Our data also suggest that the amnion cell layer may provide an effective barrier against infection of the amniotic fluid.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Streptococcus agalactiae strains or group B streptococci (GBS) are the leading cause of bacterial pneumoniae, sepsis, and meningitis in neonates. GBS are also a major cause of bacteremia in pregnant women and immunocompromised adults (4). Colonization of the human rectovaginal tract with GBS is a risk factor associated with chorioamnionitis, premature rupture of the placental membrane, and transmission to the infant (3, 4, 34). Amniotic fluid infections have occurred through apparently intact chorioamnionic membranes (31), and it has been suggested that the organism may actively invade through intact placental membranes and grow to high concentrations in the amniotic fluid (9, 46). Neonatal exposure to high concentrations of GBS in utero leads to colonization of the lung airways and subsequent pneumonia, sepsis, and meningitis (38).

The chorioamnion is a multilayered structure comprised of a monolayer of chorion epithelial cells that line the maternal side of the placenta, a thick proteinaceous stromal layer sandwiched between the epithelial cell basement membranes, and a monolayer of amnion epithelial cells that line the neonatal side of the membrane. There has been little work examining GBS interactions with the chorioamnionic membrane in a quantitative and comprehensive manner. Recently, our laboratory has applied tissue culture techniques to grow primary human chorion and amnion cells isolated from term cesarean-section placentas. This has enabled us to study the adherence and invasion properties of GBS in the laboratory with a biologically relevant model system. We found that GBS adhered to the chorion cell surface, invaded, and survived within these cells long enough to transcytose through intact chorion cell monolayers. GBS adhered to primary amnion cells, consistent with previous reports (15), but failed to invade, suggesting that these cells may provide a barrier to penetration of the amniotic membrane.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial strains and growth conditions. GBS strain A909 is a type Ia polysaccharide clinical isolate (27); GBS strain COH1 is a type III polysaccharide clinical isolate (28). Type Ia and type III polysaccharide GBS isolates were used in these studies because they cause a significant proportion of GBS disease. The capsule-negative COH1::Delta cpsA mutant contains a kanamycin resistance gene in the cpsA gene (37). GBS were grown in Todd-Hewitt (TH) broth and aerated at 37°C. Streptococcus gordonii Challis (26) was grown as nonaerated cultures in TH medium at 37°C. Escherichia coli DH5alpha [F- phi 80dlacZDelta M15 recA1 endA1 gyrA96 thi-1 hsdR17 (rK- mK-) supE44 relA1 deoR Delta (lacZTA argF) U169] was grown in aerated Luria-Bertani (LB) medium at 37°C.

Chorion cell and amnion cell isolation and growth conditions. Normal, term placentas of cesarean-section procedures were obtained immediately following delivery from Swedish Medical Center (Seattle, Wash.). The isolation of chorion and amnion cells was based on modifications of previously described protocols (6, 10, 25). The chorioamnionic membrane was cut from the placenta; aseptically placed in phosphate-buffered saline (PBS), pH 7.5, which contained penicillin (10 µg/ml), streptomycin (100 µg/ml), vancomycin (40 µg/ml), and amphotericin B (2.5 µg/ml); and incubated for 20 min at room temperature. The chorion was separated from the amnion by blunt dissection. The respective layers were cut into 1- by 5-cm strips and placed into separate 150- by 15-mm sterile petri dishes which contained Dulbecco modified Eagle (DME)-Ham's F-12 medium with 0.5% (wt/vol) trypsin (1:250; trypsin T4799; Sigma Chemical Co., St. Louis, Mo.) and 100 µM EDTA. Petri dishes containing the membrane strips were then incubated for 45 min at 37°C under 5% CO2. Membrane strips were then placed in fresh DME-Ham's F-12-trypsin-EDTA solution, and incubation was continued for an additional 1.5 and 2.5 h for amnion and chorion strips, respectively. Membrane tissues were then transferred to individual 50-ml conical centrifuge tubes which contained DME-Ham's F-12 medium with 5% fetal bovine serum that had been heat treated for 10 min at 65°C. The conical tubes were vortexed thoroughly to release cell monolayers from the basement membranes, the monolayers were transferred to new 50-ml conical tubes, and cells were pelleted by centrifugation at 1,500 × g for 10 min at 6°C. Both amnion and chorion cell pellets were individually suspended in fresh cell growth medium. Cell growth medium contained DME-Ham's F-12 medium with 5% (vol/vol) heat-treated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (2.5 µg/ml), 1× ITS (mixture of 5 µg of insulin per ml, 5 µg of transferrin per ml, and 5 ng of selenium per ml; Sigma Chemical Co.), 10 ng of epidermal growth factor (Sigma Chemical Co.) per ml, and 2 mM L-glutamine.

Cells were seeded into 75-cm2 tissue culture-treated flasks or 24-well tissue culture-treated plates at a density of 105 cells/cm2 and incubated at 37°C under 5% CO2. Cell growth medium was changed every 3 days. Cell monolayers generally reached confluence within 5 days.

Electron micrographs. Transmission electron microscopy of cells infected with GBS was performed as described previously (39).

Adherence assays. Assessment of bacterial attachment to cell monolayers was performed by a modification of a previously described protocol (42). Bacteria were radiolabeled while at exponential growth phase in either methionine- or leucine-deficient medium that contained [35S]methionine (Trans label; ICN Pharmaceuticals, Costa Mesa, Calif.) or [3H]leucine (Amersham Life Sciences, Inc., Arlington Heights, Ill.), respectively. Radiolabeled bacteria were then washed with PBS, pelleted by centrifugation, suspended in TH or LB medium with 30% glycerol, and frozen in aliquots at -70°C.

Chorion or amnion cell monolayers were grown to confluence in 24-well tissue culture plates (as described above) and washed four times with PBS. Five hundred microliters of PBS that contained 1.0% (wt/vol) bovine serum albumin (BSA-PBS) was added per well, and plates were incubated at 4°C for 2 h.

One milliliter of TH medium was added to each thawed aliquot of radiolabeled bacteria and incubated at 37°C for 15 min. Bacteria were then pelleted by centrifugation, washed once in PBS, and centrifuged again, and bacterial pellets were suspended in BSA-PBS and placed on ice for 2 h.

The BSA-PBS was aspirated off cell monolayers, and dilutions of bacteria in 200-µl volumes of BSA-PBS were added. Bacteria were brought into contact with the cell surface by centrifugation at 800 × g for 10 min followed by a 2-h incubation at 4°C. Plates were then washed six times with PBS and vortexed thoroughly each time. Two hundred microliters of 2 M NaOH was then added to each well and incubated for 20 min at 65°C to lyse cells and adherent bacteria. Fifty microliters of the lysate suspension was added to Ready-Caps (Beckman Instruments, Inc., Fullerton, Calif.) for determination of cell-associated counts per minute of radioactivity with a Beckman LS6001C scintillation counter (Beckman Instruments, Inc.).

Background activity due to nonspecific adherence of bacteria was determined by including an identically treated control plate that contained no chorion or amnion cells. The level of nonspecific binding of bacteria to the plastic tissue culture wells was less than 1% of the specific adherence values.

A CFU/counts per minute ratio was derived for each dilution of inoculum, and the total cell-associated counts per minute per well was converted into CFU bound per well. All samples were assayed in triplicate, and each assay was repeated at least three times.

Adherence assays which included protease-treated GBS were performed by incubating radiolabeled bacteria in 10 µg of erythromycin per ml for 15 min at 37°C to terminate bacterial growth. Bacteria were pelleted by centrifugation and suspended in 0.1 M NaCl-0.025 M Tris-HCl (pH 8.0). One thousand units of trypsin was added per ml of bacterial suspension. At the indicated time points, a 3 M excess of soybean trypsin inhibitor was added. Trypsin and soybean trypsin inhibitor were added to the control sample at time zero. Bacteria were washed once with PBS and suspended in 5% BSA-PBS, and the standard adherence assay was performed as described above. Counts per minute were derived for each GBS sample following the trypsin incubation to derive CFU/counts per minute ratios. Background activity was monitored as described above. All samples were assayed in triplicate.

Invasion assays. Quantitative determinations of intracellular bacteria were performed by a modification of the gentamicin-penicillin protection assay procedure described by Rubens et al. (39). Primary cell cultures were grown to confluence in 24-well tissue culture-treated plates as described above. Cell monolayers were washed three times with sterile PBS, and then 340 µl of cell growth medium without antibiotics or fungicide was added per well. Stationary-phase cultures of bacteria were pelleted by centrifugation, washed once with sterile PBS, and suspended in chorion cell growth medium. Unless otherwise indicated, bacterial suspensions were diluted so that approximately 5 × 105 CFU in a 60-µl volume was added per well. Initial contact of the bacteria with the cell monolayer was aided by centrifugation at 800 × g for 10 min, at 6°C. Plates were then incubated at 37°C, in 5% CO2, for 2 h.

Extracellular bacteria were removed by washing cell monolayers four times with PBS. Five hundred microliters of cell growth medium, which contained 10 µg of penicillin per ml and 100 µg of gentamicin per ml, was added to each well to kill adherent extracellular bacteria. Plates were again incubated at 37°C, in 5% CO2, for 2 h. The medium was gently aspirated off the cell monolayers, and the cells were subsequently washed five times with sterile PBS.

Two hundred microliters of Hank's balanced salt solution containing 100 N-alpha -benzoyl-L-arginine ethyl ester units of trypsin and 400 µM EDTA solution (1×; Sigma Chemical Co.) was added per well, and the plates were incubated at 37°C for 30 min to release the adherent cell layer. To disrupt chorion cell integrity, 800 µl of 0.025% (vol/vol) Triton X-100 was added per well and the plates were incubated at 37°C for 30 min. To accelerate cell lysis, samples were drawn up and down in a 1-ml pipette, added to individual microcentrifuge tubes, and vortexed thoroughly for at least 12 s each. Fifty-microliter aliquots of the lysates were spread onto TH or LB agar plates to determine the number of intracellular CFU.

The total number of intracellular bacteria was divided by the inoculum CFU and multiplied by 100 to determine the percent invasion of the inoculum. All samples were assayed in triplicate.

To determine cellular processes required for GBS uptake by chorion cells, both the chorion cells and the inoculum bacteria were incubated with chemical inhibitors for 30 min. Cycloheximide, cytochalasin D, and colchicine were used at final concentrations of 50, 0.5, and 50 µg/ml, respectively. These concentrations of inhibitors had no effect on bacterial viability (data not shown).

Assays that measured intracellular survival of GBS over time were performed by a modification of the chorion cell invasion assay. As described above, extracellular penicillin-gentamicin was added to all cell monolayers at 2 h postinfection to kill extracellular bacteria. However, chorion cells were then washed and lysed at multiple time points to determine intracellular CFU over a 24-h period.

For experiments that examined GBS invasion of EGTA-treated amnion cells, both amnion cells and bacteria were incubated in cell growth medium that contained various concentrations of EGTA for 30 min prior to inoculation of bacteria onto cell monolayers. There was no reduction in GBS viability with concentrations of EGTA up to 10 mM (data not shown).

Transcytosis assays. Transwell membrane inserts (Costar, Cambridge, Mass.) with 3.0-µm pores were seeded with 3 × 105 chorion cells in 100 µl of cell growth medium onto the apical side of each Transwell insert. Inserts either had been collagen coated by the manufacturer (no. 3492 inserts) or were coated (no. 3452 inserts) with rat tail collagen isolated by a modification of the procedure of Cereijido et al. (5). One milliliter of cell growth medium was added to each bottom well of the 24-well plates which held the inserts. The medium was changed every 3 days, and chorion cells were cultured for at least 9 days prior to each experiment. Sixteen hours prior to an assay, chorion cell layers were washed three times with sterile PBS and incubated in chorion cell growth medium without antibiotics at 37°C under 5% CO2. Stationary-phase bacterial cultures were used in all assays. Bacteria were washed once with PBS, pelleted by centrifugation, and suspended in cell growth medium. Approximately 5 × 106 CFU each of GBS and E. coli DH5alpha , added to monitor monolayer integrity, was applied simultaneously to the apical side of the same chorion cell monolayer. At the indicated time points, the membrane inserts were aseptically transferred to a new 24-well plate containing fresh cell growth medium prewarmed to 37°C. The basal media of the previous wells were sampled immediately to eliminate growth in the well as a contributing factor to quantitation of CFU. Aliquots from the bottom wells were spread onto TH and LB agar and incubated overnight at 37°C to assess the quantity of bacteria that had penetrated through to the basolateral side of the chorion cell monolayer. E. coli DH5alpha and GBS were readily distinguishable from each other on the basis of colonial morphology, and the CFU of each strain was determined. As an additional method of monitoring monolayer integrity in some experiments, 6 × 106 cpm of [14C]dextran (Amersham Life Sciences, Inc.) also was added to the apical side of the chorion cell layer at time zero. The counts per minute of [14C]dextran recovered from the basal side of the cell monolayers was determined with a Beckman LS6001C scintillation counter (Beckman Instruments) and Ready Caps (Beckman Instruments) as scintillant. The percentage of [14C]dextran recovered over time was determined by dividing the total counts per minute detected in the medium on the basal side of the membrane by the total counts per minute added to the apical side and multiplying by 100.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Based on pathological and epidemiological studies as well as our prior observations of GBS invasion of eukaryotic cells, we hypothesized that penetration of the intact chorioamnionic membrane was a potential route for GBS to access the amnionic cavity. Primary human chorion and amnion epithelial cells were isolated; monolayers were grown to confluence in culture; and in vitro studies which examined the ability of GBS to adhere to, invade, and transcytose these physiologically relevant barriers were performed.

Growth of primary chorion and amnion cells in vitro. Monolayers of primary chorion and amnion cells were isolated and grown in 24-well tissue culture-treated plates and Transwell membrane inserts for all experiments performed in these studies. The purity of the cell cultures was determined by light microscopic observation, and typical cellular morphology (19) was also confirmed for several experiments by transmission electron microscopy (Fig. 1 and its legend). Preparations usually contained no more than 2% fibroblasts, based on morphology, and a single passage of the cells generally eliminated fibroblasts. Preparations which contained excessive fibroblasts were discarded. Cells in culture were passaged only once.


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  		FIG. 1.   Electron micrographs of uninfected human chorion cells and amnion cells. Monolayers of primary cells were grown in vitro on collagen-coated membranes and prepared for transmission electron microscopy as described in Materials and Methods. Chorion cells were oblong in shape whereas amnion cells were more cuboidal and uniform in size (19). The dark spheres within cells were presumably lipid droplets or secretory granules. (A) Chorion cell (magnification, ×5,000); (B) chorion cell (magnification, ×7,500); (C and D) amnion cells (magnification, ×6,000). AS, apical surface; BS, basal surface; N, nucleus; CM, cytoplasmic membrane.

GBS adherence to chorion cells. GBS adherence to the chorion cell layer is a prerequisite for chorioamnion colonization and for potential invasion of the host chorion cell. Adherence assays were performed with radiolabelled bacteria and primary human chorion cells grown in vitro. In the results from the representative experiment shown in Fig. 2A, two strains of GBS, A909 and COH1, adhered to monolayers of chorion cells over a broad range of inocula (Fig. 2A). A maximum of 1.8 × 106 CFU of GBS strain COH1 bound per well (0.92%), approximately fivefold more than the 3.6 × 105 CFU of strain A909 (0.36%), which suggested strain differences in numbers of receptors expressed and/or receptor types. A maximum of 2.0 × 105 CFU (0.38%) and 1.4 × 106 CFU (3.4%) of S. gordonii and E. coli DH5alpha , respectively, per well bound to chorion cells. The high levels of adherence by S. gordonii and laboratory strain E. coli DH5alpha were consistent with prior studies which demonstrated that S. gordonii adheres to a variety of cell types (23) and that clinical isolates of E. coli attach to the chorioamnion in vitro (12).


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  		FIG. 2.   GBS strains A909 and COH1, S. gordonii, and E. coli DH5alpha adherence to primary human chorion cells. (A) Dilutions of radiolabeled bacteria were added to monolayers of primary chorion cells (CFU offered) and assessed for adherence (CFU bound) as described in Materials and Methods. (B) Adherence of GBS to chorion cells was decreased by trypsin treatment of GBS. GBS were incubated in the presence of trypsin for the periods indicated on the x axis prior to the addition of trypsin inhibitor. Trypsin inhibitor was added immediately to the time zero sample. The time zero sample was normalized to 100% adherence. Error bars indicate standard deviations; n = 3 for all experiments.

The adherence assay results varied up to 2.5-fold between placental cell preparations; however, relative binding trends among GBS strains and the other bacterial species remained qualitatively consistent. There was no visible change in chorion cell morphology following the incubation and subsequent washing steps of the procedure. Additionally, the presence of divalent cations (Ca2+ and Mg2+) during incubation and wash steps of the adherence assay had no effect on the levels of GBS adherence to chorion cells (data not shown). These studies demonstrated that a broad range of bacterial species are able to adhere to these cells.

To examine whether the GBS ligand(s) responsible for the adherence phenotype was a proteinaceous component(s) rather than another molecule(s) such as capsule, bacteria were incubated in the presence of trypsin prior to exposure to chorion cell monolayers. After a 5-min incubation with trypsin, adherence of GBS strain A909 was reduced by 87% from that of untreated A909 (Fig. 2B). Longer incubation times did not result in further reduction of adherence. Only the 1-h treatment was performed for strain COH1, which resulted in a 75% reduction of adherence to the chorion cells relative to that of untreated COH1. However, a capsule-negative isogenic mutant of COH1 retained levels of adherence equivalent to those of the parent strain (data not shown). These results indicated that the GBS ligand(s) responsible for the majority of chorion cell adherence is attributable to a GBS surface protein(s).

GBS invasion of chorion cells. Subsequent to bacterial attachment, invasion into chorion cells could enable the bacteria to penetrate the first protective layer of the chorioamnion, evade the host response, and potentially establish an infection of the stroma between epithelial linings. Following inoculation of GBS onto the apical surface of chorion cell monolayers, antibiotic protection assays revealed viable GBS in the chorion intracellular compartment (Fig. 3A). Up to 6.0 and 3.1% of the inoculum of strain A909 and COH1, respectively, were recovered with an inoculum of 2.4 × 105 CFU per well. Neither E. coli DH5alpha , a bacterium known to be noninvasive of other cell types, nor S. gordonii, a gram-positive bacterium which is an oral pathogen, invaded to a significant degree in these experiments (Fig. 3A), even though both species were shown to adhere to chorion cells (Fig. 2A). Growth rates of S. gordonii in tissue culture medium were equivalent to those of GBS over the 2-h incubation period of the invasion assay (data not shown), eliminating the possibility that low invasion by S. gordonii was due to reduced bacterial viability. When the inoculum was diluted serially (Fig. 3B), 6% (673 CFU) of A909 was recovered at a 104-CFU inoculum compared to 1.7% (2.2 × 103 CFU) and 0.1% (1.1 × 103 CFU) of the 106- and 107-CFU-per-well inocula, respectively. In contrast, the corresponding inocula of 104, 106, and 107 CFU of S. gordonii per well yielded only 0.03% (3 CFU), 0.007% (233 CFU), and 0.007% (670 CFU) of the intracellular CFU recovered, respectively. These data indicated that invasion by A909 of chorion cells was saturable, whereas S. gordonii invaded poorly regardless of the inoculum.


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  		FIG. 3.   GBS invaded primary human chorion cells by a microtubule-dependent process and were able to survive intracellularly for at least 24 h. GBS strains A909 and COH1, S. gordonii, and E. coli DH5alpha were inoculated onto confluent chorion cell monolayers, and invasion assays were performed as described in Materials and Methods. (A) Invasion assay was performed with bacterial inocula of 2.4 × 105 CFU per well. The percent invasion was defined as the number of CFU recovered from cellular lysates divided by the initial inoculum multiplied by 100. (B) Invasion assay performed with the indicated 3-log-fold range of inocula. The y axis indicates the total intracellular CFU recovered per well of chorion cells. (C) Invasion assay performed with GBS strain A909 and chorion cells that had been preincubated with the indicated inhibitors. The percent invasion by GBS of chorion cells incubated with medium only was normalized to 100%. (D) GBS intracellular survival in chorion cells. The total intracellular CFU recovered per well is indicated on the y axis. Error bars indicate standard deviations; n = 3 for all experiments.

The degree of GBS invasion was variable from strain to strain, but invasion was a shared GBS phenotype. Saturation of GBS invasion and variable invasive ability among strains have been previously observed for other cell types (13, 39). Interestingly, in this system, there was no correlation between the ability to adhere to chorion cells and invasion. Along with the observation that S. gordonii adheres but does not invade, these data suggested that there may be unique factors expressed by GBS for adherence and invasion which trigger bacterial entry into chorion cells.

The quantity of intracellular CFU varied among experiments, as certain batches of primary chorion cells from different placentas were more susceptible to bacterial invasion than were others. However, a minimum of 1 to 4% of GBS was routinely recovered with an inoculum of 2.4 × 105 CFU per well, and recovery was always significantly greater than that with either negative control strain.

Studies that compared invasion of GBS strains A909 and COH1 and control strains E. coli DH5alpha and S. gordonii in the presence of fresh growth medium, growth medium preincubated with chorion monolayers for 16 h, or human amniotic fluid (Sigma Chemical Co.) demonstrated no differences in invasion rate (data not shown). Furthermore, the addition of human epidermal growth factor to the assay also had no effect on the rate of GBS invasion. These data suggested that secreted cellular factors may not stimulate bacterial uptake by chorion cells or induce GBS factors required for invasion.

To examine the cellular processes important for GBS invasion of chorion cells, cells were pretreated with either cycloheximide, colchicine, or cytochalasin D to inhibit protein synthesis, microtubule formation, or actin polymerization, respectively. As shown previously, the inhibitors did not affect GBS viability at the concentrations used (39), nor did the inhibitors affect monolayer integrity. Cycloheximide had no significant effect on invasion by GBS strain A909 (Fig. 3C) or strain COH-1 (data not shown), which suggested that active cellular protein synthesis was not required for GBS invasion. A 31% reduction of invasion in the presence of colchicine suggested a partial requirement for microtubules, but microtubules certainly were not essential. In contrast, treatment with cytochalasin D abolished invasion by both GBS strains, indicating that actin polymerization was essential for GBS uptake by chorion cells.

We examined whether GBS could persist or replicate within chorion cells following invasion, although this phenotype has not been shown for other cell types (33, 39, 43, 45). GBS viability within chorion cells was assessed over a 24-h period (Fig. 3D) in a standard antibiotic protection assay modified to allow sampling of wells incubated in the presence of antibiotics over time. Intracellular CFU increased 4.1- and 1.7-fold for strain A909 and strain COH1, respectively, during the first 9 h postinvasion, followed by a decrease in intracellular bacterial CFU from 9 to 24 h. It was possible that GBS were exocytosing through the apical side of the cell membrane, so that the decrease in recovered CFU was due to loss of intracellular bacteria rather than to intracellular bacterial death. The results indicated that GBS could survive and undergo limited replication intracellularly; however, GBS probably do not reside or survive within chorion cells for long periods of time.

In addition to the recovery of live intracellular bacteria from chorion cells by the antibiotic protection assay, GBS were also visually identified within vacuoles of infected cells via transmission electron microscopy (Fig. 4). Figure 4A shows two adherent GBS bacteria which appear to be undergoing phagocytosis by a chorion cell. The appearance of the chorion cell membrane engulfing bacteria was consistent with the experimental requirement for actin polymerization during bacterial uptake. There were several bacteria in the single vacuole shown in Fig. 4B, which could be indicative of the limited intracellular replication demonstrated by the experiment whose results are shown in Fig. 3D.


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  		FIG. 4.   Infected chorion cells contained intracellular GBS. Monolayers of primary cells were grown in vitro on collagen-coated membranes. The transcytosis assay was performed with GBS as the inoculum. Chorion cells were then fixed and prepared for transmission electron micrographs as described in Materials and Methods. (A) Apical surface of chorion cell that appeared to be engulfing two GBS (magnification, ca. ×4,000); (B) several GBS within a single intracellular vacuole (magnification, ca. ×15,000); (C) two vacuoles containing GBS midway through a single chorion cell (magnification, ca. ×4,000); (D) two intracellular GBS near the apical surface of a chorion cell (magnification, ca. ×15,000). Adherent and intracellular GBS are indicated by the arrows.

The ability of GBS to efficiently enter into the chorion cell layer of the placenta and survive is potentially the first step in establishing chorioamnionitis.

GBS transcytosis of chorion cells. The demonstration that GBS survive intracellularly for at least 24 h, together with the observation of GBS within vacuoles throughout chorion cells (Fig. 4C and D), led us to hypothesize that GBS may be able to traverse a chorion cell monolayer without disruption of intracellular junctions. This phenotype had been reported for GBS in the strain I MDCK transformed cell line (24). Such a mechanism would enable the bacteria to reach the basement membrane and stromal layers of the chorioamnionic membrane in vivo. Approximately 5 × 106 CFU of E. coli DH5alpha , a noninvasive control (Fig. 3A), was coinoculated onto the same monolayers as an equivalent inoculum of GBS to evaluate monolayer integrity during the assay (11). In some experiments, [14C]dextran was added to the apical side of the monolayer at time zero, in addition to E. coli DH5alpha , as a second control for cell monolayer integrity (20). A control well, indicated by a plus sign in Fig. 5A, did not contain chorion cells and demonstrated that the majority of [14C]dextran passed through the membrane pores within the first hour. Similarly, GBS and E. coli DH5alpha immediately traversed a membrane-only control as well (data not shown).


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  		FIG. 5.   GBS penetrated intact chorion cell monolayers. Chorion cells were grown to confluence on collagen-coated porous membrane inserts placed in 24-well tissue culture plates. GBS and E. coli DH5alpha were inoculated simultaneously into the apical side of the chorion cells. At the indicated times, the membranes were transferred to wells which contained fresh medium and aliquots of medium were assessed for CFU. (A) Representative data samples of transcytosis assays where [14C]dextran was added at time zero simultaneously with GBS and E. coli DH5alpha . The control well contained no chorion cell layer, and only [14C]dextran was added. (B) Representative data samples from transcytosis assays which examined GBS strain A909 and E. coli DH5alpha . (C) Representative data samples of transcytosis assays which examined GBS strain COH1 and E. coli DH5alpha .

As early as 4 h postinoculation, 2.1 × 103 CFU of strain A909 was recovered from the basal medium of one well, which increased to 1 × 104 CFU by 7 h (Fig. 5A). In a second well, a maximum of 2 × 104 CFU of strain A909 translocated by 8 h postinoculation. Similarly, 120 CFU of strain COH-1 was detected at 6 h and reached a maximal translocation rate of 1 × 104 by 10 h. The minimal (1% or less) [14C]dextran that was detected at each time point (Fig. 5A) and the absence of E. coli DH5alpha recovered (data not shown in Fig. 5A) indicated that the chorion cell intracellular junctions remained intact. Thus, GBS were able to transcytose through an intact cell monolayer.

Representative samples from a different experiment with GBS strain A909 and E. coli DH5alpha or strain COH1 and E. coli DH5alpha are shown in Fig. 5B and C, respectively. In these experiments, 960 CFU of strain A909 was detected on the basolateral side of the membrane at 6.5 h postinoculation, and transcytosis was detected in all wells by 8 h with a maximum of 3.2 × 104 CFU recovered at 10 h (Fig. 5B). Similarly, in Fig. 5C, strain COH1 demonstrated transcytosis through intact cell monolayers by 4 h postinoculation. Two of the four samples shown in Fig. 5C contained just under 100 CFU in the basal medium, which increased to 103 CFU by 6 h, and yielded a maximum of 1.0 × 104 CFU (well 1) at 10 h. E. coli DH5alpha , represented by the filled symbols in Fig. 5B and C, was not detected on the basal side of these intact chorion monolayers.

In summary, GBS could be detected on the basal side of a chorion cell monolayer by 3 to 5 h postinoculation with maximal CFU (up to 3.4 × 104 CFU) recovered by 7 to 10 h. Variations in the degree of GBS transcytosis from well to well, and experiment to experiment (Fig. 5), may be attributable to the use of primary cells which were isolated from different placentas for each experiment. These experiments were repeated eight times. The ability of GBS to invade and transcytose across chorion cells, in vitro, may be a mechanism which the bacterium utilizes to penetrate through the chorion epithelium in vivo to establish infection within the chorioamnionic membrane.

GBS interaction with primary amnion cells. The amnion epithelial cell monolayer is the final barrier to the amnionic fluid encountered by an invading pathogen. Studies which examined the interaction of GBS with primary amnion cells were performed by the methods described above for chorion cells. Adherence assays revealed that, at the lowest inoculum of 1.8 × 106 CFU, 25% (4.4 × 105 CFU) of GBS strain COH1 bound to the amnion cell surface and 4.4% (6.4 × 104 CFU) of strain A909 bound when 1.5 × 106 CFU was added in the experiment whose results are shown in Fig. 6A. Strain COH1 had a 10-fold-greater level of adherence to amnion monolayers than did strain A909 in the same experiment, with maximum binding values of 4.3 × 106 CFU and 4.2 × 105 CFU, respectively. Similar to adherence of GBS to chorion cells, these same strains adhered to primary human amnion cells, in vitro.


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  		FIG. 6.   GBS were adherent but did not invade amnion cells. (A) GBS strains A909 and COH1, S. gordonii, and E. coli DH5alpha adhered to primary human amnion cells. Primary amnion cells were isolated and grown to confluence in 24-well tissue culture plates. Dilutions of radiolabeled bacteria were added to monolayers and assessed for adherence as described in Materials and Methods. (B) GBS did not invade amnion cells to a significant degree. Primary amnion cells were grown to complete or partial confluence as indicated. Cells were preincubated either in medium alone or with the final concentration of EGTA in medium as indicated on the x axis. The percentage of intracellular CFU recovered per well is indicated on the y axis. Error bars indicate standard deviations; n = 3 for all experiments.

Surprisingly, however, even though GBS adhered to these cells, there was no significant invasion by GBS of confluent primary amnion cell monolayers (Fig. 6B). The negative controls, S. gordonii and E. coli DH5alpha , also did not significantly invade these cells. Preconditioning amnion cells with human amniotic fluid or tissue culture medium for 16 h had no significant effect on the number of intracellular bacteria recovered (data not shown). It is possible that adherence and invasion are independent processes and that amnion cells lack the molecule(s) required to trigger cellular uptake. Alternatively, the cellular receptor used by GBS for chorion cell adherence may be different from the receptor for amnion cell attachment. If so, GBS may not bind to amnion cells with avidity high enough to activate cellular phagocytosis.

GBS would normally encounter the basolateral surface of amnion cell monolayers during a natural invasive infection. Therefore, the gentamicin-penicillin protection assays were also performed with nonconfluent monolayers of amnion cells and cells pretreated with EGTA to investigate basolateral entry as has been performed previously to examine Shigella flexneri invasion (29). At concentrations of 1 mM EGTA, amnion cell junctions became visibly detached from each other, but cells remained adherent to the tissue culture wells throughout the assay in up to 10 mM EGTA. Semiconfluent monolayers also remained intact during the assay. S. gordonii and E. coli DH5alpha were again used as negative control strains in these experiments. The concentrations of EGTA used had no effect on GBS viability; however, the increased amnion cell surface area did not increase the degree of intracellular invasion by GBS. Strains A909 and COH1, as well as the negative control strains, were unable to enter amnion cells under any condition tested. These data suggest that amnion epithelial cells provided an effective barrier to GBS entry. It is therefore conceivable that an intact amnion monolayer could be an effective initial barrier to GBS infection of the amniotic fluid in vivo.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

GBS are a causative agent of premature rupture of placental membranes and chorioamnionitis, which can result in amniotic fluid infections and neonatal sepsis (4). Risk for development of chorioamnionitis and the incidence of neonatal sepsis and meningitis increase with the degree of GBS colonization (47, 48). Furthermore, GBS have been cultured from under the chorion epithelial cell layer in placental membranes of almost 20% of patients with chorioamnionitis (3, 30). Neonatal deaths which have resulted from bacterial amniotic fluid infections can occur with apparently intact chorioamnionic membranes (31). Several investigators have hypothesized that GBS may actively invade through the placental membrane, resulting in growth of the organism in the amniotic fluid (9, 30, 46); however, this has not been confirmed to date.

A wide assortment of transformed epithelial and endothelial cell lines have been shown to be susceptible to GBS invasion (24, 33, 39, 43, 45), and GBS have been observed within lung epithelial, endothelial, and fibroblast cells of infected Macaca nemestrina primates (38). Additionally, a recent study reported that GBS was able to translocate through strain I MDCK epithelial cells (24). These data coupled with epidemiological and histopathological studies of chorioamnionitis lead us to speculate that GBS may be able to invade and transcytose through the two epithelial cell layers of the human placenta.

The chorion and amnion epithelial cells form the outer and inner layers of the chorioamnion, respectively. These layers are separated by a collagen-rich stroma and by basement membranes. We felt that it was crucial to examine GBS-chorioamnion interaction with each epithelial layer independently to understand the distinct interactions of the organism with each cell type. Our laboratory has applied tissue culture techniques to grow primary chorion and amnion cells from the human placental membrane based on previously described methods (6, 10, 25). In this report, we have characterized the adherence and invasive properties of GBS with these biologically relevant cell types rather than using transformed cell lines, which may have altered or atypical phenotypes, or the intact chorioamnionic membrane.

In vitro studies have been performed with sections of whole placental membrane, mounted in chambers, to examine effects of GBS on membrane integrity and bacterial penetration of the chorioamnionic membranes (7, 12, 16, 40, 41, 46). However, it is difficult to distinguish between gross membrane damage and specific bacterial penetration in the intact membrane studies due to the multilayered structure of the chorioamnionic membrane, the extremely high inocula used, and the long incubation times. Cells grown in vitro are easier to manipulate, and bacterial quantitation is less difficult. Scraping chorion cells off the chorioamnion basement membrane has been another technique used to examine bacterial adherence to cells in suspension (17). However, scraping may damage the chorion cells, and the cells do not polarize in suspension and may not express receptors similar to those of polarized cells. In our studies, primary cell monolayers grown in vitro appeared to provide undamaged, intact apical monolayer surfaces.

Colonization of host tissues is the initial phase of bacterial and host interaction, and GBS have been shown to adhere to intact chorioamnionic membranes in vitro by electron microscopy (12). The adherence assay performed here demonstrated that primary human chorion cells grown in vitro also provide an analogous colonizable surface for this organism (Fig. 2A). Furthermore, a significant reduction in adherence of trypsin-treated GBS to chorion cells indicated that surface protein(s) or another molecule(s) linked to a surface protein of GBS plays a role in cellular adherence. Although divalent cations have been shown to be required for adherence of some species of bacteria to integrin proteins (22), the presence of divalent cations had no effect on GBS adherence to chorion cells.

Upon attachment to the chorion cell layer, all GBS strains tested subsequently invaded the chorion cells as determined by the antibiotic protection experiments. Uptake of GBS by chorion cells was dependent upon active actin polymerization by the cells, and transmission electron micrographs indicated that intracellular GBS were within vacuoles. There was no correlation between GBS adherence to chorion cells and the degree of invasion by the strains used in this study. Therefore, the possibility remains that more than one surface structure or mechanism may be required for adherence and invasion, similar to what has been observed for Salmonella typhimurium (14).

A previous study concluded that GBS strains isolated from infected adults and neonates showed increased cellular invasion in a transformed epithelial cell line relative to that by isolates from colonized adults and neonates (44). We have found differences in chorion cell invasion rates among different GBS strains. However, preliminary studies in our laboratory comparing GBS isolated from mother and infant pairs have not yet detected differences in chorion cell invasion rates (data not shown). This would suggest that a more invasive subset of a single population is not necessarily induced by exposure to the chorioamnion.

Intracellular survival of GBS in chorion cells and limited intracellular bacterial replication were consistent with results of studies which have used transformed cell lines (39, 45). Exocytosis of GBS to the antibiotic-containing supernatant, leakage of the extracellular antibiotics, decreased bacterial access to nutrients, or modest intracellular killing by chorion cells may account for the slow decrease in CFU recovered over time. This latter observation may be less critical since transcytosis usually began within 3 to 5 h of apical contact.

The transcytosis experiments demonstrated that GBS can penetrate the chorion cell layer without disruption of the cellular junctions which provide monolayer integrity. There was variability in the time required for GBS to traverse replicate chorion cell monolayers from the same placenta within one experiment. Additionally, the degree of transcytosis by GBS was highly variable among various chorion cell preparations, ranging from no translocation to the maximum levels of 3.4 × 104 CFU per hour, suggesting that specific host cell factors may influence invasion and transcytosis.

The final epithelial barrier of the chorioamnionic membrane for GBS to penetrate is the amnion cell layer. Similar to GBS adherence to chorion cells, these bacteria specifically attached to primary human amnion cells, which is consistent with the results of previous studies (15, 32). Interestingly, GBS did not invade amnion cells beyond that observed for the negative controls, S. gordonii and E. coli, under the conditions used in these studies. It is possible that GBS may be able to invade the basolateral surface but that the methods used in these studies were not sufficient to demonstrate this process. The bacterial ligand(s) and/or the cellular receptor(s) required for adherence and invasion into chorion cells may be different from that required for amnion cells. Alternatively, the ligand-receptor interaction may not have been of high enough avidity with amnion cells to trigger active cellular uptake under our conditions.

Our studies suggest that the amnion epithelial cell monolayer may provide an effective barrier against entry to the nutrient-rich amniotic fluid and may partially explain why the rate of amniotic fluid infections associated with chorioamnionitis is not higher (31, 48). It is possible, however, that if amnion cells are actively being sloughed off into the amniotic fluid, any adherent GBS may passively acquire access to the amnion. Only a minimal number of CFU would need to penetrate the entire membrane, since the amniotic fluid has been shown to provide an excellent growth medium for rapid GBS replication (18, 38). Alternatively, once the organism gains access to the membrane stroma, GBS colonization may induce a host inflammatory response which could affect amnion epithelial integrity, allowing GBS to breach this barrier. Alterations in synthesis and release of cytokines as a host response to bacterial infections have previously been hypothesized to stimulate parturition prematurely (1, 21, 36). In support of this hypothesis, amnion cells exposed to GBS culture supernatants and heat-killed GBS caused elevated prostaglandin E2 release (2) and upregulation of interleukin 6 (IL-6) and IL-8 (35), respectively. Primary chorion cells released higher levels of macrophage inflammatory protein 1alpha and IL-8, which are monocyte and neutrophil chemoattractants, respectively, in the presence of heat-killed GBS (8). These inflammatory responses may weaken membrane integrity, allowing GBS to ultimately breach the amnion cell layer.

The chorion and amnion cells form two important cellular barriers protecting the neonate from bacterial infection. The ability of GBS to adhere to, invade, and translocate through intact chorion cell monolayers, reported here, correlates well with the epidemiology of chorioamnionitis (4) and the results of histopathological studies which identified viable GBS from between the chorion-amnion layers of infected placentas (3, 30). Genetic techniques can now be applied to dissect the adherence and invasion phenotypes of GBS which may be crucial to the pathogenesis of placental membrane and fetal infections.

    ACKNOWLEDGMENTS

We thank David Luthy at Swedish Hospital Perinatal Center and the nursing staff in Labor and Delivery for their assistance in providing the cesarean-section placentas.

This investigation was supported by NIH grant AI30068.

    FOOTNOTES

* Corresponding author. Mailing address: Children's Hospital and Regional Medical Center, Division of Pediatrics, Department of Infectious Diseases, Box 5371/CH-32, Seattle, WA 98105. Phone: (206) 526-2073. Fax: (206) 527-3890. E-mail: cruben{at}chmc.org.

Editor:   V. A. Fischetti

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Abstract
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Materials & Methods
Results
Discussion
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Infection and Immunity, October 1998, p. 4932-4941, Vol. 66, No. 10
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