Effects of chemically modified heparin on Chlamydia trachomatis serovar L2 infection of eukaryotic cells in culture

Hiromitsu Yabushita2, Yasuyuki Noguchi2, Hiroko Habuchi3, Satoko Ashikari3, Ken Nakabe2, Masaru Fujita2, Masayoshi Noguchi2, Jeffrey D. Esko4 and Koji Kimata1,3

2 Department of Obstetrics and Gynecology, Research Center for Infectious Disease, School of Medicine, Aichi Medical University, 21 Karimata, Nagakute-cho, Aichi-gun, Aichi 480-1195, Japan; 3 Institute for Molecular Science of Medicine, Research Center for Infectious Disease, School of Medicine, Aichi Medical University, 21 Karimata, Nagakute-cho, Aichi-gun, Aichi 480-1195, Japan; and 4 University of California, San Diego, Department of Cellular & Molecular Medicine, Glycobiology Research and Training Center, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA

Received on December 5, 2001; revised on February 20, 2002. accepted on February 20, 2002.


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The mechanism and inhibitors of Chlamydia trachomatis serovar L2 infection of eukaryotic host cells were studied using a tissue culture model infection system. Potent inhibition of infectivity was observed when elementary bodies (EBs) were exposed to heparin or when HeLa 229 cells were treated with heparinase. No significant inhibition was seen the other way around. The same potent inhibition was observed when EBs were exposed to chemically 2-O-desulfated heparin (2-ODS heparin), which is composed of repeating disaccharide units of IdoA-GlcNS(6S), but not when exposed to chemically 6-ODS heparin or completely desulfated and N-resulfated heparin, which is composed of repeating disaccharide units of IdoA(2S)-GlcNS or IdoA-GlcNS, respectively. The inhibitory effects of 2-ODS heparin could be seen only with oligosaccharides longer than dodecasaccharides. The mutant Chinese hamster ovary (CHO) cell line 677, which is deficient in the biosynthesis of heparan sulfate, was less sensitive to C. trachomatis infection than were wild-type CHO cells. F-17 cells, deficient in 2-O-sulfation of heparan sulfate, had the same sensitivity to infection as wild-type CHO cells did. These data suggest that infection of host cells by EBS results from the specific binding of ligand molecules with affinity for heparin on the EB surface to heparan sulfate proteoglycans on the host cell surface. This binding may depend on host cell heparan sulfate chains that are 6-O-sulfated and longer than dodecasaccharides. The 2-ODS heparin oligosaccharides may be a potential agent for the prevention of C. trachomatis infection.

Key words: chemically modified heparin/chlamydial attachment/heparan sulfate/heparinase/proteoglycans


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Genital tract infection with the obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial sexually transmitted disease (STD) in Japan (Noguchi et al., 1999Go) and the United States (Schachter, 1978Go). In women, ascending infections of the genital tract can produce serious sequelae that include pelvic inflammatory disease, ectopic pregnancy, and reproductive disability (Jones et al., 1982Go; Brunham et al., 1985Go, 1986; Chow et al., 1990Go; Noguchi et al., 1999Go). Currently, control of chlamydial STDs has focused on two intervention strategies: (1) improved diagnosis and treatment of subclinical infections and (2) behavioral modification. In the future, control of chlamydial STDs may be achievable with vaccination and topical microbicides. Because many chlamydial infections are subclinical, early antibiotic intervention has not been entirely effective in controlling them. Despite increasing understanding of the immune mechanisms that confer protection against chlamydial genital tract infection in animal models, no efficacious vaccine for use in humans is available yet. On the other hand, promising results are beginning to be reported, suggesting that topical microbicides may be useful in prevention of chlamydial infection.

Previous reports describe that heparin and related glycosaminoglycans are potent inhibitors of chlamydial infection for cultured human cervical epithelial cells (Zhang and Stephens, 1992Go; Zaretzky et al., 1995Go; Su et al., 1996Go). Although currently controversial, Su et al. (1996)Go have suggested that chlamydial infection of host cells is mediated through the specific binding of the chlamydial major outer membrane protein to heparan sulfate proteoglycans on host epithelial cells. It is likely that chlamydial attachment to epithelial cells is the critical initial step in the pathogenesis of infection. Inhibition of chlamydial adherence to cervical epithelial cells by intravaginally administered heparin-related glycosaminoglycans or structurally similar compounds may represent a plausible approach to the prevention of chlamydial infections. However, Su and Caldwell (1998)Go have shown that despite potent in vitro antichlamydial activity of heparin, sulfated polysaccharides, and synthetic sulfated polymer, neither heparin nor dextran sulfate was effective in inhibiting C. trachomatis mouse pneumonitis infectivity in an in vivo murine model of vaginal infection. There are several possible explanations for the disparate results between in vitro and in vivo experiments. The structural requirements for the specific binding of the chlamydial major outer membrane protein to the host cell surface heparan sulfate proteoglycans need to be elucidated to resolve this issue.

In this study, we firstly confirmed the infection of C. trachomatis to eukaryotic host cells via an interaction between heparin-binding molecules on the chlamydial outer membrane and heparan sulfates of the proteoglycans on the host cell surface using cultured HeLa 229 cells and mutant Chinese hamster ovary (CHO) cells with deficient or modified capacity to produce heparan sulfate proteoglycans. We next determined minimal structures of heparan sulfates for specific binding to inhibit infection and found that dodecasaccharides derived from chemically 2-O-desulfated (2-ODS) heparin significantly reduce the infectivity in culture. The potential usage of such oligosaccharides as antichlamydial topical microbicides is discussed.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Effects of treatment with heparin or heparinase-I on the infectivity of C. trachomatis serovar L2
We attempt to detect chlamydia-infected cells by the method using flow cytometry, which is not common. The population of infected cells measured by this method was correlated closely with the inclusion-forming unit (IFU; Figure 1). Because a large number of cells were examined quickly and objectively, this method is a useful tool for the purpose of this study. When various concentrations of heparin or heparinase-I were added to the incubation media for the infection, the infectivity of C. trachomatis was decreased in a dose-dependent manner (Figure 2). Neither the addition of chondroitin sulfate C or hyaluronan (100 µg/ml), nor that of chondroitinase ABC or Streptomyces hyaluronidase (1 U/ml) to the incubation media for the infection showed any effect on the infectivity (statistically not significantly different, vs. untreated; data not shown).



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Fig. 1. Correlation between the population of infected cells and IFUs. Percents of the infected cells and IFU were measured by flow cytometry and by the Micro Trak direct test, respectively. Bars indicate two IFU values individually measured at each cell population. Statistical values are shown inside.

 


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Fig. 2. Effect of treatment with heparin (A) or heparinase-I (B) on Chlamydia trachomatis infectivity in HeLa 229 cells. Varying concentrations of heparin or heparinase-I were added to the incubation media for the infection of EBS of C. trachomatis. The infectivity of was measured as described in Materials and methods. Each assay was performed in triplicate, and bars indicate standard deviation. Probability values (P) are shown inside.

 
We then examined whether heparin exerted its effect on the elementary bodies (EBs) or HeLa 229 cells. EBs or HeLa 229 cells were pretreated with various concentrations of heparin. Infectivity was inhibited in a dose-dependent manner when pretreated EBs were added to untreated monolayered HeLa 229 cells. However, no inhibition was observed when untreated EBs were added to pretreated HeLa 229 cells (Figure 3). We also examined whether heparinase-I affects EBs or HeLa 229 cells. On the contrary, infectivity was decreased in a dose-dependent manner when untreated EBs were added to pretreated HeLa 229 cells, but no inhibition was seen when pretreated EBs were added to untreated HeLa cells (Figure 4). Digestion of HeLa cells with heparinase-I (1 IU/ml) eliminated approximately 80% of the heparan sulfate from the cell surface of Hela 229 cells (data not shown).



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Fig. 3. Effect of pretreatment of EBs (squares) or HeLa 229 cells (circles) with varying concentrations of heparin on Chlamydia trachomatis infectivity. Either HeLa 229 cells or EBs were pretreated with heparin at indicated concentrations. Each assay was performed in triplicate, and bars indicate standard deviation. P values are shown inside.

 


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Fig. 4. Effect of pretreatment of EBs (squares) or HeLa 229 cells (circles) with varying concentrations of heparinase-1 on Chlamydia trachomatis infectivity. Either HeLa 229 cells or EBs were pretreated with heparinase-I at the indicated concentrations. Each assay was performed in triplicate, and bars indicate standard deviation. P values are shown inside.

 
To confirm the different effects of heparin and heparinase-I on EBs and HeLa cells, we examined effect of various combinations of those treatments on infectivity. Significant inhibition of infection was observed when EBs were pretreated by heparin and when HeLa 229 cells were pretreated by heparinase-I. Unexpectedly, infectivity was increased when EBs and HeLa cells were pretreated with heparinase-I and heparin, respectively (Figure 5). The inhibitory effects of heparin and heparinase-1 on the infection support the idea that membrane proteins with the heparin-binding properties can act as ligand heparan sulfate receptors on the cell surface (Su et al., 1996Go).



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Fig. 5. Chlamydia trachomatis infectivity when HeLa 229 cells and/or EBs were pretreated with 100 µg/ml heparin and/or 1 IU/ml heparinase-I. Each assay was performed in triplicate; closed rectangles show mean values, and bars indicate standard deviation. P values are shown under.

 
Involvement of host cell surface heparan sulfate proteoglycans in the infection
To confirm that host cell surface heparan sulfate proteoglycans were acting as receptors, we conducted infection studies using CHO cells and mutant lines that are abnormal or deficient in heparan sulfate biosynthesis. The infectivity of EBs to the line 677 cells, which are deficient in heparan sulfate synthesis, was reduced to about one-third compared with that of wild CHO cells. On the contrary, F-17 cells which are deficient in 2-O-sulfation of heparan sulfate showed no significant reduction or increase of C. trachomatis infectivity, compared with the wild CHO cell line (Figure 6).



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Fig. 6. Chlamydia trachomatis infectivity for wild-type CHO cells and mutant CHO cell lines, F-17 cells, and 677 cells. Each assay was performed in triplicate. Closed rectangles indicate mean values, and bars indicate standard deviation. P for 677 cells versus other cells, 0.001.

 
Structural requirements of heparin for inhibition of Ctrachomatis infectivity
The lack of any effect of 2-O-sulfation suggested that heparan sulfate chains on the host cell surface proteoglycans that bind to C. trachomatis may be somewhat unusual. Taking advantage of the new methods for the chemically desulfating heparin at specific locations (Ishihara et al., 1997Go; Kariya et al., 2000Go), we examined how altering the structure of heparin affected its ability to inhibit infection. 6-ODS heparin and completely desulfated and N-resulfated (CDSNS) heparin had no effect on infectivity, but 2-ODS heparin inhibited infection with the same dose dependence as heparin, with 100% inhibition occurring at 100 µg/ml (Figure 7). This inhibitory effect was observed when EBs were pretreated with 2-ODS, but not when HeLa 229 cells were pretreated with this compound (Figure 8). To determine the minimal chain size of 2-ODS heparin for the inhibition, we prepared 2-ODS heparin oligosaccharides, which ranged from 4 to ~18 monosaccharide units in length, and tested this inhibitory activity. Each oligosaccharide fraction was added to the HeLa 229 cell culture at the same time EBs were added. Significant inhibition of C. trachomatis infection was only observed when 2-ODS heparin oligosaccharides consisting of more than 12 sugar residues (average 18 monosaccharide units) were added (Figure 9). The results suggest that proteoglycans having heparan sulfate chains composed of more than six repeating disaccharide units rich in 6-O-sulfation play a role on the host cell surfaces as receptors for C. trachomatis infection.



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Fig. 7. Chlamydia trachomatis infectivity on HeLa 229 cells in the presence of various heparin derivatives. The cells were incubated with EBs in the presence of heparin (open squares), 6-ODS heparin (closed squares), 2-ODS heparin (open circles), or CDSNS heparin (closed circles). Each assay was performed in triplicate; bars indicate standard deviation. P values are shown inside.

 


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Fig. 8. Effects of pretreatments with various heparin derivatives on Chlamydia trachomatis infectivity. HeLa 229 cells (closed rectangles) or EBs (open rectangles) were pretreated with 100 µg/ml of heparin, 6-ODS heparin, 2-ODS heparin, or CDSNS heparin. Each assay was performed in triplicate, and bars indicate standard deviation. P values are shown inside.

 


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Fig. 9. Effect of 2-ODS heparin oligosaccharides with various sizes on Chlamydia trachomatis infectivity. The oligosaccharide with more than 12 monosaccharide units primarily consisted of 18 monosaccharide units. Each assay was performed in triplicate, and bars indicate standard deviation. P values are shown inside.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The initial event in chlamydial infection is the attachment of the organism to epithelial cells. Identification of chlamydial surface components that mediate attachment to the host cell and the receptors that chlamydial ligands bind is key to understanding the pathogenesis of chlamydial infection and may provide a rational target for the prevention of chlamydial infection.

Heparan sulfate proteoglycans are a group of ubiquitous cell surface components and are usually anchored to the plasma membranes via transmembrane core proteins or glycosylphosphatidylinositol (Kuo and Grayston, 1976Go). Heparan sulfate proteoglycans bind to a large spectrum of cellular ligands, including matrix components, growth factors, lipolytic enzymes, protease inhibitors, and transcriptional regulators (Bernfield et al., 1992Go). In addition, heparan sulfate proteoglycans have been shown to function as receptors for a variety of infectious agents. Viruses that utilize heparan sulfate proteoglycans as receptors include herpes simplex (WuDunn and Spear, 1989Go), cytomegalovirus (Neyts et al., 1992Go), pseudorabies (Mettenleiter et al., 1990Go), varicella zoster (Zhu et al., 1995Go), and retroviruses (Mitsuya et al., 1998Go). The protozoan parasite Trypanosoma cruzi (Ortega-Barria and Pereira, 1991Go) and bacterial mucosal pathogen Neisseria gonorrhoeae (van Putten and Paul, 1995Go) have also been shown to bind heparan sulfate proteoglycans. Thus, a diverse group of infectious agents has evolved common strategies that utilize heparan sulfate proteoglycans for their interaction with eukaryotic host cells.

Little has been known about the precise mechanisms how chlamydiae bind to eukaryotic cells. Binding of chlamydiae to host cell receptors was saturable and sensitive to trypsin treatment of host cells (Moulder, 1991Go). The interaction of the host cell receptor with the chlamydial ligand was found to be inhibited by exogenously adding heparin or heparan sulfate (Kuo and Grayston, 1976Go). Zhang and Stephens (1992)Go showed from studies on the dose-response kinetics for heparin inhibition of chlamydial infectivity that low concentrations of heparin or heparan sulfate could increase the degree of attachment between C. trachomatis and mammalian cells, and they suggested a novel model for chlamydia–host cell interaction in which a chlamydial heparan sulfate–like glycosaminoglycan mediated attachment and perhaps subsequent infectivity. Actually, Chen et al. (1996)Go demonstrated the direct binding of heparin to EBs using 3H-labeled heparin and a correlation between the binding heparin to EBs and the inhibitory effect on infection. However, they proposed that chlamydiae could synthesize a heparan sulfate–like molecule that interacts with the membrane surfaces of EBs and bridges chlamydiae to eukaryotic cells (Zhang and Stephens, 1992Go; Chen et al., 1996Go). Su et al. (1996)Go subsequently suggested that chlamydial attachment to the host cell is mediated through the specific binding of the chlamydial major outer membrane protein to host cell heparan sulfate proteoglycans.

Our study has shown that the infection of EBs to host cells is inhibited when EBs were treated with heparin and also when host cells were treated with heparinase-I. Mutant CHO cells deficient in heparan sulfate synthesis were less sensitive to infection than wild CHO cells, as reported previously (Zhang and Stephens, 1992Go). The results support the idea that surface molecules with an affinity for heparin present on the pathogen are able to bind to heparan sulfate proteoglycans on the host cell surfaces. Furthermore, the experiments using chemically modified heparins showed that the 2-ODS heparin effectively inhibited chlamydial attachment to host cells and thus suggested that this inhibition appears to depend on the heparan sulfate size and structure (12 or more monosaccharides modified by 6-O-sulfation). Although previous studies (Chen et al., 1996Go; Su et al., 1996Go) have suggested chylamydial membrane surface molecules, such as the major outer membrane protein, as a candidate acceptor for the binding of the heparan sulfate to the EBs, molecular properties of the interaction of heparan sulfates of such sizes and O-sulfation with EB membrane surface molecules remain to be investigated.

Interestingly, infectivity was not inhibited completely in EBs treated with heparin and in host cells treated with heparinase-I simultaneously. In addition, 677 cells, the mutant CHO cell line deficient in heparan sulfate synthesis, also succumbed to chlamydial infection. These results also suggest that C. trachomatis EBs can adhere and invade host cells via heparin-independent pathways. Thus, the organism may use a two-step process for infection, much like herpes simplex virus utilizes both heparan sulfate and proteinaceous receptors, herpes virus entry mediator (HVEM) receptors, for attachment and invasion (Shukla et al., 1999Go).

The 2-ODS heparin preparation that effectively inhibited chlamydial attachment has greatly reduced anticoagulant activity (the periodate oxidation used in its preparation destroys both anticoagulant activity and the binding to FGF-2; unpublished data). The 2-ODS heparin oligosaccharides (longer than 12 monosaccharide units) presumably has similar properties. Although Su and Caldwell (1998)Go reported that heparin was not effective in inhibiting infectivity in an in vivo murine model system, 2-ODS heparin could be a more optimal compound for the prevention of C. trachomatis infection and should be tested in the murine model of vaginal infection. Such prevention, particularly in women, could be easily administered intravaginally by a suppository composed of 2-ODS heparin.

C. trachomatis consists of three goups of biovars, serovars A to C, serovars D to K, and lymphogranuloma venereum (LGV), which differ in their infectivity in vivo and in vitro (Chen et al., 1996Go; Davis and Wyrick, 1997Go). In the present article, we have examined the effect of 2-ODS heparin on chlamydial attachment of the LGV biovar but not the other trachoma biovars. Because chlamydial STD is more frequently caused by serovars D to K, it may be important to determine if these biovars also exhibit sensitivtiy to heparin and heparin oligosaccharides. In addition, the infection mechanism and the structures involved in the chlamydial attachment might vary among host cells (Carabeo and Hackstadt, 2001Go; Taraktchoglou et al., 2001Go). Therefore, comparisons of chlamydial infectivity among several types of host cells should be also examined. Conceivably, the tropism of each biovar may vary according to the structure of the heparan sulfate chain present on the host cell, which is known to vary in different cell types.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cell lines and chlamydial strain
HeLa 229 cell lines were grown in CMGA medium (Eagle’s minimal essential medium [MEM] supplemented with 10% fetal calf serum, 0.5% glucose, 0.3 mg/ml glutamine, 0.05 mg/ml streptomycin, 0.1 mg/ml vancomycin, and 2 IU/ml nystatin). The C. trachomatis strain L2/432/Bu was grown in monolayer cultures of HeLa 229 cells. Chlamydiae grow only within eukaryotic host cells and have a developmental cycle characterized by two forms of the organism, a metabolically active intracellular form and a metabolically inactive extracellular form (Moulder, 1991Go). The latter form is known as the EBs and is capable of infecting mammalian cells (Caldwell et al., 1981Go). Chlamydial EBs were isolated by sonic treatment of cell suspensions and purified by centrifugation through discontinuous Renografin density gradients (Caldwell et al., 1981Go). EBs were washed with Hank’s balanced salt solution (HBSS), resuspended in sucrose-phosphate-glutamic acid buffer (0.2 M sucrose, 0.02 M phosphate, and 0.72 mg/ml glutamic acid; pH 7.4) and stored at –70°C until used. The purity of EBs was confirmed by examining if its IFU/ml was within the value reported previously (Caldwell et al., 1981Go).

The characteristics of mutant CHO cell lines F-17 and 677 have been described elsewhere (Lidholt et al., 1992Go; Bai and Esko, 1996Go; Bai et al., 1997Go; Wei et al., 2000Go). Briefly, the F-17 (pgsF) cell line is deficient in expression of uronosyl 2-O-sulfotransferase and does not produce any 2-O-sulfated groups in heparan sulfate. The 677 cell line (pgsD) is deficient in heparan sulfate synthesis due to mutations in EXT1, the copolymerase. The mutant cell lines and wild CHO cells were maintained in a 1:1 mixture of Dulbecco’s MEM and F-12 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum, 0.05 mg/ml streptomycin, and 200 IU/ml penicillin G.

Chemically modified heparin and heparinase
Sodium heparin were purchased from Nakarai (Osaka, Japan). The following chemically modified heparin preparations were prepared and provided by Seikagaku (Tokyo); CDSNS heparin, consisting of 86% [IdoA-GlcNS] unit; 2-ODS heparin, prepared by selective 2-O-desulfation (Ishihara et al., 1997Go) of the derivatives obtained from heparin by oxidation with periodate and subsequent reduction, consisting of 60% [IdoA-GlcNS(6S)] and 10% [IdoA-GlcNS] units; 6-ODS heparin, prepared according to a reported method (Kariya et al., 2000Go), consisting of 53% [IdoA(2S)-GlcNS], 27% [IdoA-GlcNS], and 12% [IdoA-GlcNAc] units.

Oligosaccharides of 2-ODS heparin were prepared by the digestion with heparitinase II (Seikagaku), and those of various molecular sizes composed of 4, 6, 8, 10, 12, and more than 12 monosaccharide units were obtained by repeated gel filtration on Superdex pg 30 column (Pharmacia, Uppsala, Sweden). Each oligosaccharide contained less than 10% of the higher and shorter oliosaccharides. Oligosaccharide composed of more than 12 monosaccharide units were further fractionated by gel filtration on the Superdex pg 75 column, and the fraction composed of primarily 18 monosaccharide units were obtained. The larger oligosaccharide fractions, which had a definite size with the high purity, could hardly be obtained because of the difficulty in the separation on the gel filtration.

Heparinase-I was purchased from Sigma (St. Louis, MO). Chondroitin sulfate C (shark cartilage), hylauronan (human umbilical cord), chondroitinase ABC, and bacterial hyaluronidase (Streptomyces hyalurolyticus) were from Seikagaku.

Infectivity assay
C. trachomatis infectivity was determined using cultured cell monolayers on 24-well tissue culture plate (Falcon 3034, Becton Dickinson, NJ). Cells were plated and grown for 24 h to become confluent monolayers. Two hundred fifty microliters of C. trachomatis EBs suspension, which is equivalent to 1 x 106 IFU/ml, was then added to triplicate monolayers, followed by the incubation at 37°C. After 1 h monolayers were washed three times with HBSS and then incubated in CMGA medium containing 10 µg/ml cyclohexamide for 48 h at 37°C. Cells were harvested with trypsinization and fixed with 100% ethanol for 5 min. After hydration and washing three times with phosphate buffered saline, cells were incubated with C. trachomatis species-specific murine monoclonal antibody conjugated with fluorescein isothiocyanate (Syva, Palo Alto, CA). The population of the cells containing C. trachomatis inclusions was measured by the flow cytometric analysis using FACS Calibur (Becton Dickinson). In this analysis, 1 x 104 cells were counted for each sample, and the infected cells were detected by fluorescence intensity set on a log scale after removing clumps of cells. The infected cells with inclusions obtained by cell sorting system were identified under the fluorescence microscopic examination. The population of infected cells measured by flow cytometry was correlated closely with IFU/ml measured by the Micro Trak direct test (Uyeda et al., 1984Go) (Figure 1). Control cultures usually showed 25–40% of the cells containing C. trachomatis inclusions in this analysis.

For the infectivity inhibition assay, EBs were pretreated with heparin, chemically modified heparin, or heparinase-I for 1 h at 37°C, and then washed three times with HBSS following centrifugation for 30 min at 8000 x g. After washes and centrifugations, EBs were resuspended at the adequate concentration in HBSS. HeLa 229 cells cultured in monolayer were pretreated with heparin, chemically modified heparin, or heparinase-I for 1 h at 37°C and then washed three times with HBSS. After the pretreatment, HeLa 229 cells were incubated with EBs for 1 h at 37°C, washed three times with HBSS, and then continued to be cultured for 48 h at 37°C. Following the culture, the population of infected cells were assayed as described.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Makoto Naruse for his assistance in flow cytometric analysis. This work was supported by a preparatory grant for the research at the Research Center for Infectious Disease, Aichi Medical University; by grants-in-aid from the Ministry of Education, Science, Sport and Culture of Japan; and by grant R37GM33063 from the National Institutes of Health, USA.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CDSNS, completely desulfated and N-resulfated; CHO, Chinese hamster ovary; CMGA, MEM supplemented with 10% fetal calf serum, 0.5% glucose, 0.3 mg/ml glutamine, 0.05 mg/ml streptomycin, 0.1 mg/ml vancomycin, and 2 IU/ml nystatin; EB, elementary body; GlcN5, N-sulfated glucosamine; HBSS, Hank’s balanced salt solution; IdoA, iduronic acid; IFU, inclusion forming unit; LGV, lymphogranuloma venereum; MEM, Eagle’s modified essential medium; ODS, O-desulfated; STD, sexually transmitted disease.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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