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.
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Abstract |
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Key words: chemically modified heparin/chlamydial attachment/heparan sulfate/heparinase/proteoglycans
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Introduction |
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Previous reports describe that heparin and related glycosaminoglycans are potent inhibitors of chlamydial infection for cultured human cervical epithelial cells (Zhang and Stephens, 1992; Zaretzky et al., 1995
; Su et al., 1996
). Although currently controversial, Su et al. (1996)
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)
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.
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Results |
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Discussion |
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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, 1976). 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., 1992
). 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, 1989
), cytomegalovirus (Neyts et al., 1992
), pseudorabies (Mettenleiter et al., 1990
), varicella zoster (Zhu et al., 1995
), and retroviruses (Mitsuya et al., 1998
). The protozoan parasite Trypanosoma cruzi (Ortega-Barria and Pereira, 1991
) and bacterial mucosal pathogen Neisseria gonorrhoeae (van Putten and Paul, 1995
) 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, 1991). 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, 1976
). Zhang and Stephens (1992)
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 chlamydiahost cell interaction in which a chlamydial heparan sulfatelike glycosaminoglycan mediated attachment and perhaps subsequent infectivity. Actually, Chen et al. (1996)
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 sulfatelike molecule that interacts with the membrane surfaces of EBs and bridges chlamydiae to eukaryotic cells (Zhang and Stephens, 1992
; Chen et al., 1996
). Su et al. (1996)
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, 1992). 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., 1996
; Su et al., 1996
) 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., 1999).
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) 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., 1996; Davis and Wyrick, 1997
). 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, 2001
; Taraktchoglou et al., 2001
). 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.
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Materials and methods |
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The characteristics of mutant CHO cell lines F-17 and 677 have been described elsewhere (Lidholt et al., 1992; Bai and Esko, 1996
; Bai et al., 1997
; Wei et al., 2000
). 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 Dulbeccos 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., 1997) 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., 2000
), 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., 1984) (Figure 1). Control cultures usually showed 2540% 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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