J Med Microbiol 57 (2008), 261-266; DOI: 10.1099/jmm.0.47237-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Is lipopolysaccharide a factor in infectivity of Chlamydia trachomatis?
Sanaa Fadel and
Adrian Eley
Henry Wellcome Laboratories for Medical Research, Unit of Infection and Immunity, School of Medicine and Biomedical Sciences, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK
Correspondence
Adrian Eley
a.r.eley{at}sheffield.ac.uk
Received 18 February 2007
Accepted 9 November 2007
Lipopolysaccharide (LPS) is a major surface component of Chlamydia trachomatis, as with all Gram-negative bacteria. The effect of C. trachomatis LPS on C. trachomatis infectivity of human epithelial cells was investigated. C. trachomatis LPS and C. trachomatis LPS antibody significantly reduced infectivity, mostly in a dose-dependent manner. As the structure of LPS in C. trachomatis is simple and consists only of lipid A and 3-deoxy-D-manno-octulosonic acid (Kdo), we investigated whether lipid A or Kdo was inhibitory to chlamydial infectivity. Polymyxin B, as a lipid A inhibitor, and Kdo considerably reduced C. trachomatis infectivity. With all the LPS inhibitors used, there was greater inhibition against serovar E than serovar LGV. These results suggest a role for LPS in chlamydial infectivity. Elucidation of how LPS acts in infectivity and identification of host-cell receptors would help in understanding pathogenicity.
Abbreviations: EB, elementary body; GAG, glycosaminoglycan; Kdo, 3-deoxy-D-manno-octulosonic acid; PmB, polymyxin B; TC, tissue culture.
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INTRODUCTION
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Several mechanisms by which Chlamydia trachomatis attaches to and infects host cells have been described. Proposed ligands include the major outer-membrane protein (MOMP) (Su et al., 1990; Kuo et al., 1996), heat-shock protein 70 (Raulston et al., 1993) and glycosaminoglycans (GAGs) (Zhang & Stephens, 1992). For the last there is some debate as to whether the GAG is on C. trachomatis or is a host molecule (Taraktchoglou et al., 2001; Fadel & Eley, 2004). However, recent work has shown that interaction of C. trachomatis with mammalian cells may be independent of host-cell heparan sulphate GAGs (Stephens et al., 2006). There has also been interest in the entry of C. trachomatis into host cells involving cholesterol-rich membrane domains or lipid ‘rafts’ which appear to be serovar-dependent (Norkin et al., 2001; Stuart et al., 2003). Use of a chlamydial glycolipid exo-antigen vaccine (GLXA; unrelated to lipopolysaccharide) in a vaccine has shown some protection against genital chlamydial infection in a mouse model (Whittum-Hudson et al., 2001), although in other experimental conditions, it enhances infection (Vora & Stuart, 2003). However, little evidence has been reported on whether C. trachomatis lipopolysaccharide (LPS) might be implicated in infectivity. This is surprising, as LPS is a major surface component of C. trachomatis (Brade, 1999) and has been widely used as the target molecule in laboratory diagnosis by enzyme immunoassay (EIA) (Jones et al., 1984; Chernesky et al., 2001). The present study investigated whether C. trachomatis LPS might be involved in infectivity towards human epithelial cells.
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METHODS
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Cell lines and C. trachomatis serovars.
The Hec-1B cell line (endometrial carcinoma) was obtained from the American Type Culture Collection (ATCC) and maintained according to the supplier's instructions. Serovar LGV1 was kindly provided by M. Ward (University of Southampton), and C. trachomatis reference strain E/UW-5/CX was a kind gift of D. Dean (Children's Hospital, Oakland Research Institute). Chlamydiae were grown in semiconfluent McCoy cells for 48–72 h in maintenance medium [minimum essential medium Eagle (EMEM) supplemented with cycloheximide (2 µg ml–1)]. The cell suspension containing elementary bodies (EBs) was harvested, and sonicated twice for 10 s at 15 µm amplitude. Cell debris was removed by centrifugation at 500 g for 15 min, and the remaining suspension was further purified by centrifugation at 30 000 g for 1 h at 4 °C. The resulting crude EB pellet was resuspended in 8 ml PBS, sonicated as described above, layered over 30 % urografin (Schering) and centrifuged at 30 000 g. Urografin is commonly used to purify EBs in a discontinuous density gradient. The final pellet was resuspended in sucrose phosphate buffer (SPG; 5 mM glutamine, 0.2 M sucrose, 0.2 M phosphate buffer, pH 7.4) and stored at –70 °C for further use.
Infectivity inhibition assays.
Inocula of C. trachomatis serovars LGV and E were adjusted so that not more than 
50 inclusions were present in each field at x400 magnification [5x105 inclusion forming units (i.f.u.)] in 100 µl PBS. The inocula were mixed with dilutions of C. trachomatis LPS, C. trachomatis LPS antibody, polymyxin B (PmB) or synthetic 3-deoxy-D-manno-octulosonic acid (Kdo), and used to infect confluent Hec-1B cell monolayers in 24-well tissue culture (TC) trays, which we have used in previous infectivity studies (Taraktchoglou et al., 2001). C. trachomatis LGV LPS was extracted by the method of Nurminen et al. (1985) and quantified using the Limulus amoebocyte lysate (LAL) test (Cambrex Biosciences). Fig. 1
shows the purity of the extracted LPS after electrophoresis through a 14 % acrylamide gel and staining with the Bio-Rad Silver Stain kit. Re mutant LPS from Salmonella enterica 595 (Sigma) was used as a control for C. trachomatis LPS. C. trachomatis LPS mAb was obtained from Biogenesis and shows activity against all C. trachomatis serovars. Isotype IgG2A (Sigma) was used as a control for C. trachomatis LPS antibody. Both PmB and synthetic Kdo were obtained from Sigma, and PmB nonapeptide (Sigma) and D-glucuronic acid (Sigma) were used as their controls, respectively. The cell monolayers were incubated for 1 h at 37 °C in 5 % CO2, and then washed with PBS three times before adding 1 ml EMEM TC fluid supplemented with cycloheximide to each well. After incubation at 37 °C in 5 % CO2 for 48 h, infected monolayers were fixed, stained, and counted by direct immunofluorescence microscopy using the C. trachomatis culture confirmation test (Trinity Biotech). The mean number of inclusion bodies for 25 fields (x400 magnification) was determined, and compared to the control to calculate the percentage infectivity.
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Fig. 1. LPS extracted from C. trachomatis LGV1 (lane 1) and a control E. coli O55 : B5 LPS (lane 2) electrophoresed through a 14 % acrylamide gel and stained with silver stain.
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Examination of the binding of LPS to Hec-1B cells using immunohistochemistry.
Semiconfluent Hec-1B cell monolayers in 24-well TC trays were incubated with C. trachomatis LPS for 2 h, washed twice with PBS then incubated with C. trachomatis LPS horseradish peroxidase (HRP)-conjugated antibody for 1 h, and the reaction was developed using 4-chloro-1-naphthol. The reaction was viewed with a light microscope at x1000 magnification. For comparison, the reaction was carried out using Escherichia coli LPS mAb (Biogenesis). The reaction performed in the absence of C. trachomatis LPS was used as a negative control. A positive control included Hec-1B cells infected with serovar LGV1, incubated directly with C. trachomatis LPS antibody for 1 h at room temperature, and developed using the same method.
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RESULTS AND DISCUSSION
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Effect of chlamydial LPS on infectivity
LPS from serovar LGV inhibited the infectivity of C. trachomatis in a dose-dependent manner (Fig. 2). At all concentrations of LPS used, there was greater inhibition against serovar E than with serovar LGV, such that at 10 µg ml–1 there was 33 and 48 % inhibition with serovars LGV and E, respectively (P <0.0001). As a control, we used a Re mutant LPS of S. enterica, which more closely resembled the overall composition of C. trachomatis LPS. Although S. enterica LPS is closely related to C. trachomatis LPS, the latter has a family-specific epitope (a 2–8 linkage in the core trisaccharide Kdo) which results in a 100-fold decrease in endotoxicity (Kosma, 1999) and is presumably responsible for the difference in infectivity inhibition. It is appreciated that LPS from only one C. trachomatis serovar was tested; however, it is widely believed that the structure of LPS from different serovars is in fact very similar (Heine et al., 2003). C. trachomatis LPS was not toxic to Hec-1B cells at a concentration of 10 µg ml–1.
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Fig. 2. Infectivity inhibition assay using C. trachomatis LGV LPS. The assay was performed with C. trachomatis serovar LGV or serovar E using Hec-1B cells at 37 °C. Re mutant LPS was used as a control. The figure represents the mean of six experiments, and error bars represent SEM. Results were statistically analysed using one-way-paired ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. To compare inhibitory effects on serovars E and LGV, an unpaired t test was used. Using 10 µg ml–1 LPS, the two-tailed P value was <0.0001.
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Effect of anti-C. trachomatis antibody on infectivity
Similar findings were obtained with a commercial C. trachomatis LPS antibody (Fig. 3). However, for serovar E at anti-LPS concentrations between 12.5 and 75 µg ml–1, there appeared to be more of a plateau than dose-dependent effect. At a concentration of 100 µg ml–1, the LPS antibody inhibited infectivity of serovars LGV and E by 36 and 54 % respectively. We used an isotype IgG2A antibody as a control. The isotype showed no inhibition of infectivity at the highest antibody concentration used (100 µg ml–1), confirming that the inhibitory effect of anti-LPS antibody was not due to a non-specific reaction.
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Fig. 3. Infectivity inhibition assay using C. trachomatis LPS antibody. The assay was performed with C. trachomatis serovar LGV or serovar E using Hec-1B cells at 37 °C. Isotype IgG2A was used as a control. The figure represents the mean of six experiments, and error bars represent SEM. Results were statistically analysed using one-way-paired ANOVA. *, P<0.05, **, P<0.01; ***, P<0.001; NS, non-significant.
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Effect of PmB and Kdo on infectivity
PmB is often used as an LPS inhibitor as it specifically binds to lipid A (Morrison & Jacobs, 1976). However, as it also has some antibacterial activity, its use in infectivity experiments could be misleading. Nevertheless, as the drug acts almost exclusively on extracellular pathogens, an antibiotic effect on C. trachomatis, which is obligately intracellular, seems unlikely (Redecke et al., 1998). Moreover, we used PmB nonapeptide as a control. This cationic cyclic peptide is derived from PmB. Although it has extremely low antimicrobial activity, it is still capable, like PmB, of binding to LPS (Tsubery et al., 2002). At 100 µg ml–1, PmB showed inhibition of infectivity of serovars LGV and E by 49 and 65 %, respectively (Fig. 4), which again is suggestive of LPS, especially lipid A, involvement. Similarly, PmB nonapeptide showed a comparable inhibitory effect, confirming that inhibition by PmB was due to LPS binding, and not due to antibacterial activity. As the structure of LPS in C. trachomatis is simple and consists only of lipid A and Kdo, we next investigated whether Kdo was inhibitory. Unfortunately, Kdo from C. trachomatis was unavailable, so instead we used synthetic Kdo. Interestingly, at 100 µg ml–1 Kdo, there was inhibition of serovars LGV and E by 39 and 57 %, respectively (Fig. 5). As a control, we used another synthetic carbohydrate, D-glucuronic acid, which like Kdo is negatively charged and is also found in chlamydial LPS; this showed no inhibitory effect and confirmed the specificity of Kdo.
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Fig. 4. Infectivity inhibition assay using PmB. The assay was performed with C. trachomatis serovar LGV or serovar E using Hec-1B cells at 37 °C. PmB nonapeptide was used as a control. The figure represents the mean of three experiments, and error bars represent SEM. Results were statistically analysed using one-way-paired ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant.
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Fig. 5. Infectivity inhibition assay using synthetic Kdo. The assay was performed with C. trachomatis serovar LGV or serovar E using Hec-1B cells at 37 °C. D-Glucuronic was used as a control. The figure represents the mean of three experiments, and error bars represent SEM. Results were statistically analysed using one-way-paired ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant.
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Immunohistochemistry
Another approach to investigate the role of LPS in chlamydial adherence was by immunohistochemistry using a specific C. trachomatis LPS HRP-conjugated antibody. As shown in Fig. 6, incubation of Hec-1B cells with chlamydial LPS followed by C. trachomatis LPS antibody showed a positive reaction in the form of a deep-purple colour distributed evenly at the edges of the cells. Hec-1B cells infected with LGV1 serovar and then incubated with C. trachomatis LPS antibody showed that most of the cells displayed dark staining. This was used as a positive control. The reaction carried out in the absence of LPS was negative.
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Fig. 6. Examination of the binding of C. trachomatis LPS to Hec-1B cells using immunohistochemistry. Cells were incubated with chlamydial LPS for 2 h, washed, then incubated with C. trachomatis HRP-conjugated antibody for 1 h, and the reaction was developed and immediately viewed with a light microscope (x1000). (a) Negative control in which C. trachomatis LPS was omitted. (b) Hec-1B cells incubated with C. trachomatis LPS, followed by C. trachomatis antibody. (c) Positive control, in which Hec-1B cells infected with serovar LGV1 were incubated with C. trachomatis antibody. Bar, 10 µm.
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In this study, all the findings suggest that C. trachomatis LPS plays a role in infectivity towards epithelial cells, although some of our data support the idea that infectivity is complex and likely involves more than one moiety. That C. trachomatis LPS might be playing a role in infectivity was demonstrated using LPS, C. trachomatis LPS antibody, PmB and Kdo as inhibitors. To the best of our knowledge, this is the first report of experiments with PmB nonapeptide and D-glucuronic acid. Immunohistochemistry experiments also suggested that C. trachomatis LPS binds to Hec-1B cells. However, the nature of the receptor to which LPS binds remains unclear. An approach using heparin affinity chromatography showed that LPS did not bind to heparin, indicating that the host receptor is unlikely to belong to the GAGs (data not shown). Nevertheless, different results have been shown by other workers (Su et al., 1990; Byrne et al., 1993) in C. trachomatis LPS antibody experiments, and although their C. trachomatis LPS antibody showed little inhibitory effect, there were several key differences in methodology. The serovar tested was B, the cells used were HaK (Syrian hamster kidney), and the inoculum size was significantly larger. All these differences make it extremely difficult to compare findings. We also realize that the Kdo we used in our experiments was synthetic and that inhibition by Kdo could have been due, at least in part, to its negative charge; however, these results do suggest that further work should be initiated.
We should also make it clear that even though we used a standard method for LPS quantification, it is known that C. trachomatis LPS has very low endotoxic activity (Kosma, 1999), and therefore the actual amounts of LPS used in our experiments are likely to have been higher than those measured.
Also of note in our experiments was the fact that unlike many other inhibition studies with C. trachomatis, especially those using GAG inhibitors, in which serovar LGV is almost always inhibited to a greater degree than serovar E (Taraktchoglou et al., 2001; Fadel & Eley, 2004), the converse was true when LPS was the target for infectivity. It may suggest that this possible target of infectivity is more important for serovar E than serovar LGV.
LPS, or in some cases lipooligosaccharide (LOS), has been reported to be important in bacterial adherence (Jacques, 1996), and more specifically in attachment of several bacterial pathogens of the genital and respiratory tracts, including Neisseria gonorrhoeae (Harvey et al., 2000), Haemophilus ducreyi (Alfa & DeGagne, 1997) and Actinobacillus pleuropneumoniae (Belanger et al., 1990). For the last organism, further evidence shows that its adherence to porcine tracheal cells is inhibited by A. pleuropneumoniae LPS antibody (Paradis et al., 1999). The same workers have shown that the active component of its LPS in adherence is more likely to be Kdo than lipid A (Paradis et al., 1994). Our data also show that Kdo from C. trachomatis LPS may play a significant role in C. trachomatis infectivity that is therefore not unique.
Although it is premature to consider if lipid ‘rafts’ might be related in any way to the role of LPS in infectivity, recent evidence suggests that A. pleuropneumoniae LPS binds to the phospholipid phosphatidylethanolamine (PE) of the host (Jeannotte et al., 2003). This is of interest, as back in 1991, C. trachomatis was found to bind PE as well as asialo-GM1 and asialo-GM2, and at that time these molecules were considered to be putative host-cell receptors (Krivan et al., 1991).
Several studies have reported that chlamydial LPS is present on the surface of infected cells (Campbell et al., 1994; Wyrick et al., 1994), albeit in low quantities; therefore, it may be that C. trachomatis, like A. pleuropneumoniae, uses LPS as an adhesin. However, whether surface exposure of LPS is greater on chlamydial reticulate bodies (RBs) than on EBs (Collett et al., 1989) remains an important question in determining whether LPS is more likely to be implicated in infectivity mechanisms other than adherence. Further work is therefore warranted to investigate the role of C. trachomatis LPS in infectivity, including the use of chlamydial lipid A and Kdo, despite limitations caused by difficulties in their extraction.
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INTRODUCTION
METHODS
