Susceptibility of Chlamydia trachomatis to the Excipient Hydroxyethyl Cellulose: pH and Concentration Dependence of Antimicrobial Activity

Chlamydia trachomatis causes pelvic inflammatory disease, ectopic pregnancy, and reproductive disability and is the most common bacterial sexually transmitted infection in the world (4). However, chlamydial infection is asymptomatic in most men and women infected with the pathogen (9). Since new preventative antimicrobials could prevent chlamydial infection, there is much effort to develop microbicides and related compounds (7, 20). Recently, a number of reports have highlighted the in vitro antichlamydial properties of polysaccharide-based chemotherapeutics and excipients (2, 8, 10, 19). Studies on the basis of the antimicrobial properties of polysaccharides highlight the need for the use of excipient-only controls when the effectiveness of new antimicrobials is tested and contribute to our understanding of the molecular mechanism of chlamydial adhesion to polysaccharides or glycoproteins on the surface of host cells. The studies reported here were designed to interrogate the potential antichlamydial effects of the common polysaccharide-based excipient hydroxyethyl cellulose (HEC) as a function of both concentration and pH.

Excipients are an inherent part of drug delivery systems used for both topical vaginal medications and contraceptives and fall into four principal classes: antioxidants, preservatives, acidifying agents, and gelling agents (8). Gelling agents are frequently polysaccharides, which are also adhesins through which bacterial pathogens bind to host cells. As such, these molecules can have competitive inhibitory effects on bacterial adhesion. However, it is possible that these excipients can play a more complex role in host-pathogen interactions. Although a number of potential host cell adhesins have been proposed to play a role in the initial stages of mammalian infection by C. trachomatis (3, 14, 18), it appears possible that membrane-localized calcium concentrations could also play a role in promoting infection.

In the current study, three experimental parameters were adjusted to determine the effects of a commonly used excipient, HEC, upon infection of cervical epithelial cells (HeLa cells) by the lymphogranuloma (LGV/L2) serovar of C. trachomatis. HeLa 229 cells were chosen as a model host system, since they are immortalized cervical epithelial cells and are germane to in vitro modeling of vaginal infection by C. trachomatis. The effects of the HEC concentration, the average molecular mass, and pH were explored in order to test the hypothesis that the HEC monomer concentration is the primary determinant of antimicrobial potency. Both HeLa 229 cells and the LGV/L2 serovar strain of C. trachomatis were obtained from American Type Culture Collection (Manassas, VA). The serovar D strain of C. trachomatis was a gracious gift from Deborah Dean (CHORI, Oakland, and University of California, Berkeley). HeLa cells were maintained in a humidified incubator at 37°C with 5% CO₂ in Dulbecco modified Eagle medium (Glutamax-1; Life Technologies, Inc., Rockville, MD) supplemented with 10% heat-inactivated fetal calf serum. C. trachomatis was grown and the multiplicity of infection (MOI) was calculated as described previously (1). The excipient solutions were prepared by dissolving 20 mg of the HEC polymers in 10 ml of deionized water with stirring and while heating. Two average molecular masses of 1.3 MDa and 90 kDa were used (Aldrich, Milwaukee, WI). The resulting solution was then diluted 1:1 with 0.2 M phosphate (pH 7) or 0.2 M acetate (pH 5) to give a final concentration of 1 mg/ml. This solution was diluted 1:100 in 0.1 M phosphate (pH 7) or 0.1 M acetate (pH 5) to give a final concentration of 10 μg/ml. The solutions were then filtered through sterile 0.22-μm-pore-size polyvinylidene difluoride filters (Fisher Scientific, Houston, TX). One control consisted of the addition of 20 μl of sterile deionized water to 80 μl of chlamydiae to a final MOI of 0.75. The two other controls were buffered with 20 μl of sterile 100 mM acetate or phosphate buffer at pH 5 and 7, respectively, added to the same volume of chlamydiae. The concentrations of HEC listed in Fig. 1 and 2 are final concentrations after dilution into the chlamydial stock. The buffer concentrations were chosen so that the resulting pH during chlamydial infection was indistinguishable from that in the stock excipient solution.

The potencies of the HEC solutions as antimicrobials were tested by mixing 20 μl of the (5×) HEC solution with 80 μl of C. trachomatis such that the final MOI was 0.75. This mixture was incubated at room temperature for 20 min and then added to six-well plates of HeLa cells grown to a density of approximately 5 × 10⁵ cells/well (∼70% confluence). The plates were incubated at 37°C for 24 h. The media were then aspirated from the wells; and the cells fixed with chilled methanol for 8 to 10 min, washed with phosphate-buffered saline, and stained with Hoechst stain and fluorescein isothiocyanate-conjugated chlamydial antibody (Argene, Varilhes, France). The percentage of infected cells was quantified by counting the chlamydial inclusions in several fields of a fluorescence microscope (Leica Microsystems, Bannockburn, IL).

Figure 1 illustrates the dose dependence of the response of C. trachomatis serovar LGV/L2 inclusion formation versus the HEC polymer concentration at pH 5. These concentrations of HEC also inhibit infection by the more clinically relevant serovar D strain at each pH tested (data not shown). Interestingly, the effectiveness of the excipient is not directly proportional to the concentration of individual HEC monomer units in the infection assay. This finding is in contrast to the possibility that there could be a concentration dependence based on the individual monomer concentration or polymer concentration. In fact, because the monomer is not specifically substituted with hydroxyethyl at a single site, different monomer sites might be anticipated to yield different affinities for binding sites on chlamydiae. However, substitution is expected to be stochastic, and both the large and the small HEC polymers should have the same substitution distributions. Surprisingly, Fig. 2 shows an inverse polymer concentration dependence at neutral pH. The lowest final polymer concentration (2 μg/ml of 1.3-MDa HEC) attenuates C. trachomatis serovar LGV/L2 infection by 80% of the level of infection for the control. Notably, the phosphate and acetate buffer controls showed similar percentages of infection. Thus, the observed effect is unlikely to be due simply to the hydronium ion concentration.

The buffered pH levels used in the current study were chosen to provide a point of comparison with the work of Lampe et al. (8). While studies performed at pH 4 also demonstrated inhibition for serovar LGV/L2 (data not shown), the effect was less pronounced than that at pH 5 or 7.

The results obtained for the infection of HeLa cells by C. trachomatis at pH 5 (Fig. 1) could be explained in terms of chlamydial binding to host cell glycolipids and glycoproteins. By using ¹²⁵I-labeled chlamydiae, bacteria were observed to bind to the phosphatidylethanolamine, asialo-GM₁, and asialo-GM₂ moities (6). Specifically, it is thought that C. trachomatis binds to the GalNAcβ1-4Galβ1-4Glc pattern. HEC utilizes a repeating Glcβ1-4Glc unit that is nonspecifically substituted with hydroxyethyl or hydroxyethoxyethyl repeats. The extent of substitution is constant between the two polymers used in this study. Given the similarity of GalNAcβ1-4Galβ1-4Glc and Glcβ1-4Glc in structure and charge neutrality, it is possible that HEC acts as a competitive inhibitor for host recognition sites on the chlamydial surface. At each pH, two comparisons can be made regarding the monomer concentration. For the measurements obtained with 200 μg/ml HEC, there was a distinct and statistically significant difference between the inhibition efficiencies. This implies that the effect observed is not proportional to the monomer concentration. Furthermore, Fig. 2 shows statistically significant differences between the infection percentages for loadings of both 2 μg/ml and 200 μg/ml. Likewise, at each pH, there are four different polymer concentrations. The nonlinear dependence of inhibition on the HEC polymer concentration implies that the polymer with an average molecular mass of 1.3 MDa can inhibit more host recognition sites on the cell membrane of chlamydiae than the smaller 90-kDa polymer, since 2 μg/ml 90-kDa HEC is 22 nM in concentration, while 200 μg/ml 1.3-MDa HEC is 1.5 nM. Thus, while the larger polymer is present at <10% the concentration of the smaller polymer, it has slightly more inhibitory activity.

The percent inhibition at pH 7 is counter to what might be expected, since the inhibitory activity of HEC varies inversely and nonlinearly with the polymer concentration. This effect could be explained by the [Ca²⁺] requirements of chlamydial infection. It has been reported that extracellular Ca²⁺ facilitates both the attachment and endocytosis of chlamydiae (5, 13). While the participation of locally high concentrations of calcium in invasion by C. trachomatis has not yet been shown, microcrystalline calcium phosphate can mediate the attachment of adenovirus to HeLa cells. Thus, coprecipitation of adenovirus with calcium phosphate elicited a more than fourfold increase in infectivity over that achieved with bare adenovirus (17). In the adenovirus studies, it was hypothesized that the microcrystalline calcium phosphate destabilized the membranes of HeLa cells. Similarly, HeLa cells treated with basic calcium phosphate crystals and a luciferase reporter plasmid showed luciferase activity of about 25% of that achieved with Lipofectamine (15).

Since the membrane localization of calcium likely occurs during infection by C. trachomatis, a hypothetical role of HEC in aiding infection can be envisioned. Two processes are operative during the precipitation of supersaturated solutions: nucleation and crystal growth. Nucleation is the formation of a small collection of highly organized molecules or ions that is capable of forming a suitable template for further crystal growth. The entity formed in this process is known as the critical nucleus, which is defined by its equivalent tendency to disperse into solution or aggregate into morphologically defined crystals (11). It has been shown that the presence of HEC facilitates the nucleation of calcium phosphate in supersaturated solutions at pH 7.4 (12). Interestingly, when the calcium-carrying capacity of HEC was studied as a function of pH, it was found that HEC promoted the mineralization of calcium phosphate at pH 7 and, consequently, released calcium phosphate at pH 4.8 with no significant hysteresis upon repeated cyclings of pH change, implying that HEC does not undergo chemical degradation at low pH (16). The pH dependence of HEC-promoted mineralization indicates that increased local membrane calcium phosphate would not be expected in the experiments whose results are shown in Fig. 1 but could be attained in the experiments whose results are shown in Fig. 2. While the results gathered in the current study are not conclusive, the tendency for HEC to promote nucleation in a concentration-dependent manner may explain the results observed in Fig. 2.

While the current study was initially designed to characterize the antichlamydial effects of a common class of excipients, the data collected have also provided insight into factors that mediate the in vitro infectivity of C. trachomatis. While it appears likely that neutral polysaccharides such as HEC are capable of competitively interfering with adhesion, the current study emphasizes the necessity for proper buffering in vaginal excipients. The current study also highlights a heretofore neglected property of excipient components: that of providing a template for calcium phosphate nucleation.

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FIG. 1.

Number of epithelial cells infected by the LGV/L2 serovar of C. trachomatis in the presence of two concentrations of HEC of two different average molecular masses at pH 5. All samples were buffered with 0.1 M acetate (pH 5). The averages and standard deviations are representative of the results from four replicates evaluated in two separate experiments. P values for relevant comparisons: *, 0.52; **, 0.11.

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FIG. 2.

Number of epithelial cells infected by the LGV/L2 serovar of C. trachomatis in the presence of two concentrations of HEC of two different average molecular masses at pH 7. All samples were buffered with 0.1 M phosphate (pH 7). The averages and standard deviations are representative of the results from two separate experiments. P values for relevant comparisons: *, 0.14; **, 0.10.

• Copyright © 2008 American Society for Microbiology