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Examination of Chlamydia trachomatis Infection in Environments Mimicking Normal and Abnormal Vaginal pH

YASIN, BUSHRA PhD*; PANG, MABEL BS*; WAGAR, ELIZABETH A. MD*; LEHRER, ROBERT I. MD†

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From the *Department of Pathology and Laboratory Medicine and Department of Medicine, UCLA School of Medicine, Los Angeles, California

The authors thank Ms. Rena Sonshine for her review of the manuscript.

Supported by grant 1 PO1 AI-37945 from the National Institutes of Health.

Reprint requests: Elizabeth A. Wagar, MD, Associate Professor, Department of Pathology and Laboratory Medicine, UCLA School of Medicine, CHS 1P-244, Los Angeles, CA 90095-1731. E-mail: ewagar@mednet.ucla.edu

Received for publication September 4, 2001,

revised November 7, 2001, and accepted November 13, 2001.

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Abstract

Background: It has long been assumed that a healthy acidic vaginal environment inhibits infection by Chlamydia trachomatis. The research objectives were to evaluate the effect of pH on C trachomatis infection by two in vitro methods, to assess pH effect at different serial dilutions of C trachomatis elementary bodies (EBs), and to examine protection by an antibiotic peptide, protegrin (PG-1), over a pH range.

Goals: The goals of this study were to test the hypothesis that acidic pH inhibits C trachomatis infection and to determine the ability of PG-1 to provide protection at acidic and neutral pH.

Study Design: The effect of pH on C trachomatis was examined using two pH-adjusted preincubation shell vial assays. C trachomatis EBs (serovars L2, D, and E) were exposed to pH-adjusted media, with and without PG-1, and infection was assessed by inclusion forming unit (IFU) formation in McCoy cell monolayers.

Results: Acidic pH in preincubation media markedly decreased IFUs by both in vitro methods. Serial dilution experiments showed a 3- to 10-fold reduction in IFUs for C trachomatis (L2 and E) at pH 5.0, compared with those at pH 7.5. C trachomatis (D) showed a 17- to 23-fold reduction in IFUs (serial dilutions 1:1–1:4). PG-1 protected McCoy cell monolayers from infection by C trachomatis after exposure to varied pH environments.

Conclusion: Acidic pH exposure significantly reduced C trachomatis infection in vitro. Our results support the hypothesis that a healthy acidic vaginal environment protects women from C trachomatis infection. In addition, antibiotic peptides may provide protection as topical microbicides, regardless of vaginal pH.

THE GRAM-NEGATIVE BACTERIAL PATHOGEN Chlamydia trachomatis is sexually transmitted and widely prevalent. 1–4 It requires an intracellular environment in eukaryotic cells for bacterial replication. 5,6 The ability of C trachomatis to infect eukaryotic cells in various vaginal environments has not been documented. Recent studies indicate, however, that bacterial vaginosis, a condition of increased vaginal pH and altered microbial profile, is associated with sexually transmitted infections 7,8 and upper genital tract disease. 9 Bacterial vaginosis is also a common condition during the fertile period. 10 Bacteria associated with bacterial vaginosis include Gardnerella vaginalis, Mobiluncus sp, and various anaerobes such as Prevotella sp. 11–13 Increasing the vaginal pH from an acidic pH to a neutral pH results in a loss of normal vaginal flora, in particular, Lactobacillus sp. 14

The vaginal mucosa and normal vaginal flora, including Lactobacillus sp, contribute to a relatively low vaginal pH. 15 This environment additionally contains antimicrobial secretions that are protective, such as lysozyme, zinc, lactoferrin, and peroxidase. A 36-amino-acid antimicrobial peptide, human β-defensin-1, is also present in the epithelial layers of the vagina and is thought to contribute to natural protection. 16Lactobacillus sp are an important component of the normal flora. Ninety-five percent of patients without bacterial vaginosis, compared with only 60% of patients with bacterial vaginosis, have Lactobacillus sp present. 17 There may be a coaggregation of Lactobacillus sp and other bacteria that provides a protective vaginal surface. In addition, menses is associated with variation in the vaginal ecology, including a decreased predominance of Lactobacillus sp and an increased risk for sexually transmitted infections.

It has been widely assumed that the naturally acidic vaginal environment inhibits infection by common sexually transmitted agents. 18 However, no attempts have been made to scientifically test this hypothesis. Recently described preincubation assays 19,20 allow assessment of the effect of pH on chlamydial inclusion forming unit (IFU) formation. The goals of this investigation were to determine if pH affects C trachomatis infectivity in vitro, by employing preincubation exposure to both acidic and neutral pH; to verify these observations by using two described preincubation assays over a pH range; and to quantitatively assess the decrease in infectivity induced by an acidic pH, by using serial dilutions. In addition, we examined the ability of an antibiotic peptide, protegrin (PG-1), 21 to provide protection in both acidic and neutral pH environments. Antibiotic peptides are important candidates for vaginal topical microbicides that may effectively treat chlamydial infections in a variety of vaginal environments.

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Materials and Methods

Chlamydia

C trachomatis (L2) (L2/434/Bu) seed was prepared as previously described. 22 The titer was determined at the time of preparation and adjusted to produce reproducible IFU counts. All reported experiments were repeated at least two or more times with duplicate or triplicate coverslips, unless otherwise indicated. The D strain was acquired from the American Type Culture Collection (VR-885; ATCC, Rockville, MD). C trachomatis serovar E (mtw447) was graciously provided by Dr. Mary Lampe, University of Washington, Seattle.

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Peptides

PG-1 amide was synthesized (SynPep, Dublin, CA) and purified as previously described. 19 Peptides used in these studies were at least 98% pure, as judged by analytical reverse-phase high-performance liquid chromatography (RP-HPLC) and acid–urea–polyacrylamide gel electrophoresis (PAGE). The purified peptides were prepared and stored as 1 mg/ml stock solutions in sterile acidified water (0.01% glacial acetic acid).

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Effect of pH on C trachomatis Infection in Vitro in Preincubation Assays (Comparison of Two Methods)

The effect of pH on chlamydial infection was determined with use of two preincubation shell vial assays. 19,20 For each method, coverslipped monolayers of McCoy cells (Bartels Diagnostics, Deerfield, IL) grown in shell vials containing 1 ml Eagle's minimal essential medium with 10% fetal bovine serum (EMEM/BS) were used. The concentrated C trachomatis elementary body (EB) seed stock was stored at −85 °C in a mixture of 0.2 mol/l sucrose, 0.004 mol/l KH2PO4, 0.009 mol/l Na2HPO4, and 0.004 mol/l glutamic acid (SPG).

Approximately 900 or 300 chlamydial EBs were mixed with SPG (adjusted to pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5) in a total volume of 50 μl and incubated for 2 hours at room temperature. SPG (adjusted to pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5) without EBs was included as controls. In the first method, EMEM/BS was aspirated from the McCoy cells and replaced with the preincubated C trachomatis (50 μl) or controls, and the shell vials were centrifuged at 1500 g for 1 hour at 20 °C. After removal of the inocula, the monolayers were washed twice with EMEM/BS containing 1 mg/ml cycloheximide (Bio Whittaker, Walkersville, MD) and incubated at 35 °C for 48 hours in 1 ml of the cycloheximide-containing EMEM/BS.

Subsequent fixation with ethanol, staining with Microtrak fluorescein isothiocyanate–linked monoclonal antibody (Syva Company, San Jose, CA), and fluorescence microscopy were performed as previously described. 19 A second preincubation method was also employed. 20 Following the 2-hour preincubation step, 5-μl aliquots of preincubated C trachomatis were mixed with 195 μl SPG, and 100 μl of this 200-μl mixture was inoculated to the McCoy cell monolayers. 20 The remainder of the procedure was identical to that described above.

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Serial Dilution Analysis of C trachomatis Infection

The effect of pH on C trachomatis infection was also examined by serial dilution analysis, to determine how pH altered IFU counts when inoculum size varied. Serial twofold dilutions of C trachomatis (L2, D, or E) were prepared in SPG (adjusted to pH 5.0 and pH 7.5). The dilutions ranged from an inoculum size expected to yield approximately 400 EBs to that expected to yield 12.5 EBs. Five μl of each dilution of C trachomatis inoculum was mixed with 45 μl SPG (adjusted to pH 5.0 and pH 7.5) and incubated at room temperature for 2 hours. SPG-only controls (pH-adjusted) were incubated along with the test samples. At completion of the incubation period, an additional 150 μl SPG was added per vial. After careful aspiration of the EMEM/BS from the McCoy cell monolayers and replacement of it with the chlamydial dilutions, coverslips were incubated for 48 hours in EMEM/BS containing 1 mg/ml cycloheximide. The shell vial coverslips were then fixed and stained with fluorescein isothiocyanate–linked antibody to C trachomatis.

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Effect of Antimicrobial Peptides on C trachomatis Infection at Neutral and Acidic pH

We examined the effect of an antimicrobial peptide, PG-1, against C trachomatis infection in a pH range to determine if PG-1 was able to provide protection at both acidic and neutral pH. Experiments were conducted at pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 with use of pH-adjusted SPG. We examined the activity of PG-1 at different concentrations on chlamydial EB infectivity with use of the two modified versions of the standard shell vial assay. Approximately 900 or 300 viable chlamydial EBs were mixed with SPG (pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5) and added to 2.5 μl peptide (12.5, 25.0, or 50.0 μg/ml final concentration) in acidified water, in a total volume that was adjusted to 50 μl by the addition of SPG (pH, 4.5–7.5). The mixtures were incubated for 2 hours at room temperature. Appropriate C trachomatis–only controls were included at each pH. The remainder of the procedures were as described above.

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Effect of Seminal Plasma on C trachomatis Infection at Neutral and Acidic pH

With approval from the UCLA Institutional Review Board, fresh-frozen human semen that had been obtained from anonymous normal donors was purchased (Biologic Specialty Corporation, Colmar, PA). These semen specimens were stored at −80 °C. Before use, they were placed in a 37 °C water bath for 1 hour to allow the seminal plasma to liquefy. Next, the specimens were centrifuged at 10,000 g for 10 minutes at 4 °C to deposit sperm and other cellular elements. The supernatant plasma was removed and stored at −80 °C until used. Pooled seminal plasma used in these studies was derived from four to six individual donors. The seminal plasma was used immediately after thawing, within a short period of incubation.

To determine the pH effect of the male ejaculate on C trachomatis infection, serial dilutions of seminal plasma (adjusted to pH 4.5 and 7.5) in SPG (adjusted to pH 4.5 and pH 7.5) were incubated with 300 viable C trachomatis (L2, E) EBs at room temperature for 2 hours. At completion of the incubation period, the EMEM/BS was aspirated from the McCoy cell monolayers and replaced with the seminal plasma/chlamydial dilutions, and coverslips were incubated for 48 hours in EMEM/BS containing 1 mg/ml cycloheximide. The shell vial coverslips were then fixed and stained with fluorescein isothiocyanate–linked antibody to C trachomatis, as previously described.

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Results

Effect of pH on C trachomatis Infection in Vitro in Preincubation Assays (Comparison of Two Methods)

The in vitro pH environment markedly influenced the ability of chlamydial EBs to infect an in vitro McCoy cell monolayer by both methods (Figure 1, A–C). Experiments were performed with two different methods to determine whether reproducible patterns occurred over a range of pH adjustments. It was anticipated that quantitative variation would occur between different serovars of C trachomatis because of the different procedures used in the two methods. However, the pattern of decreased IFUs in acidic pH was consistent by both methods. IFUs were markedly decreased when preincubated at acidic pH versus neutral pH. Decreased numbers of IFUs were observed at acidic pH for all three C trachomatis serovars with both methods. At pH 6.0 to 6.5, the number of IFUs was ≥75% of IFUs at neutral pH for C trachomatis L2 (Figure 1A). A similar pattern was observed for C trachomatis D and E (Figure 1, B and C). At pH 5.5 to 6.0, C trachomatis D and E again infected McCoy cells at ≥75% of IFUs observed at neutral pH.

Fig. 1
Fig. 1
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Serial Dilution Analysis of C trachomatis Infection

Data from serial dilutions showed a decrease in C trachomatis (L2, D, and E) IFUs because of acidic pH over a range of dilutions. Serial dilutions also mimic the anticipated variation in inoculum size in naturally occurring sexually transmitted diseases. The data confirm that at pH 5.0, C trachomatis L2 and E IFUs decreased threefold to 10-fold in comparison with IFUs at pH 7.5. The effect of acidic pH was more significant for C trachomatis D than for C trachomatis L2 and E (Figure 2, A–C). At pH 5.0, C trachomatis D, IFUs decreased 17- to 23-fold in comparison with IFUs at pH 7.5 (serial dilutions, 1:1–1:4;Figure 2B). However, this decrease was still within the same log as those experiments performed with serovars L2 and E.

Fig. 2
Fig. 2
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Effect of PG-1 on C trachomatis Infection at Neutral and Acidic pH

At a concentration of 12.5 μg/ml, PG-1 provided protection against C trachomatis L2 in pH environments ranging from 4.5 to 7.5 (27.2–70.7% reduction in IFUs by method 1 and 11.1–72.8% by method 2;Table 1). A lesser degree of PG-1 protection was seen versus C trachomatis D (0–61.6% reduction in IFUs) and E (0–48.9% reduction in IFUs) at all PG-1 concentrations tested (12.5, 25.0, and 50.0 μg/ml). These results were confirmed for all three serovars and both inocula sizes, by means of the two preincubation assays (data not shown).

Table 1
Table 1
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Effect of Seminal Plasma on C trachomatis Infection at Neutral and Acidic pH

Seminal plasma, when in an acidic environment, was associated with a marked decrease in the ability of chlamydial EBs to infect an in vitro McCoy cell monolayer. These results were comparable to those in experiments assessing acidic pH without seminal plasma. Preincubation of C trachomatis L2 with seminal plasma in an acidic environment decreased the number of IFUs in comparison with that with preincubation with seminal plasma at neutral pH (pH 4.5: 37–92% reduction in IFUs; pH 7.5: 14–58% reduction in IFUs). A similar pattern was observed for C trachomatis E. There may be as yet unidentified protective elements in seminal plasma, accounting for some decrease in IFUs at neutral pH, as well as at acidic pH.

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Discussion

There is much speculation about the effect of the vaginal acidic environment on sexually transmitted infections. 7,8,14 Although chlamydial infections may affect the mucosal cells in the cervical channel, which has a pH higher than that of the vagina, the cervical os and exposed external cervical surfaces are bathed by the vaginal environment. Thus, the cervical opening is directly exposed to the pH of the vagina. The effect of pH on infection over a determined pH range has not been thoroughly analyzed for C trachomatis.

In this investigation, we analyzed the ability of C trachomatis to infect a eukaryotic monolayer after exposure to various pH environments in vitro with use of two preincubation assays. We demonstrated the following: (1) infections are decreased in acidified preincubation experiments by both methods (Figure 1, A–C); (2) when EBs are serially diluted, the effect of acidic pH reduces infection 3- to 10-fold for C trachomatis L2 and E and 17- to 23-fold for C trachomatis D (Figure 2, A–C); and (3) a representative antibiotic peptide, PG-1, has antimicrobial activity after exposure to both acidic pH and neutral pH preincubation (Table 1).

The theory that the neutral pH environment of bacterial vaginosis better supports infection by C trachomatis may be supported by our data. However, other variables of the vaginal environment need to be examined. When the entire range of pH from 4.5 to 7.5 is examined for the three serovars, pH 5.5 to 6.0 appears to be the cutoff point at which ≥65% of IFUs are recovered, in comparison with neutral pH. This is true for all three C trachomatis serovars, L2, D, and E. There are some differences noted in the response of C trachomatis L2 and C trachomatis D when the preincubation stage is acidified. The serial dilution studies indicate that C trachomatis D is much more sensitive to acidification in the preincubation stage. This variation, however, is within the same log for most experiments. Further pH analyses with other serovars may determine whether this is a significant difference. Seminal plasma experiments suggested that acidification was protective regardless of the presence of seminal plasma.

Protegrin (PG-1) has been shown to have a marked antimicrobial effect against C trachomatis at pH 7.5. 19,23 Our current findings demonstrate that PG-1 is active against C trachomatis L2 in acidic and normal pH environments (Table 1). Our data also provide an interesting profile of the PG-1 susceptibility of three different C trachomatis serovars. In addition, these data show the ability of PG-1 to provide topical antimicrobial protection vaginally, regardless of pH. On the basis of this information, it appears that antibiotic peptide structure can be optimized for enhanced antimicrobial function with use of pH-adjusted assays.

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