The Cervicovaginal Microbiota-Host Interaction Modulates Chlamydia trachomatis Infection

Abstract

The mechanism(s) by which Lactobacillus-dominated cervicovaginal microbiota provide a barrier to Chlamydia trachomatis infection remain(s) unknown. Here we evaluate the impact of different Lactobacillus spp. identified via culture-independent metataxonomic analysis of C. trachomatis-infected women on C. trachomatis infection in a three-dimensional (3D) cervical epithelium model. Lactobacillus spp. that specifically produce d(-) lactic acid were associated with long-term protection against C. trachomatis infection, consistent with reduced protection associated with Lactobacillus iners, which does not produce this isoform, and with decreased epithelial cell proliferation, consistent with the observed prolonged protective effect. Transcriptomic analysis revealed that epigenetic modifications involving histone deacetylase-controlled pathways are integral to the cross talk between host and microbiota. These results highlight a fundamental mechanism whereby the cervicovaginal microbiota modulates host functions to protect against C. trachomatis infection.IMPORTANCE The vaginal microbiota is believed to protect women against Chlamydia trachomatis, the etiologic agent of the most prevalent sexually transmitted infection (STI) in developed countries. The mechanism underlying this protection has remained elusive. Here, we reveal the comprehensive strategy by which the cervicovaginal microbiota modulates host functions to protect against chlamydial infection, thereby providing a novel conceptual mechanistic understanding. Major implications of this work are that (i) the impact of the vaginal microbiota on the epithelium should be considered in future studies of chlamydial infection and other STIs and (ii) a fundamental understanding of the cervicovaginal microbiota's role in protection against STIs may enable the development of novel microbiome-based therapeutic strategies to protect women from infection and improve vaginal and cervical health.

Keywords: Lactobacillus; epigenetic; lactic acid; microbiome; proliferation; sexually transmitted infection.

FIG 1

Effect of Lactobacillus spp. on C. trachomatis infectivity. (A) Frequency of vaginal CSTs in controls, at visit 1 (C. trachomatis positive) and visit 2 (C. trachomatis cleared). (B) Comparison of the proportions of each CST between control, visit 1, and visit 2. (C) Azithromycin and doxycycline MICs determined for strains of G. vaginalis, L. crispatus, L. gasseri, L. iners, and L. jensenii. (D) Secondary infection assay performed after A2EN 3D epithelial cells were preexposed prior to C. trachomatis infection to either cell culture medium (control), NYC III bacterial culture medium (control), or sterile-filtered culture supernatants from L. crispatus, L. jensenii, L. iners alone, and L. iners supplemented with 1% d(−) or l(+) lactic acid at pH 4 and visualized by fluorescence microscopy. Epithelial cell nuclei are stained blue while C. trachomatis inclusions are stained green. Representative images are shown. (E) Percentage of C. trachomatis-infected epithelial cells in secondary infection assays performed after A2EN 3D epithelial cell models were preexposed to control medium or culture supernatants from L. crispatus, L. jensenii, and L. iners prior to C. trachomatis infection. Results are from three independent experiments. (F) d(−) and l(+) lactic acid concentrations in the culture supernatants obtained from Lactobacillus spp. and chemical solutions of 1% d(−) and l(+) lactic acid. (G) Percentage of C. trachomatis-infected epithelial cells in secondary infection assays from A2EN 3D epithelial cell models preexposed prior to C. trachomatis infection to L. iners culture supernatant alone and L. iners culture supernatant supplemented with 1% d(−) or l(+) lactic acid at pH 4. Results are from three independent experiments. Statistical significance is shown as follows: one dot, P value < 0.05; two dots, P value < 0.01; three dots, P value < 0.001; and four dots, P value < 0.0001. Colored dots indicate pairwise comparison to A2EN medium, while black dots represent statistical significances obtained from ANOVAs comparing all four treatments.

FIG 2

Effect of d(−), d/l racemic mixture, and l(+) lactic acid on C. trachomatis infectivity. (A) Secondary infection assay of A2EN 3D epithelial cells preexposed prior to C. trachomatis infection to lactic acid or HCl at various concentrations and hence pHs. Representative images are shown. (B) Percentages of C. trachomatis-infected epithelial cells in secondary infection assays are shown; results are from three independent experiments. (C) Percentage of C. trachomatis-infected epithelial cells in secondary infection assays after A2EN 3D epithelial cell models were either preexposed to d(−) or l(+) lactic acid at pH 4 or 7 and infected with C. trachomatis or not exposed but infected with C. trachomatis preexposed to d(−) or l(+) lactic acid at pH 4 or 7 or both C. trachomatis and cells were preexposed to d(−) or l(+) lactic acid at pH 4 or 7. Results are from three independent experiments. Statistical significance is shown as follows: one dot, P value < 0.05; two dots, P value < 0.01; three dots, P value < 0.001; and four dots, P value < 0.0001.

FIG 3

Long-term effect of lactic acid and Lactobacillus culture supernatants on C. trachomatis infectivity. Percentage of C. trachomatis-infected epithelial cells in secondary infection assays after A2EN 3D epithelial cell models were preexposed to lactic acid; HCl; or L. crispatus, L. jensenii, and L. iners culture supernatants prior to C. trachomatis infection performed at 0 h, 24 h, and 48 h. Results are from three independent experiments. Statistical significance is shown as follows: one dot, P value < 0.05; two dots, P value < 0.01; three dots, P value < 0.001; and four dots, P value < 0.0001. Colored dots correspond to pairwise comparison to A2EN medium, and black dots represent statistical significances obtained from ANOVAs comparing the four treatments.

FIG 4

The composition of the vaginal microbiota affects cell cycling functions and C. trachomatis infection. (A) Normalized miR-193b expression in relation to in vivo Lactobacillus relative abundance. Each point is colored according to the CST of the samples from which it is derived. (B) Percentage of miR-193b gene targets mapped to Gene Ontology Direct major processes. Evaluation of the effect of culture supernatant on migration as evaluated by scratch assay of VK2 epithelial cells. (C) Percent filled scratch area (compared to 0 h) after exposure to L. crispatus, L. jensenii, L. iners, and G. vaginalis culture supernatants or lactic acid for 22 h. (D) Evaluation of the effect of culture supernatant on proliferation evaluated by EdU assay. Percentage of VK2 epithelial cells positive for EdU nucleobases at each time point across the 22-h assay. (E) Percentage of A2EN epithelial cells positive for EdU nucleobases after exposure to CDK4 inhibitors CAS 546102-60-7 and Fascaplysin or cell culture medium and evaluation by EdU assay. (F) Percentage of C. trachomatis-infected cells in the primary 2D A2EN epithelial cells preexposed to CDK4 inhibitors or cell culture medium performed on coverslips. Statistical significance is shown as follows: single dot, P value < 0.05. For panels C and D, statistically significant time points (P value < 0.05) are represented with black points.

FIG 5

Longitudinal relative expression patterns for selected cell cycle and chromatin remodeling genes. Shown are gene log₂ fold change values for 4 h, 13 h, and 22 h in VK2 cells exposed to L. crispatus, L. jensenii, L. iners, or G. vaginalis culture supernatants or cell culture medium. Horizontal lines indicate log₂ fold changes of −1, 0, and 1. Statistical significance is shown as follows: single dot, FDR < 0.01.