Members of Prevotella Genus Distinctively Modulate Innate Immune and Barrier Functions in a Human Three-Dimensional Endometrial Epithelial Cell Model

Abstract

Background: Prevotella species are commonly isolated from the reproductive tract of women with obstetric/gynecologic health complications. However, contributions of this genus to changes in local microenvironment are not well characterized. Our objective was to evaluate species-specific effects of Prevotella on the human endometrial epithelium.

Methods: Thirteen Prevotella strains, originally isolated from the human oral cavity, amniotic fluid, endometrium, or vagina (including women with bacterial vaginosis), were obtained from BEI and ATCC resources. Bacteria were evaluated in silico and in vitro using human endometrial epithelial cells (EEC) grown as monolayers or a 3-dimensional (3D) model.

Results: Genomic characterization illustrated metabolic and phylogenetic diversity of Prevotella genus. Among tested species, P. disiens exhibited cytotoxicity. Scanning electron microscopy analysis of the 3D EEC model revealed species-specific colonization patterns and alterations of ultracellular structures. Infection with sialidase-producing P. timonensis resulted in elongated microvilli, and increased MUC3 and MUC4 expression. Infections with Prevotella species, including P. bivia, did not result in significant proinflammatory activation of EEC.

Conclusions: Collectively, findings indicate that Prevotella species are metabolically diverse and overall not cytotoxic or overtly inflammatory in EEC; however, these bacteria can form biofilms, alter barrier properties of the endometrial epithelium, and ultimately impact colonization of secondary colonizers.

Keywords: ascending infection; bacterial vaginosis; biofilm; colonizer; epithelial layer; genital inflammation; membrane-associated mucin; pathway prediction; sialidase; upper reproductive tract infection.

Figure 1.

Prevotella isolates are genetically and metabolically diverse and share genetic elements based on isolation source. A, Phylogenetic tree constructed based on 16S rRNA gene sequences of the Prevotella strains using unweighted pair group method with arithmetic mean algorithm. The 16S rRNA amplicons were downloaded from the Silva database (https://www.arb-silva.de) and the sequences were aligned using the Multiple Alignment using Fast Fourier Transform (MAFFT) version 7 program (https://mafft.cbrc.jp/alignment/server/) [25]. Number of genes and number of metabolic reactions were extracted from the MetaCyc database (https://metacyc.org) [27]. The outer grey circles represent number of genes detected in each strain. The size of the inner circle represents number of genes with known functions. The color intensity of the inner circle represents number of metabolic reactions. P. bivia strains were phylogenetically more similar to P. amnii and P. timonensis than the other vaginal strains in the analysis. P. disiens and P. denticola strains were more metabolically diverse and clustered with the oral strains. P. bivia species had relatively smaller genomes, number of genes with known functions, and metabolic reactions in comparison to other Prevotella species isolated from female reproductive tract or oral cavity. B, Number of metabolic pathways and shared metabolic pathways among the Prevotella species based on predictions from the MetaCyc database. Oral isolates had the highest number of pathways whereas P. bivia strain had the greatest number of shared pathways. Abbreviation: BV, bacterial vaginosis.

Figure 2.

Among the Prevotella strains tested, P. disiens was dramatically cytotoxic to the endometrial epithelial cells grown as monolayers as well as in the 3D model. Human EEC models (monolayers and 3D) were incubated with Prevotella strains for 24 hours at MOI 10. Monolayer cultures were used to screen 13 Prevotella strains (Supplementary Figure 1) and 3D model was incubated with selected Prevotella strains for more robust evaluation of cytotoxicity. A, Crystal violet staining of the endometrial epithelial cells grown as monolayers and (B) light microscopy images of the 3D EEC aggregates visualized the damage on endometrial epithelial cells due to P. disiens infection (× 100). Light microscopy images were converted to grayscale using the same brightness and contrast filters. C, Crystal violet staining densities of the cells in the monolayer model were quantified using Fiji (ImageJ) software. The comparisons were performed relative to PBS control. All the incubations were performed under the same conditions. D, Viabilities of the endometrial epithelial cells were significantly lower when the 3D EEC aggregates were incubated with P. disiens compared to other Prevotella species. Viability calculations were based on trypan blue exclusion in comparison to PBS control. Student t test **** P < .001. Data are representative of at least 3 independent experiments. C and D, Error bars represent standard deviation from the mean. Abbreviations: 3D, 3-dimensional; BV, bacterial vaginosis; EEC, endometrial epithelial cell; MOI, multiplicity of infection; PBS, phosphate-buffered saline.

Figure 3.

Prevotella isolates colonize human 3D endometrial epithelial cell model in a species-specific manner. The 3D EEC aggregates were incubated with Prevotella species at MOI 10 for 4 hours under anaerobic conditions. The SEM images depict PBS treatment and colonization with P. timonensis CRIS 5C-B1, P. disiens DNF00882, P. bivia DNF00188, P. bivia VPI6822, and P. corporis MJR7716. SEM images also demonstrated viability of Prevotella strains. Prevotella species induced species-specific microvilli remodeling and cellular secretion patterns. The images were pseudocolored using Adobe Photoshop. The images presented were selected from a minimum of 3 representative SEM images. Abbreviations: 3D, 3-dimensional; EEC, endometrial epithelial cell; MOI, multiplicity of infection; PBS, phosphate-buffered saline; SEM, scanning electron microscopy.

Figure 4.

The molecular interactions between P. timonensis and human 3-dimensional endometrial epithelial cell model rely on mucin metabolism. Relative expression levels of membrane-associated mucins (A) MUC3 and (B) MUC4 in comparison to phosphate-buffered saline (PBS) treated cells. P. timonensis, P. buccae, and P. corporis induced significantly higher levels of MUC3 and MUC4. C, Sialidase activity of the selected Prevotella strains. P. timonensis had more than 1000-times greater sialidase activity in comparison to the representative Prevotella strains. P. bivia VPI6822 was selected to represent Prevotella strains. Data are representative of at least 3 independent experiments. D, Predictions of enzymatic reactions involved in mucin degradation or metabolism based on Prevotella genomes in MetaCyc database. P. denticola and P. timonensis strains are predicted to contain genes that can metabolize mucins. P. timonensis harbors another gene that encodes transformation of mucins. Strain designations: P. amnii CRIS 21A-A, P. buccae D17, P. bivia VPI6822, P. corporis MJR7716, P. denticola DNF00960, and P. timonensis CRIS 5C-B1. Tukey’s multiple comparisons results: *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5.

Prevotella strains do not robustly induce an inflammatory response in the human 3D endometrial epithelial cell model. The expression of proinflammatory genes in the human 3D EEC following infection with Prevotella species for 24 hours. Expression levels of IL8, TNF, and CCL20 genes were normalized to expression levels of GAPDH gene and log₁₀-transformed prior to the hierarchal clustering analysis. The clustering was performed based on Euclidean distances. FLA and poly(I:C) were used as positive controls for induction of proinflammatory genes and Gardnerella vaginalis infections were used to compare the response from Prevotella infections. Data are representative of at least 3 independent experiments. Abbreviations: 3D, 3-dimensional; EEC, endometrial epithelial cell; FLA, flagellin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL8, interleukin-8; PBS, phosphate-buffered saline; TNF, tumor necrosis factor.