Mobiluncus mulieris alters the transcriptomic profile of cervicovaginal epithelial cells, shedding light on molecular drivers of adverse reproductive outcomes

Abstract

The vaginal microbiota is critical for reproductive health, and its disruption, particularly the loss of Lactobacillus spp. and dominance of anaerobes such as Mobiluncus mulieris (community state type IV, CST IV), is associated with bacterial vaginosis, sexually transmitted infections, and adverse reproductive outcomes, including preterm birth (PTB). While Gardnerella spp. have been widely studied, the role of M. mulieris remains poorly understood. This study used an unbiased discovery approach to examine host-microbe interactions driven by M. mulieris across distinct epithelial barriers of the lower reproductive tract. RNA sequencing revealed that live bacteria, cell-free supernatant, and bacterial extracellular vesicles (bEVs) each induced unique transcriptional responses in epithelial cells. All three components activated immune and inflammatory pathways, with bEVs eliciting the strongest response, particularly via toll-like receptor (TLR) 2 and TLR5 signaling. M. mulieris also altered extracellular matrix (ECM) remodeling pathways, including upregulation of matrix metalloproteinase 9 (MMP9), a key mediator linked to PTB. These findings were supported by clinical data showing elevated MMP9 in pregnant women with M. mulieris-containing vaginal microbiota. Collectively, these results highlight the broad impact of M. mulieris on epithelial responses and identify mechanisms by which specific anaerobes contribute to inflammation and ECM disruption in adverse reproductive outcomes.

Fig. 1. Overview of pathway over-representation analysis and DEGs results under different M. mulieris exposures.

a Heatmap showing the number of entities associated with each main pathway category in the network view of the pathway over-representation analysis. Color intensity is proportional to the number of entities in each pathway, with darker colors representing higher numbers of entities. Venn diagrams representing the number of DEGs identified in response to three M. mulieris exposure in b Ect, c End, and d VK2 cell lines.

Fig. 2. GSEA, pathway analysis, and their DEGs for key biological pathways.

a A summary of the results from GSEA, visualizing the impact of M. mulieris on pathways at the highest hierarchical level. Fold changes (FCs) were calculated by comparing to the control group. Only cell line and exposure sets showing significant alterations are included in the plot. Different shapes represent cell lines (circle for Ect, square for End, and triangle for VK2), and colors represent the three M. mulieris exposures (orange for live, blue for BFS, and green for bEV exposure). b Heatmap showing the sub-pathways under the main pathway “Cell-cell communication” and c the DEGs involved in these sub-pathways. d Heatmap for sub-pathways under “Extracellular matrix organization” and e the DEGs involved in these sub-pathways. f Heatmap for sub-pathways under the “Immune system” and g the DEGs involved in these sub-pathways. For all heatmaps, darker colors indicate higher FC compared to the control group. Annotations representing significant alterations are visualized while non-significant changes are left blank (white) in the plots, and p values from VK2 treated with M. mulieris bEV group are summarized in Supplementary Data 13 (pathways) and Supplementary Data 14 (DEGs).

Fig. 3. Measurement of inflammation markers in Ect, End, and VK2 cells in response to M. mulieris (MM) exposure with or without hTLR2 and/or hTLR5 inhibitors.

a IL-6, b IL-8, and c CCL20 levels in response to live M. mulieris exposure; d IL-6, e IL-8, and f CCL20 levels in response to BFS M. mulieris exposure; g IL-6, h IL-8, and i CCL20 levels in response to bEV M. mulieris exposure. The inhibitors for hTLR2 and hTLR5 are written as TLR2i and TLR5i. Bar plots represent the mean, the error bars for the standard deviation, and the dots represent the technical replicate (n = 3). One-way ANOVA was used, followed by Tukey’s multiple comparison test, with statistical significance denoted by “*” (p < 0.05), “**” (p < 0.01), “***” (p < 0.001), “****” (p < 0.0001).

Fig. 4. In vitro analysis of MMP9 protein expression in response to live, BFS, and bEV M. mulieris (MM) exposures.

MMP9 protein levels were measured in the culture media of VK2 cells exposed to a live, b BFS, and c bEV M. mulieris exposures, with and without the addition of hTLR2 and/or hTLR5 inhibitors (denoted as TLR2i and TLR5i). One-way ANOVA was used to compare MMP9 concentrations between groups. For statistically significant results (p < 0.05), post-hoc tests were applied. d MMP9 protein levels measured in the culture media of THP1 wild-type cells in response to d live, e bEV, and f BFS M. mulieris exposures. Unpaired t-tests were used to compare MMP9 concentrations between control and M. mulieris exposures. g MMP9 protein levels measured in the culture media of THP1 wild type and THP-TLR2KO exposed to g live and h bEV. mulieris exposures. White bars represent the results from THP1 cells, and black bars represent the results from THP1-TLR2KO cells. Bar plots represent the mean, the error bars for the standard deviation, and the dots represent the technical replicate (n = 3). Two-way ANOVA was applied to assess the impact of TLR2 knockout. The statistical significance was denoted by “*” (p < 0.05), “**” (p < 0.01), and “***” (p < 0.001).

Fig. 5. MMP9 concentrations in vaginal swab samples from subjects in the high L. crispatus (LC) and high M. mulieris (MM) groups.

a A bar plot showing the MMP9 concentrations measured in vaginal swab samples from subjects in the high LC and high MM groups. Bar plots represent the mean and the error bars for the standard deviation. The Mann–Whitney test was applied to compare MMP9 levels between the two groups, with statistical significance indicated by “***” (p < 0.001). b Within the high MM group, MMP9 concentrations were visualized by finer CST groupings.

Fig. 6. Schematic summary of the key findings from this study.

This figure provides a visual overview of the main results, illustrating the relationships between M. mulieris exposure and gene expression changes in immune system pathways, cell-cell communication, and extracellular matrix organization within the cervicovaginal space. These changes are compared to the condition with healthy vaginal microbiota, and the potential clinical relevance of these findings is also highlighted. All annotations are based on DEGs and significantly enriched pathways. Annotations in gray represent genes or pathways with a fold change of less than 1.5 compared to the control groups. This figure was generated with BioRender.com.