Computational Microbiome Pharmacology Analysis Elucidates the Anti-Cancer Potential of Vaginal Microbes and Metabolites.

The vaginal microbiome's role in risk, progression, and treatment of female cancers has been widely explored. Yet, there remains a need to develop methods to understand the interaction of microbiome factors with host cells and to characterize their potential therapeutic functions. To address this challenge, we developed a systems biology framework we term the Pharmacobiome for microbiome pharmacology analysis. The Pharmacobiome framework evaluates similarities between microbes and microbial byproducts and known drugs based on their impact on host transcriptomic cellular signatures. Here, we apply our framework to characterization of the Anti-Gynecologic Cancer Vaginal Pharmacobiome. Using published vaginal microbiome multi-omics data from the Partners PrEP clinical trial, we constructed vaginal epithelial gene signatures associated with each profiled vaginal microbe and metabolite. We compared these microbiome-associated host gene signatures to post-drug perturbation host gene signatures associated with 35 FDA-approved anti-cancer drugs from the Library of Integrated Network-based Cellular Signatures database to identify vaginal microbes and metabolites with high statistical and functional similarity to these drugs. We found that Lactobacilli and their metabolites can regulate host gene expression in ways similar to many anti-cancer drugs. Additionally, we experimentally tested our model prediction that taurine, a metabolite produced by L. crispatus, kills cancerous breast and endometrial cancer cells. Our study shows that the Pharmacobiome is a powerful framework for characterizing the anti-cancer therapeutic potential of vaginal microbiome factors with generalizability to other cancers, microbiomes, and diseases.

Cortical astrocyte N-methyl-D-aspartate receptors influence whisker barrel activity and sensory discrimination in mice.

Astrocytes express ionotropic receptors, including N-methyl-D-aspartate receptors (NMDARs). However, the contribution of NMDARs to astrocyte-neuron interactions, particularly in vivo, has not been elucidated. Here we show that a knockdown approach to selectively reduce NMDARs in mouse cortical astrocytes decreases astrocyte Ca2+ transients evoked by sensory stimulation. Astrocyte NMDAR knockdown also impairs nearby neuronal circuits by elevating spontaneous neuron activity and limiting neuronal recruitment, synchronization, and adaptation during sensory stimulation. Furthermore, this compromises the optimal processing of sensory information since the sensory acuity of the mice is reduced during a whisker-dependent tactile discrimination task. Lastly, we rescue the effects of astrocyte NMDAR knockdown on neurons and improve the tactile acuity of the animal by supplying exogenous ATP. Overall, our findings show that astrocytes can respond to nearby neuronal activity via their NMDAR, and that these receptors are an important component for purinergic signaling that regulate astrocyte-neuron interactions and cortical sensory discrimination in vivo.

Increased genital mucosal cytokines in Canadian women associate with higher antigen-presenting cells, inflammatory metabolites, epithelial barrier disruption, and the depletion of L. crispatus.

BACKGROUND: Cervicovaginal inflammation has been linked to negative reproductive health outcomes including the acquisition of HIV, other sexually transmitted infections, and cervical carcinogenesis. While changes to the vaginal microbiome have been linked to genital inflammation, the molecular relationships between the functional components of the microbiome with cervical immunology in the reproductive tract are understudied, limiting our understanding of mucosal biology that may be important for reproductive health. RESULTS: In this study, we used a multi'-omics approach to profile cervicovaginal samples collected from 43 Canadian women to characterize host, immune, functional microbiome, and metabolome features of cervicovaginal inflammation. We demonstrate that inflammation is associated with lower amounts of L. crispatus and higher levels of cervical antigen-presenting cells (APCs). Proteomic analysis showed an upregulation of pathways related to neutrophil degranulation, complement, and leukocyte migration, with lower levels of cornified envelope and cell-cell adherens junctions. Functional microbiome analysis showed reductions in carbohydrate metabolism and lactic acid, with increases in xanthine and other metabolites. Bayesian network analysis linked L. crispatus with glycolytic and nucleotide metabolism, succinate and xanthine, and epithelial proteins SCEL and IVL as major molecular features associated with pro-inflammatory cytokines and increased APCs. CONCLUSIONS: This study identified key molecular and immunological relationships with cervicovaginal inflammation, including higher APCs, bacterial metabolism, and proteome alterations that underlie inflammation. As APCs are involved in HIV transmission, parturition, and cervical cancer progression, further studies are needed to explore the interactions between these cells, bacterial metabolism, mucosal immunity, and their relationship to reproductive health. Video Abstract.

Vaginal epithelial dysfunction is mediated by the microbiome, metabolome, and mTOR signaling.

Bacterial vaginosis (BV) is characterized by depletion of Lactobacillus and overgrowth of anaerobic and facultative bacteria, leading to increased mucosal inflammation, epithelial disruption, and poor reproductive health outcomes. However, the molecular mediators contributing to vaginal epithelial dysfunction are poorly understood. Here we utilize proteomic, transcriptomic, and metabolomic analyses to characterize biological features underlying BV in 405 African women and explore functional mechanisms in vitro. We identify five major vaginal microbiome groups: L. crispatus (21%), L. iners (18%), Lactobacillus (9%), Gardnerella (30%), and polymicrobial (22%). Using multi-omics we show that BV-associated epithelial disruption and mucosal inflammation link to the mammalian target of rapamycin (mTOR) pathway and associate with Gardnerella, M. mulieris, and specific metabolites including imidazole propionate. Experiments in vitro confirm that type strain G. vaginalis and M. mulieris supernatants and imidazole propionate directly affect epithelial barrier function and activation of mTOR pathways. These results find that the microbiome-mTOR axis is a central feature of epithelial dysfunction in BV.

Nonoptimal bacteria species induce neutrophil-driven inflammation and barrier disruption in the female genital tract.

Neutrophil recruitment and activation within the female genital tract are often associated with tissue inflammation, loss of vaginal epithelial barrier integrity, and increased risk for sexually transmitted infections, such as HIV-1. However, the direct role of neutrophils on vaginal epithelial barrier function during genital inflammation in vivo remains unclear. Using complementary proteome and immunological analyses, we show high neutrophil influx into the lower female genital tract in response to physiological surges in progesterone, stimulating distinct stromal, immunological, and metabolic signaling pathways. However, despite the release of extracellular matrix-modifying proteases and inflammatory mediators, neutrophils contributed little to physiological mucosal remodeling events such as epithelial shedding or re-epithelialization during transition from diestrus to estrus phase. In contrast, the presence of bacterial vaginosis-associated bacteria resulted in a rapid and sustained neutrophil recruitment, resulting in vaginal epithelial barrier leakage and decreased cell-cell junction protein expression in vivo. Thus, neutrophils are important mucosal sentinels that rapidly respond to various biological cues within the female genital tract, dictating the magnitude and duration of the ensuing inflammatory response at steady state and during disease processes.

Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling.

BACKGROUND: The microbiota of the lower female genital tract plays an important role in women's health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT. METHODS: DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition. RESULTS: DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.

Vaginal microbiome-hormonal contraceptive interactions associate with the mucosal proteome and HIV acquisition.

Alterations to the mucosal environment of the female genital tract, such as genital inflammation, have been associated with increased HIV acquisition in women. As the microbiome and hormonal contraceptives can affect vaginal mucosal immunity, we hypothesized these components may interact in the context of HIV susceptibility. Using previously published microbiome data from 685 women in the CAPRISA-004 trial, we compared relative risk of HIV acquisition in this cohort who were using injectable depot medroxyprogesterone acetate (DMPA), norethisterone enanthate (NET-EN), and combined oral contraceptives (COC). In women who were Lactobacillus-dominant, HIV acquisition was 3-fold higher in women using DMPA relative to women using NET-EN or COC (OR: 3.27; 95% CI: 1.24-11.24, P = 0.0305). This was not observed in non-Lactobacillus-dominant women (OR: 0.95, 95% CI: 0.44-2.15, P = 0.895) (interaction P = 0.0686). Higher serum MPA levels associated with increased molecular pathways of inflammation in the vaginal mucosal fluid of Lactobacillus-dominant women, but no differences were seen in non-Lactobacillus dominant women. This study provides data suggesting an interaction between the microbiome, hormonal contraceptives, and HIV susceptibility.

The neovaginal microbiome of transgender women post-gender reassignment surgery.

BACKGROUND: Gender reassignment surgery is a procedure some transgender women (TW) undergo for gender-affirming purposes. This often includes the construction of a neovagina using existing penile and scrotal tissue and/or a sigmoid colon graft. There are limited data regarding the composition and function of the neovaginal microbiome representing a major gap in knowledge in neovaginal health. RESULTS: Metaproteomics was performed on secretions collected from the neovaginas (n = 5) and rectums (n = 7) of TW surgically reassigned via penile inversion/scrotal graft with (n = 1) or without (n = 4) a sigmoid colon graft extension and compared with secretions from cis vaginas (n = 32). We identified 541 unique bacterial proteins from 38 taxa. The most abundant taxa in the neovaginas were Porphyromonas (30.2%), Peptostreptococcus (9.2%), Prevotella (9.0%), Mobiluncus (8.0%), and Jonquetella (7.2%), while cis vaginas were primarily Lactobacillus and Gardnerella. Rectal samples were mainly composed of Prevotella and Roseburia. Neovaginas (median Shannon's H index = 1.33) had higher alpha diversity compared to cis vaginas (Shannon's H = 0.35) (p = 7.2E-3, Mann-Whitney U test) and were more similar to the non-Lactobacillus dominant/polymicrobial cis vaginas based on beta diversity (perMANOVA, p = 0.001, r2 = 0.342). In comparison to cis vaginas, toll-like receptor response, amino acid, and short-chain fatty acid metabolic pathways were increased (p < 0.01), while keratinization and cornification proteins were decreased (p < 0.001) in the neovaginal proteome. CONCLUSIONS: Penile skin-lined neovaginas have diverse, polymicrobial communities that show similarities in composition to uncircumcised penises and host responses to cis vaginas with bacterial vaginosis (BV) including increased immune activation pathways and decreased epithelial barrier function. Developing a better understanding of microbiome-associated inflammation in the neovaginal environment will be important for improving our knowledge of neovaginal health. Video Abstract.