An In Vitro A-431 Epithelial Cell Infection Model for Studying Fungal Pathogenicity and Immune Responses Associated...

Vulvovaginal candidiasis (VVC), also known as vaginal thrush, is an infection of the vulvovaginal mucosa caused by fungi of the Candida genus. Particularly for patients suffering from recurrent infection, the disease has a significant impact on their quality of life. The still unknown aspects of disease pathogenesis, as well as factors driving the development of infections and recurrence, represent a challenge for both clinical practitioners and patients. Mouse models and patient studies have suggested important roles of the microbiome, deployment of fungal pathogenicity mechanisms in the vagina, and dysregulated immune responses for VVC pathology. Dissecting their individual contributions can reveal specific processes associated with infection and may inspire novel therapeutic strategies. Epithelial in vitro infection models have been playing a key role in dissecting a crucial interaction during VVC, the invasion and infection of the vaginal mucosa. They have been instrumental in characterizing candidalysin as a fungal toxin that damages epithelial cells and elicits initial inflammatory responses to catalyze downstream inflammation. Moreover, they have also revealed potential protective immune pathways. Such a standardized epithelial cell infection model offers high versatility and compatibility with different downstream assays to link epithelial responses with other processes during VVC. This protocol describes a general A-431 vulvovaginal epithelial cell-Candida infection model in detail and provides several adaptations, such as live-cell imaging and mRNA silencing, as well as possible follow-up readouts, like the quantification of cytokine release, cytotoxicity, and neutrophil recruitment to study diverse processes relevant to VVC research. Key features • This protocol describes the use of the A-431 epidermoid carcinoma cell line as an in vitro infection model of vulvovaginal candidiasis. • The protocol can be adapted to answer research questions relevant to vulvovaginal candidiasis (epithelial damage, release of inflammatory cytokines, recruitment and activation of neutrophils). • Live-cell imaging can be used to study dynamic infection processes. • mRNA silencing can be applied to interrogate the function of genes of interest in the host-pathogen interaction. This protocol is used in.