Abstract
Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually transmitted bacterium Neisseria gonorrhoeae, or gonococcus (GC) infects the female reproductive tract primarily from the endocervical epithelium. Due to lack of an infection model that mimics all aspects of human infections in the female reproductive tract, GC pathogenesis is poorly understood. This protocol takes advantage of the viability and functional integrity of human cervical tissues propagated in culture to generate an ex vivo infection model. This tissue model maintains the nature of the infection target and environment without any manipulation such as immortalization of epithelial cells by viruses. Using immunofluorescence microscopy, the interaction of GC with the endocervical epithelium was analyzed.
Keywords: Neisseria gonorrhoeae, Gonorrhea, Infection, ex vivo, Endocervix, Immunofluorescence staining
Background
Neisseria gonorrhoeae (GC) infects human genital epithelium causing gonorrhea, a common sexually transmitted infection. Infections in women can lead to severe complications, such as pelvic inflammatory disease, causing fallopian tube scarring and blockage and predisposition to ectopic pregnancy or infertility. Gonorrhea has reemerged as a critical public health issue due to increased prevalence of antibiotic-resistant strains. Because humans are the only host for GC, a lack of an infection model that mimics all aspects of human infections has been a major obstacle to advance our understanding of GC pathogenesis. We have established a human endocervical tissue explant model and immunofluorescence microscopic analysis to examine the mechanism by which GC infect the human endocervix, the primary site for GC infection in women. This ex vivo model maintains the normal cytoarchitecture and tissue integrity of the endocervical epithelium. Using this model and immunofluorescence analysis, we demonstrate that GC colonizes and penetrates into the endocervical tissue, where they potentially cause symptomatic and disseminated gonococcal infection. GC penetration is enabled by the junction disruption and exfoliation of endocervical epithelial cells in response to GC infection. Taken together, our data show that GC infection in endocervical tissue explants resembles GC infection in vivo observed using patients’ biopsies. In combination with immunofluorescent microscopy, this infection model removes an important roadblock to fully understanding the pathogenesis of GC.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
In our published studies, we quantitatively analyzed the penetration of GC into the subepithelium of the endocervix and the exfoliation of endocervical epithelial cells.
Recipes
Acknowledgments
We would like to thank NDRI for tissue collection. This work was supported by NIH grants (RO1 AI068888, R21 AI103797, and RO1 AI123340 to WS and DCS). This protocol was partially adapted from previously published work by Merbah et al. (2011) and Schürch et al. (1978). The authors declare no conflicts of interest within this work.
References
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