Abstract
Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions.
Highlights
Neisseria gonorrhoeae is a Gram-negative diplococcus and a causative agent of the second most prevalent sexually transmitted infection in the world
We have developed novel tissue models based on small intestinal submucosa (SIS) scaffold that are comprised of two types of cells, primary human dermal fibroblasts (HDFib) and target epithelial cells of N. gonorrhoeae
We established different mucosal tissue models for gonococcal infection: a tight epithelial barrier model with highly polarized cells based on the colon carcinoma T84 cell line, a model representing female reproductive tract mucosal tissue using endometrial adenocarcinoma cell line HEC-1-B, and a model for the male urogenital tissue consisting of the immortalized uroepithelial cell line SV-HUC-1
Summary
Neisseria gonorrhoeae is a Gram-negative diplococcus and a causative agent of the second most prevalent sexually transmitted infection in the world. In a small number of cases, the bacteria can cross the endothelial barrier leading to disseminated gonococcal infection (DGI) (Eisenstein and Masi, 1981). N. gonorrhoeae contain different types of virulence factors including lipooligosaccharide (LOS), type IV pili, opacityassociated (Opa) proteins, and an outer membrane porin PorB, which enable the bacteria to attach to the epithelial cells, invade them, or survive in the presence of serum. Opa proteins mediate an efficient invasion into the host cells (Makino et al, 1991). N. gonorrhoeae express one of the two subtypes of PorB, PorBIA, or PorBIB, of which PorBIA has been implicated in gonococcal resistance to serum and in DGI (Ram et al, 1999; Rechner et al, 2007)
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