Biomimetic Human Tissue Model for Long-Term Study of Neisseria gonorrhoeae Infection

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.

Folliculin Controls the Intracellular Survival and Trans-Epithelial Passage of Neisseria gonorrhoeae

“…To reach deeper tissues, gonococci have to overcome the epithelial barrier during infection, of which the cell-cell communication and cell polarization are critical markers (Wang et al, 2017 ; Heydarian et al, 2019 ). To investigate the role of FLCN in this process, we seeded the UOK cells on transwell support to induce cell polarization.…”

“…Models were grown 10 days under submerged static conditions at 37°C/5% CO 2 for further experiments. The integrity of the monolayer was determined using 4 kDa FITC-dextran (Sigma-Aldrich/Merck, St. Louis, USA) permeability assay as described before (Heydarian et al, 2019 ).…”

“…Infections of cells grown on Transwell inserts were performed in DMEM plus 10% FBS at MOI50. To determine the transmigration of bacteria across the polarized monolayer after different time points of infection, 25 μl of medium were plated on GC-agar plate or the medium was centrifuged shortly at 2,400 × g and the pellet resuspended in 25 μl of medium was plated on GC agar as described (Heydarian et al, 2019 ). Infected or non-infected samples were fixed with 4% PFA at RT for 30 min and decorated with anti-E-cadherin and anti-ZO-1 (Proteintech, Manchester, United Kingdom) to observe cell junction proteins and polarization.…”

“…The epithelium is sealed by the adherence junction, which contains catenins and E-cadherin (E-cad) and the tight junction, containing claudin, occluding and zonula occludens (ZO) proteins 1 and 2 and others, to prevent the transmigration of pathogens and toxins (Miyoshi and Takai, 2005 ; Hartsock and Nelson, 2008 ; Niessen and Gottardi, 2008 ). Studies using polarized cell culture models developed on Transwell® inserts, rotating wall vessel bioreactor and porcine small intestinal submucosa (SIS) scaffold, respectively showed the disruption of apical junctions upon N. gonorrhoeae infection (Stein et al, 2015 ; Łaniewski et al, 2017 ; Heydarian et al, 2019 ).…”

Immunoinformatics Prediction of an Epitope Based Peptide Vaccine for Neisseria Gonorrhea Dihydrolipoamide Acetyltransferase Protein

“…Neisseria Gonorrhea which is the causative agent of Gonorrhea, is a gram-negative diplococcus. It poses a growing threat to the public health concern, as evident by the estimated 78 million infections per year [5].N. Gonorrhea infections usually start at the urogenital, rectal, and nasopharyngeal mucosa.…”