Infection of cornea models with different Neisseria gonorrhoeae derivatives to study the importance of the Type IV pilus for bacterial adherence and infection outcome

Neisseria gonorrhoeae (Ngo) is a human obligate pathogen and is the main cause of ophthalmia neonatorum, a type of conjunctivitis that affects newborns, which untreated can lead to blindness (1). One of the most important virulence factors of Ngo is the Type IV pilus, filamentous protein structures that facilitate the adhesion of the bacteria to their target tissue.

In this project, we studied the importance of the Type IV pilus in Ngo infection of cornea tissue models, derived either from cell lines or from primary cells (2). We infected the models with MS11 Ngo derivatives: MS11 F3 (Pili+, RecA+), MS11 N159 (Pili+, RecA-, Adh+cornea) and MS11 N191 (Pili+, RecA-, Adh-cornea). We observed that the bacterial adherence was highest for N159, which could be confirmed using fluorescence microscopy and SEM. However, no significant difference in the tissue integrity between the non-infected and infected models was detected. Cytotoxicity assessment by measuring LDH in the supernatant showed that the highest cytotoxicity was present after 72 h of infection with N159 Ngo. In the primary-cell-derived models the cytotoxicity was comparable between the different derivatives. Measuring of the cytokines showed that IL-8 was the most secreted cytokine; its concentration was the highest in N159-infected cell line-derived models after 72 h, suggesting a specific response that could be possibly pilus-dependent. However, there was no significant difference in the IL-8 secretion between primary-cell derived models infected with F3 and N159 after 72 h. To challenge the pilus relevance, the cell-line derived models were treated with trifluoperazine, a drug that has shown an effect on pilus retraction. We observed a reduction of more than 50% of the bacterial adherence when treated in an early stage of the infection. There was also a reduction of the secretion of IL-8, suggesting a possible decrease also on the severity of the infection outcome. These results show that cornea models can be used for studying Ngo infection, as well as the importance of the functional Type IV pilus and its role not only in bacterial adherence but also in the outcome of the infection.

1. Tjia, K., van Putten, J., Pels, E., & Zanen, H. (1988). The interaction between Neisseria gonorrhoeae and the human cornea in organ culture. Ophtalmology, 341-345

2. Lotz, C., et al. (2016). Alternative methods for the replacement of eye irritation testing. ALTEX - Alternatives to animal experimentation. 55-67