Invasion of Human Middle Ear Epithelial Cells by Nonencapsulated Streptococcus pneumoniae Expressing Green Fluorescent Protein
Jay T. Nguyen1, Jessica L. Bradshaw2, Cecile Snell2, Courtney D. Thompson2, Mary D. Jackson2, and Larry S. McDaniel2
1Mississippi INBRE Research Scholar, The University of Southern Mississippi, Hattiesburg, MS
2Department of Microbiology and Immunology, University of Mississippi Medical Center, Jackson, MS
Streptococcus pneumoniae (pneumococcus) colonizes the human nasopharynx and causes human infections including pneumonia, conjunctivitis, and otitis media (OM). Currently licensed pneumococcal vaccines have reduced the incidence of invasive pneumococcal disease but have not effectively reduced mucosal infections such as OM. Nonecapsulated Streptococcus pneumoniae (NESp) have been isolated from up to 8% of OM cases, and the incidence of NESp-associated OM has increased over the past decade. In a chinchilla model of OM, we have shown that NESp MNZ41 causes high bacterial burden resulting in invasive disease. However, the mechanism by which MNZ41 is able to invade middle ear epithelial cells (MEECs) to cause invasive disease is unknown. To determine the host-pathogen interactions between MNZ41 and MEECs, we aimed to isolate human MEECs containing intracellular bacteria. We hypothesized NESp expression of green fluorescent protein (GFP) will allow detection of hMEEC containing intracellular NESp. To test this hypothesis, we transformed MNZ41 into GFP-expressing bacterium and verified the presence of the gfp cassette using PCR. Expression of GFP in MNZ41 was confirmed by fluorescent microscopy and flow cytometry. Then GFP-expressing MNZ41 cells were allowed for adhesion and invasion to hMEEC before removing extracellular bacteria. We were able to detect hMEEC containing intracellular GFP-expressing MNZ41. These cells were collected for downstream dual-RNA sequencing of both hMEEC and intracellular MNZ41 to reveal interactions between NESp and the host. Overall, this study will allow for a better understanding of NESp virulence mechanisms during OM.