Describing the dual orientation of vaccine candidate P6

Nontypable Haemophilus influenzae (NTHi) are pathogenic bacteria that cause several illnesses, including otitis media (ear infections). The outer membrane protein P6 is currently a top vaccine candidate for NTHi. Past studies have demonstrated that P6 can interact with molecules both inside AND outside of the cell, but its sequence and structure suggests that it does not cross the outer cellular membrane. P6 is a peptidoglycan associated lipoprotein and therefore known to interact with peptidoglycan inside the cell. To demonstrate surface exposure of P6, we utilized flow cytometry and confocal microscopy. In both experiments, whole NTHi cells were incubated with P6 monoclonal antibody and a secondary antibody conjugated to a fluorophore. Fluorescently labeled cells were detected using flow cytometry and visualized via confocal microscopy. Results from our flow cytometry experiments demonstrate that ~17% of NTHi cells express surface exposed P6. Confocal microscopy corroborates that P6 is indeed on the cell surface of intact cells. We conclude that P6 is expressed in two orientations in NTHi: one in which it is surface exposed and able to interact with extracellular antibodies, and one in which it is internally localized and able to interact with peptidoglycan. This study was funded by the Rochester Institute of Technology, NIH NIDCD RO1 08671 (to MEP), and an ASBMB UAN Undergraduate Research Award to Joy Snyder.