Protease digestion of P6: Demonstrating a novel dual orientation of P6 in Nontypable Haemophilus influenzae

Abstract

Nontypable Haemophilus influenzae (NTHi) causes otitis media (ear infections) and other respiratory illnesses. Currently, NTHi's outer membrane protein P6 is a leading vaccine candidate for the pathogenic bacteria. We have recently demonstrated that P6 is surface exposed, but not a transmembrane protein. In this study, we performed a protease digestion experiment to show that P6 is also present on the inside of NTHi cells. We exposed whole and lysed NTHi cells to a serine protease called Proteinase K and collected time resolved samples to test for the digestion of P6. P6 present on the inside of whole cells were protected from Proteinase K digestion, while P6 in lysed cells were readily digested by Proteinase K. This experiment, in light of other studies from our lab, has demonstrated that P6 exists in two orientations in the outer membrane of NTHi. Additionally, we have made progress toward quantifying the amount of P6 present on the inside versus the outside of the cell. This study was funded by the Rochester Institute of Technology and by NIH NIDCD RO1 08671 (to MEP).