Biological Significance of Dual Oriented P6 from Nontypeable Haemophilus influenzae: Assessing surface P6 levels in different strains of NTHi, cultured under various physiologically relevant environmental conditions

Nontypeable Haemophilus influenzae (NTHi) or nonencapsulated Haemophilus influenzae has been connected to such diseases as pneumonia, bacteremia, meningitis and sepsis in adults. NTHi is also linked to 25-35% of the roughly 25 million annual cases (within The United States) of acute otitis media (ear infections) in children. Vaccines against encapsulated strains of Haemophilus influenzae have been proven effective; yet, no vaccines have been produced to protect against NTHi infection. The 16-kDa outer membrane lipoprotein P6 has been shown to be nearly homologous between NTHi strains, making it one of the leading vaccine candidates for NTHi. However, it was recently demonstrated, using flow cytometry, confocal microscopy, and other biochemical methods, that P6 exhibits dual orientation in the outer membrane of NTHi. Specifically, a small percentage of cells contain P6 which faces out of the cell while a much larger percentage of cells contain P6 which faces in toward the periplasm. Nonetheless, these studies were only performed on a single strain of NTHi, which was cultured in a laboratory under aerobic conditions. In order to gain insight into P6's in vivo orientation(s), similar studies were performed on multiple clinically relevant strains of NTHi, cultured under microaerophilic and anaerobic conditions as well as physiological pH conditions which more closely resemble the environment of the middle ear. We conclude that all tested strains of NTHi (cultured under various pH and oxygen conditions) contain at least a small percentage of cells which exhibit surface P6.