The majority of outer membrane (OM) lipoproteins in Gram-negative bacteria are tethered to the membrane via an attached lipid moiety and oriented facing in toward the periplasmic space; a few lipoproteins have been shown to be surface exposed. The outer membrane lipoprotein P6 from the Gram-negative pathogenic bacterium nontypeable Haemophilus influenzae (NTHi) is surface exposed and a leading vaccine candidate for prevention of NTHi infections. However, we recently found that P6 is not a transmembrane protein as previously thought (L. V. Michel, B. Kalmeta, M. McCreary, J. Snyder, P. Craig, M. E. Pichichero, Vaccine 29:1624 -1627, 2011). Here we pursued studies to show that P6 has a dual orientation, existing infrequently as surface exposed and predominantly as internally oriented toward the periplasmic space. Flow cytometry using three monoclonal antibodies with specificity for P6 showed surface staining of whole NTHi cells. Confocal microscopy imaging confirmed that antibodies targeted surface-exposed P6 of intact NTHi cells and not internal P6 in membrane-compromised or dead cells. Western blots of two wild-type NTHi strains and a mutant NTHi strain that does not express P6 showed that P6 antibodies do not detect a promiscuous epitope on NTHi. Depletion of targets to nonlipidated P6 significantly decreased bactericidal activity of human serum. Protease digestion of surface-exposed P6 demonstrated that P6 is predominantly internally localized in a manner similar to its homologue Pal in Escherichia coli. We conclude that P6 of NTHi is likely inserted into the OM in two distinct orientations, with the predominant orientation facing in toward the periplasm.T he outer membrane (OM) of Gram-negative bacteria is asymmetrically structured with lipopolysaccharides in its outer leaflet and phospholipids in its inner leaflet (1). The OM is also comprised of numerous lipoproteins that are typically tethered to the inner leaflet of the OM (via their N-terminally attached lipid moieties) and oriented toward the periplasmic space of the cell (1-4). However, a subset of lipoproteins, including the outer membrane protein P6 from nontypeable Haemophilus influenzae (NTHi), are surface exposed (5-8).Since its discovery in the mid-1980s, P6 has been a leading vaccine candidate for prevention of NTHi infections in humans (acute otitis media, sinusitis, acute exacerbations of chronic bronchitis, and pneumonia). P6 is a strong vaccine candidate because it is immunogenic in children and adults, it is surface exposed, and it is highly conserved among pathogenic strains (5,(8)(9)(10)(11)(12)(13)(14)(15)(16).Previous work demonstrated noncovalent binding of P6 to the peptidoglycan layer of the cell (17-19). Therefore, P6 was thought to be a transmembrane protein, able to access both intracellular and extracellular molecules by physically spanning the OM. In 2011, we demonstrated that P6 could not be a transmembrane protein based on structural and computational studies (20) utilizing the nuclear magnetic resonance (NMR) solution structu...

P6 has been a vaccine candidate for nontypable Haemophilus influenzae (NTHi) based on its location on the outer membrane and immunogenicity. Because P6 is attached to the inner peptidoglycan layer of NTHi, and is putatively surface exposed, it must be a transmembrane protein. We examined the P6 structure using computational modeling, a P6 modified by sitedirected mutagenesis to study monoclonal antibody attachment to P6, and nuclear magnetic resonance spectroscopy. We found that P6 cannot be a transmembrane protein, and therefore may not be surface exposed. We conclude that there may be another protein on the surface of NTHi that has epitopes similar if not identical to P6.

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Nontypable Haemophilus influenzae (NTHi) causes otitis media and other respiratory diseases in people of all ages, conferring the need for a vaccine against the bacteria. P6, a lipoprotein vaccine candidate for NTHi, was recently discovered to exhibit two populations: one facing out of the cell and one facing in towards the periplasm. We hypothesized that dual orientation is a novel, widespread phenomenon among bacterial lipoproteins. To corroborate our hypothesis, we performed a biotinylation experiment on Pal, homolog to P6, in Escherichia coli (E. coli). In the experiment, whole cells were incubated with a cell‐impermeable biotinylating reagent (NHS‐LC‐LC‐biotin) in order to label all surface exposed proteins. The labeled, outer proteins were separated from the unlabeled, internal proteins via streptavidin‐agarose beads. Standard protein detection and immunoblotting techniques confirmed that Pal exists in two orientations in E. coli, with the majority of Pal facing in toward the periplasmic space. Only one other protein (Lpp of E. coli) has been shown to exhibit dual orientations. The results of this experiment suggest that lipoprotein dual orientation may be more widespread than originally thought, which could have important implications in the field of bacterial lipoprotein biology. This study was funded by the Rochester Institute of Technology and NIH NIDCD RO1 08671 (to MEP).

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.