Nontypable Haemophilus influenzae (NTHi) causes acute otitis media, sinusitis, and acute exacerbations of chronic bronchitis. My interest in NTHi relates to its role in acute otitis media (ear infections) in children. Otitis media is the most common childhood illness for which pediatricians prescribe antibiotics [1-3] and current treatments and lost wages total $5 billion annually . The incidence and health care burden of ear infections in the United States points to the urgent need for a vaccine against NTHi.
The outer membrane lipoprotein P6, discovered almost 30 years ago, is currently one of the leading vaccine candidates for NTHi [5-7]. Although P6 has been studied for its immunological properties, not much was known about its structural orientation or localization in NTHi. It is critical for a vaccine candidate to be surface exposed in the bacterial cell such that it is accessible to antibodies and can initiate the appropriate immune response upon infection.
Our group has shown, using recombinant DNA technologies, recombinant protein expression/purification, enzyme-linked immunosorbent assays (ELISA), nuclear magnetic resonance (NMR) spectroscopy, and computational structural analysis, that P6 is not a transmembrane protein . However, past studies suggested that P6 interacted with the peptidoglycan layer inside of the cell [9-12] and monoclonal antibodies outside of the cell [12-16]. Thus, an apparent contradiction was presented. If P6 did not span the outer membrane, how could P6 interact with both intracellular and extracellular molecules? To address this apparent contradiction, I proposed the following hypotheses: P6 exists in two orientations in the outer membrane (see Figure): one in which P6 is facing out of the cell and one in which P6 is facing in towards the periplasm, P6 is not surface exposed,or P6 does not interact with peptidoglycan inside the cell.Only one other protein (Lpp of Escherichia coli) had been shown to exist in two forms, one form being surface exposed and the other internally localized within the cell . This was the only characterized example of a lipoprotein that was able to occupy two distinct subcellular locations and membrane orientations.
Our group showed that P6 was surface exposed in intact whole NTHi cells (using flow cytometry, a bactericidal assay, and confocal microscopy) and on the inside of NTHi (using Proteinase K digestion experiments). These experiments demonstrated that P6 was localized both inside and outside of NTHi (Figure 1A), pointing to a relatively novel biological phenomenon which we have called dual orientation. The results of this study were published in the Journal of Bacteriology in 2013 .
The goal of our next study was to determine whether or not P6’s homologue, Pal, in Escherichia coli (E. coli) was also dual oriented. Pal’s sequence and structure were highly similar to those of P6. Pal’s periplasmic binding partners have also been well established. Pal is one of the key components in the Tol-Pal complex interacting noncovalently with the peptidoglycan layer , outer membrane proteins OmpA  and Lpp , periplasmic protein TolB  and the inner membrane protein TolA . While its periplasmic interactions were well established, we considered the possibility that Pal might have a second orientation allowing for surface exposure (similar to P6). In this study, we utilized confocal microscopy, flow cytometry, and a biotinylation labeling technique to detect the surface exposed population of Pal. We discovered that Pal was also dual oriented, although the exposed population of Pal was smaller than the periplasmic population of Pal .
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