Dual Orientation of the Outer Membrane Lipoprotein P6 of Nontypeable Haemophilus influenzae

Abstract

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...

Dual orientation of the outer membrane lipoprotein Pal in Escherichia coli

“…The discovery that the homologue of Pal in NTHi, P6, is expressed both on the cell surface and in the periplasm (Michel et al, 2013) led us to the hypothesis that Pal might also be dual oriented. As corroborative evidence, it was also suggested that both Lpp and Pal epitopes were exposed on the surface of E. coli that had been incubated in human serum (Hellman et al, 1997).…”

“…Bacterial samples were cultured as described above and used for surface expression analysis by flow cytometry as described in the literature (Michel et al, 2013). Briefly, approximately 250 ml of cells (OD 600 of approximately 0.8) were washed twice with PBS/1% BSA pH 7.2, and then incubated with 250 ml of primary antibody (anti-Pal) in serum at a 1:200 dilution for 1 h at room temperature.…”

“…We have recently shown that a homologue protein of Pal called P6 in nontypable Haemophilus influenzae (NTHi) has a dual orientation, existing as a surface-exposed protein and a periplasmic protein (Michel et al, 2013). Cowles et al (2011) were the first to describe a bacterial lipoprotein existing in two distinct orientations.…”

The molecular mechanism of bacterial lipoprotein modification—How, when and why?

“…The last amino acids of the C-terminus form a hydrophobic capping motif that is important for binding to PG and possibly involved in the recognition of lipoprotein by ligase YbiS (Zhang and Wu 1992;Shu et al, 2000). It is thus likely that Lpp has a dual orientation, similar as described for peptidoglycan-associated Lipoprotein P6 of Haemophilus influenza (Michel et al, 2013), although the correlation between surface-exposed orientation and biological function is not yet known. Surface-exposed lipoproteins are abundant in Borrelia, which lacks LPS in the OM (for a recent review see, Zuckert 2014).…”

Revisiting the Gram-Negative Lipoprotein Paradigm

“…(9)(10)(11). Most OM lipoproteins are thought to face into the periplasm; however, it is apparent that a few lipoproteins are exposed on the outer surface of the OM and face the extracellular milieu (12,13). An exception to this is found with the spirochete Borrelia burgdorferi, in which the majority of OM lipoproteins are surface exposed (14).…”