P6 has been reported to comprise 1–5% (by weight) of the total protein in the outer membrane of NTHi [5
]. Several experimental approaches have been utilized to demonstrate the surface exposure of P6. Monoclonal and polyclonal antibodies to P6 have been shown to stain the entire organism by immunofluorescence and immunoelectron microscopy [1
]. NTHi strain 3524 was radio-labeled with 125
I, and then after disruption of the bacterial outer membrane, the OMPs were dispersed on an agarose gel. The presence of a band at ~16 kDa that was extrinsically labeled suggested that P6 was surface exposed [1
]. Adsorbed antiserum was shown to immunoprecipitate P6 [19
], P6 was shown to be accessible to bactericidal antibodies [7
] and the anti-P6 antibody was shown to be eluted from the surface of intact NTHi [5
]. It is also generally accepted that NTHi P6 interacts with the peptidoglycan layer in the periplasmic space of the bacterial cell [11
There have been several key immunological experiments involving monoclonal antibodies to P6 that have suggested that P6 is surface exposed [5
]. Apicella et al
. performed immunoelectron microscope experiments that utilized the P6 monoclonal antibodies 3B9 and 4G4 [5
]. The presence of gold-labeled secondary antibodies on NTHi cells (visualized via an electron microscope) suggested that both monoclonal antibodies bound to P6 on the surface of the cells [5
]. A third monoclonal antibody, 7F3, was proposed to bind to the same or a closely related epitope on P6 as the 4G4 antibody [6
]. Pre-incubation of NTHi with monoclonal antibody 7F3 inhibited human serum bactericidal killing, suggesting that P6 was indeed surface exposed and the target of bactericidal activity [20
]. While the specific amino acids that make up the epitope for 7F3 and 4G4 have not been fully identified, aspartic acid at position 59 has been implicated in antibody binding to P6 [21
]. The results of our experiments demonstrate that D59 does indeed participate in the binding of P6 to both 7F3 and 4G4 monoclonal antibodies. The OM typically measures between 7.5 and 10 nm thick. At its longest measurement, P6 is only ~7 nm long, making it unlikely that P6 would be able to interact with monoclonal antibodies on the cell surface via residue 59 (which is located in the middle of the protein) and span the OM such that it can also interact with the peptidoglycan layer ().
Transmembrane proteins typically demonstrate a pattern of hydrophobic/hydrophilic amino acids which can be used to predict the probable “membrane spanning” regions. In the case of NTHi P6, seven out of seven transmembrane prediction programs (available on the ExPASy server) did not recognize any membrane spanning regions within the P6 amino acid sequence (). These results, along with our protein structure analysis of P6 and the NTHi OM, suggest that P6 cannot possibly span the OM in order to interact with monoclonal antibodies (at residue 59) on the cell surface and with the peptidoglycan layer in the interior of the bacterial cell.
It is important to mention that the experiments presented in this work were performed on recombinant non-lipidated P6 protein in which the leader sequence containing the first 19 amino acids was removed. During in vivo processing of P6, the N-terminal leader sequence is removed and replaced by a lipid moiety. The ELISA and NMR experiments were attempted on the lipidated version of P6; however, purified lipidated P6 unfolds and/or aggregates such that NMR spectroscopy cannot be performed on the protein (data not shown). The lipid moiety of a bacterial lipoprotein anchors it to the membrane by insertion into the inner or outer leaflet of the lipid bilayer. Since P6 has to interact with antibodies and peptidoglycan through its amino acids (and not through its lipid moiety), using the non-lipidated version of P6 does not prevent us from making conclusions about the length of native P6 or the location of its epitopes.
The structural similarity between NTHi P6 and E. coli
Pal is notable. The NMR structure of NTHi P6 was determined in 2006 [11
]. It showed a high degree of similarity between NTHi P6 and E. coli
]. The N-terminally truncated 108-residue Pal crystal structure can be overlaid onto the NTHi P6 NMR structure (). The overall folds of the proteins are highly similar, suggesting the potential for similar binding partners and/or similar functions in vivo
. Although the functions of Pal in E. coli
are not definitive, Pal is known to be anchored to the inner leaflet (i.e.
, the inner lipid layer) of the OM of E. coli
via its lipid moiety and is proposed to interact with the peptidoglycan layer as well as several Tol proteins and outer membrane protein A (OmpA) [12
]. Importantly, there is no controversy that E. coli
Pal is attached to the OM via its lipid moiety, but is otherwise defined as non-membrane spanning protein. In other words, with the exception of the N-terminal lipid, Pal does not span either layer of the OM and faces in towards the intermembrane (i.e.
, periplasmic) space of E. coli
. Although there is no definitive evidence demonstrating a similar orientation for P6 in the NTHi OM, the remarkable similarity between the structures of NTHi P6 and E. coli
Pal points to the possibility of similar membrane orientations.
Fig. 3 Backbone representations of truncated P6 from NTHi (blue, PDB ID 2AIZ) and Pal from E. coli (red, PDB ID 1OAP) overlay with an RMSD of ~1.2 Å. The graphical representation was prepared using the Visual Molecular Dynamics program .
In conclusion, our results show that NTHi P6 is not a transmembrane protein since it is not of sufficient length to be surface-exposed and interact with the peptidoglycan layer on the inside of the bacterial cell [11
]. In order for P6 to interact with the peptidoglycan layer and
interact with monoclonal/bactericidal antibodies on the cell surface (as shown by immunological experiments), then P6 must either be inserted into the OM via two distinct “flipped” orientations or there is another OMP on the surface of NTHi that expresses an epitope similar to P6. Since the former has never been described for an OMP of any bacterial species, we are now pursuing studies to identify the OMP of NTHi that has structural epitopes similar to P6 but that is not P6.