Peptidoglycan associated lipoprotein (Pal) of Escherichia coli (E. coli) is a characteristic bacterial lipoprotein, with an N-terminal lipid moiety anchoring it to the outer membrane. Since its discovery over three decades ago, Pal has been well studied for its participation in the TolPal complex which spans the periplasm and has been proposed to play important roles in bacterial survival, pathogenesis and virulence. Previous studies of Pal place the lipoprotein in the periplasm of E. coli, allowing it to interact with Tol proteins and the peptidoglycan layer. Here, we describe for the first time, a subpopulation of Pal which is present on the cell surface of E. coli. Flow cytometry and confocal microscopy detect anti-Pal antibodies on the surface of intact E. coli cells. Interestingly, Pal is surface exposed in an 'all or nothing' manner, such that most of the cells contain only internal Pal, with fewer cells (,20 %) exhibiting surface Pal.
Nontypable Haemophilus influenzae (NTHi) causes otitis media and other respiratory diseases in people of all ages, conferring the need for a vaccine against this bacterium. P6 is a lipoprotein and a vaccine candidate for protection against NTHi. Recent studies show that P6 is inserted into the outer membrane of NTHi in two distinct orientations‐ facing out toward the extracellular space and in toward the periplasm (Michel et al., J of Bacteriology 195: 3252). To further investigate the two orientations of P6, we utilized a cell‐impermeable biotinylating reagent (NHS‐LC‐LC‐biotin) and streptavidin‐agarose beads to separate the outward and inward facing P6. We then used standard protein detection methods to quantify the two populations. Results showed that the great majority of P6 faces in toward the periplasmic space of NTHi. Similar methods were used to quantify the two populations of Pal, P6’s homologue in Escherichia coli. Our results showed that, like P6, Pal is mostly oriented facing in toward the periplasmic space of the bacterial cell. We propose that, in light of our results, there may be more dual oriented lipoproteins in biology. Grant Funding Source: This study was supported by NIH NIDCD RO1 08671 (to MEP) and the Rochester Institute of Technology.
Pal is a well‐characterized peptidoglycan‐associated lipoprotein found in the periplasm of Escherichia coli (E. coli). Although the biological function of Pal is not understood, the protein has been shown to interact with Tol B, Lpp, and several other periplasmic proteins. We recently discovered that Pal’s homologue, P6 of nontypable Haemophilus Influenzae (NTHi), is inserted into the outer membrane in two orientations—one facing in toward the periplasm and one facing out toward the extracellular space. One other lipoprotein, Lpp of E. coli, was also shown to exhibit two orientations in the outer membrane. In an effort to collect data in support of our hypothesis that lipoprotein dual orientation is a more widespread phenomenon, we utilized confocal microscopy and flow cytometry to assess the localization and orientation(s) of Pal in E. coli. Our results demonstrate that Pal is indeed present on the surface of E. coli, suggesting that Pal is also a dual oriented lipoprotein. Grant Funding Source: Supported by the Camille and Henry Dreyfus Special Grant Program in the Chemical Sciences.
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