Investigation into the Interaction of the Phosphoporin PhoE with Outer Membrane Lipids: Physicochemical Characterization and Biological Activity

J.      Andra; H.   d.   Cock; P.      Garidel; J.      Howe; K.      Brandenburg

Abstract

Outer membrane pore proteins such as phosphoporin (PhoE) are important constituents of Gram-negative bacteria such as Escherichia coli. We have studied the interaction of PhoE with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein:lipid interaction corresponding to the outer membrane system as well as the activity of LPS:PhoE complexes in the infected host after release from the bacterial surface. The interaction of the lipids PE, PG, and LPS with PhoE was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface groups (ester), and polar groups (phosphates) applying Fourier-transform infrared spectroscopy (FTIR), and by analysing the phase transition behaviour of the lipids using FTIR and differential scanning calorimetry (DSC). The activity of PhoE and LPS:PhoE complexes was investigated in biological test systems (human mononuclear cells and Limulus amebocyte lysate assay) and with phospholipid model membranes using fluorescence resonance energy transfer spectroscopy (FRET). The results show a strong influence of PhoE on the mobility of the lipids leading to a considerable fluidization of the acyl chains of LPS, but much less to those from phospholipids: PhoE released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which is not found in the LPSspecific Limulus amebocyte lysate test, indicates an LPS-independent mechanism of cell activation.

Keywords: phoe, lps, cytokine induction, membrane fluidity, outer membrane

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