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Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

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Abstract

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein–lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.

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Acknowledgements

We are indebted to G. von Busse and C. Hamann for performing the IR spectroscopic and fluorescence spectroscopic measurements. The expert help of B. Fölting for performing the DSC and U. Diemer, the LAL measurements, is kindly acknowledged. This work was financially supported by the Commission of the European Communities, specific RTD programme Quality of Life and Management of Living Resources, QLK-CT-2002-01001, “Antimicrobial endotoxin neutralizing peptides to combat infectious diseases”.

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Authors and Affiliations

  1. Forschungszentrum Borstel, Div. of Biophysics, Leibniz-Institut für Medizin und Biowissenschaften, Parkallee 10, 23845 , Borstel, Germany

    Klaus Brandenburg, Andra B. Schromm, Jörg Andrä & Andre Wiese

  2. Institut für Physikalische Chemie, Martin-Luther Universität Halle-Wittenberg, 06108 , Halle, Germany

    Patrick Garidel

  3. Centre for Biomembranes and Lipid Enzymology, Universiteit Utrecht, Utrecht, The Netherlands

    Arjen Kramer & Maarten Egmond

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  1. Klaus Brandenburg
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Correspondence to Klaus Brandenburg.

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Brandenburg, K., Garidel, P., Schromm, A.B. et al. Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes. Eur Biophys J 34, 28–41 (2005). https://doi.org/10.1007/s00249-004-0422-3

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  • DOI: https://doi.org/10.1007/s00249-004-0422-3

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