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

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.

Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state

“…It is difficult to understand the modulation pattern of OmpT and lipoprotein 28 (NlpA) observed in this work (Table 1). Brandenburg et al . (2005) and McCarter et al .…”

“…It is difficult to understand the modulation pattern of OmpT and lipoprotein 28 (NlpA) observed in this work (Table 1). Brandenburg et al (2005) and McCarter et al (2004) have shown OmpT to be a major defence molecule in such Gram-negative bacteria as E. coli, whose function is to cleave antimicrobial peptides. Bodero et al (2007) and Yamaguchi & Inouye (1988) found that lipoprotein 28 is nonessential for cell growth under laboratory conditions, but they did not rule out the possibility that lipoprotein 28 plays an important role under certain condition.…”

Temperature-Induced Changes in the Lipopolysaccharide ofYersinia pestisAffect Plasminogen Activation by the Pla Surface Protease

“…The motif is made up by basic amino acids and shared by a number of prokaryotic and eukaryotic proteins which bind to lipopolysaccharide (LPS) (16); these include the Escherichia coli porin FhuA (17) and the human MD-2 protein that presents LPS to Toll-like receptor 4 (TLR4) (70). Indeed, the presence of LPS is needed to reactivate enzymatic function of the purified omptins OmpT of E. coli (6,37,38) and Pla of Y. pestis (39) reconstituted in detergent micelles, strongly suggesting that interaction with LPS is important for omptin functions in general. Further, the modification of LPS structure inside the mouse macrophage was recently found important for the high degree of in vivo activity of the PgtE omptin of Salmonella enterica (42).…”

Fibrinolytic and coagulative activities of Yersinia pestis

“…Omptins are unique proteases in that they require rough (short O side chain) LPS to be active (Kramer et al, 2000, 2002; Kukkonen et al, 2001, 2004; Brandenburg et al, 2005; Suomalainen et al, 2010). A consensus protein motif for binding to 4′-phosphate in lipid A (Ferguson et al, 2000) is present in the omptin barrel (Vandeputte-Rutten et al, 2001) and consists of R138 and R171 in Pla.…”

“…Pla could potentiate growth of Y. pestis in the lungs through cleavage of pulmonary antimicrobial peptides (Galvan et al, 2008). In this context, it is interesting that OmpT has been suggested to induce cytokine responses independently of the omptin-bound LPS (Brandenburg et al, 2005). …”