International Journal of Medical Microbiology 294 (2004) 373–381 OmpA of a septicemic Escherichia coli O78 – secretion and convergent evolution Uri Gophna, Diana Ideses, Ran Rosen, Adam Grundland, Eliora Z. Ron à Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Received 7 June 2004; received in revised form 24 August 2004; accepted 26 August 2004 Abstract OmpA is an important constituent of the outer membrane of Gram-negative bacteria. OmpA is involved in a variety of host–bacteria interactions, including crossing of the blood–brain barrier by E. coli strains causing newborn meningitis, and elicits a significant response by the immune system of the host. The bactericidal effect of neutrophil elastase (NE) is also attributed to degradation of the bacterial OmpA. Here we examined the OmpA of septicemic E. coli O78 strains and show that two surface-exposed loops are conserved among invasive strains of E. coli and other pathogenic Enterobacteriaceae. In addition, there is evidence for convergent evolution, implying the existence of selective pressure. Our results also indicate that large quantities of OmpA are secreted into the medium during all phases of growth, where it is present both in secreted vesicles and as a soluble secreted protein. We assume that secreted OmpA can play a role in protection of bacteria from NE by competitive inhibition. Support for this assumption was obtained from experiments indicating that addition of exogenous, purified OmpA reduces killing of bacteria by NE. r 2004 Elsevier GmbH. All rights reserved. Keywords: OmpA; Septicemic E. coli; Molecular evolution; Protein secretion; Outer membrane proteins Introduction OmpA – outer membrane protein A – is a major constituent of outer membranes of Gram-negative bacteria, and is required for the structural integrity of the cell surface. The best studied ompA gene variant, that of E. coli K-12, encodes a 325-amino-acid polypeptide (Chen et al., 1980) targeted to the mem- brane by a 21-amino-acid signal peptide. The N- terminal domain of the mature protein crosses the membrane eight times, creating four surface-exposed loops. The C-terminal region of OmpA is located in the periplasm and contains a peptidoglycan-association motif (Koebnik, 1995; Singh et al., 2003). OmpA is a prime target of the host immune system: its binding activates macrophages (Soulas et al., 2000) and induces dendritic cell maturation (Jeannin et al., 2000). The degradation of OmpA by neutrophil elastase (NE) promotes killing of E. coli, and ompA À mutants are immune to the effect of NE (Belaaouaj et al., 2000). OmpA has been implicated in the pathogenicity of encapsulated E. coli strains (K1) causing newborn meningitis (NBM), a disease in which bacteremia is followed by bacterial crossing of the blood–brain barrier. The E. coli K1 OmpA was shown to be involved in invasion of brain microvascular endothelial cells (an in vitro model for NBM) by a receptor–ligand mecha- nism (Prasadarao et al., 1996b), mediated by the first ARTICLE IN PRESS www.elsevier.de/ijmm 1438-4221/$-see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.08.004 à Corresponding author. Tel.: +972(3)6409379; fax: +972(3)6414138. E-mail address: eliora@post.tau.ac.il (E.Z. Ron).