Entry and Intracellular Replication ofEscherichia coliK1 in Macrophages Require Expression of Outer Membrane Protein A

Abstract

Interactions between Escherichia coli K1, which causes meningitis in neonates, and macrophages have not been explored well. In this study we found that E. coli K1 was able to enter, survive, and replicate intracellularly in both murine and human macrophage cell lines, as well as in monocytes and macrophages of newborn rats. In addition, we demonstrated that OmpA ؉ E. coli also enters and replicates in human peripheral blood monocytes in vitro. Outer membrane protein A (OmpA) expression on E. coli contributes to binding to macrophages, phagocytosis, and survival within macrophages. Opsonization with either complement proteins or antibody is not required for uptake and survival of the bacteria within the macrophages. Transmission electron microscopy and immunocytochemistry studies with the infected macrophages indicated that OmpA ؉ E. coli multiplies enormously in a single phagosome and bursts the cell. Internalization of OmpA ؉ E. coli by RAW 264.7 cells occurred by both actin-and microtubule-dependent processes, which are independent of RGD-mediated integrin receptors. Internalization and intracellular survival within phagocytic cells thus may play an important role in the development of bacteremia, which is crucial for E. coli crossing of the blood-brain barrier.Escherichia coli is one of the leading gram-negative bacteria that cause neonatal meningitis. The rates of mortality and neurologic sequelae remain high despite advances in antimicrobial therapy (17,19). The two important steps in the pathogenesis of neonatal meningitis are the development of bacteremia with intravascular growth and passage of bacteria across the blood-brain barrier. Studies have shown that a certain threshold of bacteremia is necessary for the development of meningitis in an experimental rat model of hematogenous meningitis (14, 29). Thus, E. coli must avoid host defense mechanisms and proliferate either in the blood or in tissues to maintain a high level of bacteremia, which leads to the onset of meningitis. Invasion and intracellular survival of E. coli, therefore, represent an important pathogenicity mechanism in this infection. Although several E. coli structures and/or genes have been shown to be essential for interaction with brain microvascular endothelial cells (BMEC) (14,30,36,42), a single layer of cells lining the blood-brain barrier, very little is known about the interaction with phagocytic cells.Studies with BMEC have suggested that outer membrane protein A (OmpA) of E. coli plays an important role in the invasion by interacting with a 96-kDa glycoprotein on human BMEC (HBMEC) (25,27). Interaction of OmpA with its receptor induces actin condensation at the E. coli binding site (28). The actin reorganization induced by E. coli depends on activation of several host proteins involved in signaling, including focal adhesion kinase, PI3-kinase, PKC-␣, and caveolin-1 (32,33,37,38). The activated molecules accumulate along with actin during the invasion of HBMEC. In addition, it has recently been shown that OmpA also binds to a c...

Serum Amyloid A Protein Binds to Outer Membrane Protein A of Gram-negative Bacteria

“…Properties vary between bacteria (16). It is not clear what role OmpA plays in invasion and survival within cells; it may depend on the context, because OmpA-deficient bacteria have been reported to survive better in macrophages (25) and it has been shown that neutrophil elastase can attack OmpA (26). However, OmpA has been shown to be required for interaction and for crossing the brain microvascular endothelial cell barrier (27,28), and OmpA may reduce complement-mediated attack (29).…”

Deciphering the Roles of Outer Membrane Protein A Extracellular Loops in the Pathogenesis of Escherichia coli K1 Meningitis

“…Neonates having lower than the threshold levels of C4bp may be at a higher risk to E. coli K1 meningitis as evidenced when the bacterium treated with adult serum, which contained higher amounts of C4bp, could not invade HBMEC compared with newborn serum treatment (18). Nonetheless, neither of the sera prevents the entry of E. coli K1 into macrophages or dendritic cells (DCs) for which OmpA expression is also needed, indicating that various epitopes of OmpA are involved at different stages of pathogenesis of E. coli K1 meningitis (19,20). Therefore, in this study it is our goal to delineate the roles of different domains of OmpA interacting with various cells in vitro and their role in a well established newborn mouse model of meningitis (7,(21)(22)(23).…”

Outer Membrane Protein A Expression in Escherichia coli K1 Is Required to Prevent the Maturation of Myeloid Dendritic Cells and the Induction of IL-10 and TGF-β

“…Studies have shown that a high degree of bacteremia is required for E. coli to cross the blood-brain barrier, indicating that the bacterium must evade host defense mechanisms and survive in the blood stream (6,7). Our studies in the newborn rat model of hematogenous meningitis have demonstrated that E. coli enters monocytes and macrophages and then multiplies in a time-dependent fashion (8). Therefore, the ability of E. coli to survive in host immune cells like monocytes, macrophages, and neutrophils may represent an important step in the vicious cycle of this organism.…”