Outer membrane protein A (OmpA) has been implicated as an important virulence factor in several Gram-negative bacterial infections such as Escherichia coli K1, a leading cause of neonatal meningitis associated with significant mortality and morbidity. In this study, we generated E. coli K1 mutants that express OmpA in which three or four amino acids from various extracellular loops were changed to alanines, and we examined their ability to survive in several immune cells. We observed that loop regions 1 and 2 play an important role in the survival of E. coli K1 inside neutrophils and dendritic cells, and loop regions 1 and 3 are needed for survival in macrophages. Concomitantly, E. coli K1 mutants expressing loop 1 and 2 mutations were unable to cause meningitis in a newborn mouse model. Of note, mutations in loop 4 of OmpA enhance the severity of the pathogenesis by allowing the pathogen to survive better in circulation and to produce high bacteremia levels. These results demonstrate, for the first time, the roles played by different regions of extracellular loops of OmpA of E. coli K1 in the pathogenesis of meningitis and may help in designing effective preventive strategies against this deadly disease.Escherichia coli K1 is a prominent Gram-negative bacterium that causes meningitis in neonates with case fatality rates ranging from 5 to 30% of infected infants (1-4). Those who survive are often left with permanent neurological dysfunction such as hearing loss, mental retardation, and cortical blindness (5, 6). Despite the use of advanced antibiotics, the morbidity and mortality rates associated with E. coli K1 meningitis remain unchanged over the last few decades (7,8). In addition, because of a recent surge in antibiotic-resistant E. coli K1 strains, the mortality rates will further increase significantly (9, 10). Therefore, new modes of prevention are warranted for which the understanding disease pathophysiology is clearly necessary. Studies from this laboratory have demonstrated that E. coli K1 interacts with human brain microvascular endothelial cells (HBMEC) 2 to enter the central nervous system (11,12). The interaction of the bacterium with HBMEC is mediated by outer membrane protein A (OmpA) of E. coli K1 and a glycoprotein receptor, Ecgp96, on HBMEC (13, 14). OmpA initially binds to GlcNAc1-4GlcNAc epitopes of Ecgp96, followed by the peptide portion of the receptor (15). Of note, synthetic peptides that represent loops 1 and 2 of OmpA prevented the E. coli K1 invasion of HB-MEC (15). OmpA has been shown to be responsible for conferring serum resistance by binding to a complement regulator protein, C4b-binding protein (C4bp) (16, 17). 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 (...