The interaction of N-glycans in Fcγ receptor I α-chain with Escherichia coli K1 outer membrane protein A for entry into macrophages: experimental and computational analysis.

Neonatal meningitis, caused by Escherichia coli K1, is a serious central nervous system disease. We have established that macrophages serve as permissive niches for E. coli K1 to multiply in the host and for attaining a threshold level of bacterial load, which is a prerequisite for the onset of the disease. Here, we demonstrate experimentally that three N-glycans in FcγRIa interact with OmpA of E. coli K1 for binding to and entering the macrophages. Adoptive transfer of FcγRIa(-/-) bone marrow-derived macrophages transfected with FcγRIa into FcγRIa(-/-) newborn mice renders them susceptible to E. coli K1-induced meningitis. In contrast, mice that received bone marrow-derived macrophages transfected with FcγRIa in which N-glycosylation sites 1, 4, and 5 are mutated to alanines exhibit resistance to E. coli K1 infection. Our molecular dynamics and simulation studies predict that N-glycan 5 exhibits strong binding at the barrel site of OmpA formed by loops 3 and 4, whereas N-glycans 1 and 4 interact with loops 1, 3, and 4 of OmpA at tip regions. Molecular modeling data also suggest no role for the IgG binding site in the invasion process. In agreement, experimental mutations in IgG binding site had no effect on the E. coli K1 entry into macrophages in vitro or on the onset of meningitis in newborn mice. Together, this integration of experimental and computational studies reveals how the N-glycans in FcγRIa interact with the OmpA of E. coli K1 for inducing the disease pathogenesis.

Deciphering the roles of outer membrane protein A extracellular loops in the pathogenesis of Escherichia coli K1 meningitis.

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.

Novel model to study virulence determinants of Escherichia coli K1.

It is shown here for the first time that locusts can be used as a model to study Escherichia coli K1 pathogenesis. E. coli K-12 strain HB101 has very low pathogenicity to locusts and does not invade the locust brain, whereas the injection of 2 x 10(6) E. coli K1 strain RS218 (O18:K1:H7) kills almost 100% of locusts within 72 h and invades the brain within 24 h of injection. Both mortality and invasion of the brain in locusts after injection of E. coli K1 require at least two of the known virulence determinants shown for mammals. Thus, deletion mutants that lack outer membrane protein A or cytotoxic necrotizing factor 1 have reduced abilities to kill locusts and to invade the locust brain compared to the parent E. coli K1. Interestingly, deletion mutants lacking FimH or the NeuDB gene cluster are still able to cause high mortality. It is argued that the likely existence of additional virulence determinants can be investigated in vivo by using this insect system.

Molecular epidemiology of Escherichia coli causing neonatal meningitis.

Escherichia coli is the second cause of neonatal meningitis which is a major cause of neonatal mortality and is associated with a high incidence of neurological sequelae. E. coli neonatal meningitis (ECNM) strains, as other extraintestinal pathogenic E. coli, mainly belong to the phylogenetic group B2 and to a lesser extent to group D, but are distributed in fewer clonal groups. One of these, the O18:K1:H7 clone is worldwide distributed meanwhile others such as O83:K1 and O45:K1 are restricted to some countries. Over the past few years, major progress has been made in the understanding of the pathophysiology of E. coli O18:K1:H7 neonatal meningitis. In particular, specific virulence factors have been identified and are known to be carried by ectochromosomal DNA in most cases. Molecular epidemiological studies, including characterization of virulence genotypes and phylogenetic analysis are important to lead to a comprehensive picture of the origins and spread of virulence factors within the population of ECNM strains. To date, all the known genetic determinants obtained in ECNM strains are not sufficient to explain their virulence in their globality and further studies on clonal groups different from the archetypal O18:K1:H7 clone are needed. These studies would serve to find common pathogenic mechanisms among different ECNM clonal groups that may be used as potential target for a worldwide efficacious prevention strategy.

Differential regulation of Toll-like receptor mRNAs in experimental murine central nervous system infections.

Toll-like receptors (TLR) play a key role in the recognition of microbial components. We investigated the differential regulation of TLR mRNA expression in bacterial and viral mouse models of central nervous system infection. Streptococcus pneumoniae meningitis led to an enhanced expression of TLR2, TLR4 and TLR9 mRNA. In Escherichia coli meningitis, TLR2, TLR4 and TLR7 mRNA expression was increased and Herpes simplex encephalitis caused a rise of TLR4 mRNA. In organotypic hippocampal cultures treatment with S. pneumoniae R6 led to increased expression of TLR2 and TLR3 mRNA. Our data provide evidence that regulation of TLR mRNA is not fully specific for the molecular patterns of the infectious pathogen. The TLR mRNA regulation observed probably represents a combination of specific response to the causative pathogen and non-specific activation of the innate immune system.