Escherichia coli outer membrane protein A adheres to human brain microvascular endothelial cells

Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis. The outer membrane protein A (OmpA) assembles a beta-barrel structure having four surface-exposed loops in E. coli outer membrane. OmpA of meningitis-causing E. coli K1 is shown to contribute to invasion of the human brain microvascular endothelial cells (HBMEC), the main cellular component of the blood-brain barrier (BBB). However, the direct evidence of OmpA protein interacting with HBMEC is not clear. In this study, we showed that OmpA protein, solubilized from the outer membrane of E. coli, adhered to HBMEC surface. To verify OmpA interaction with the HBMEC, we purified N-terminal membrane-anchoring beta-barrel domain of OmpA and all surface-exposed loops deleted OmpA proteins, and showed that the surface-exposed loops of OmpA were responsible for adherence to HBMEC. These findings indicate that the OmpA is the adhesion molecule with HBMEC and the surface-exposed loops of OmpA are the determinant of this interaction.     

A novel genetic island of meningitic Escherichia coli K1 containing the ibeA invasion gene (GimA): functional annotation and carbon-source-regulated invasion of human brain microvascular endothelial cells

The IbeA (ibe10) gene is an invasion determinant contributing to E. coli K1 invasion of the blood-brain barrier. This gene has been cloned and characterized from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1, strain RS218 (018:K1: H7). In the present study, a genetic island of meningitic E. coli containing ibeA (GimA) has been identified. A 20.3-kb genomic DNA island unique to E. coli K1 strains has been cloned and sequenced from an RS218 E. coli K1 genomic DNA library. Fourteen new genes have been identified in addition to the ibeA. The DNA sequence analysis indicated that the ibeA gene cluster was localized to the 98 min region and consisted of four operons, ptnIPKC, cglDTEC, gcxKRCI and ibeRAT. The G+C content (46.2%) of unique regions of the island is substantially different from that (50.8%) of the rest of the E. coli chromosome. By computer-assisted analysis of the sequences with DNA and protein databases (GenBank and PROSITE databases), the functions of the gene products could be anticipated, and were assigned to the functional categories of proteins relating to carbon source metabolism and substrate transportation. Glucose was shown to enhance E. coli penetration of human brain microvascular endothelial cells and exogenous cAMP was able to block the stimulating effect of glucose, suggesting that catabolic regulation may play a role in control of E. coli K1 invasion gene expression. Our data suggest that this genetic island may contribute to E. coli invasion of the blood-brain barrier through a carbon-source-regulated process. Electronic Publication     

Identification of a surface protein on human brain microvascular endothelial cells as vimentin interacting with Escherichia coli invasion protein IbeA

Escherichia coli K1 is the most common gram-negative bacteria that cause meningitis during the neonatal period. The ibeA gene product in E. coli K1 has been characterized as a virulence factor that contributes to the binding to and invasion of brain microvascular endothelial cells (BMEC). Here, we identified a surface protein on human BMEC, vimentin, that interacts with the E. coli invasion protein IbeA. The binding sites of the IbeA-vimentin interaction are located in the 271-370 residue region of IbeA and the vimentin head domain. The regulatory protease factor Xa is able to cleave IbeA between R297 and K298 residues, and this cleavage abolishes the IbeA-vimentin interaction.     

A novel interaction of outer membrane protein A with C4b binding protein mediates serum resistance of Escherichia coli K1

Escherichia coli is an important pathogen that causes meningitis in neonates. The development of bacteremia preceding the traversal across the blood-brain barrier is a prerequisite for this pathogen that obviously must survive the bactericidal activity of serum. Here we report that outer membrane protein A (OmpA) of Escherichia coli contributes to serum resistance by binding to C4b binding protein (C4bp), a complement fluid phase regulator. C4bp contains seven identical alpha-chains and one beta-chain linked together with disulfide bridges. We found that OmpA binds the alpha-chain of C4bp, which is composed of eight homologous complement control protein (CCP) modules. Binding studies using mutants of recombinant C4bp that lack one CCP at a time suggest that CCP3 is the major site of interaction with OmpA. Furthermore, we demonstrate that the N terminus of OmpA interacts with C4bp. Binding of C4bp to OmpA is not significantly inhibited in the presence of either C4b or heparin and is not salt sensitive, implying that it is hydrophobic in nature, suggesting a novel interaction between OmpA and C4bp. A compelling observation in this study is that synthetic peptides corresponding to CCP3 sequences block the binding of C4bp to OmpA and also significantly enhance serum bactericidal activity.     

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.     

Responses of brain and non-brain endothelial cells to meningitis-causing Escherichia coli K1

Bacterial interaction with specific host tissue may contribute to its propensity to cause an infection in a particular site. In this study, we examined whether meningitis-causing Escherichia coli K1 interaction with human brain microvascular endothelial cells, which constitute the blood-brain barrier, differed from its interaction with non-brain endothelial cells derived from skin and umbilical cord. We showed that E. coli K1 association was significantly greater with human brain microvascular endothelial cells than with non-brain endothelial cells. In addition, human brain microvascular endothelial cells maintained their morphology and intercellular junctional resistance in response to E. coli K1. In contrast, non-brain endothelial cells exhibited decreased transendothelial electrical resistance and detachment from the matrix upon exposure to E. coli K1. These different responses of brain and non-brain endothelial cells to E. coli K1 may form the basis of E. coli K1's propensity to cause meningitis.     

Ca2+/calmodulin-dependent invasion of microvascular endothelial cells of human brain by Escherichia coli K1

Escherichia coli K1 invasion of microvascular endothelial cells of human brain (HBMEC) is required for E. coli penetration into the central nervous system, but the microbial-host interactions that are involved in this invasion of HBMEC remain incompletely understood. We have previously shown that FimH, one of the E. coli determinants contributing to the binding to and invasion of HBMEC, induces Ca²⁺ changes in HBMEC. In the present study, we have investigated in detail the role of cellular calcium signaling in the E. coli K1 invasion of HBMEC, the main constituents of the blood-brain barrier. Addition of the meningitis-causing E. coli K1 strain RS218 (O18:K1) to HBMEC results in transient increases of intracellular free Ca²⁺. Inhibition of phospholipase C with U-73122 and the chelating of intracellular Ca²⁺ by BAPTA/AM reduces bacterial invasion of HBMEC by approximately 50%. Blocking of transmembrane Ca²⁺ fluxes by extracellular lanthanum ions also inhibits the E. coli invasion of HBMEC by approximately 50%. In addition, E. coli K1 invasion is significantly inhibited when HBMEC are pretreated by the calmodulin antagonists, trifluoperazine or calmidazolium, or by ML-7, a specific inhibitor of Ca²⁺/calmodulin-dependent myosin light-chain kinase. These findings indicate that host intracellular Ca²⁺ signaling contributes in part to E. coli K1 invasion of HBMEC. This work was supported by the American Heart Association (grant SDG 0435177N to Y.K.) and by NIH grants (to K.S.K.).     

Outer membrane protein A of Escherichia coli K1 selectively enhances the expression of intercellular adhesion molecule-1 in brain microvascular endothelial cells

Escherichia coli K1 meningitis is a serious central nervous system disease with unchanged mortality and morbidity rates for last few decades. Intercellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule involved in leukocyte trafficking toward inflammatory stimuli at the vascular endothelium; however, the effect of E. coli invasion of endothelial cells on the expression of ICAM-1 is not known. We demonstrate here that E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) selectively up-regulates the expression of ICAM-1, which occurs only in HBMEC invaded by the bacteria. The interaction of outer membrane protein A (OmpA) of E. coli with its receptor, Ecgp, on HBMEC was critical for the up-regulation of ICAM-1 and was depend on PKC-alpha and PI3-kinase signaling. Of note, the E. coli-induced up-regulation of ICAM-1 was not due to the cytokines secreted by HBMEC upon bacterial infection. Activation of NF-kappaB was required for E. coli mediated expression of ICAM-1, which was significantly inhibited by over-expressing the dominant negative forms of PKC-alpha and p85 subunit of PI3-kinase. The increased expression of ICAM-1 also enhanced the binding of THP-1 cells to HBMEC. Taken together, these data suggest that localized increase in ICAM-1 expression in HBMEC invaded by E. coli requires a novel interaction between OmpA and its receptor, Ecgp.     

Genetic suppression demonstrates interaction of TonB protein with outer membrane transport proteins in Escherichia coli.

Energy-coupled reactions of the Escherichia coli outer membrane transport proteins BtuB and Cir require the tonB product. Some point mutations in a region of btuB and cir that is highly conserved in TonB-dependent transport proteins led to loss of TonB-coupled uptake of vitamin B12 and colicin Ia, whereas binding was unaffected. Most other point mutations in this region had no detectable effect on transport activity. Mutations in tonB that suppressed the transport defect phenotype of these btuB mutations were isolated. All carried changes of glutamine 165 to leucine, lysine, or proline. The various tonB mutations differed markedly in their suppression activities on different btuB or cir mutations. This allele specificity of suppression indicates that TonB interacts directly with the outer membrane transport proteins in a manner that recognizes the local conformation but not specific side chains within this conserved region. An effect of the context of the remainder of the protein was seen, since the same substitution (valine 10----glycine) in btuB and cir responded differently to the suppressors. This finding supports the proposal that TonB interacts with more of the transport proteins than the first conserved domain alone.     

Effect of rpoS mutations on stress-resistance and invasion of brain microvascular endothelial cells in Escherichia coli K1

Escherichia coli K1 strains are predominant in causing neonatal meningitis. We have shown that invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for E. coli K1 crossing of the blood-brain barrier. BMEC invasion by E. coli K1 strain RS218, however, has been shown to be significantly greater with stationary-phase cultures than with exponential-phase cultures. Since RpoS participates in regulating stationary-phase gene expression, the present study examined a possible involvement of RpoS in E. coli K1 invasion of BMEC. We found that the cerebrospinal fluid isolates of E. coli K1 strains RS218 and IHE3034 have a nonsense mutation in their rpoS gene. Complementation with the E. coli K12 rpoS gene significantly increased the BMEC invasion of E. coli K1 strain IHE3034, but failed to significantly increase the invasion of another E. coli K1 strain RS218. Of interest, the recovery of E. coli K1 strains following environmental insults was 10-100-fold greater on Columbia blood agar than on LB agar, indicating that growing medium is important for viability of rpoS mutants after environmental insults. Taken together, our data suggest that the growth-phase-dependent E. coli K1 invasion of BMEC is affected by RpoS and other growth-phase-dependent regulatory mechanisms.     

Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein

To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane-spanning segments are essential for the assembly process.     

Regulation of protein kinase C in Escherichia coli K1 invasion of human brain microvascular endothelial cells.

Escherichia coli is one of the most important pathogens involved in the development of neonatal meningitis in many parts of the world. Traversal of E. coli across the blood-brain barrier is a crucial event in the pathogenesis of E. coli meningitis. Our previous studies have shown that outer membrane protein A (OmpA) expression is necessary in E. coli for a mechanism involving actin filaments in its passage through the endothelial cells. Focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) have also been activated in host cells during the process of invasion. In an attempt to elucidate the mechanisms leading to actin filament condensation, we have focused our attention on protein kinase C (PKC), an enzyme central to many signaling events, including actin rearrangement. In the current study, specific PKC inhibitors, bisindolmaleimide and a PKC-inhibitory peptide, inhibited E. coli invasion of human brain microvascular endothelial cells (HBMEC) by more than 75% in a dose-dependent manner, indicating a significant role played by this enzyme in the invasion process. Our results further showed that OmpA+ E. coli induces significant activation of PKC in HBMEC as measured by the PepTag nonradioactive assay. In addition, we identified that the PKC isoform activated in E. coli invasion is a member of the conventional family of PKC, PKC-alpha, which requires calcium for activation. Immunocytochemical studies have indicated that the activated PKC-alpha is associated with actin condensation beneath the bacterial entry site. Overexpression of a dominant negative mutant of PKC-alpha in HBMEC abolished the E. coli invasion without significant changes in FAK phosphorylation or PI3K activity patterns. In contrast, in HBMEC overexpressing the mutant forms of either FAK or PI3K, E. coli-induced PKC activation was significantly blocked. Furthermore, our studies showed that activation of PKC-alpha induces the translocation of myristoylated alanine-rich protein kinase C substrate, an actin cross-linking protein and a substrate for PKC-alpha, from the membrane to cytosol. This is the first report of FAK- and PI3K-dependent PKC-alpha activation in bacterial invasion related to cytoskeletal reorganization.     

A novel outer membrane protein, Wzi, is involved in surface assembly of the Escherichia coli K30 group 1 capsule

Escherichia coli group 1 K antigens form a tightly associated capsule structure on the cell surface. Although the general features of the early steps in capsular polysaccharide biosynthesis have been described, little is known about the later stages that culminate in assembly of a capsular structure on the cell surface. Group 1 capsule biosynthesis gene clusters (cps) in E. coli and Klebsiella pneumoniae include a conserved open reading frame, wzi. The wzi gene is the first of a block of four conserved genes (wzi-wza-wzb-wzc) found in all group 1 K-antigen serotypes. Unlike wza, wzb, and wzc homologs that are found in gene clusters responsible for production of exopolysaccharides (i.e., predominantly cell-free polymer) in a range of bacteria, wzi is found only in systems that assemble capsular polysaccharides. The predicted Wzi protein shows no similarity to any other known proteins in the databases, but computer analysis of Wzi predicted a cleavable signal sequence. Wzi was expressed with a C-terminal hexahistidine tag, purified, and used for the production of specific antibodies that facilitated localization of Wzi to the outer membrane. Circular dichroism spectroscopy indicates that Wzi consists primarily of a beta-barrel structure, and dynamic light scattering studies established that the protein behaves as a monomer in solution. A nonpolar wzi chromosomal mutant retained a mucoid phenotype and remained sensitive to lysis by a K30-specific bacteriophage. However, the mutant showed a significant reduction in cell-bound polymer, with a corresponding increase in cell-free material. Furthermore, examination of the mutant by electron microscopy showed that it lacked a coherent capsule structure. It is proposed that the Wzi protein plays a late role in capsule assembly, perhaps in the process that links high-molecular-weight capsule to the cell surface.     

Phosphatidylinositol 3-kinase activation and interaction with focal adhesion kinase in Escherichia coli K1 invasion of human brain microvascular endothelial cells

Invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli K1. We have previously demonstrated the requirement of cytoskeletal rearrangements and activation of focal adhesion kinase (FAK) in E. coli K1 invasion of human BMEC (HBMEC). The current study investigated the role of phosphatidylinositol 3-kinase (PI3K) activation and PI3K interaction with FAK in E. coli invasion of HBMEC. PI3K inhibitor LY294002 blocked E. coli K1 invasion of HBMEC in a dose-dependent manner, whereas an inactive analogue LY303511 had no such effect. In HBMEC, E. coli K1 increased phosphorylation of Akt, a downstream effector of PI3K, which was completely blocked by LY294002. In contrast, non-invasive E. coli failed to activate PI3K. Overexpression of PI3K mutants Deltap85 and catalytically inactive p110 in HBMEC significantly inhibited both PI3K/Akt activation and E. coli K1 invasion of HBMEC. Stimulation of HBMEC with E. coli K1 increased PI3K association with FAK. Furthermore, PI3K/Akt activation was blocked in HBMEC-overexpressing FAK dominant-negative mutants (FRNK and Phe397FAK). These results demonstrated the involvement of PI3K signaling in E. coli K1 invasion of HBMEC and identified a novel role for PI3K interaction with FAK in the pathogenesis of E. coli meningitis.     

Topology of outer membrane pore protein PhoE of Escherichia coli. Identification of cell surface-exposed amino acids with the aid of monoclonal antibodies

Monoclonal antibodies which recognize the cell surface-exposed part of outer membrane protein PhoE of Escherichia coli were used to select for antigenic mutants producing an altered PhoE protein. The selection procedure was based on the antibody-dependent bactericidal action of the complement system. Using two distinct PhoE-specific monoclonal antibodies, seven independent mutants with an altered PhoE protein were isolated. Among these seven mutants, five distinct binding patterns were observed with a panel of 10 monoclonal antibodies. DNA sequence analysis revealed the following substitutions in the 330-residue-long PhoE protein: Arg-201----His (three isolates), Arg-201----Cys, Gly-238----Ser, Gly-275----Ser and Gly-275----Asp. It is argued that amino acid residues 201, 238, and 275 are most likely directly involved in antibody binding and, therefore, exposed at the cell surface. Together with Arg-158, which was previously shown to be cell surface exposed as it is changed in phage TC45-resistant phoE mutants, these four positions show a remarkably regular spacing, being approximately 40 residues apart. A model is suggested in which the PhoE polypeptide repeatedly traverses the outer membrane in an antiparallel beta-pleated sheet structure, exposing eight areas to the outside which are all separated by approximately 40 residues.     

Identification of an outer membrane protein of Escherichia coli, with a role in the coordination of deoxyribonucleic acid replication and cell elongation.

Protein G of molecular weight 15,000 is the fourth commonest protein in the outer membrane of Escherichia coli B/r. From experiments described here on the relationship of protein G production to cell elongation and septation, the hypothesis is proposed that protein G is a structural protein of cell elongation. Furthermore, a surplus of protein G is produced when deoxyribonucleic acid synthesis is arrested and septation is thereby prevented. Thus protein G may be an important coordination protein in E. coli for integration of deoxyribonucleic acid synthesis, cell envelope elongation, and septation. Inhibition of normal cell elongation in a rod configuration in E. coli B/r by the novel amidinopenicillanic acid FL1060 was accompanied by changes in the rate of appearance of protein G and several other outer membrane proteins. The rate of appearance of protein G decreased some 70% within 60 min, in parallel with termination of rounds of normal cell elongation. Filament-inducing concentrations of nalidixic acid increased dramatically the rate of appearance of protein G. After 30 min a plateau level some 250% higher than the control value was reached. Similar kinetics were observed in parallel with filament formation induced by incubation of a dnaB mutant of E. coli at the nonpermissive temperature. No change in the rate of appearance of protein G was observed during cephalexin- or benzylpenicillin-induced filament formation, indicating that increased protein G production was not a secondary consequence of filamentation. Cells treated with FL1060 lost their ability to be induced for protein G formation, with nalidixic acid, in parallel with their loss of ability to initiate rounds of normal cell elongation. A pulse-chase experiment demonstrated that the protein G appearing in the outer membrane as a consequence of inhibition of deoxyribonucleic acid synthesis was the result of de novo synthesis rather than of interconversion from previously synthesized protein species. A preliminary characterization of protein G revealed several similarities with the well-characterized lipoprotein of the outer membrane of E. coli. A comparison of the incorporation of several 14C-labeled amino acids into protein G and the lipoprotein revealed substantial differences, however, perhaps ruling out a simple relationship between these two proteins.     

Identification of EaeA protein in the outer membrane of attaching and effacing Escherichia coli O45 from pigs

Abstract We have previously reported that the production of attaching and effacing lesions by Escherichia coli O45 isolates from pigs is associated with the eaeA ( E. coli attaching and effacing) gene. In the present study, expression of the EaeA protein, the eaeA gene product, among swine O45 E. coli isolates was examined. The majority (20/22) of attaching and effacing positive, eaeA⁺ E. coli O45 isolates, but none of ten attaching and effacing negative, eaeA⁻ or eaeA⁺ isolates, expressed a 97-kDa outer membrane protein as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Amino-terminal amino acid sequencing demonstrated a high homology between this 97-kDa protein of swine E. coli O45 and the EaeA protein (intimin) of human enteropathogenic E. coli and enterohemorrhagic E. coli . In addition, a serological relationship between the EaeA proteins of swine O45, rabbit (RDEC-1) and human (E2348/69) attaching and effacing E. coli strains was observed. Our results indicate an association between expression of the EaeA protein and attaching and efficacing activity among O45 E. coli isolates. The data also suggest an antigenic relatedness of the EaeA proteins of swine, rabbit, and human attaching and effacing E. coli .