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OmpT is one of the members of the outer membrane protein family that has been identified as a virulence factor in most of the uropathogenic Escherichia coli (UPEC). However, the exact role of OmpT in the urinary tract infections (UTIs) remains unclear. To determine the role of OmpT in the pathogenesis of UPEC, an isogenic deletion mutant of ompT (COTD) was constructed by the λ Red recombination. Human bladder epithelial cell line 5637(HBEC 5637) was used to evaluate the ability of bacterial adhesion/invasion. A murine model of UTI was established to study the formation of intracellular bacterial communities (IBCs) in the process of UTIs. The cytokines were also examined during the pathogenesis. The results showed that the COTD strain was deficient in bacterial adhesion and invasion as well as in IBC formation compare to the parent strain. ELISA quantification analysis of cytokines showed that the levels of TNF-α, IL-6 and IL-8 in the serum, bladder and kidney tissues of the mice infected with COTD were lower than that of the CFT073 group. In summary, these results suggest that OmpT plays a multifaceted role in pathogenesis of UTI, including increased bacterial adhesiveness/invasiveness, formation of IBCs and upregulated proinflammatory cytokines., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aim: Polyphosphate kinase 1 (PPK1), encoded by the ppk1 gene, is one of the major enzymes to reversibly catalyze the synthesis of polyphosphate (poly P) from the terminal phosphate of ATP. Poly P confers resistance to stress in a number of bacterial species but its role in the virulence of meningitic bacterial pathogens is unknown. The aim of this study was to determine the role of PPK1 in the pathogenesis of Escherichia coli meningitis., Materials & Methods: An isogenic in-frame ppk1 deletion mutant (PD44) of E. coli K1 strain E44 was constructed and characterized. Human brain microvascular endothelial cells and neonatal rats were used as the in vitro and in vivo models, respectively, to evaluate bacterial adhesion/invasion and the abilities of bacteria crossing the blood-brain barrier (BBB) to cause meningitis. The survival of PD44 and E44 under osmotic and acid stress conditions were also examined., Results: Poly P levels in E44 were clearly higher than those in PD44, especially at the stationary phase (SP). The ppk1 deletion mutant PD44 also showed poor survival rates during osmotic shock and acidic challenge, which the bacteria would face during pathogenesis. In vitro and in vivo assays revealed that PD44 was defective in bacterial adhesion and translocation across the BBB. By using the Evans blue method, we found that E44-induced permeability of the BBB in neonatal rats was significantly higher than that of the animals infected with PD44. Cytokine ELISA results showed that the TNF-α and IL-1β levels in the serum and brain tissues of the neonatal rats infected with PD44 were lower than that of the E44 group. A more obvious meningeal inflammation could be observed in the brain tissues of the rats infected with E44 when compared with that of the PD44 group by histopathological examination. Furthermore, the mRNA expression of IbeR, which is an RpoS-like regulator contributing to the SP regulation in E44, was found to be decreased in PD44 when compared with the parent strain. PD44 was also deficient in mRNA expression of the invasin IbeA, the adhesin FimH and the outer member protein A, which contributes to E44 penetration across BBB and resistance to the stimulations of low pH and high osmolarity., Conclusion: These results indicate that ppk1 plays an important role in stress adaption and virulence in meningitic E. coli K1 strain E44, and controls the relevant phenotypes by modulating the expression of the SP regulatory gene ibeR and the virulence genes ibeA, fimH and ompA.

Background: IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PSF is required for meningitic E. coli K1 penetration and leukocyte transmigration across the blood-brain barrier (BBB), which are the hallmarks of bacterial meningitis. However, it is unknown how vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB, which are required for bacteria-mediated pathogenicities., Methodology/principal Findings: IbeA-induced E. coli K1 invasion, polymorphonuclear leukocyte (PMN) transmigration and IKK/NF-κB activation are blocked by Caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB. IKKα/β phosphorylation is blocked by ERK inhibitors. Co-immunoprecipitation analysis shows that vimentin forms a complex with IκB, NF-κB and tubulins in the resting cells. A dissociation of this complex and a simultaneous association of PSF with NF-κB could be induced by IbeA in a time-dependent manner. The head domain of vimentin is required for the complex formation. Two cytoskeletal components, vimentin filaments and microtubules, contribute to the regulation of NF-κB. SiRNA-mediated knockdown studies demonstrate that IKKα/β phosphorylation is completely abolished in HBMECs lacking vimentin and PSF. Phosphorylation of ERK and nuclear translocation of NF-κB are entirely dependent on PSF. These findings suggest that vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB activation. PSF is essential for translocation of NF-κB and ERK to the nucleus., Conclusion/significance: These findings reveal previously unappreciated facets of the IbeA-binding proteins. Cooperative contributions of vimentin and PSF to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB may represent a new paradigm in pathogen-induced signal transduction and lead to the development of novel strategies for the prevention and treatment of bacterial meningitis.

Transmigration of neutrophil [polymorphonuclear neutrophil (PMN)] across the blood-brain barrier (BBB) is a critical event in the pathogenesis of bacterial meningitis. We have shown that IbeA is able to induce meningitic Escherichia coli invasion of brain microvascular endothelial cells (BMECs), which constitutes the BBB. In this report, we provide evidence that IbeA and its receptor, vimentin, play a key role in E. coli-induced PMN transmigration across BMEC. In vitro and in vivo studies indicated that the ibeA-deletion mutant ZD1 was significantly less active in stimulating PMN transmigration than the parent strain E44. ZD1 was fully complemented by the ibeA gene and its product. E. coli-induced PMN transmigration was markedly inhibited by withaferin A, a dual inhibitor of vimentin and proteasome. These cellular effects were significantly stimulated and blocked by overexpression of vimentin and its head domain deletion mutant in human BMEC, respectively. Our studies further demonstrated that IbeA-induced PMN migration was blocked by bortezomib, a proteasomal inhibitor and correlated with upregulation of endothelial ICAM-1 and CD44 expression through proteasomal regulation of NFκB activity. Taken together, our data suggested that IbeA and vimentin contribute to E. coli K1-stimulated PMN transendothelial migration that is correlated with upregulation of adhesion molecule expression at the BBB., (© 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology.)

Escherichia coli OmpT, located in the outer membrane, has been characterized as a plasminogen activator, with the ability to hydrolyze protamine and block its entry. In this investigation, a complex of low molecular weight cationic peptides purified from human urine by a combination of membrane ultrafiltration and weak cation exchange chromatography was characterized. The impact of OmpT on E. coli resistance to urinary cationic peptides was investigated by testing ompT knockout strains. The ompT mutants were more susceptible to urinary cationic peptides than ompT(+) strains, and this difference was abolished by complementation of the mutants with pUC19 carrying the ompT gene. The urinary protease inhibitor ulinastatin greatly decreased the resistance of the ompT(+) strains. Overall, the data indicate that OmpT may help E. coli persist longer in the urinary tract by enabling it to resist the antimicrobial activity of urinary cationic peptides.

IbeA in meningitic Escherichia coli K1 strains has been described previously for its role in invasion of BMECs (brain microvascular endothelial cells). Vimentin was identified as an IbeA-binding protein on the surface of HBMECs (human BMECs). In the present study, we demonstrated that vimentin is a primary receptor required for IbeA+ E. coli K1-induced signalling and invasion of HBMECs, on the basis of the following observations. First, E44 (IbeA+ E. coli K1 strain) invasion was blocked by vimentin inhibitors (withaferin A and acrylamide), a recombinant protein containing the vimentin head domain and an antibody against the head domain respectively. Secondly, overexpression of GFP (green fluorescent protein)-vimentin and GFP-VDM (vimentin head domain deletion mutant) significantly increased and decreased bacterial invasion respectively. Thirdly, bacterial invasion was positively correlated with phosphorylation of vimentin at Ser82 by CaMKII (Ca2+/calmodulin-dependent protein kinase II) and IbeA+ E. coli-induced phosphorylation of ERK (extracellular-signal-regulated kinase). Blockage of CaMKII by KN93 and inhibition of ERK1/2 phosphorylation by PD098059 resulted in reduced IbeA+ E. coli invasion. Fourthly, IbeA+ E. coli and IbeA-coated beads induced the clustering of vimentin that was correlated with increased entry of bacteria and beads. Lastly, IbeA+ E. coli K1 invasion was inhibited by lipid-raft-disrupting agents (filipin and nystatin) and caveolin-1 siRNA (small interfering RNA), suggesting that caveolae/lipid rafts are signalling platforms for inducing IbeA-vimentin-mediated E. coli invasion of HBMECs. Taken together, the present studies suggest that a dynamic and function-related interaction between IbeA and its primary receptor vimentin at HBMEC membrane rafts leads to vimentin phosphorylation and ERK-mediated signalling, which modulate meningitic E. coli K1 invasion.

Objective: To purify IbeA-binding protein from intestinal epithelial Caco-2 cells., Methods: Recombinant IbeA was purified, and 1, 5, and 10 microg/ml His-IbeA and bovine serum albumin (control) were preincubated with confluent Caco-2 monolayer for 30 min at 4degrees celsius;. Gentamicin protection assay was used to test the invasion of E. coli K1 pathogenic isolate E44 in Caco-2 cells. The binding proteins were purified from Caco-2 by IbeA-Cu(2+) sepharose affinity chromatography, and validated by Far-Western blotting. The N-terminal amino acid sequence of the binding protein was determined using Edman assay., Results: E44 invasion in Caco-2 cells was blocked by the recombinant IbeA in a dose-dependent manner. Two binding bands were obtained with His pull-down, and the binding specificity was demonstrated by Far-Western blotting. The N-terminal amino acid sequence of IBP200 was MASITKLP with an isoelectric point of about 5.0., Conclusion: Two novel Caco-2 proteins interacting with IbeA of E. coli have been purified and identified.

IbeR is a regulator present in meningitic Escherichia coli strain E44 that carries a loss-of-function mutation in the stationary-phase (SP) regulatory gene rpoS. In order to determine whether IbeR is an SP regulator in E44, two-dimensional gel electrophoresis and LC-MS were used to compare the proteomes of a noninvasive ibeR deletion mutant BR2 and its parent strain E44 in the SP. Four up-regulated (TufB, GapA, OmpA, AhpC) and three down-regulated (LpdA, TnaA, OpmC) proteins in BR2 were identified when compared to E44. All these proteins contribute to energy metabolism or stress resistance, which is related to SP regulation. One of the down-regulated proteins, tryptophanase (TnaA), which is regulated by RpoS in other E. coli strains, is associated with SP regulation via production of a signal molecule indole. Our studies demonstrated that TnaA was required for E44 invasion, and that indole was able to restore the noninvasive phenotype of the tnaA mutant. The production of indole was significantly reduced in BR2, indicating that ibeR is required for the indole production via tnaA. Survival studies under different stress conditions indicated that IbeR contributed to bacteria stress resistance in the SP. Taken together, IbeR is a novel regulator contributing to the SP regulation.

IbeT is a downstream gene of the invasion determinant ibeA in the chromosome of a clinical isolate of Escherichia coli K1 strain RS218 (serotype 018:K1:H7). Both ibeT and ibeA are in the same operon. Our previous mutagenesis and complementation studies suggested that ibeT may coordinately contribute to E. coli K1 invasion with ibeA. An isogenic in-frame deletion mutant of ibeT has been made by chromosomal gene replacement with a recombinant suicide vector carrying a fragment with an ibeT internal deletion. The characteristics of the mutant in meningitic E. coli infection were examined in vitro [cell culture of human brain microvascular endothelial cells (HBMEC)] and in vivo (infant rat model of E. coli meningitis) in comparison with the parent strain. The ibeT deletion mutant was significantly less adhesive and invasive than its parent strain E. coli E44 in vitro, and the adhesion- and invasion-deficient phenotypes of the mutant can be complemented by the ibeT gene. Recombinant IbeT protein is able to block E. coli E44 invasion of HBMEC. Furthermore, the ibeT deletion mutant is less capable of colonizing intestine and less virulent in bacterial translocation across the blood-brain barrier (BBB) than its parent E. coli E44 in vivo. These data suggest that ibeT-mediated E. coli K1 adhesion is associated with the bacterial invasion process.

During the development of Escherichia coli K1 meningitis, interaction between E. coli invasion protein IbeA and surface protein(s) on human brain microvascular endothelial cells (HBMEC) is required for invasion and IbeA-mediated signaling. Here, an IbeA-binding protein was identified as polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF). The specific binding was confirmed by ligand overlay assay. The cell surface-expressed PSF was verified by the confocal microscopy. Recombinant PSF blocked E. coli K1 invasion of HBMEC effectively. Overexpression of PSF in the lentivirus-transducted HBMEC significantly enhanced E. coli K1 invasion. These results suggest that IbeA interacts with PSF for the E. coli K1 invasion of HBMEC.

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.

The presence of ibeA, a gene encoding a known virulence factor of Escherichia coli strains responsible for neonatal meningitis in humans, was investigated in the genome of 213 avian pathogenic E. coli (APEC) strains and 55 non-pathogenic E. coli strains of avian origin. Fifty-three strains were found to be ibeA(+), all of which belonged to the APEC group. The ibeA gene is therefore positively linked to the pathogenicity of strains (P<0.0001). Analysis of the serogroup of strains revealed a positive association of ibeA with serogroups O18, O88 and O2. On the contrary, only 1/59 O78 strains are ibeA(+), indicating a negative association of ibeA with this serogroup (P<0.0001). The role of ibeA in the virulence of the APEC strain BEN 2908 was investigated by constructing an ibeA mutant. Challenge assays on 3-week-old chickens showed a reduced virulence for the ibeA mutant. Furthermore, the APEC strain BEN 2908 was able to invade brain microvascular epithelial cells, this invasion being significantly reduced upon inactivation of ibeA. Altogether, these results suggest a role of ibeA in the pathogenicity of some APEC strains and confirm the close relationship between APEC and other human extraintestinal pathogenic E. coli isolates.