Escherichia coli K1 Modulates Peroxisome Proliferator–Activated Receptor γ and Glucose Transporter 1 at the Blood-Brain Barrier in Neonatal Meningitis

Escherichia coli K1 meningitis continues to be a major threat to neonatal health. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with endothelial cell glycoprotein 96 (Ecgp96) in the blood-brain barrier to enter the central nervous system. Here we show that the interaction between OmpA and Ecgp96 downregulates peroxisome proliferator–activated receptor γ (PPAR-γ) and glucose transporter 1 (GLUT-1) levels in human brain microvascular endothelial cells, causing disruption of barrier integrity and inhibition of glucose uptake. The suppression of PPAR-γ and GLUT-1 by the bacteria in the brain microvessels of newborn mice causes extensive pathophysiology owing to interleukin 6 production. Pretreatment with partial or selective PPAR-γ agonists ameliorate the pathological outcomes of infection by suppressing interleukin 6 production in the brain. Thus, inhibition of PPAR-γ and GLUT-1 by E. coli K1 is a novel pathogenic mechanism in meningitis, and pharmacological upregulation of PPAR-γ and GLUT-1 levels may provide novel therapeutic avenues

Escherichia coli K1 Modulates Peroxisome Proliferator–Activated Receptor γ and Glucose Transporter 1 at the Blood-Brain Barrier in Neonatal Meningitis

Escherichia coli K1 meningitis continues to be a major threat to neonatal health. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with endothelial cell glycoprotein 96 (Ecgp96) in the blood-brain barrier to enter the central nervous system. Here we show that the interaction between OmpA and Ecgp96 downregulates peroxisome proliferator–activated receptor γ (PPAR-γ) and glucose transporter 1 (GLUT-1) levels in human brain microvascular endothelial cells, causing disruption of barrier integrity and inhibition of glucose uptake. The suppression of PPAR-γ and GLUT-1 by the bacteria in the brain microvessels of newborn mice causes extensive pathophysiology owing to interleukin 6 production. Pretreatment with partial or selective PPAR-γ agonists ameliorate the pathological outcomes of infection by suppressing interleukin 6 production in the brain. Thus, inhibition of PPAR-γ and GLUT-1 by E. coli K1 is a novel pathogenic mechanism in meningitis, and pharmacological upregulation of PPAR-γ and GLUT-1 levels may provide novel therapeutic avenues

BAAV Transcytosis Requires an Interaction with β-1-4 Linked- Glucosamine and gp96

Cell surface carbohydrates play an important role in virus entry and intracellular trafficking. Bovine Adeno-Associated Virus (BAAV) uses plasma membrane gangliosides for transduction and infection. In addition, independent of the infectious pathway, BAAV also has the ability to pass through barrier epithelia and endothelia using a transcytosis pathway dependent upon the presence of cell surface carbohydrates. Thus, in order to better define the carbohydrate interactions that are necessary for BAAV infection or transcytosis, a glycan microarray composed of both natural and synthetic carbohydrates was probed with HA-tagged BAAV particles. This identified chitotriose, a trimer of β-1-4-linked N-acetyl glucosamine, as having an interaction with BAAV. Competition experiments showed that the BAAV interaction with this carbohydrate is not necessary for infection but is instead important in the transcytosis pathway. The β-1-4-linked N-acetyl glucosamine modification has been reported on gp96, a glycoprotein involved in the transcytosis of bacteria and toxins. Significantly, immunoprecipitation and competition experiments with an anti-gp96 antibody and a soluble form of gp96, respectively, showed this glycoprotein can also interact with BAAV to serve as a receptor for its transcytosis

Fcγ Receptor I Alpha Chain (CD64) Expression in Macrophages Is Critical for the Onset of Meningitis by Escherichia coli K1

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa−/− mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa−/− macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa−/− mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1

Aggregatibacter actinomycetemcomitans Omp29 Is Associated with Bacterial Entry to Gingival Epithelial Cells by F-Actin Rearrangement

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29+/OmpA− E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29−/OmpA− E. coli. While the entry of Aa and Omp29+/OmpA− E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway

The Soluble Recombinant Neisseria meningitidis Adhesin NadAΔ351–405 Stimulates Human Monocytes by Binding to Extracellular Hsp90

The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadAΔ351–405, devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadAΔ351–405 cellular effects in monocytes. We show that NadAΔ351–405 (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadAΔ351–405 cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadAΔ351–405 /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadAΔ351–405 and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadAΔ351–405 determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadAΔ351–405 alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by anti-TLR2 antibodies. We propose that hsp90-dependent recruitment into an hsp90/hsp70/TLR4 transducing signal complex is necessary for the immune-stimulating activity of NadAΔ351–405 anti-MenB vaccine candidate

Outer Membrane Vesicles as a Candidate Vaccine against Edwardsiellosis

Infection with Edwardsiella tarda, a Gram-negative bacterium, causes high morbidity and mortality in both marine and freshwater fish. Outer membrane vesicles (OMVs) released from Gram-negative bacteria are known to play important roles in bacterial pathogenesis and host immune responses, but no such roles for E. tarda OMVs have yet been described. In the present study, we investigated the proteomic composition of OMVs and the immunostimulatory effect of OMVs in a natural host, as well as the efficacy of OMVs when used as a vaccine against E. tarda infection. A total of 74 proteins, from diverse subcellular fractions, were identified in OMVs. These included a variety of important virulence factors, such as hemolysin, OmpA, porin, GAPDH, EseB, EseC, EseD, EvpC, EvpP, lipoprotein, flagellin, and fimbrial protein. When OMVs were administrated to olive flounder, significant induction of mRNAs encoding IL-1β, IL-6, TNFα, and IFNγ was observed, compared with the levels seen in fish injected with formalin-killed E. tarda. In a vaccine trial, olive flounder given OMVs were more effectively protected (p<0.0001) than were control fish. Investigation of OMVs may be useful not only for understanding the pathogenesis of E. tarda but also in development of an effective vaccine against edwardsiellosis

Interaction of Variable Bacterial Outer Membrane Lipoproteins with Brain Endothelium

Previously we reported that the variable outer membrane lipoprotein Vsp1 from the relapsing fever spirochete Borrelia turicatae disseminates from blood to brain better than the closely related Vsp2 [1]. Here we studied the interaction between Vsp1 and Vsp2 with brain endothelium in more detail.We compared Vsp1 to Vsp2 using human brain microvascular endothelial cell (HBMEC) association assays with aminoacid radiolabeled Vsp-expressing clones of recombinant Borrelia burgdorferi and lanthanide-labeled purified lipidated Vsp1 (LVsp1) and Vsp2 (LVsp2) and inoculations of the lanthanide-labeled proteins into mice. The results showed that heterologous expression of LVsp1 or LVsp2 in B. burgdorferi increased its association with HBMEC to a similar degree. Purified lanthanide-labeled lipidated Vsp1 (LVsp1) and LVsp2 by themselves were capable of associating with HBMEC. The association of LVsp1 with brain endothelium was time-dependent, saturable, and required the lipidation. The association of Vsp1 with HBMEC was inhibited by incubation at lower temperature or with excess unlabeled LVsp1 or LVsp2 but not with excess rVsp1 or mouse albumin or an anti Vsp1 monoclonal antibody. The association of LVsp2 with HBMEC and its movement from blood to brain parenchyma significantly increased in the presence of LVsp1.Variable bacterial outer membrane lipoproteins interact with brain endothelium differently; the lipidation and variable features at the protein dome region are key modulators of this interaction

A Computational and Experimental Study of the Regulatory Mechanisms of the Complement System

The complement system is key to innate immunity and its activation is necessary for the clearance of bacteria and apoptotic cells. However, insufficient or excessive complement activation will lead to immune-related diseases. It is so far unknown how the complement activity is up- or down- regulated and what the associated pathophysiological mechanisms are. To quantitatively understand the modulatory mechanisms of the complement system, we built a computational model involving the enhancement and suppression mechanisms that regulate complement activity. Our model consists of a large system of Ordinary Differential Equations (ODEs) accompanied by a dynamic Bayesian network as a probabilistic approximation of the ODE dynamics. Applying Bayesian inference techniques, this approximation was used to perform parameter estimation and sensitivity analysis. Our combined computational and experimental study showed that the antimicrobial response is sensitive to changes in pH and calcium levels, which determines the strength of the crosstalk between CRP and L-ficolin. Our study also revealed differential regulatory effects of C4BP. While C4BP delays but does not decrease the classical complement activation, it attenuates but does not significantly delay the lectin pathway activation. We also found that the major inhibitory role of C4BP is to facilitate the decay of C3 convertase. In summary, the present work elucidates the regulatory mechanisms of the complement system and demonstrates how the bio-pathway machinery maintains the balance between activation and inhibition. The insights we have gained could contribute to the development of therapies targeting the complement system.Singapore. Ministry of Education (Grant T208B3109)Singapore. Agency for Science, Technology and Research (BMRC 08/1/21/19/574)Singapore-MIT Alliance (Computational and Systems Biology Flagship Project)Swedish Research Counci

Yersinia enterocolitica Serum Resistance Proteins YadA and Ail Bind the Complement Regulator C4b-Binding Protein

Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host