Role of gut-derived bacterial lipopolysaccharide and peripheral TLR4 in immobilization stress-induced itch aggravation in a mouse model of atopic dermatitis.

Psychological stress and intestinal leakage are key factors in atopic dermatitis (AD) recurrence and exacerbation. Here, we demonstrate the mechanism underlying bacterial translocation across intestinal epithelial barrier damaged due to stress and further aggravation of trimellitic anhydride (TMA)-induced itch, which remain unclear, in AD mice. Immobilization (IMO) stress exacerbated scratching bouts and colon histological damage, and increased serum corticosterone and lipopolysaccharide (LPS). Orally administered fluorescein isothiocyanate (FITC)-dextran and surgically injected (into the colon) Cy5.5-conjugated LPS were detected in the serum and skin after IMO stress, respectively. The relative abundance of aerobic or facultative anaerobic bacteria was increased in the colon mucus layer, and Lactobacillus murinus, E. coli, Staphylococcus nepalensis, and several strains of Bacillus sp. were isolated from the spleens and mesenteric lymph nodes. Oral antibiotics or intestinal permeability blockers, such as lubiprostone (Lu), 2,4,6-triaminopyrimidine (TAP) and ML-7, inhibited IMO stress-associated itch; however, it was reinduced through intradermal or i.p. injection of LPS without IMO stress. I.p. injection of TAK-242 (resatorvid), a TLR4 inhibitor, abrogated IMO stress-associated itch, which was also confirmed in TLR4-KO mice. IMO stress alone did not cause itch in naïve mice. IMO stress-induced itch aggravation in TMA-treated AD mice might be attributed to the translocation of gut-derived bacterial cells and LPS, which activates peripheral TLR4 signaling.

The Modification of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Melt Blending.

Crystalline and noncrystalline poly(3-hyroxybutylate-co-4-hyroxybutylate) (P(3HB-co-4HB)) were melt blended to obtain mixtures of P(3HB-co-4HB) copolymers. The mixtures and P(3HB-co-4HB) copolymers of different 4HB contents were compared to study the effect of 4HB content on the properties of the copolymers and mixtures. P(3HB-co-4HB) copolymer mixtures, having various 4HB content, have been successfully made by melt blending instead of bacterial biosynthesis. In the case of copolymers, they were noncrystalline when the 4HB content was over 16%, while the P(3HB-co-4HB) mixtures at the same 4HB content were crystalline. The mixtures had a higher glass transition temperature, suggesting that their chain mobility is relatively low compared with the copolymer having the same 4HB content. Due to this effect, the mixture is expected to have a higher melt viscosity and a lower loss tangent to exhibit better melt processing properties. The mechanical properties of the mixtures show a similar behavior to the copolymers in that the tensile strength and the modulus decreases and elongation at the break increases with an increase in the 4HB content.

Supercritical CO2 Foaming of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate).

The supercritical carbon dioxide foaming characteristics of the biodegradable polymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) are studied for environmentally friendly packaging materials. The effect of the 4HB composition of the P(3HB-co-4HB) copolymers on the foaming conditions such as pressure and temperature is studied and the density and the expansion ratio of the resulting P(3HB-co-4HB) foam are together evaluated. The increase in the 4HB content reduces the crystallinity and tan δ value of P(3HB-co-4HB) required for the growth of the foam cells. Therefore, the foaming temperature needs to be lower to retain a suitable tan δ value of P(3HB-co-4HB) for foaming. It was found that P(3HB-co-4HB) with less crystallinity showed better formability and cell uniformity. However, foaming is not possible regardless of the foaming temperature when the 4HB content of P(3HB-co-4HB) is over 50%, due to the high tan δ value. A lower foam density and higher expansion ratio can be obtained with crystalline P(3HB-co-4HB) of low 4HB content, compared with non-crystalline P(3HB-co-4HB) of high 4HB content. The expansion ratio of P(3HB-co-4HB) foams can be increased slightly by using a chain extender, due to the lowing of crystallinity and tan δ. This is most effective in the case of P(3HB-co-4HB), whose 4HB content is 16%.

Identification of SpiA that interacts with Corynebacterium glutamicum WhcA using a two-hybrid system.

The Corynebacterium glutamicum whcA gene is known to play a negative role in the expression of genes responding to oxidative stress. The encoded protein contains conserved cysteines, which likely coordinate the redox-sensitive Fe-S cluster. To identify proteins which may interact with WhcA, we employed a two-hybrid system utilizing WhcA as 'bait'. Upon screening, several partner proteins were isolated from the C. glutamicum genomic library. Sequencing analysis of the isolated clones revealed out-of-frame peptide sequences, one of which showed high sequence homology with a dioxygenase encoded by NCgl0899. In vivo analysis of protein interaction using real-time quantitative PCR, which monitors his3 reporter gene expression, demonstrated that the interaction between NCgl0899-encoded protein and WhcA was specific. The interaction was labile to oxidants, such as diamide and menadione. Based on these data, NCgl0899 was named spiA (stress protein interacting with WhcA). Physical association and dissociation of the purified His(6)-WhcA and GST-SpiA fusion proteins, as assayed by in vitro pull-down experiments, were consistent with in vivo results. These data indicated that the interaction between WhcA and SpiA is not only specific but also modulated by the redox status of the cell and the functionality of the WhcA protein is probably modulated by the SpiA protein.

Human brain endothelial ATP synthase beta-subunit is mannose-insensitive binding target of FimH.

Binding of meningitis-causing Escherichia coli K1 to human brain microvascular endothelial cells (HBMEC) contributes to traversal of the blood-brain barrier, which occurs in part by the mannose-sensitive binding of FimH. In this study, we showed that FimH also binds to HBMEC, independent of mannose, and identified ATP synthase beta-subunit and actin proteins from the surface biotinylated HBMEC as the mannose-insensitive binding targets for FimH. Co-immunoprecipitation experiments in the presence of alpha-methyl mannose verified the binding of FimH to ATP synthase beta-subunit of HBMEC. ATP synthase beta-subunit antibody decreased E. coli K1 binding to HBMEC in the presence of alpha-methyl mannose. Taken together, these findings demonstrate that FimH of E. coli K1 binds to HBMEC in both mannose-sensitive and -insensitive manner.

Acetate consumption activity directly determines the level of acetate accumulation during Escherichia coli W3110 growth.

Escherichia coli excretes acetate during aerobic growth on glycolytic carbon sources, which has been explained as an overflow metabolism when the carbon flux into the cell exceeds the capacity of central metabolic pathways. Nonacetogenic growth of E. coli on gluconeogenic carbon sources like succinate or in carbon-limited slow growth conditions is believed an evidence for the explanation. However, we found that a strain defected in the acs (acetyl Co-A synthetase) gene, the product of which is involved in scavenging acetate, accumulated acetate even in succinate medium and in carbon-limited low growth rate condition, where as its isogenic parental strain did not. The acs promoter was inducible in noncatabolite repression condition, whereas the expression of the ackA-pta operon encoding acetate kinase and phosphotransacetylase for acetate synthesis was constitutive. Results in this study suggest that E. coli excretes and scavenges acetate simultaneously in the carbon-limited low growth condition and in nonacetogenic carbon source, and the activity of the acetate consumption pathway directly affects the accumulation level of acetate in the culture broth.

FimH-mediated Escherichia coli K1 invasion of human brain microvascular endothelial cells.

Adhesion to brain microvascular endothelial cells, which constitute the blood-brain barrier is considered important in Escherichia coli K1 bacterial penetration into the central nervous system. Type 1 fimbriae are known to mediate bacterial interactions with human brain microvascular endothelial cells (HBMEC). Here, we demonstrate that type 1 fimbriae, specifically FimH adhesin is not only an adhesive organelle that provides bacteria with a foothold on brain endothelial cells but also triggers signalling events that promote E. coli K1 invasion in HBMEC. This is shown by our demonstrations that exogenous FimH increases cytosolic-free-calcium levels as well as activates RhoA. Using purified recombinant mannose-recognition domain of FimH, we identified a glycosylphosphatidylinositol-anchored receptor, CD48, as a putative HBMEC receptor for FimH. Furthermore, E. coli K1 binding to and invasion of HBMEC were blocked by CD48 antibody. Taken together, these findings indicate that FimH induces host cell signalling cascades that are involved in E. coli K1 invasion of HBMEC and CD48 is a putative HBMEC receptor for FimH.

Effects of ompA deletion on expression of type 1 fimbriae in Escherichia coli K1 strain RS218 and on the association of E. coli with human brain microvascular endothelial cells.

We have previously shown that outer membrane protein A (OmpA) and type 1 fimbriae are the bacterial determinants involved in Escherichia coli K1 binding to human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In investigating the role of OmpA in E. coli K1 binding to HBMEC, we showed for the first time that ompA deletion decreased the expression of type 1 fimbriae in E. coli K1. Decreased expression of type 1 fimbriae in the ompA deletion mutant was largely the result of driving the fim promoter toward the type 1 fimbrial phase-OFF orientation. mRNA levels of fimB and fimE were found to be decreased with the OmpA mutant compared to the parent strain. Of interest, the ompA deletion further decreased the abilities of E. coli K1 to bind to and invade HBMEC under the conditions of fixing type 1 fimbria expression in the phase-ON or phase-OFF status. These findings suggest that the decreased ability of the OmpA mutant to interact with HBMEC is not entirely due to its decreased type 1 fimbrial expression and that OmpA and type 1 fimbriae facilitate the interaction of E. coli K1 with HBMEC at least in an additive manner.

Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells.

Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.

RhoA and Rac1 contribute to type III group B streptococcal invasion of human brain microvascular endothelial cells.

Type III group B streptococcus (GBS) has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, but the underlying mechanisms remain incompletely understood. In the present study, we showed that the geranylgeranyl transferase I inhibitor, GGTI-298, not the farnesyltransferase inhibitor, FTI-277 inhibited type III GBS invasion of HBMEC. The substrates for GGTI-298 include Rho family GTPases, and we showed that RhoA and Rac1 are involved in type III GBS invasion of HBMEC. This was shown by the demonstration that infection with type III GBS strain K79 increased the levels of activated RhoA and Rac1 and GBS invasion was inhibited in HBMEC expressing dominant-negative RhoA and Rac1. Of interest, the level of activated Rac1 in response to type III GBS was decreased in HBMEC expressing dominant-negative RhoA, while the level of activated RhoA was not affected by dominant-negative Rac1. These findings indicate for the first time that activation of geranylgeranylated proteins including RhoA and Rac1 is involved in type III GBS invasion of HBMEC and RhoA is upstream of Rac1 in GBS invasion of HBMEC.

FimH adhesin of Escherichia coli K1 type 1 fimbriae activates BV-2 microglia.

The generation of intense inflammation in the subarachnoid space in response to meningitis-causing bacteria contributes to brain dysfunction and neuronal injury in bacterial meningitis. Microglia, the major immune effector cells in the central nervous system (CNS), become activated by bacterial components to produce proinflammatory immune mediators. In this study, we showed that FimH adhesin, a tip component of type 1 fimbriae of meningitis-causing Escherichia coli K1, activated the murine microglial cell line, BV-2, which resulted in the production of nitric oxide and the release of tumor necrosis factor-alpha. Mitogen-activated protein kinases, ERK and p-38, and nuclear factor-kappaB were involved in FimH adhesin-mediated microglial activation. These findings suggest that FimH adhesin contributes to the CNS inflammatory response by virtue of activating microglia in E. coli meningitis.

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.

Escherichia coli K1 RS218 interacts with human brain microvascular endothelial cells via type 1 fimbria bacteria in the fimbriated state.

Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC.

Outer membrane protein A and cytotoxic necrotizing factor-1 use diverse signaling mechanisms for Escherichia coli K1 invasion of human brain microvascular endothelial cells.

Escherichia coli K1 invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for penetration into the central nervous system. We previously have shown that outer membrane protein A (OmpA) and cytotoxic necrotizing factor-1 (CNF1) contribute to E. coli K1 invasion of BMEC. In this study we constructed a double-knockout mutant by deleting ompA and cnf1. We demonstrated that the double-knockout mutant was significantly less invasive in human BMEC as compared with its individual Delta ompA and Delta cnf1 mutants, suggesting that the contributions of OmpA and CNF1 to BMEC invasion are independent of each other. In addition, we showed that OmpA treatment of human BMEC resulted in phosphatidylinositol 3-kinase (PI3K) activation with no effect on RhoA, while CNF1 treatment resulted in RhoA activation with no effect on PI3K, supporting the concept that OmpA and CNF1 contribute to E. coli K1 invasion of BMEC using different mechanisms. This concept was further confirmed by using both PI3K inhibitor (LY294002) and Rho kinase inhibitor (Y27632), which exhibited additive effects on inhibiting E. coli K1 invasion of BMEC. We isolated a 96KD OmpA interacting human BMEC protein by affinity chromatography using purified OmpA, which was identified as gp96 protein, a member of the HSP90 family. This receptor differed from the CNF1 receptor (37LRP) identified from human BMEC. Taken together, these data indicate that OmpA and CNF1 contribute to E. coli K1 invasion of BMEC in an additive manner by interacting with different BMEC receptors and using diverse host cell signaling mechanisms.

37-kDa laminin receptor precursor modulates cytotoxic necrotizing factor 1-mediated RhoA activation and bacterial uptake.

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRP-overexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells.