ARHGEF18 can promote BVDV NS5B activation of the host NF-κB signaling pathway by combining with the NS5B-palm domain.

Rho guanine nucleotide exchange factor 18 (ARHGEF18) is a member of the Rho guanine nucleotide exchange factor (RhoGEF) family. RhoGEF plays an important role in the occurrence of tumors and neurological diseases; however, its involvement in host cell resistance against pathogenic microorganisms is mostly unknown. Herein, we report that bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) can activate the nuclear factor kappa B (NF-κB) signaling pathway to induce an immune response. To clarify the functional domains of NS5B that activate NF-κB signaling, the six structural domains of NS5B were expressed separately: NS5B-core, NS5B-finger, NS5B-palm, NS5B-thumb, NS5B-N and NS5B-c domain. We preliminarily determined that the functional domains of NS5B that activate NF-κB signaling are the finger and palm domains. We used a bovine kidney cell cDNA library and yeast two-hybrid technology to identify that the host protein ARHGEF18 interacts with NS5B. Co-immunoprecipitation assays showed that ARHGEF18 interacts strongly with NS5B-palm. Interestingly ARHGEF18 could promote NF-κB signaling activation by BVDV NS5B. In addition silencing ARHGEF18 significantly inhibited NS5B-palm activation of NF-κB signaling. We concluded that ARHGEF18 can bind to BVDV NS5B through the palm domain to activate the NF-κB pathway. These findings provide direct evidence that BVDV NS5B induces immune responses by activating NF-κB signaling.

Bisphenols induce cardiotoxicity in zebrafish embryos: Role of the thyroid hormone receptor pathway.

Bisphenols are frequently found in the environment and have been of emerging concern because of their adverse effects on aquatic animals and humans. In this study, we demonstrated that bisphenol A, S, and F (BPA, BPS, BPF) at environmental concentrations induced cardiotoxicity in zebrafish embryos. BPA decreased heart rate at 96 hpf (hours post fertilization) and increased the distance between the sinus venosus (SV) and bulbus arteriosus (BA), in zebrafish. BPF promoted heart pumping and stroke volume, shortened the SV-BAdistance, and increased body weight. Furthermore, we found that BPA increased the expression of the dio3b, thrß, and myh7 genes but decreased the transcription of dio2. In contrast, BPF downregulated the expression of myh7 but upregulated that of thrß. Molecular docking results showed that both BPA and BPF are predicted to bind tightly to the active pockets of zebrafish THRß with affinities of -4.7 and -4.77 kcal/mol, respectively. However, BPS did not significantly affect dio3b, thrß, and myh7 transcription and had a higher affinity for zebrafish THRß (-2.13 kcal/mol). These findings suggest that although BPA, BPS, and BPF have similar structures, they may induce cardiotoxicity through different molecular mechanisms involving thyroid hormone systems. This investigation provides novel insights into the potential mechanism of cardiotoxicity from the perspective of thyroid disruption and offer a cautionary role for the use of BPA substitution.

Experimental Study of the Dynamic Characteristics of a New Antidrainage Subgrade Structure for High-Speed Railways in Diatomaceous Earth Areas.

The experience needed to carry out engineering and construction in diatomaceous earth areas is currently lacking. This project studies the new Hang Shaotai high-speed railway passing through a diatomaceous earth area in Shengzhou, Zhejiang Province, and analyzes the hydrological and mechanical properties of diatomaceous earth on the basis of a field survey and laboratory. Moreover, a new antidrainage subgrade structure was proposed to address the rainy local environment, and field excitation tests were performed to verify the antidrainage performance and stability of the new subgrade structure. Finally, the dynamic characteristics and deformation of the diatomaceous earth roadbed were examined. The hydrophysical properties of diatomaceous earth in the area are extremely poor, and the disintegration resistance index ranges from 3.1% to 9.0%. The antidrainage subgrade structure has good water resistance and stability under dynamic loading while submerged in water. After 700,000 loading cycles, the dynamic stress and vibration acceleration of the surface of the subgrade bed stabilized at approximately 6.37 kPa and 0.94 m/s2, respectively. When the number of excitations reached 2 million, the settlement of the diatomaceous earth foundation was 0.08 mm, and there was basically negligible postwork settlement of the diatomaceous earth foundation. These results provide new insights for engineering construction in diatomaceous earth areas.

In vitro evaluation of the potential for resistance development to ceragenin CSA-13.

OBJECTIVES: Though most bacteria remain susceptible to endogenous antimicrobial peptides, specific resistance mechanisms are known. As mimics of antimicrobial peptides, ceragenins were expected to retain antibacterial activity against Gram-positive and -negative bacteria, even after prolonged exposure. Serial passaging of bacteria to a lead ceragenin, CSA-13, was performed with representative pathogenic bacteria. Ciprofloxacin, vancomycin and colistin were used as comparators. The mechanisms of resistance in Gram-negative bacteria were elucidated. METHODS: Susceptible strains of Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii were serially exposed to CSA-13 and comparators for 30 passages. MIC values were monitored. Alterations in the Gram-negative bacterial membrane composition were characterized via mass spectrometry and the susceptibility of antimicrobial-peptide-resistant mutants to CSA-13 was evaluated. RESULTS: S. aureus became highly resistant to ciprofloxacin after <20 passages. After 30 passages, the MIC values of vancomycin and CSA-13 for S. aureus increased 9- and 3-fold, respectively. The Gram-negative organisms became highly resistant to ciprofloxacin after <20 passages. MIC values of colistin for P. aeruginosa and A. baumannii increased to ≥100 mg/L after 20 passages. MIC values of CSA-13 increased to ∼20-30 mg/L and plateaued over the course of the experiment. Bacteria resistant to CSA-13 displayed lipid A modifications that are found in organisms resistant to antimicrobial peptides. CONCLUSIONS: CSA-13 retained potent antibacterial activity against S. aureus over the course of 30 serial passages. Resistance generated in Gram-negative bacteria correlates with modifications to the outer membranes of these organisms and was not stable outside of the presence of the antimicrobial.

PagP activation in the outer membrane triggers R3 core oligosaccharide truncation in the cytoplasm of Escherichia coli O157:H7.

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the first outer core glucose unit of the R3 core oligosaccharide. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagPDelta5-14 allele, which mostly eliminates a periplasmic amphipathic alpha-helical domain but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier.

Phosphoethanolamine substitution in the lipid A of Escherichia coli O157 : H7 and its association with PmrC.

This study shows that lipid A of Escherichia coli O157 : H7 differs from that of E. coli K-12 in that it has a phosphoform at the C-1 position, which is distinctively modified by a phosphoethanolamine (PEtN) moiety, in addition to the diphosphoryl form. The pmrC gene responsible for the addition of PEtN to the lipid A of E. coli O157 : H7 was inactivated and the changes in lipid A profiles were assessed. The pmrC null mutant still produced PEtN-modified lipid A species, albeit in a reduced amount, indicating that PmrC was not the only enzyme that could be used to add PEtN to lipid A. Natural PEtN substitution was shown to be present in the lipid A of other serotypes of enterohaemorrhagic E. coli and absent from the lipid A of E. coli K-12. However, the cloned pmrC(O157) gene in a high-copy-number plasmid generated a large amount of PEtN-substituted lipid A species in E. coli K-12. The occurrence of PEtN-substituted lipid A species was associated with a slight increase in the MICs of cationic peptide antibiotics, suggesting that the lipid A modification with PEtN would be beneficial for survival of E. coli O157 : H7 in certain environmental niches. However, PEtN substitution in the lipid A profiles was not detected when putative inner-membrane proteins (YhbX/YbiP/YijP/Ecf3) that show significant similarity with PmrC in amino acid sequence were expressed from high-copy-number plasmids in E. coli K-12. This suggests that these potential homologues are not responsible for the addition of PEtN to lipid A in the pmrC mutant of E. coli O157 : H7. When cells were treated with EDTA, the amount of palmitoylated lipid A from the cells carrying a high-copy-number plasmid clone of pmrC(O157) that resulted in significant increase of PEtN substitution was unchanged compared with cells without PEtN substitution, suggesting that the PEtN moiety substituted in lipid A does not compensate for the loss of divalent cations required for bridging neighbouring lipid A molecules.

Lipid trafficking controls endotoxin acylation in outer membranes of Escherichia coli.

The biogenesis of biological membranes hinges on the coordinated trafficking of membrane lipids between distinct cellular compartments. The bacterial outer membrane enzyme PagP confers resistance to host immune defenses by transferring a palmitate chain from a phospholipid to the lipid A (endotoxin) component of lipopolysaccharide. PagP is an eight-stranded antiparallel beta-barrel, preceded by an N-terminal amphipathic alpha-helix. The active site is localized inside the beta-barrel and is aligned with the lipopolysaccharide-containing outer leaflet, but the phospholipid substrates are normally restricted to the inner leaflet of the asymmetric outer membrane. We examined the possibility that PagP activity in vivo depends on the aberrant migration of phospholipids into the outer leaflet. We find that brief addition to Escherichia coli cultures of millimolar EDTA, which is reported to replace a fraction of lipopolysaccharide with phospholipids, rapidly induces palmitoylation of lipid A. Although expression of the E. coli pagP gene is induced during Mg2+ limitation by the phoPQ two-component signal transduction pathway, EDTA-induced lipid A palmitoylation occurs more rapidly than pagP induction and is independent of de novo protein synthesis. EDTA-induced lipid A palmitoylation requires functional MsbA, an essential ATP-binding cassette transporter needed for lipid transport to the outer membrane. A potential role for the PagP alpha-helix in phospholipid translocation to the outer leaflet was excluded by showing that alpha-helix deletions are active in vivo. Neither EDTA nor Mg(2+)-EDTA stimulate PagP activity in vitro. These findings suggest that PagP remains dormant in outer membranes until Mg2+ limitation promotes the migration of phospholipids into the outer leaflet.

Enzymology of lipid A palmitoylation in bacterial outer membranes.

The enzymology of palmitate addition to lipid A can be traced to the early discovery of monosaccharide lipid A precursors, but the functional importance of lipid A palmitoylation in bacterial resistance to the host immune response has emerged only recently. Lipid A palmitoylation in enterobacteria is determined by a PhoP/PhoQ-activated gene pagP, which encodes an unusual outer membrane enzyme of lipid A biosynthesis. PagP structure and dynamics have now been elucidated by both NMR spectroscopy and X-ray crystallography. PagP is an 8-stranded antiparallel beta-barrel preceded by an N-terminal amphipathic alpha-helix. The PagP barrel axis is uniquely tilted by 30 degrees with respect to the membrane normal. An interior hydrophobic pocket in the upper half of the molecule functions as a hydrocarbon ruler, which allows the enzyme to distinguish palmitate from other acyl chains found in phospholipids. Internalization of a phospholipid palmitoyl group within the barrel appears to occur by lateral diffusion from the outer leaflet through non-hydrogen bonded regions between beta-strands. The MsbA-dependent trafficking of lipids from the inner membrane to the outer membrane outer leaflet is necessary for lipid A palmitoylation in vivo. Efforts to determine the PagP catalytic mechanism may lead to the development of inhibitors for the treatment of infections.

An msbB homologue carried in plasmid pO157 encodes an acyltransferase involved in lipid A biosynthesis in Escherichia coli O157:H7.

Escherichia coli O157:H7 carries a chromosomal msbB1 and a plasmid-encoded msbB2 gene. We characterized msbB2 function as a homologue of msbB1 by examination of wild-type organisms and mutant strains that lacked functional msbB1, msbB2, and both msbB1 and msbB2. The msbB double-mutant strain generated pentaacyl lipid A, while the single-mutant strains synthesized hexaacyl lipid A. Complementation with overexpressed msbB2 converted pentaacyl into hexaacyl lipid A in the double-mutant strain. The transcription of both msbB genes occurred simultaneously. Lack of MsbB2 activity slightly increased the microheterogeneity of the lipid A species. These results suggest that the msbB2 gene plays a role not only in the routine generation of fully hexaacylated lipid A but also in suppressing the microheterogeneity of lipid A species, the endotoxic determinant of the organism.