Characterization of thiobarbituric acid derivatives as inhibitors of hepatitis C virus NS5B polymerase

In an effort to find chemicals inhibiting the enzymatic activity of the hepatitis C virus (HCV) NS5B polymerase, a series of thiobarbituric acid derivatives were selected from a library provided by Korea Research Institute of Chemical Technology and characterized. The selected compounds exhibited IC50 values ranging from 1.7 to 3.8 μM, and EC50 values ranging from 12.3 to 20.7 μM against NS5B polymerase of type 1b strain. They showed little effect against type 2a polymerase. One of the compounds, G05, was selected and further characterized. It inhibited the synthesis of RNA by recombinant HCV NS5B polymerase in a dose dependent manner. The CC50 value was 77 μM. The inhibition was in a noncompetitive manner with the substrate UTP. The compound did not inhibit the elongation step of RNA synthesis in a single-cycle processive polymerization assay. It inhibited the binding of NS5B polymerase to the template RNA in a dose-dependent manner

Motility increase of adherent invasive Escherichia coli (AIEC) induced by a sub-inhibitory concentration of recombinant endolysin LysPA90

Endolysins are bacteriophage enzymes required for the eruption of phages from inside host bacteria via the degradation of the peptidoglycan cell wall. Recombinant endolysins are increasingly being seen as potential antibacterial candidates, with a number currently undergoing clinical trials. Bacteriophage PBPA90 infecting Pseudomonas aeruginosa harbors a gene encoding an endolysin, lysPA90. Herein, recombinant LysPA90 demonstrated an intrinsic antibacterial activity against Escherichia coli in vitro. It was observed that a sub-inhibitory concentration of the recombinant protein induced the upregulation of genes related to flagella biosynthesis in a commensal E. coli strain. Increases in the number of bacterial flagella, and in motility, were experimentally substantiated. The treatment caused membrane stress, leading to the upregulation of genes rpoE, rpoH, dnaK, dnaJ, and flhC, which are upstream regulators of flagella biosynthesis. When adherent invasive Escherichia coli (AIEC) strains were treated with subinhibitory concentrations of the endolysin, bacterial adhesion and invasion into intestinal epithelial Caco-2 cells was seen to visibly increase under microscopic examination. Bacterial counting further corroborated this adhesion and invasion of AIEC strains into Caco-2 cells, with a resultant slight decrease in the viability of Caco-2 cells then being observed. Additionally, genes related to flagella expression were also upregulated in the AIEC strains. Finally, the enhanced expression of the proinflammatory cytokine genes TNF-α, IL-6, IL-8, and MCP1 in Caco-2 cells was noted after the increased invasion of the AIEC strains. While novel treatments involving endolysins offer great promise, these results highlight the need for the further exploration of possible unanticipated and unintended effects

The Lipid-Containing Bacteriophage PR4: Posttranslational Processing of the Major Capsid Protein and Lipid Selection in Particle Assembly

115 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1992.PR4 is a bacteriophage that contains lipid bilayer membrane within its protein capsid. The hosts include Escherichia coli and Salmonella typhimurium.I describe an investigation showing that capsid proteins P16 and P18 of bacteriophage PR4 are formed by post-translational processing of a portion of the major capsid protein P2. A polyclonal antibody raised against purified P2 reacted with P16 and P18 as well as P2. A monoclonal antibody reacted with both P2 and P18. The amino acid sequences of N-termini of P2 and P18 exactly matched indicating that P18 is the N-terminal fragment of processed P2. These data were confirmed by the analysis of the proteins encoded by various P2 nonsense and missense mutants. The 3129 bp MnlI-C fragment of PR4 genome was shown to encode P2 and the nucleotide sequence of this fragment was obtained and shown to contain a single continuous ORF encoding P2, thus excluding the possibility of introns or other post-transcriptional processing in production of P16 and P18. The MnlI-C DNA segment contained 7 ORFs sized >200 bp in addition to P2 gene and the genes encoding proteins P6 and P6A were mapped by marker rescue analysis.Phage PR4 contains a lipid bilayer within the protein capsid. The phospholipids of the bilayer are derived from those of the host. I report that phage morphogenesis selects against the unusually bulky phospholipids synthesized by Escherichia coli grown in the presence of various sugar alcohols. These data indicate that assembly of the PR4 lipid bilayer is a selective process rather than the bulk appropriation of host membrane lipids. I also demonstrate that phage PR4 morphogenesis is compatible with the incorporation of several abnormal lipids, monoacylglycerol, diacylglycerol, and phosphatidylinositol into the phage particle.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Degradation of AIMP1/p43 induced by hepatitis C virus E2 leads to upregulation of TGF-β signaling and increase in surface expression of gp96.

Hepatitis C virus (HCV) causes chronic hepatitis leading to liver fibrosis and autoimmune diseases. AIMP1/p43 is a multifunctional protein initially known as a cofactor of aminoacyl tRNA synthetase complex. Its function includes negative regulation of TGF-β signaling and suppression of Lupus-like autoimmune disease by inhibition of surface expression of gp96. HCV E2 was shown to directly interact with AIMP1/p43 by GST pulldown assay and coimmunoprecipitation. Their subcellular colocalization was observed in an immunofluorescence confocal microscopy. We showed that HCV E2 led to degradation of AIMP1/p43 in two ways. First, in the presence of HCV E2, endogenous AIMP1/p43 was shown to be degraded in an ubiquitin-dependent proteasome pathway. Second, grp78, an ER chaperone, was shown to interact with and stabilize AIMP1/p43. And HCV E2 inhibited this interaction leading to reduction of cellular AIMP1/p43. The degradation of AIMP1/p43 by HCV E2 resulted in increase of TGF-β signaling and cell surface expression of gp96. Thus we suggest that these are novel mechanisms responsible for liver fibrosis and autoimmune diseases caused by HCV

HCV E2 promotes cell surface expression of gp96 and TGF-β signaling.

<p>A. Palsmid expressing HCV E2, NS3, NS5A, or NS5B was transfected to Huh-7 cells and grown for 24 hours. Cell surface gp96 was probed with anti-gp96 antibody in flow cytometry. B. The same experiment with HCV E2 was carried out in Bjab cells. C. JFH1 particle (HCVcc) was infected to Huh7.5 cells. Cell surface gp96 was probed with anti-gp96 antibody using flow cytometry. D. HCV pseudoparticle (HCVpp) was infected to Huh7.5 cells. Cell surface gp96 was probed with anti-gp96 antibody using flow cytometry. E. A reporter plasmid, p3TP-lux, was transfected into Huh-7 cells along with 0.5, 1.0, or 2.0 µg of plasmid expressing HCV E2 and luciferase activity was measured in a luminometer. Transfection efficiency was normalized with cotransfection of pCH110 harboring LacZ gene and measuring β-galactosidase activity. Experiment was done in a triplicate. F. Plasmid expressing HCV E2 (either 0.5 µg or 2 µg) were used to transfect Huh-7 cells and PAI-1 expression was observed. TGF-β was added at the concentration of 4 ng/ml for 20 hours (lanes 4, 5, and 6) and PAI-1 expression was detected using western blotting with anti-PAI-1 antibody. EV; empty vector.</p

HCV E2 induced ubiquitin-mediated degradation of AIMP1/p43.

<p>A. Plasmid expressing HCV E2 was transfected to Huh-7 cells. Medium was changed 6 hours after transfection and MG132 was added at the concentration of 50 µM in 20 hours. Western blot analysis was done 6 hours after the treatment. β-tubulin was used as a loading control. EV; empty vector. B. Plasmid expressing HA-tagged ubiquitin and plasmid expressing HCV E2 were transfected to Huh-7 cells. Medium was changed 6 hours after transfection and MG132 was added at the concentration of 50 µM in 20 hours. Cell lysate was subjected to immunoprecipitation using anti-AIMP1/p43 antibody and protein G.</p

Comparison of Enterococcus faecalis Biofilm Removal Efficiency among Bacteriophage PBEF129, Its Endolysin, and Cefotaxime

Enterococcus faecalis is a Gram-positive pathogen which colonizes human intestinal surfaces, forming biofilms, and demonstrates a high resistance to many antibiotics. Especially, antibiotics are less effective for eradicating biofilms and better alternatives are needed. In this study, we have isolated and characterized a bacteriophage, PBEF129, infecting E. faecalis. PBEF129 infected a variety of strains of E. faecalis, including those exhibiting antibiotic resistance. Its genome is a linear double-stranded DNA, 144,230 base pairs in length. Its GC content is 35.9%. The closest genomic DNA sequence was found in Enterococcus phage vB_EfaM_Ef2.3, with a sequence identity of 99.06% over 95% query coverage. Furthermore, 75 open reading frames (ORFs) were functionally annotated and five tRNA-encoding genes were found. ORF 6 was annotated as a phage endolysin having an L-acetylmuramoyl-l-alanine amidase activity. We purified the enzyme as a recombinant protein and confirmed its enzymatic activity. The endolysin’s host range was observed to be wider than its parent phage PBEF129. When applied to bacterial biofilm on the surface of in vitro cultured human intestinal cells, it demonstrated a removal efficacy of the same degree as cefotaxime, but much lower than its parent bacteriophage

Characterization of Two Novel Bacteriophages Infecting Multidrug-Resistant (MDR) Acinetobacter baumannii and Evaluation of Their Therapeutic Efficacy in Vivo

Acinetobacter baumannii is emerging as a challenging nosocomial pathogen due to its rapid evolution of antibiotic resistance. We report characterization of two novel bacteriophages, PBAB08 and PBAB25, infecting clinically isolated, multidrug-resistant (MDR) A. baumannii strains. Both phages belonged to Myoviridae of Caudovirales as their morphology observed under an electron microscope. Their genomes were double stranded linear DNAs of 42,312 base pairs and 40,260 base pairs, respectively. The two phages were distinct from known Acinetobacter phages when whole genome sequences were compared. PBAB08 showed a 99% similarity with 57% sequence coverage to phage AB1 and PBAB25 showed a 97% similarity with 78% sequence coverage to phage IME_AB3. BLASTN significant alignment coverage of all other known phages were &lt;30%. Seventy six and seventy genes encoding putative phage proteins were found in the genomes of PBAB08 and PBAB25, respectively. Their genomic organizations and sequence similarities were consistent with the modular theory of phage evolution. Therapeutic efficacy of a phage cocktail containing the two and other phages were evaluated in a mice model with nasal infection of MDR A. baumannii. Mice treated with the phage cocktail showed a 2.3-fold higher survival rate than those untreated in 7 days post infection. In addition, 1/100 reduction of the number of A. baumannii in the lung of the mice treated with the phage cocktail was observed. Also, inflammatory responses of mice which were injected with the phage cocktail by intraperitoneal, intranasal, or oral route was investigated. Increase in serum cytokine was minimal regardless of the injection route. A 20% increase in IgE production was seen in intraperitoneal injection route, but not in other routes. Thus, the cocktail containing the two newly isolated phages could serve as a potential candidate for therapeutic interventions to treat A. baummannii infections