scholarly journals Full Capacity of Recombinant Escherichia coliHeat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity

2000 ◽  
Vol 68 (7) ◽  
pp. 4064-4074 ◽  
Author(s):  
Isabelle Batisson ◽  
Maurice Der Vartanian ◽  
Brigitte Gaillard-Martinie ◽  
Michel Contrepois
Keyword(s):  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Biological Properties ◽  
Leader Peptide ◽  
Dependent Manner ◽  
Reporter Protein ◽  
E Coli ◽  
Heat Stable

ABSTRACT We have successfully used the major subunit ClpG ofEscherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH2 or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface ofE. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

Protective effects of Cathelicidin against glomerulonephritis through promotion of host defense

2014 ◽  
Vol 2 (3) ◽  
pp. 189-198
Author(s):  
Ajay H. Bahl ◽  
Wanda Lee
Keyword(s):  
Host Defense ◽  
Disulfide Bonds ◽  
Helical Conformation ◽  
Antimicrobial Protein ◽  
Protective Effects ◽  
In Vivo Models ◽  
E Coli ◽  
Risk Of Death

Cathelicidin-related antimicrobial peptides are a family of polypeptides found in lysosomes of macrophages and polymorphonuclear leukocytes (PMNs). Some of these peptides can assume an alpha-helical conformation, others contain one or two disulfide bonds, still others are Pro- and Arg-rich, or Trp-rich. Higher levels of human cathelicidin antimicrobial protein (hCAP18), which are up-regulated by vitamin D, appear to significantly reduce the risk of death from infection in dialysis patients. Using in vitro and in vivo models of kidney infection, we demonstrate key antimicrobial and host immunomodulatory properties of cathelicidins. To directly assess the role of endogenous cathelicidin in the development of glomerulonephritis, WT and mCRAMP KO mice were provided with 5% DSS to induce glomerulonephritis. Some mice groups were administered with E. coli DNA I.P. Our findings showed that mCRAMP KO mice develop more severe glomerulonephritis. These data demonstrate key roles for cathelicidins in host defense against glomerulonephritis and the potential to inform the development of synthetic analogues to modulate specific host-pathogen interactions as novel antimicrobial therapeutics.

scholarly journals Curcumin Oxidation Is Required for Inhibition of Helicobacter pylori Growth, Translocation and Phosphorylation of Cag A

2021 ◽  
Vol 11 ◽  
Author(s):  
Ashwini Kumar Ray ◽  
Paula B. Luis ◽  
Surabhi Kirti Mishra ◽  
Daniel P. Barry ◽  
Mohammad Asim ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Growth Inhibition ◽  
Mrna Expression ◽  
Host Protein ◽  
Dependent Manner ◽  
Gene Encoding ◽  
E Coli ◽  
H Pylori

Curcumin is a potential natural remedy for preventing Helicobacter pylori-associated gastric inflammation and cancer. Here, we analyzed the effect of a phospholipid formulation of curcumin on H. pylori growth, translocation and phosphorylation of the virulence factor CagA and host protein kinase Src in vitro and in an in vivo mouse model of H. pylori infection. Growth of H. pylori was inhibited dose-dependently by curcumin in vitro. H. pylori was unable to metabolically reduce curcumin, whereas two enterobacteria, E. coli and Citrobacter rodentium, which efficiently reduced curcumin to the tetra- and hexahydro metabolites, evaded growth inhibition. Oxidative metabolism of curcumin was required for the growth inhibition of H. pylori and the translocation and phosphorylation of CagA and cSrc, since acetal- and diacetal-curcumin that do not undergo oxidative transformation were ineffective. Curcumin attenuated mRNA expression of the H. pylori virulence genes cagE and cagF in a dose-dependent manner and inhibited translocation and phosphorylation of CagA in gastric epithelial cells. H. pylori strains isolated from dietary curcumin-treated mice showed attenuated ability to induce cSrc phosphorylation and the mRNA expression of the gene encoding for IL-8, suggesting long-lasting effects of curcumin on the virulence of H. pylori. Our work provides mechanistic evidence that encourages testing of curcumin as a dietary approach to inhibit the virulence of CagA.

scholarly journals IN VITRO ANTIBACTERIAL ACTIVITY OF ETHANOLIC EXTRACT OF SEEDPOD AND QUERCETIN OF NELUMBO NUCIFERA GAERTN

2019 ◽  
Vol 12 (1) ◽  
pp. 164
Author(s):  
Ruvanthika Pn ◽  
Manikandan S
Keyword(s):  
Antibacterial Activity ◽  
Ethanolic Extract ◽  
Antibacterial Activities ◽  
Nelumbo Nucifera ◽  
Diffusion Method ◽  
Dependent Manner ◽  
E Coli ◽  
In Vitro Antibacterial Activity

Objective: The objective of the study was to evaluate whether ethanolic extracts of Nelumbo nucifera (EENN) seedpod and quercetin (active component of NN) possess antibacterial proprieties against Gram (-) bacteria such as Escherichia coli and Pseudomonas aeruginosa and Gram (+) bacteria such as Staphylococcus aureus. Methods: Antibacterial activities of EENN seedpod and quercetin were investigated using disc diffusion method, minimum inhibitory concentration against E. coli and P. aeruginosa and Gram (+) bacteria such as S. aureus. Results: The antibacterial activity of both EENN seedpod and quercetin was found to be increased in dose-dependent manner. The maximum zone of inhibition was exhibited by both EENN seedpod and quercetin against E. coli (14 mm and 15 mm) and P. aeruginosa (13 mm and 15 mm). Gram-negative bacteria were more susceptible to the EENN seedpod extract and quercetin than Gram-positive bacteria.Conclusion: The results of the present study suggested that the effect of EENN seedpod and quercetin against the tested bacteria in vitro may contribute to the in vivo activities of the EENN seedpod and quercetin.

scholarly journals Nuclear Import of c-Jun Is Mediated by Multiple Transport Receptors

2007 ◽  
Vol 282 (38) ◽  
pp. 27685-27692 ◽  
Author(s):  
Inga Waldmann ◽  
Sarah Wälde ◽  
Ralph H. Kehlenbach
Keyword(s):  
Transcription Factor ◽  
Nuclear Import ◽  
Leucine Zipper ◽  
Dependent Manner ◽  
Reporter Protein ◽  
Permeabilized Cells ◽  
Import Receptors ◽  
Factor C

c-Jun and c-Fos are major components of the transcriptional complex AP-1. Here, we investigate the nuclear import pathway(s) of the transcription factor c-Jun. c-Jun bound specifically to the nuclear import receptors importin β, transportin, importin 5, importin 7, importin 9, and importin 13. In digitonin-permeabilized cells, importin β, transportin, importin 7, and importin 9 promoted efficient import of c-Jun into the nucleus. Importin α, by contrast, inhibited nuclear import of c-Jun in vitro. A single basic region preceding the leucine zipper of c-Jun functions as a nuclear localization signal (NLS) and was required for interaction with all tested import receptors. In vivo, nuclear import of a c-Jun reporter protein lacking the leucine zipper strictly depended on this NLS. In a leucine zipper-dependent manner, c-Jun with mutations in its NLS was still imported into the nucleus in a complex with endogenous leucine zipper proteins or, for example, with cotransfected c-Fos. Together, these results explain the highly efficient nuclear import of the transcription factor c-Jun.

scholarly journals Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment.

1995 ◽  
Vol 131 (4) ◽  
pp. 913-927 ◽  
Author(s):  
M Lussier ◽  
A M Sdicu ◽  
T Ketela ◽  
H Bussey
Keyword(s):  
Membrane Protein ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Dependent Manner ◽  
Reporter Protein ◽  
Golgi Localization ◽  
Golgi Compartment ◽  
Membrane Spanning

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.

In vitro and in vivo Evaluation of 111In-labeled E. coli Heat-Stable Enterotoxin Analogs for Specific Targeting of Human Breast Cancers

2006 ◽  
Vol 98 (1) ◽  
pp. 7-15 ◽  
Author(s):  
Michael F. Giblin ◽  
Hariprasad Gali ◽  
Gary L. Sieckman ◽  
Nellie K. Owen ◽  
Timothy J. Hoffman ◽  
...  
Keyword(s):  
Breast Cancers ◽  
Human Breast ◽  
In Vivo Evaluation ◽  
E Coli ◽  
Specific Targeting ◽  
Heat Stable

P074 HUMAN-DERIVED CLOSTRIDIUM VE202 STRAINS REDUCE ENTEROBACTERIACEAE AND FUSOBACTERIA AND REVERSE EXPERIMENTAL COLITIS INDUCED BY HUMAN GUT MICROBIOTA

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S36-S37
Author(s):  
Akihiko Oka ◽  
Yoshiyuki Mishima ◽  
Gerold Bongers ◽  
Andrew Baltus ◽  
Bo Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Protective Mechanism ◽  
Dependent Manner ◽  
Lipocalin 2 ◽  
Healthy Human ◽  
Human Donor ◽  
E Coli ◽  
Colitis Model

Abstract Background 17 human-derived Clostridium strains (VE202), belonging to a bacterial cluster under-represented in active IBD, induce colonic IL-10-producing FOXP3+ regulatory T cells and prevent colitis in murine IBD models (Nature 2013). The VE202 consortium is a promising live biotherapeutic product for clinical application. However, its 1) therapeutic benefit on established colitis and 2) effect on intestinal microbiota composition remain unclear. Methods Models: We established colitis in ex-germ-free (exGF) Rag2−/− mice with naïve T cell transfer (Hu-nT) and Il10−/− (Hu-Il10−/−) mice colonized with healthy human fecal microbiota, using stool with low abundance of Clostridiales. In Hu-Il10−/−, we tested two other healthy donor stools (one with low Clostridiales and another with Fusobacteria, which is a potential pathobiont in IBD). For a selected bacteria-colonized model, we selected three aggressive Enterobacteriaceae strains cultured from the human donor stool used for the previous studies, based on colonic Th1 response and colitis in monoassociated exGF-Il10−/−. We then established an aggressive Klebsiella (K), Enterobacter (E) and E. coli (E) strain -colonized exGF Il10−/− colitis model (KEE-colitis). In addition, we established a human IBD-derived E.coli and Fusobacterium varium consortium-colonized Il10−/− (EF-colitis) model. In vitro, we anaerobically co-cultured human stools with VE202 and analyzed bacterial composition. Therapy: We treated mice by oral administration of VE202 or vehicle twice weekly for 2–4 weeks. Evaluation: Colitis severity was assessed by blinded histological score, fecal lipocalin-2, stool consistency score and colonic IFNγ +CD4+ T cells. Bacteria profiles in cecal contents were analyzed by metagenomics or qPCR. Results Hu-nT and Hu-Il10−/− (Fig.1A, B): VE202 significantly reversed histological colitis and other inflammatory endpoints compared to vehicle. Consistently VE202 attenuated colitis induced by three different donor stools. Bacteria profiling revealed that VE202 attenuated the colitis-associated bloom of Enterobacteriaceae and Fusobacteria. In KEE-colitis (Fig.1C) VE202 reversed colitis with reduction of all Enterobacteriaceae strains. In EF-colitis (Fig.1D) VE202 reversed colitis with reduction of E. coli and F. varium. Co-culture (Fig.1E): VE202 reduced abundance of Enterobacteriaceae in a dose dependent manner. Conclusion Our findings suggest a novel IL-10-independent protective mechanism for human Clostridium VE202 strains, i.e. correction of dysbiosis with reduction of levels of Enterobacteriaceae and Fusobacteria. In addition, VE202 treatment is consistently effective for different human donor microbiota in vivo and in vitro. These results provide a rationale and target for therapeutic use of rationally selected resident protective bacterial cocktails in IBD patients.

In vitro and in vivo evaluation of 177Lu- and 90Y-labeled E. coli heat-stable enterotoxin for specific targeting of uroguanylin receptors on human colon cancers

2006 ◽  
Vol 33 (4) ◽  
pp. 481-488 ◽  
Author(s):  
Michael F. Giblin ◽  
Gary L. Sieckman ◽  
Tiffani D. Shelton ◽  
Timothy J. Hoffman ◽  
Leonard R. Forte ◽  
...  
Keyword(s):  
Human Colon ◽  
In Vivo Evaluation ◽  
E Coli ◽  
Specific Targeting ◽  
Colon Cancers ◽  
Heat Stable

Molecular and biochemical characterisation of a novel type II peroxiredoxin (XvPrx2) from the resurrection plant Xerophyta viscosa

2016 ◽  
Vol 43 (7) ◽  
pp. 669 ◽  
Author(s):  
Kershini Govender ◽  
Jennifer A. Thomson ◽  
Sagadevan Mundree ◽  
Abdelaleim Ismail ElSayed ◽  
Mohammed Suhail Rafudeen
Keyword(s):  
Type Ii ◽  
Dna Protection ◽  
Reporter Protein ◽  
Significant Similarity ◽  
Fluorescent Reporter ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Biochemical Characterisation

A type II peroxiredoxin gene (XvPrx2) was isolated from a Xerophyta viscosa (Baker) cDNA cold-stress library. The polypeptide displayed significant similarity to other plant type II peroxiredoxins, with the conserved amino acid motif (PGAFTPTCS) proposed to constitute the active site of the enzyme. Northern blot analyses showed that XvPrx2 gene was stress-inducible in response to abiotic stresses while gel analyses revealed that XvPrx2 homologues exist within the X. viscosa proteome. Using a yellow fluorescent reporter protein, the XvPrx2 protein localised to the cytosol. A mutated protein (XvV7) was generated by converting the valine at position 76 to a cysteine and an in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV7, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred to Escherichia coli cells overexpressing either XvPrx2 or XvV7. The XvPrx2 activity was maximal with DTT as electron donor and H2O2 as substrate. Using E. coli thioredoxin, a 2–15-fold lower enzyme activity was observed. The XvPrx2 activity with glutathione was significantly lower and glutaredoxin had no measurable effect on this reaction. The XvV7 protein displayed significantly lower activity compared with XvPrx2 for all substrates assessed.

scholarly journals Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC

2003 ◽  
Vol 185 (9) ◽  
pp. 2920-2926 ◽  
Author(s):  
Wilson B. Muse ◽  
Christopher J. Rosario ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Assimilation ◽  
Cytosine Deaminase ◽  
In Vitro Transcription ◽  
Dependent Manner ◽  
E Coli ◽  
Different Response ◽  
Control Protein

ABSTRACT Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. β-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position −59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions −120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions −83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

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