scholarly journals Identification of a Two-Partner Secretion Locus of Enterotoxigenic Escherichia coli

2006 ◽  
Vol 74 (4) ◽  
pp. 2245-2258 ◽  
Author(s):  
James M. Fleckenstein ◽  
Koushik Roy ◽  
Julia F. Fischer ◽  
Michael Burkitt
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Epithelial Cells ◽  
High Molecular Weight ◽  
Cloning And Expression ◽  
Peptide Sequence ◽  
Enterotoxigenic Escherichia Coli ◽  
Virulence Plasmid ◽  
E Coli

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) remains a formidable cause of diarrheal illness worldwide. At present, there is no vaccine that provides broad-based protection against ETEC. A ′phoA-based self-cloning mutagenesis system, TnphoA.ts, employed to identify novel ETEC surface antigens, led to identification of an ETEC two-partner secretion locus (etpBAC) on the pCS1 virulence plasmid of prototype strain H10407. Cloning and expression of etpBAC in recombinant E. coli LMG194(pJY019) resulted in secretion of a high-molecular-weight (HMW) glycosylated exoprotein. This glycoprotein, EtpA, exhibits linear peptide sequence and predicted structural homologies with known HMW adhesins produced by other two-partner secretion loci. Antibodies directed against recombinant EtpA (anti-rEtpA.6H) recognized an HMW protein in culture supernatants of ETEC strains H10407 and LMG194(pJY019) but not in culture supernatant of strain H10407-P, which lacks the 92-kb pCS1 plasmid, or an isogenic etpA mutant. etpA mutants were deficient in adherence to intestinal epithelial cells in vitro, and anti-rEtpA.6H antibodies inhibited association of H10407 with target epithelial cells. Cloning and expression of etpB in recombinant E. coli were sufficient to confer adherence. Screening of multiple ETEC isolates for the etpBAC locus by colony hybridization and by EtpA immunoblotting suggested that EtpA is one of the most common antigens secreted by these pathogens. Together, these results indicate that the newly identified ETEC two-partner secretion locus directs the secretion of a high-molecular-weight glycosylated protein, EtpA, that in concert with the putative EtpB transporter participates in adherence of H10407 to epithelial cells, thereby expanding the repertoire of potential ETEC virulence proteins and vaccine candidates.

Cloning and expression of Xylella fastidiosa antigens in Escherichia coli and Erwinia stewartii

1989 ◽  
Vol 35 (4) ◽  
pp. 487-491 ◽  
Author(s):  
Paul H. Goodwin
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Genomic Dna ◽  
Xylella Fastidiosa ◽  
Gene Bank ◽  
Cloning And Expression ◽  
Western Blots ◽  
E Coli ◽  
Cosmid Vector ◽  
Erwinia Stewartii

Xylella fastidiosa DNA, partially digested with Sau3A, was ligated into the cosmid vector, pUCD615. Approximately 4500 ampicillin-resistant Escherichia coli colonies were obtained. The frequency of complementation of leucine auxotrophy in transfected E. coli indicated that the cosmid gene bank was representative of X. fastidiosa genomic DNA. Colonies were lysed directly onto nitrocellulose membranes using a thermo-inducible λ lysogen and screened for expression of X. fastidiosa antigens. Approximately 16.5% of a random sample of clones were found to express X. fastidiosa antigens as determined by Western blots. These proteins comigrated with proteins of X. fastidiosa and ranged in molecular weight from 10 000 to 160 000. Conjugation of several of the plasmids into Erwinia stewartii resulted in expression of the similar molecular weight cloned proteins with similar levels of expression as in E. coli.Key words: Xylella fastidiosa, Pierce's disease, immunological clone screening, thermo-inducible lysogeny.

scholarly journals In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions

2021 ◽  
Vol 9 (9) ◽  
pp. 1869
Author(s):  
Joanna Kaczorowska ◽  
Eoghan Casey ◽  
Gabriele A. Lugli ◽  
Marco Ventura ◽  
David J. Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Galleria Mellonella ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Composite Mixture ◽  
Ph Conditions ◽  
The Stability ◽  
Higher Sensitivity

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

scholarly journals A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack.

1992 ◽  
Vol 118 (5) ◽  
pp. 1015-1026 ◽  
Author(s):  
M G Waters ◽  
D O Clary ◽  
J E Rothman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Protein Transport ◽  
Gel Filtration ◽  
Bovine Liver ◽  
Peptide Sequence ◽  
Golgi Stack ◽  
Transport Assay ◽  
Weight Protein

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.

scholarly journals Comparative Analysis of EspF Variants in Inhibition of Escherichia coli Phagocytosis by Macrophages and Inhibition of E. coli Translocation through Human- and Bovine-Derived M Cells

2011 ◽  
Vol 79 (11) ◽  
pp. 4716-4729 ◽  
Author(s):  
Amin Tahoun ◽  
Gabriella Siszler ◽  
Kevin Spears ◽  
Sean McAteer ◽  
Jai Tree ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cellular Localization ◽  
M Cells ◽  
Binding Assays ◽  
M Cell ◽  
Content Type ◽  
E Coli ◽  
Coculture System

ABSTRACTThe EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagicEscherichia coli(EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspFO127is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspFO127has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of differentespFalleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. TheespFgene fromE. coliserotype O157 (espFO157) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibitE. colitranslocation through a human-derivedin vitroM-cell coculture system in comparison toespFO127andespFO26. In contrast,espFO157was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

scholarly journals Repression of the Inner Membrane Lipoprotein NlpA by Rns in Enterotoxigenic Escherichia coli

2006 ◽  
Vol 189 (5) ◽  
pp. 1627-1632 ◽  
Author(s):  
Maria D. Bodero ◽  
M. Carolina Pilonieta ◽  
George P. Munson
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Enterotoxigenic Escherichia Coli ◽  
Start Codon ◽  
Inner Membrane ◽  
E Coli ◽  
Inner Membrane Protein

ABSTRACT The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions −152 and −195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.

scholarly journals Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

2004 ◽  
Vol 186 (20) ◽  
pp. 6845-6854 ◽  
Author(s):  
Koichi Mori ◽  
Reiko Bando ◽  
Naoki Hieda ◽  
Tetsuo Toraya
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Protein S ◽  
Amino Acid Residues ◽  
Permeabilized Cells ◽  
E Coli ◽  
Cell Extracts ◽  
Auxiliary Protein

ABSTRACT The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5′ end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

scholarly journals Effects of Multiple Deletions of Murein Hydrolases on Viability, Septum Cleavage, and Sensitivity to Large Toxic Molecules in Escherichia coli

2002 ◽  
Vol 184 (22) ◽  
pp. 6093-6099 ◽  
Author(s):  
Christoph Heidrich ◽  
Astrid Ursinus ◽  
Jürgen Berger ◽  
Heinz Schwarz ◽  
Joachim-Volker Höltje
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
High Molecular Weight ◽  
Macconkey Agar ◽  
Lytic Transglycosylases ◽  
E Coli ◽  
Murein Hydrolase ◽  
Outer Membrane Permeability

ABSTRACT The multiplicity of murein hydrolases found in most bacteria presents an obstacle to demonstrating the necessity of these potentially autolytic enzymes. Therefore, Escherichia coli mutants with deletions in multiple murein hydrolases, including lytic transglycosylases, amidases, and dd-endopeptidases, were constructed. Even a mutant from which seven different hydrolases were deleted was viable and grew at a normal rate. However, penicillin-induced lysis was retarded. Most of the mutants were affected in septum cleavage, which resulted in the formation of chains of cells. All three enzymes were shown to be capable of splitting the septum. Failure to cleave the septum resulted in an increase in outer membrane permeability, and thus the murein hydrolase mutants did not grow on MacConkey agar plates. In addition, the hydrolase mutants not only could be lysed by lysozyme in the absence of EDTA but also were sensitive to high-molecular-weight antibiotics, such as vancomycin and bacitracin, which are normally ineffective against E. coli.

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