scholarly journals Intracellular Expression of the Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli

2003 ◽  
Vol 71 (9) ◽  
pp. 5364-5370 ◽  
Author(s):  
Bao Quan Sui ◽  
Pinaki R. Dutta ◽  
James P. Nataro
Keyword(s):  
Escherichia Coli ◽  
Cell Elongation ◽  
Morphological Changes ◽  
Intracellular Localization ◽  
Toxic Effects ◽  
Expression Vectors ◽  
Passenger Domain ◽  
Mammalian Expression ◽  
Site Of Action ◽  
Intracellular Expression

ABSTRACT The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a serine protease autotransporter that acts as an enterotoxin and cytotoxin. When applied to epithelial cells in culture, purified toxin induces cell elongation and rounding, followed by exfoliation of cells from the substratum. These effects are accompanied by loss of actin stress fibers and electrophysiologic changes. Although it has been hypothesized that Pet has an intracellular site of action, evidence for this is indirect. In addition, Pet has recently been shown to cleave spectrin in vitro and in vivo. If Pet requires intracellular localization to execute its toxic effects, then intracellular expression of the protein could induce cytopathic effects similar to those observed when the toxin is applied to the cell surface. To test this hypothesis, we expressed the mature Pet toxin (comprising only the passenger domain of the Pet precursor) in the cytoplasm of HEp-2 cells by using mammalian expression vectors. Separately, we expressed the Pet passenger domain mutated at the catalytic serine (PetS260A), a construct that has been reported to lack toxic effects. Forty-eight hours after transient transfection of pcDNA3.1-pet in HEp-2 cells, we observed cell elongation and other morphological changes similar to those induced by applied toxin. Cells transfected with pcDNA3.1 vector alone appeared normal, while cells expressing the PetS260A mutant displayed similar (though less pronounced) changes compared with those in cells expressing pcDNA3.1-pet. Notably, intracellular expression of Pet was accompanied by condensation of the spectrin cytoskeleton. These studies corroborate an intracellular site of action for the Pet toxin, further implicate a role for spectrin in Pet intoxication, and provide a powerful tool for Pet structure and function analyses.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

Interaction of Gram-Negative Bacteria with the Lysosomal Fraction of Polymorphonuclear Leukocytes II. Changes in the Cell Envelope of Escherichia coli

1970 ◽  
Vol 1 (3) ◽  
pp. 311-318
Author(s):  
D. Friedberg ◽  
I. Friedberg ◽  
M. Shilo
Keyword(s):  
Escherichia Coli ◽  
Polymorphonuclear Leukocytes ◽  
Cytoplasmic Membrane ◽  
Morphological Changes ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Intact Cells ◽  
Lysosomal Fraction ◽  
E Coli ◽  
Absorbing Material

Interaction of lysosomal fraction with Escherichia coli caused damage to the cell envelope of these intact cells and to the cytoplasmic membrane of E. coli spheroplasts. The damage to the cytoplasmic membrane was manifested in the release of 260-nm absorbing material and β-galactosidase from the spheroplasts, and by increased permeability of cryptic cells to O -nitrophenyl-β- d -galactopyranoside; damage to the cell wall was measured by release of alkaline phosphatase. Microscope observation showed morphological changes in the cell envelope.

scholarly journals Escherichia coli Morphological Changes and Lipid A Removal Induced by Reduced Pressure Nitrogen Afterglow Exposure

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0116083 ◽  
Author(s):  
Hayat Zerrouki ◽  
Virginie Rizzati ◽  
Corinne Bernis ◽  
Anne Nègre-Salvayre ◽  
Jean Philippe Sarrette ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Morphological Changes ◽  
Reduced Pressure

scholarly journals SITE OF ACTION OF 3',5'-CYCLIC ADENOSINE MONOPHOSPHATE IN PRODUCTION OF TRYPTOPHANASE IN ESCHERICHIA COLI

Genetics ◽  
1972 ◽  
Vol 70 (1) ◽  
pp. 175-180
Author(s):  
LaDonna Immken ◽  
David Apirion
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Polypeptide Chain ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Chain Elongation ◽  
Site Of Action

ABSTRACT 3″,5″ cyclic-AMP (cAMP) will stimulate the rate of tryptophanase synthesis in Escherichia coli cultures induced with tryptophan. Adding cAMP after the initiation of messenger RNA synthesis was blocked by rifampicin, did not stimulate tryptophanase synthesis. This indicates that cAMP acts at initiation of either transcription or translation and not at the level of chain elongation of either the messenger or the polypeptide chain.

Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal

1993 ◽  
Vol 105 (2) ◽  
pp. 481-488 ◽  
Author(s):  
J.V. Frangioni ◽  
B.G. Neel
Keyword(s):  
Amino Acid ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Protein Targeting ◽  
General Purpose ◽  
Expression Vectors ◽  
Intracellular Protein ◽  
Mammalian Expression ◽  
Nuclear Lamin ◽  
Localization Signal

We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.

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