Cloning, sequencing, and expression in ficoll-generated minicells of an Escherichia coli heat-stable enterotoxin gene

Stieglitz, Heather; Cervantes, Lourdes; Robledo, Renato; Fonseca, Rodrigo Nunes da; Covarrubias, Luis; Bolívar, Francisco; Kupersztoch, Yankel M.

doi:10.1016/0147-619x(88)90006-6

Cited by 25 publications

(21 citation statements)

References 30 publications

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“…(9,21). However, there is evidence that in E. coli cyclic AMP and cyclic AMP receptor proteins are involved in glucose-mediated catabolite repression of Pap piliation (36) and heatstable toxin expression (73). Perhaps the presence of glucose is used by the organism to detect the transition from the mucosal surface to the bloodstream.…”

Section: Cholerae (70)mentioning

confidence: 99%

Environmental signals controlling expression of virulence determinants in bacteria

1992

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Section: Cholerae (70)mentioning

confidence: 99%

Environmental signals controlling expression of virulence determinants in bacteria

1992

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“…Both STas share an almost identical carboxy-terminal sequence (23,26). Four STa genes (one STp gene and three STh genes) have been cloned, and the nucleotide sequences have been determined (6,23,24). Sequencing revealed that two of the three STh genes are identical and that the nucleotide sequence of the remaining STh gene is very similar to that of the two identical STh genes.…”

mentioning

confidence: 99%

Synthesis of Escherichia coli heat-stable enterotoxin STp as a pre-pro form and role of the pro sequence in secretion

Okamoto

Takahara

1990

J Bacteriol

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Escherichia cofi heat-stable enterotoxin STp is presumed from its DNA sequence to be synthesized in vivo as a 72-amino-acid residue precursor that is cleaved to generate mature STp consisting of the 18 carboxy-terminal amino acid residues. There are two methionine residues in the inferred STp sequence in addition to the methionine residue at position 1. In order to confirm production of the STp 72-amino-acid residue precursor, we substituted the additional methionine residues by oligonucleotide-directed site-specific mutagenesis. Since these substitutions did not cause a significant change in STp production, it can be concluded that STp is normally synthesized as the 72-amino-acid residue precursor. The length of the STp precursor indicates the existence of a pro sequence between the signal peptide and the mature protein. In order to identify the pro sequence and determine its role in protein secretion, deletion and fusion proteins were made. A deletion mutant in which the gene fragment encoding amino acid residues 22 to 53 of STp was removed was made. STp activity was found in the culture supernatant of ceDls. Amino acid sequence analysis of the purified STp deletion mutant revealed that the pro sequence encompasses amino acid residues 20 to 54. A hybrid protein consisting of STp amino acids 1 to 53 fused in frame from residue 53 to nuclease A was not secreted into the culture supernatant. These results indicate that the pro sequence does not function to guide periplasmic protein into the extracellular milieu.

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“…1) revealed that both possessed the estA gene coding for the STIb (STh) heat-stable toxin (11,17,30). Although both strains also possessed the LT genes, no LT was detectable in culture supernatants of WS-3504D using ELISA or functional assays (data not shown).…”

Section: Characterization Of Etec Clinical Isolates Ws-3504d and Ws-2mentioning

confidence: 99%

Generation and Characterization of a Live Attenuated Enterotoxigenic Escherichia coli Combination Vaccine Expressing Six Colonization Factors and Heat-Labile Toxin Subunit B

Turner¹,

Stephens²,

Beavis³

et al. 2011

Clin Vaccine Immunol

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Live attenuated oral enterotoxigenic Escherichia coli (ETEC) vaccines have been demonstrated to be safe and immunogenic in human volunteers and to provide a viable approach to provide protection against this important pathogen. This report describes the construction of new ETEC vaccine candidate strains from recent clinical isolates and their characterization. All known genes for ETEC toxins were removed, and attenuating deletion mutations were made in the aroC, ompC, and ompF chromosomal genes. An isolate expressing coli surface antigen 2 (CS2), CS3, heat-labile toxin (LT), heat-stable toxin (ST), and enteroaggregative Escherichia coli heat-stable toxin 1 (EAST1) was attenuated to generate ACAM2007. The subsequent insertion of the operon encoding CS1 created ACAM2017, and this was further modified by the addition of an expression cassette containing the eltB gene, encoding a pentamer of B subunits of LT (LTB), to generate ACAM2027. Another isolate expressing CS5, CS6, LT, ST, and EAST1 was attenuated to generate ACAM2006, from which a lysogenic prophage was deleted to create ACAM2012 and an LTB gene was introduced to form ACAM2022. Finally, a previously described vaccine strain, ACAM2010, had the eltB gene incorporated to generate ACAM2025. All recombinant genes were incorporated into the chromosomal sites of the attenuating mutations to ensure maximal genetic stability. The expression of the recombinant antigens and the changes in plasmids accompanying the deletion of toxin genes are described. Strains ACAM2025, ACAM2022, and ACAM2027 have been combined to create the ETEC vaccine formulation ACE527, which has recently successfully completed a randomized, double-blind, placebo-controlled phase I trial and is currently undergoing a randomized, doubleblind placebo-controlled phase II challenge trial, both in healthy adult volunteers.

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Cloning, sequencing, and expression in ficoll-generated minicells of an Escherichia coli heat-stable enterotoxin gene

Cited by 25 publications

References 30 publications

Environmental signals controlling expression of virulence determinants in bacteria

Environmental signals controlling expression of virulence determinants in bacteria

Synthesis of Escherichia coli heat-stable enterotoxin STp as a pre-pro form and role of the pro sequence in secretion

Generation and Characterization of a Live Attenuated Enterotoxigenic Escherichia coli Combination Vaccine Expressing Six Colonization Factors and Heat-Labile Toxin Subunit B

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