Crystal structure of heat-labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit

ABSTRACT Escherichia coli heat-labile enterotoxin (LT) consists of an A subunit and five B subunits. These subunits oligomerize into an assembled holotoxin within the periplasm. Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus. This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm. However, it is not known whether other regions of the A subunit contribute to the assembly. The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199. It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein. We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit. We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes. The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly. A reconstitution experiment in vitro supported the notion. Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs. These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.

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A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

Journal of Experimental Medicine ◽

10.1084/jem.180.6.2147 ◽

1994 ◽

Vol 180 (6) ◽

pp. 2147-2153 ◽

Cited By ~ 69

Author(s):

M Pizza ◽

M R Fontana ◽

M M Giuliani ◽

M Domenighini ◽

C Magagnoli ◽

...

Keyword(s):

Escherichia Coli ◽

Cholera Toxin ◽

Neutralizing Antibodies ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

E Coli ◽

Heat Labile ◽

A Subunit ◽

Adp Ribosyltransferase ◽

Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

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Crystal Structure of the B Subunit of Escherichia coli Heat-labile Enterotoxin Carrying Peptides with Anti-herpes Simplex Virus Type 1 Activity

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)87394-5 ◽

1999 ◽

Vol 274 (13) ◽

pp. 8764-8769

Author(s):

Dubravka Matković-Calogović ◽

Arianna Loregian ◽

Maria Rosa D'Acunto ◽

Roberto Battistutta ◽

Alessandro Tossi ◽

...

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

B Subunit ◽

Heat Labile ◽

Simplex Virus

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Protection against Lethal Leptospirosis after Vaccination with LipL32 Coupled or Coadministered with the B Subunit of Escherichia coli Heat-Labile Enterotoxin

Clinical and Vaccine Immunology ◽

10.1128/cvi.05720-11 ◽

2012 ◽

Vol 19 (5) ◽

pp. 740-745 ◽

Cited By ~ 35

Author(s):

André A. Grassmann ◽

Samuel R. Félix ◽

Carolina Ximendes dos Santos ◽

Marta G. Amaral ◽

Amilton C. P. Seixas Neto ◽

...

Keyword(s):

Escherichia Coli ◽

Immune Response ◽

Subunit Vaccine ◽

Lethal Dose ◽

Control Group ◽

Hamster Model ◽

Content Type ◽

B Subunit ◽

Heat Labile ◽

A Subunit

ABSTRACTLeptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species ofLeptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of theEscherichia coliheat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD50) ofLeptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P< 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.

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Differential Biological and Adjuvant Activities of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin Hybrids

Infection and Immunity ◽

10.1128/iai.69.3.1528-1535.2001 ◽

2001 ◽

Vol 69 (3) ◽

pp. 1528-1535 ◽

Cited By ~ 40

Author(s):

Christal C. Bowman ◽

John D. Clements

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Cholera Toxin ◽

The Other ◽

Toxin Gene ◽

Wild Type ◽

B Subunit ◽

Heat Labile ◽

A Subunit ◽

Ifn Γ

ABSTRACT Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A–CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A–CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-γ) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A–LT-B and native CT both produced lower levels of antigen-specific IFN-γ. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

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Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the Escherichia coli heat-labile enterotoxin

Immunology ◽

10.1046/j.1365-2567.1999.00817.x ◽

1999 ◽

Vol 97 (4) ◽

pp. 706-713 ◽

Cited By ~ 20

Author(s):

De Haan ◽

Holtrop ◽

Verweij ◽

Agsteribbe ◽

Wilschut

Keyword(s):

Escherichia Coli ◽

Adjuvant Activity ◽

Heat Labile ◽

A Subunit

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Nicking sites in a subunit of cholera toxin and Escherichia coli heat-labile enterotoxin for Vibrio cholerae hemagglutinin/protease

Toxicon ◽

10.1016/s0041-0101(97)00135-9 ◽

1998 ◽

Vol 36 (7) ◽

pp. 1001-1005 ◽

Cited By ~ 4

Author(s):

Atsuko Naka ◽

Tetsuya Iida ◽

Takeshi Ohara ◽

Koichiro Yamamoto ◽

Toshio Miwatani ◽

...

Keyword(s):

Escherichia Coli ◽

Cholera Toxin ◽

Vibrio Cholerae ◽

Heat Labile ◽

A Subunit

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Inactivation of the Escherichia coli heat-labile enterotoxin by in vitro mutagenesis of the A-subunit gene

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1989.tb14930.x ◽

1989 ◽

Vol 183 (2) ◽

pp. 311-316 ◽

Cited By ~ 32

Author(s):

Stephen HARFORD ◽

Colin W. DYKES ◽

Adrian N. HOBDEN ◽

Melanie J. READ ◽

Ishbel J. HALLIDAY

Keyword(s):

Escherichia Coli ◽

In Vitro Mutagenesis ◽

Subunit Gene ◽

Heat Labile ◽

A Subunit

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Temperature Regulation of Heat-Labile Enterotoxin (LT) Synthesis in Escherichia coli Is Mediated by an Interaction of H-NS Protein with the LT A-Subunit DNA

Journal of Bacteriology ◽

10.1128/jb.180.14.3715-3718.1998 ◽

1998 ◽

Vol 180 (14) ◽

pp. 3715-3718 ◽

Cited By ~ 17

Author(s):

Julie D. Trachman ◽

Werner K. Maas

Keyword(s):

Escherichia Coli ◽

Temperature Effect ◽

Temperature Regulation ◽

Low Temperatures ◽

Regulatory Element ◽

Mrna Levels ◽

Dna Coding ◽

Heat Labile ◽

A Subunit

ABSTRACT Protein and mRNA levels of heat-labile enterotoxin (LT) ofEscherichia coli are highest at 37°C, and they decrease gradually as temperature is decreased. This temperature effect is eliminated in an Hns− mutant. Deletion of portions of DNA coding for the LT A subunit also results in an increase in LT expression at low temperatures, suggesting that the H-NS protein causes inhibition of transcription at low temperatures by interacting with the LT A-subunit DNA. The region that interacts with H-NS is referred to as the downstream regulatory element (DRE). Plasmids in an hns strain from which the DRE has been deleted still produce elevated levels of LT at 18°C, suggesting that intact DRE is not required for transcription from the LT promoter.

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