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

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala → Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 μg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose–response experiments in mice, 20 μg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

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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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Assembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Kinetics and molecular basis of rate-limiting steps in vitro.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)35438-3 ◽

1996 ◽

Vol 271 (43) ◽

pp. 27188

Author(s):

Lloyd W. Ruddock ◽

Jeremy J.F. Coen ◽

Caroline Cheesman ◽

Robert B. Freedman ◽

Timothy R. Hirst

Keyword(s):

Escherichia Coli ◽

Molecular Basis ◽

B Subunit ◽

Heat Labile ◽

Rate Limiting

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In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli

Vaccine ◽

10.1016/j.vaccine.2009.05.027 ◽

2009 ◽

Vol 27 (32) ◽

pp. 4302-4308 ◽

Cited By ~ 28

Author(s):

Shuang Liang ◽

Kavita B. Hosur ◽

Hesham F. Nawar ◽

Michael W. Russell ◽

Terry D. Connell ◽

...

Keyword(s):

Escherichia Coli ◽

B Subunit ◽

Heat Labile

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Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

Clinical and Vaccine Immunology ◽

10.1128/cvi.00351-12 ◽

2012 ◽

Vol 19 (10) ◽

pp. 1603-1608 ◽

Cited By ~ 20

Author(s):

Koushik Roy ◽

David J. Hamilton ◽

James M. Fleckenstein

Keyword(s):

Escherichia Coli ◽

Diarrheal Disease ◽

Vaccine Development ◽

Enterotoxigenic Escherichia Coli ◽

Intestinal Colonization ◽

Content Type ◽

Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonizationin vivoand toxin delivery to epithelial cellsin vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development.

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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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Escherichia coli heat-labile enterotoxin: dan-directed in vitro synthesis and structure

Toxicon ◽

10.1016/0041-0101(79)90039-4 ◽

1979 ◽

Vol 17 ◽

pp. 37

Keyword(s):

Escherichia Coli ◽

In Vitro Synthesis ◽

Heat Labile

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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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Molecular characterization of human stathmin expressed in Escherichia coli: site-directed mutagenesis of two phosphorylatable serines (Ser-25 and Ser-63)

Biochemical Journal ◽

10.1042/bj3000331 ◽

1994 ◽

Vol 300 (2) ◽

pp. 331-338 ◽

Cited By ~ 31

Author(s):

P A Curmi ◽

A Maucuer ◽

S Asselin ◽

M Lecourtois ◽

A Chaffotte ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Kinase ◽

Glutamic Acid ◽

Protein Characterization ◽

Mitogen Activated Protein Kinase ◽

Helical Conformation ◽

In Vitro Mutagenesis ◽

Native Protein

Stathmin, a probable relay protein possibly integrating multiple intracellular regulatory signals [reviewed in Sobel (1991) Trends Biochem. Sci. 16, 301-305], was expressed in Escherichia coli at levels as high as 20% of total bacterial protein. Characterization of the purified recombinant protein revealed that it had biochemical properties very similar to those of the native protein. It is a good substrate for both cyclic AMP-dependent protein kinase (PKA) and p34cdc2, on the same four sites as the native eukaryotic protein. As shown by m.s., the difference in isoelectric points from the native protein is probably due to the absence of acetylation of the protein produced in bacteria. C.d. studies indicate that stathmin probably contains about 45% of its sequence in an alpha-helical conformation, as also predicted for the sequence between residues 47 and 124 by computer analysis. Replacement of Ser-63 by alanine by in vitro mutagenesis resulted in a ten times less efficient phosphorylation of stathmin by PKA which occurred solely on Ser-16, confirming that Ser-63 is the major target of this kinase. Replacement of Ser-25, the major site phosphorylated by mitogen-activated protein kinase in vitro and in vivo, by the charged amino acid glutamic acid reproduced, in conjunction with the phosphorylation of Ser-16 by PKA, the mobility shift on SDS/polyacrylamide gels induced by the phosphorylation of Ser-25. This result strongly suggests that glutamic acid in position 25 is able to mimic the putative interactions of phosphoserine-25 with phosphoserine-16, as well as the resulting conformational changes that are probably also related to the functional regulation of stathmin.

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