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

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.

Download Full-text

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.

Download Full-text

Enterotoxigenic Escherichia coli infection and intestinal thiamin uptake: studies with intestinal epithelial Caco-2 monolayers

AJP Cell Physiology ◽

10.1152/ajpcell.00276.2013 ◽

2013 ◽

Vol 305 (11) ◽

pp. C1185-C1191 ◽

Cited By ~ 8

Author(s):

Abhisek Ghosal ◽

Nabendu S. Chatterjee ◽

Tristan Chou ◽

Hamid M. Said

Keyword(s):

Escherichia Coli ◽

Significant Inhibition ◽

Water Soluble ◽

Enterotoxigenic Escherichia Coli ◽

Adenylate Cyclase System ◽

Mrna Levels ◽

Intestinal Epithelial ◽

E Coli ◽

Heat Labile ◽

Etec Infection

Infections with enteric pathogens like enterotoxigenic Escherichia coli ( ETEC) is a major health issue worldwide and while diarrhea is the major problem, prolonged, severe, and dual infections with multiple pathogens may also compromise the nutritional status of the infected individuals. There is almost nothing currently known about the effect of ETEC infection on intestinal absorptions of water-soluble vitamins including thiamin. We examined the effect of ETEC infection on intestinal uptake of the thiamin using as a model the human-derived intestinal epithelial Caco-2 cells. The results showed that infecting confluent Caco-2 monolayers with live ETEC (but not with boiled/killed ETEC or nonpathogenic E. coli) or treatment with bacterial culture supernatant led to a significant inhibition in thiamin uptake. This inhibition appears to be caused by a heat-labile and -secreted ETEC component and is mediated via activation of the epithelial adenylate cyclase system. The inhibition in thiamin uptake by ETEC was associated with a significant reduction in expression of human thiamin transporter-1 and -2 (hTHTR1 and hTHTR2) at the protein and mRNA levels as well as in the activity of the SLC19A2 and SLC19A3 promoters. Dual infection of Caco-2 cells with ETEC and EPEC (enteropathogenic E. coli) led to compounded inhibition in intestinal thiamin uptake. These results show for the first time that infection of human intestinal epithelial cells with ETEC causes a significant inhibition in intestinal thiamin uptake. This inhibition is mediated by a secreted heat-labile toxin and is associated with a decrease in the expression of intestinal thiamin transporters.

Download Full-text

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.

Download Full-text

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.

Download Full-text