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Deletion mutations in N-terminal alpha1 helix render heat labile enterotoxin B subunit susceptible to degradation.

ABSTRACT: Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is a heterohexameric protein consisting of an enzymatically active A subunit, LTA, and a carrier pentameric B subunit, LTB. It is clear from the crystal structure of LTB that the N-terminal alpha1 helix lies outside the core structure. However, the function of the N-terminal alpha1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the alpha1 helix on LTB synthesis. Six amino acids (PQSITE) located at positions 2-7 from the N terminus, including 4 aa from the alpha1 helix, were deleted by site-directed mutagenesis. The deletion resulted in complete inhibition of LTB expression in E. coli when expressed along with its signal sequence. A single amino acid deletion within the alpha1 helix also resulted in loss of expression. However, a single amino acid deletion outside the alpha1 helix did not affect LTB synthesis. Mutant proteins, whose synthesis was not detected in vivo, could be successfully translated in vitro by using the coupled transcription-translation system. Immunoblot analysis, Northern blot analysis, and in vitro transcription-translation data collectively indicate that the lack of synthesis of the mutant proteins is caused by the immediate degradation of the expressed product by cellular proteases rather than by faulty translation of mutant LTB mRNA. Coexpression of the LTA could not rescue the degradation of LTB mutants.

SUBMITTER: Alone PV

PROVIDER: S-EPMC2042161 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Deletion mutations in N-terminal alpha1 helix render heat labile enterotoxin B subunit susceptible to degradation.

Alone Pankaj V PV Malik Gunjan G Krishnan Anuja A Garg Lalit C LC

Proceedings of the National Academy of Sciences of the United States of America 20071002 41

Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is a heterohexameric protein consisting of an enzymatically active A subunit, LTA, and a carrier pentameric B subunit, LTB. It is clear from the crystal structure of LTB that the N-terminal alpha1 helix lies outside the core structure. However, the function of the N-terminal alpha1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the alpha1 helix on LTB synthesis. ...[more]

PMID: 17911243

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Project description:IntroductionEnterotoxigenic Escherichia coli (ETEC) is a common cause of infectious diarrhoea and a leading cause of morbidity and mortality in children living in resource-limited settings. It is also the leading cause of travellers' diarrhoea among civilian and military travellers. Its dual importance in global public health and travel medicine highlights the need for an effective vaccine. ETEC express colonization factors (CFs) that mediate adherence to the small intestine. An epidemiologically prevalent CF is coli surface antigen 6 (CS6). We assessed the safety and immunogenicity of a CS6-targeted candidate vaccine, CssBA, co-administered intramuscularly with the double-mutant heat-labile enterotoxin, dmLT [LT(R192G/L211A)].MethodsThis was an open-label trial. Fifty subjects received three intramuscular injections (Days 1, 22 and 43) of CssBA alone (5 µg), dmLT alone (0.1 µg) or CssBA (5, 15, 45 µg) + dmLT (0.1 and 0.5 µg). Subjects were actively monitored for adverse events for 28 days following the third vaccination. Antibody responses (IgG and IgA) were characterized in the serum and from lymphocyte supernatants (ALS) to CS6 and the native ETEC heat labile enterotoxin, LT.ResultsAcross all dose cohorts, the vaccine was safe and well-tolerated with no vaccine-related severe or serious adverse events. Among vaccine-related adverse events, a majority (98%) were mild with 79% being short-lived vaccine site reactions. Robust antibody responses were induced in a dose-dependent manner with a clear dmLT adjuvant effect. Response rates in subjects receiving 45 µg CssBA and 0.5 µg dmLT ranged from 50 to 100% across assays.ConclusionThis is the first study to demonstrate the safety and immunogenicity of CssBA and/or dmLT administered intramuscularly. Co-administration of the two components induced robust immune responses to CS6 and LT, paving the way for future studies to evaluate the efficacy of this vaccine target and development of a multivalent, subunit ETEC vaccine.

Project description:UnlabelledCholera toxin (CT) and the related heat-labile enterotoxins (LT) of Escherichia coli have been implicated as adjuvants in human therapies, but reactivity upon intranasal delivery dampened efforts to develop other clinical applications. However, each CT family member variant has unique biological properties that may warrant development as therapeutic platforms. In the current study, a nontoxic variant of the heat-labile enterotoxin IIa (LTIIa) was engineered to deliver heterologous, functional proteins into the cytosol of neurons. As proof of principle, the LTIIa variant delivered two cargos into neurons. LTIIa delivered β-lactamase efficiently into cells containing complex gangliosides, such as GD1b, as host receptors. LTIIa delivery of β-lactamase was sensitive to brefeldin A, an inhibitor that collapses the Golgi compartment into the endoplasmic reticulum, but not sensitive to treatment with botulinum neurotoxin D (BoNT/D), an inhibitor of synaptic vesicle cycling. LTIIa delivered a single-chain, anti-BoNT/A camelid antibody that inhibited SNAP25 cleavage during post-BoNT/A exposure of neurons. Delivery of functional, heterologous protein cargos into neurons demonstrates the potential of LTII variants as platforms to deliver therapies to inactivate toxins and microbial infections and to reverse the pathology of human neurodegenerative diseases.ImportanceThis study engineered a protein platform to deliver functional, heterologous proteins into neurons. The protein platform developed was a variant of the heat-labile enterotoxin IIa (LTIIa) which lacked the catalytic domain, yielding a nontoxic protein. As proof of principle, LTIIa variants delivered two functional proteins into neurons, β-lactamase and a camelid antibody. These studies show the utility of LTIIa variants to deliver therapies into neurons, which could be extended to inactivate toxins and microbial infections and potentially to reverse the progression of neurological diseases, such as Alzheimer's disease and Parkinson's disease.

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Deletion mutations in N-terminal alpha1 helix render heat labile enterotoxin B subunit susceptible to degradation.

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Deletion mutations in N-terminal alpha1 helix render heat labile enterotoxin B subunit susceptible to degradation.

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