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Crystallization of Escherichia coli CdtB, the biologically active subunit of cytolethal distending toxin.

ABSTRACT: Cytolethal distending toxin (CDT) is a secreted protein toxin produced by several bacterial pathogens. The biologically active CDT subunit CdtB is an active homolog of mammalian type I DNase. Internalization of CdtB and subsequent translocation into the nucleus of target cells results in DNA-strand breaks, leading to cell-cycle arrest and apoptosis. CdtB crystals were grown using microbatch methods with polyethylene glycol 8000 as the precipitant. The CdtB crystals contain one molecule of MW 30.5 kDa per asymmetric unit, belong to space group P2(1)2(1)2(1) and diffract to 1.72 A.

SUBMITTER: Hontz JS

PROVIDER: S-EPMC2197165 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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Crystallization of Escherichia coli CdtB, the biologically active subunit of cytolethal distending toxin.

Hontz Jill S JS Villar-Lecumberri Maria T MT Dreyfus Lawrence A LA Yoder Marilyn D MD

Acta crystallographica. Section F, Structural biology and crystallization communications 20060210 Pt 3

Cytolethal distending toxin (CDT) is a secreted protein toxin produced by several bacterial pathogens. The biologically active CDT subunit CdtB is an active homolog of mammalian type I DNase. Internalization of CdtB and subsequent translocation into the nucleus of target cells results in DNA-strand breaks, leading to cell-cycle arrest and apoptosis. CdtB crystals were grown using microbatch methods with polyethylene glycol 8000 as the precipitant. The CdtB crystals contain one molecule of MW 30. ...[more]

PMID: 16511299

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Project description:Typhoid fever caused by Salmonella enterica serovar Typhi (S.Typhi) continues to be a major problem, especially in developing countries. Due to the rapid emergence of multi-drug-resistant (MDR) strains, which limits the efficacy of conventional antibiotics as well as problems associated with the existing vaccines, efforts are being made to develop effective prophylactic agents. CdtB subunit of typhoid toxin was selected for assessing its vaccine potential due to its high conservation throughout the Typhi strains. In-vitro assessment of DNase activity of cloned and purified CdtB protein showed a significant decrease in the band intensity of DNA. The measure of metabolic activity and morphological alterations assessed using different cell lines in the presence of CdtB protein showed no significant signs of toxicity. These observations were further strengthened by cell cycle analysis, assessed by flow cytometry. Keeping these observations in mind, the immunoprotective potential of CdtB was assessed using S.Typhi induced mouse peritonitis model. A significant titer of IgG antibodies (>128000) against CdtB protein was recorded in the immunized mice by enzyme-linked immunosorbent assay (ELISA), which was also validated by immunoblotting. Active immunization with the protein protected 75% mice against a lethal dose of S.Typhi Ty2. The data indicated a significant (up to 5?log) reduction in the bacterial load in the spleen and liver of immunized-infected mice compared to control (unimmunized-infected) mice which might have resulted in the modulation of histoarchitecture of spleen and liver and the levels of cytokines (IL-6, TNF-? and IL-10) production; thereby indicating the effectiveness of the subunit. The observations deduced from the study give the proof of concept of immunogenic potential of protein. However, further studies involving the immunoreactivity of CdtB with the statistically significant number of sera samples obtained from the human patients would be helpful in establishing the relevance of CdtB protein in humans and for making the strategies to develop it as an effective vaccine candidate.

Dataset Information

Crystallization of Escherichia coli CdtB, the biologically active subunit of cytolethal distending toxin.

Publications

Crystallization of Escherichia coli CdtB, the biologically active subunit of cytolethal distending toxin.

Similar Datasets

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