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Crystallization and X-ray crystallographic analysis of the cholesterol-dependent cytolysin listeriolysin O from Listeria monocytogenes.

ABSTRACT: The secreted pore-forming toxin listeriolysin O (LLO) from the intracellular pathogen Listeria monocytogenes is a member of the family of cholesterol-dependent cytolysins (CDC) with broad properties in pathogenesis. Its role as a virulence factor is enigmatic: it disrupts membranes and acts as an inductor of both pro- and anti-inflammatory responses in infected cells. In addition, LLO is also a potent target for immunogenicity during infection. Natively secreted LLO from a recombinant L. innocua strain was crystallized in its water-soluble monomeric form. The crystals obtained belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 26.7, b = 85.1, c = 230.0?Å, and diffracted to beyond 2.2?Å resolution. The Matthews coefficient and the solvent content were estimated to be 2.4?Å(3)?Da(-1) and 49.2%, respectively. The structure with one molecule in the asymmetric unit was solved using Phaser employing the structure of the previously characterized CDC toxin perfringolysin O as a search model.

SUBMITTER: Koster S

PROVIDER: S-EPMC3818035 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Crystallization and X-ray crystallographic analysis of the cholesterol-dependent cytolysin listeriolysin O from Listeria monocytogenes.

Köster Stefan S Hudel Martina M Chakraborty Trinad T Yildiz Özkan Ö

Acta crystallographica. Section F, Structural biology and crystallization communications 20131017 Pt 11

The secreted pore-forming toxin listeriolysin O (LLO) from the intracellular pathogen Listeria monocytogenes is a member of the family of cholesterol-dependent cytolysins (CDC) with broad properties in pathogenesis. Its role as a virulence factor is enigmatic: it disrupts membranes and acts as an inductor of both pro- and anti-inflammatory responses in infected cells. In addition, LLO is also a potent target for immunogenicity during infection. Natively secreted LLO from a recombinant L. innocua ...[more]

PMID: 24192351

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Project description:Listeria monocytogenes is a facultative intracellular pathogen responsible for the life-threatening disease listeriosis. The pore-forming toxin listeriolysin O (LLO) is a critical virulence factor that plays a major role in the L. monocytogenes intracellular lifecycle and is indispensable for pathogenesis. LLO is also a dominant antigen for T cells involved in sterilizing immunity and it was proposed that LLO acts as a T cell adjuvant. In this work, we generated a novel full-length LLO toxoid (LLOT) in which the cholesterol-recognition motif, a threonine-leucine pair located at the tip of the LLO C-terminal domain, was substituted with two glycine residues. We showed that LLOT lost its ability to bind cholesterol and to form pores. Importantly, LLOT retained binding to the surface of epithelial cells and macrophages, suggesting that it could efficiently be captured by antigen-presenting cells. We then determined if LLOT can be used as an antigen and adjuvant to protect mice from L. monocytogenes infection. Mice were immunized with LLOT alone or together with cholera toxin or Alum as adjuvants. We found that mice immunized with LLOT alone or in combination with the Th2-inducing adjuvant Alum were not protected against L. monocytogenes. On the other hand, mice immunized with LLOT along with the experimental adjuvant cholera toxin, were protected against L. monocytogenes, as evidenced by a significant decrease in bacterial burden in the liver and spleen three days post-infection. This immunization regimen elicited mixed Th1, Th2, and Th17 responses, as well as the generation of LLO-neutralizing antibodies. Further, we identified T cells as being required for immunization-induced reductions in bacterial burden, whereas B cells were dispensable in our model of non-pregnant young mice. Overall, this work establishes that LLOT is a promising vaccine antigen for the induction of protective immunity against L. monocytogenes by subunit vaccines containing Th1-driving adjuvants.

Dataset Information

Crystallization and X-ray crystallographic analysis of the cholesterol-dependent cytolysin listeriolysin O from Listeria monocytogenes.

Publications

Crystallization and X-ray crystallographic analysis of the cholesterol-dependent cytolysin listeriolysin O from Listeria monocytogenes.

Similar Datasets

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