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Listeriolysin O as a reporter to identify constitutive and in vivo-inducible promoters in the pathogen Listeria monocytogenes.

ABSTRACT: Listeria monocytogenes is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by hly. LLO-negative mutants of L. monocytogenes are avirulent in the mouse model. We have developed a genetic system with hly as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting L. monocytogenes chromosomal fragments upstream of the promoterless hly gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this hly-based promoter trap system, combined with access to the L. monocytogenes genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the hly-based system as an in vivo expression technology tool, nine in vivo-induced loci of L. monocytogenes were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative N-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of hly as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in L. monocytogenes.

SUBMITTER: Dubail I

PROVIDER: S-EPMC97571 | biostudies-other | 2000 Jun

REPOSITORIES: biostudies-other

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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.

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Listeriolysin O as a reporter to identify constitutive and in vivo-inducible promoters in the pathogen Listeria monocytogenes.

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