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Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes.

ABSTRACT: We have isolated, by molecular cloning and genetic complementation of a listeriolysin-negative mutant, a gene required for the expression of this virulence factor in Listeria monocytogenes. The mutant strain SLCC53, which was nonhemolytic and avirulent, harbored a deletion of 450 base pairs located approximately 1500 base pairs upstream of the listeriolysin gene. No transcripts corresponding to the listeriolysin gene were detected in the mutant. DNA sequencing of this region from the hemolytic strain EGD revealed that the region deleted in the mutant would abrogate expression of a 27-kDa polypeptide. Introduction of a recombinant plasmid expressing this 27-kDa polypeptide restored hemolytic activity to the mutant and increased the hemolytic activity of the wild-type L. monocytogenes strain EGD. We have designated the gene encoding the 27-kDa polypeptide prfA, for positive regulatory factor of listeriolysin (lisA) expression. The prfA gene regulates transcription of the lisA gene positively.

SUBMITTER: Leimeister-Wachter M

PROVIDER: S-EPMC54950 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes.

Leimeister-Wächter M M Haffner C C Domann E E Goebel W W Chakraborty T T

Proceedings of the National Academy of Sciences of the United States of America 19901101 21

We have isolated, by molecular cloning and genetic complementation of a listeriolysin-negative mutant, a gene required for the expression of this virulence factor in Listeria monocytogenes. The mutant strain SLCC53, which was nonhemolytic and avirulent, harbored a deletion of 450 base pairs located approximately 1500 base pairs upstream of the listeriolysin gene. No transcripts corresponding to the listeriolysin gene were detected in the mutant. DNA sequencing of this region from the hemolytic s ...[more]

PMID: 2122460

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Project description:While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.

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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Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes.

Publications

Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes.

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