Listeria monocytogenes pathogenicity

Listeriolysin O (LLO) and ActA are essential virulence determinants for Listeria monocytogenes pathogenesis. Transcription of actA and hly , encoding LLO, is regulated by PrfA and increases dramatically during intracellular infection. The 5 ¢ untranslated regions (5 ¢ UTRs) of actA and prfA have been shown to upregulate expression of their respective gene products. Here, we demonstrate that the hly 5 ¢ UTR plays a critical role in regulating expression of LLO during intracellular infection. Deletion of the hly 5 ¢ UTR, while retaining the hly ribosome binding site, had a moderate effect on LLO production during growth in broth culture, yet resulted in a marked decrease in LLO levels during intracellular infection. The diminished level of LLO resulted in a significant defect in bacterial cell-to-cell spread during intracellular infection and a 10-fold reduction in virulence during in vivo infection of mice. Insertion of the hly 5 ¢ UTR sequence between a heterologous promoter and reporter gene sequences indicated that the hly 5 ¢ UTR functions independent of PrfA-mediated transcription and can enhance expression of cis -associated genes through a mechanism that appears to act at both a post-transcriptional and translational level. The ability of the hly 5 ¢ UTR to increase gene expression can be exploited to achieve PrfA-independent complementation of virulence genes and high-level expression of single copy heterologous genes in L. monocytogenes .