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

Listeria monocytogenes is a facultative intracellular gram-positive bacteri um capable of growing in the cytoplasm of infected host cells. Bacterial es cape from the phagosomal vacuole of infected cells is mainly mediated by th e pore-forming hemolysin listeriolysin O (LLO) encoded by hly. LLO-negative mutants of L. monocytogenes are avirulent in the mouse model. We have deve loped a genetic system with hly as a reporter gene allowing the identificat ion of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting L. monocytogenes chrom osomal fragments upstream of the promoterless hly gene cloned into gram-pos itive and gram-negative shuttle vectors and expressed in an LLO-negative mu tant strain. With this hly-based promoter trap system, combined with access to the L. monocytogenes genome database, rye identified 20 in vitro-transc ribed 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 exp ression technology tool, nine in vice-induced loci of L. monocytogenes were identified, including genes encoding (i) the previously known in vivo-indu cible phosphatidylinositol phospholipase C and (ii) a putative N-acetylgluc osamine epimerase, possibly involved in teichoic acid biosynthesis. The use of hly as a reporter is a simple and powerful alternative to classical met hods for transcriptional analysis to monitor promoter activity in L. monocy togenes.