An Inducible Cre-lox System to Analyze the Role of LLO in Listeria monocytogenes Pathogenesis

Abstract

Listeriolysin O (LLO) is a pore-forming cytolysin that allows <i>Listeria monocytogenes</i> to escape from phagocytic vacuoles and enter the host cell cytosol. LLO is expressed continuously during infection, but it has been a challenge to evaluate the importance of LLO secreted in the host cell cytosol because deletion of the gene encoding LLO (<i>hly</i>) prevents localization of <i>L. monocytogenes</i> to the cytosol. Here, we describe a <i>L. monocytogenes</i> strain (<i>hly</i>^fl) in which <i>hly</i> is flanked by <i>loxP</i> sites and Cre recombinase is under the transcriptional control of the <i>L. monocytogenes</i><i>actA</i> promoter, which is highly induced in the host cell cytosol. In less than 2 h after infection of bone marrow-derived macrophages (BMMs), bacteria were 100% non-hemolytic. <i>hly</i>^fl grew intracellularly to levels 10-fold greater than wildtype <i>L. monocytogenes</i> and was less cytotoxic. In an intravenous mouse model<i>,</i> 90% of bacteria were non-hemolytic within three hours in the spleen and eight hours in the liver. The loss of LLO led to a 2-log virulence defect in the spleen and a 4-log virulence defect in the liver compared to WT <i>L. monocytogenes</i>. Thus, the production of LLO in the cytosol has significant impact on the pathogenicity of <i>L. monocytogenes</i>.