Abstract
Listeriolysin O (LLO) is a pore-forming cytolysin that allows Listeria monocytogenes 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 (hly) prevents localization of L. monocytogenes to the cytosol. Here, we describe a L. monocytogenes strain (hlyfl) in which hly is flanked by loxP sites and Cre recombinase is under the transcriptional control of the L. monocytogenes actA 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. hlyfl grew intracellularly to levels 10-fold greater than wildtype L. monocytogenes and was less cytotoxic. In an intravenous mouse model, 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 L. monocytogenes. Thus, the production of LLO in the cytosol has significant impact on the pathogenicity of L. monocytogenes.
Highlights
The field of microbial pathogenesis and the study of virulence factors has been guided for decades by Molecular Koch’s Postulates, which stipulate that inactivation of a gene encoding a suspected virulence factor should lead to measurable loss of virulence, and replacement of the gene should restore pathogenicity [1]
LoxP sites were inserted into the L. monocytogenes chromosome to flank hly and an adjacent gene, tetL, which provides tetracycline resistance
L. monocytogenes at an at of 4 for Lactate dehydrogenase (LDH)was release was measured and values normalized accordingtotolysis lysis with dehydrogenase (LDH) release measured and values werewere normalized according with 1% Triton-X
Summary
The field of microbial pathogenesis and the study of virulence factors has been guided for decades by Molecular Koch’s Postulates, which stipulate that inactivation of a gene encoding a suspected virulence factor should lead to measurable loss of virulence, and replacement of the gene should restore pathogenicity [1]. Targeted gene deletions are invaluable in determining the function of genes and pathways, there remain circumstances in which it is not possible to generate viable deletion mutants, or deletion of a gene encoding multiple functions precludes analysis of later functions. The latter is the case for the gene encoding Listeriolysin O (hly) of Listeria monocytogenes. L. monocytogenes is a Gram-positive facultative intracellular pathogen that replicates in the cytosol of host cells. In order to reach the host cell cytosol, L. monocytogenes must first escape from the phagocytic entry vacuole, which requires the secreted pore-forming cytolysin Listeriolysin O (LLO) [2,3]. L. monocytogenes replicates and produces an actin nucleation factor (ActA)
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