| Listeria monocytogenes is a Gram-positive, food-borne, facultative intracellular pathogen of animals and humans. Although initially confined to phagosomal compartment after phagocytosis or induced uptake, L. monocytogenes is adapted for growth and replication within the cytosol. Central to the virulence of L. monocytogenes is the cholesterol-dependent pore-forming cytolysin listeriolysin O (LLO), which facilitates the escape of the pathogen from the phagosome into the cytoplasm. However, the activity of this toxin must be compartmentalized to avoid lysing the host cell membrane. Although considerable work has focused on intragenic mechanisms of LLO regulation, little is known about the extragenic factors that regulate LLO.;Here we undertook a broad, genetic screen for factors that mediate the production or activity of LLO. We describe the design and construction of a new Himar1 mariner transposon system for use in L. monocytogenes. We observed that libraries generated by this new system are more complex than those generated by the previous standard transposon system, therefore increasing the resolution of forward genetic screens. Approximately 50,000 insertion mutants were then analyzed on blood agar plates for either a hypohemolytic or hyperhemolytic phenotype. Nearly 450 mutants were identified: transposon insertions in 63 unique features resulted in a hypohemolytic phenotype and insertions into 82 unique regions resulted in a hyperhemolytic phenotype.;The hypohemolytic mutants were analyzed further, and six were found to have a plaque defect, a hemolytic defect, and an in vivo defect: lmo0964 (similar to yjbH); lmo1268 (clpX); lmo1401 (similar to ymdB); lmo1575 (similar to ytq1 ); lmo1821 (similar to prpC); and lmo2219 (prsA2). One gene in particular, prsA2, had a profound influence on virulence, and mutants were essentially avirulent. The homolog in Bacillus subtilis is responsible for folding exported substrates, and consistent with a similar role for PrsA2 in L. monocytogenes, multiple virulence factors were affected upon its deletion. In addition to LLO, a broad range phospholipase C (PC-PLC) was less active in mutant strains. Our results suggest that PrsA2 may have evolved to alleviate the increase in export stress that occurs during an infection.;In summary, we updated the genetic tools available for use in L. monocytogenes, and have begun to explore the factors involved in regulating the mechanisms necessary for virulence. |
