Intracellular methicillin selection of Listeria monocytogenes mutants unable to replicate in a macrophage cell line

To dissect the determinants of Listeria monocytogenes that are required for pathogenicity, we designed an intracellular selection protocol based on penicillin selection to isolate mutants defective for intracellular growth. Eight independent mutants obtained by insertion of Tn916 were isolated that were resistant to methicillin treatment following internalization by the J774 macrophage-like cell line. Seven mutants were absolutely defective for intracellular growth, whereas one showed abortive intracellular growth. The majority of the mutants were nonhemolytic and lacked a secreted 58-kDa polypeptide thought to be the L. monocytogenes hemolysin, listeriolysin O. Southern blot analysis indicated that one mutant contained a Tn916 insertion in hlyA, the listeriolysin O structural gene, which resulted in a truncated listeriolysin O polypeptide, whereas another mutant contained an insertion immediately upstream of hlyA, which resulted in reduced expression of listeriolysin O. The other mutants contained Tn916 insertions in genes other than hlyA, although all but one were nonhemolytic. Revertants isolated by their ability to grow within tissue culture cells regained hemolytic activity. These data show that intracellular methicillin selection facilitates isolation of mutations in genes required for intracellular growth and strengthens the premise that listeriolysin O is essential for intracellular growth.