Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes

The role of hemolysin in the virulence of Listeria monocytogenes was studied by using transposon mutagenesis. The 26-kilobase conjugative transposon Tn1545, originally found in Streptococcus pneumoniae, was transferred to a hemolytic virulent strain of L. monocytogenes. The frequency of transfer was estimated to be about 10(-8) per recipient. This allowed us to isolate a nonhemolytic mutant which most likely harbors a single copy of Tn1545. Loss of hemolysin production was associated with loss of virulence. The 50% lethal dose of the mutant was assessed to about 10(9.6) bacteria per mouse after intravenous challenge. Nonhemolytic bacteria were unable to grow in host tissues and were rapidly eliminated from the spleen and liver of infected mice. Virulence was restored in hemolysin-producing revertant obtained by spontaneous loss of transposon Tn1545. These results strongly suggest that hemolysin is a major virulence factor implicated in the intracellular growth of L. monocytogenes.