Reacquisition of virulence of haemolysin-negative Listeria monocytogenes mutants by complementation with a plasmid carrying the hlyA gene.

An haemolysin negative mutant of Listeria monocytogenes LO28 produced by insertion of Tn917 resulted in an avirulent derivative. The hlyA gene of the same strain was previously cloned in Escherichia coli and retransfered by transformation to the hly- derivative, after subcloning in the shuttle vector pMK4. The transformant strain (L828) reacquired an haemolytic activity at a similar level than the wild strain. The virulence of this hly+ transformant was estimated by determining the LD50 in Swiss mice infected intravenously. With increasing doses of bacteria a hly- control strain (transformant with only pMK4) appeared to be totally avirulent; however, no significant difference in virulence was found between the hly+ transformants and the wild strain. Seriol viable counts in the liver and spleen of infected mice demonstrated an increase in number of L828 hly+ transformants at 48 h, but the hly- control transformants were rapidly eliminated. These results confirm that the production of haemolysin is a major factor in the pathogenic capabilities of L. monocytogenes.