EFFECTS OF AN ISOGENIC ZN-METALLOPROTEASE-DEFICIENT MUTANT OF LEGIONELLA-PNEUMOPHILA IN A GUINEA-PIG PNEUMONIA MODEL

To determine the effects, if any, of the Zn-metalloprotease on virulen ce of Legionella pneumophila infection, an isogenic mutant deficient i n protease (encoded by the proA gene) was tested in an Acanthamoeba ce ll model, in guinea-pig macrophages, and in a guinea-pig pneumonia mod el. The cloned proA gene was completely inactivated by insertion of a kanamycin-resistance cassette into the protease gene of L. pneumophila AA100. This mutated gene was then introduced into the L. pneumophila chromosome by allelic exchange to form the isogenic ProA(-) mutant AA2 00. AA200 showed no difference in its ability to enter, survive, or gr ow in Acanthamoeba and explanted guinea-pig macrophages; neither light nor;electron microscopy revealed morphological differences in the euk aryotic cells infected with the protease mutant or the wild-type strai ns. The proA gene was found to be expressed in L. pneumophila during i ntracellular growth in amoebae by measuring the light produced from a truncated luxC gene fusion with the proA promoter. Virulence of the pr otease mutant was attenuated when tested in a guinea-pig model of infe ction employing the intratracheal inoculation method. AA200 was slower to cause death, grew to lower numbers in the lungs, resulted in less necrotic debris and a larger macrophage infiltrate, and was more likel y to be found in association with macrophage vacuoles than the parent strain. Although deletion of the protease was not sufficient to comple tely abolish virulence in a guinea-pig model, the mutation caused a de lay in the lethal effects of L. pneumophila and attenuated the infecti on.