IDENTIFICATION OF LEGIONELLA-PNEUMOPHILA GENES REQUIRED FOR GROWTH WITHIN AND KILLING OF HUMAN MACROPHAGES

Legionella pneumophila was mutagenized with Tn903dII/acZ, and a collec tion of mutants was screened for defects in macrophage killing (Mak(-) ). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophage s. Forty-nine of the Mak(-) mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be c omplemented for macrophage killing by a DNA fragment containing icm an d dot, two recently described L. pneumophila loci that are required fo r macrophage killing. Phenotypic analysis showed that none of the muta nts were any more sensitive than the wild type to human serum, oxidant s, iron chelators, or lipophilic reagents nor did they require additio nal nutrients for growth. The only obvious difference between the Mak( -) mutants and wild-type L. pneumophila was that almost all of the Mak (-) mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes a ppeared when Tn903dII/acZ mutations from two Mak(-) strains were trans ferred to wild-type backgrounds. However, salt sensitivity is not a re quisite for killing macrophages since a group of Mak(-) mutants contai ning a plasmid that restored macrophage killing remained resistant to NaCl. Mak(-) mutants from groups I through IX associated with HL-60 ce lls similarly to wild-type L. pneumophila. However, like the intracell ular-multiplication-defective (icm) mutant 25D, the Mak(-) mutants wer e unable to multiply within macrophages. Thus, the ability of L. pneum ophila to kill macrophages seems to be determined by many genetic loci , almost all of which are associated with sensitivity to NaCl.