IDENTIFICATION OF MACROPHAGE-SPECIFIC INFECTIVITY LOCI (MIL) OF LEGIONELLA-PNEUMOPHILA THAT ARE NOT REQUIRED FOR INFECTIVITY OF PROTOZOA

We have recently shown that many mutants of Legionella pneumophila exh ibit similar defective phenotypes within both U937 human-derived macro phages and the protozoan host Acanthamoeba (L.-Y. Gao, O. S. Harb, and Y. Abu Kwaik, Infect. Immun. 65:4738-4746, 1997). These observations have suggested that many of the mechanisms utilized by L. pneumophila to parasitize mammalian and protozoan cells are similar, but our data have not excluded the possibility that there are unique mechanisms uti lized by L. pneumophila to survive and replicate within macrophages bu t not protozoa. To examine this possibility, we screened a bank of 5,2 80 miniTn10::kan transposon insertion mutants of L. pneumophila for po tential mutants that exhibited defective phenotypes of cytopathogenici ty and intracellular replication within macrophage-like U937 cells but not within Acanthamoeba polyphaga. We identified 32 mutants with vari ous degrees of defects in cytopathogenicity, intracellular survival, a nd replication within human macrophages, and most of the mutants exhib ited wild-type phenotypes within protozoa. Six of the mutants exhibite d mild defects in protozoa. The defective loci were designated mil (fo r macrophage-specific infectivity loci). Based on their intracellular growth defects within macrophages, the mil mutants were grouped into f ive phenotypic groups. Groups I to III included the mutants that were severely defective in macrophages, while members of the other two grou ps exhibited a modestly defective phenotype within macrophages. The gr owth kinetics of many mutants belonging to groups I to III were also e xamined, and these were shown to have a similar defective phenotype in peripheral blood monocytes and a wild-type phenotype within another p rotozoan host, Hartmannella vermiformis. Transmission electron microsc opy of A. polyphaga infected by three of the mil mutants belonging to groups I and II showed that they were similar to the parent strain in their capacity to recruit the rough endoplasmic reticulum (RER) around the phagosome. In contrast, infection of macrophages showed that the three mutants failed to recruit the RER around the phagosome during ea rly stages of the infection. None of the mil mutants was resistant to NaCl, and the dot or icm NaClr mutants are severely defective within m ammalian and protozoan cells. Our data indicated that in addition to d ifferences in mechanisms of uptake of L. pneumophila by macrophages an d protozoa, there were also genetic loci required for L. pneumophila t o parasitize mammalian but not protozoan cells. We hypothesize that L. pneumophila has evolved as a protozoan parasite in the environment bu t has acquired loci specific for intracellular replication within macr ophages. Alternatively, ecological coevolution with protozoa has allow ed L. pneumophila to possess multiple redundant mechanisms to parasiti ze protozoa and that some of these mechanisms do not function within m acrophages.