Identification and subcellular localization of the Legionella pneumophila IcmX protein: a factor essential for establishment of a replicative organelle in eukaryotic host cells

The gram-negative respiratory pathogen Legionella pneumophila infects and g rows within mammalian macrophages and protozoan host cells. Upon uptake int o macrophages, L. pneumophila establishes a replicative organelle that avoi ds fusion with endocytic vesicles. There are 24 dot/icm genes on the L. pne umophila chromosome required for biogenesis of this vacuole. Many of the Do t/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems. We have been inves tigating the function of L. pneumophila dot/icm gene products that do not h ave obvious orthologs in other type TV transfer systems, since these determ inants could govern processes unique to phagosome biogenesis. The icmX gene product falls into this category. To understand the role of the IcmX prote in in pathogenesis, we have detailed interactions between an L. pneumophila icmX deletion mutant and murine bone marrow-derived macrophages. These dat a demonstrate that icmX is required for biogenesis of the L, pneumophila re plicative organelle, Immunoblot analysis indicates that the icmX gene produ ct is a polypeptide with an estimated molecular mass of 50 kDa, The IcmX pr otein was localized to the bacterial periplasm, and periplasmic translocati on was mediated by an N-terminal sec-dependent leader peptide. A truncated IcmX product was secreted into culture supernatants by wild-type L, pneumop hila growing extracellularly in liquid media; however, transport of the Icm X protein into eukaryotic host cells was not detected. Proteins similar in molecular weight to IcmX were identified in other Legionella species by imm unoblot analysis using a monoclonal antibody specific for L, pneumophila Ic mX protein. From these data, we conclude that the IcmX protein is an essent ial component of the dot/icm secretion apparatus, and that a conserved mech anism of host cell parasitism exists for members of the Legionellaceae fami ly.