SummaryLegionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV-related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein± protein interactions. Specific interactions between different Icm proteins were detected by yeast twohybrid and gel overlay analysis. These data support a model in which the IcmQ±IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS±IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.

The gram-negative respiratory pathogen Legionella pneumophila infects and grows within mammalian macrophages and protozoan host cells. Upon uptake into macrophages, L. pneumophila establishes a replicative organelle that avoids fusion with endocytic vesicles. There are 24 dot/icm genes on the L. pneumophila chromosome required for biogenesis of this vacuole. Many of the Dot/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems. We have been investigating the function of L. pneumophila dot/icm gene products that do not have obvious orthologs in other type IV transfer systems, since these determinants could govern processes unique to phagosome biogenesis. The icmX gene product falls into this category. To understand the role of the IcmX protein in pathogenesis, we have detailed interactions between an L. pneumophila icmX deletion mutant and murine bone marrow-derived macrophages. These data demonstrate that icmX is required for biogenesis of the L. pneumophila replicative organelle. Immunoblot analysis indicates that the icmX gene product is a polypeptide with an estimated molecular mass of 50 kDa. The IcmX protein was localized to the bacterial periplasm, and periplasmic translocation was mediated by an N-terminal sec-dependent leader peptide. A truncated IcmX product was secreted into culture supernatants by wild-type L. pneumophila growing extracellularly in liquid media; however, transport of the IcmX protein into eukaryotic host cells was not detected. Proteins similar in molecular weight to IcmX were identified in other Legionella species by immunoblot analysis using a monoclonal antibody specific for L. pneumophila IcmX protein. From these data, we conclude that the IcmX protein is an essential component of the dot/icm secretion apparatus, and that a conserved mechanism of host cell parasitism exists for members of the Legionellaceae family.

Summary During infection, Legionella pneumophila creates a replication vacuole within eukaryotic cells and this process requires a Type IVb secretion system (T4bSS). IcmQ is a critical component of this translocase and associates with IcmR. In this report, we show that the N-terminal domain of IcmQ (Qn) mediates IcmQ dimerization, while the C-terminal domain with a linker region promotes a stable membrane association of IcmQ. We then determined crystal structures of Qn with the interacting domain of IcmR. In this complex, each protein forms an α-helical hairpin within a parallel 4-helix bundle. We find that IcmR binding to IcmQ prevents dimerization of IcmQ and blocks membrane permeabilization. However, IcmR does not completely block membrane association of IcmQ. The amphipathic nature of Qn suggests two models for how IcmQ may permeabilize membranes. The Rm-Qn structure also suggests how hyper-variable IcmR-like proteins in other Legionellae may interact with their IcmQ partners to regulate IcmQ function.