Identification and Subcellular Localization of the Legionella pneumophila IcmX Protein: a Factor Essential for Establishment of a Replicative Organelle in Eukaryotic Host Cells

Abstract

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.

Characterization of theicmHandicmFGenes Required forLegionella pneumophilaIntracellular Growth, Genes That Are Present in Many Bacteria Associated with Eukaryotic Cells

“…As described above, the gene regions containing the icmF and icmH genes were found in one of several species that belong to the same genus or, in the case of S. enterica, in only one subspecies. To examine whether the icmF and icmH genes are also present in other Legionella species besides L. pneumophila (as was shown before for several icm/dot genes [icmD, icmE, icmG, icmX, dotA, and dotB] in Legionella micdadei and for icmX in several Legionella species [34,40]), we used lowstringency Southern hybridization with the genes. The hybridizations performed with the icmF (Fig.…”

Comparative assessment of virulence traits in Legionella spp.

“…Previous studies have shown the presence of dotA and icmX genes in L. micdadei Pittsburg, L. micdadei Tatlock, L. bozemanii, L. gratiana (Matthews & Roy, 2000), and of dotA, dotB, dotE and dotFG in L. micdadei 31B, Rivera, Camileri and D-2676 (Joshi & Swanson, 1999). The IcmX protein has been demonstrated in L. micdadei and L. gratiana (Matthews & Roy, 2000). In our studies, all strains of legionellae investigated harboured all the dot/icm loci.…”

A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells

“…All Legionella were derivatives of L. pneumophila strain Lp01 (14). Strains defective in dot͞icm genes and the ralF gene were described (20,(23)(24)(25)(26). Details of plasmid construction are given in Supporting Text and Table 3, which are published as supporting information on the PNAS web site.…”