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

Abstract

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates intracellularly within a specialized phagosome of mammalian and protozoan host cells, and the Icm/Dot type IV secretion system has been shown to be essential for this process. Unlike all the other known Icm/Dot proteins, the IcmF protein, which was described before, and the IcmH protein, which is characterized here, have homologous proteins in many bacteria (such as Yersinia pestis, Salmonella enterica, Rhizobium leguminosarum, and Vibrio cholerae), all of which associate with eukaryotic cells. Here, we have characterized the L. pneumophila icmH and icmF genes and found that both genes are present in 16 different Legionella species examined. The icmH and icmF genes were found to be absolutely required for intracellular multiplication in Acanthamoeba castellanii and partially required for intracellular growth in HL-60-derived human macrophages, for immediate cytotoxicity, and for salt sensitivity. Mutagenesis of the predicted ATP/GTP binding site of IcmF revealed that the site is partially required for intracellular growth in A. castellanii. Analysis of the regulatory region of the icmH and icmF genes, which were found to be cotranscribed, revealed that it contains at least two regulatory elements. In addition, an icmH::lacZ fusion was shown to be activated during stationary phase in a LetA-and RelA-dependent manner. Our results indicate that although the icmH and icmF genes probably have a different evolutionary origin than the rest of the icm/dot genes, they are part of the icm/dot system and are required for L. pneumophila pathogenesis.Bacterial pathogens, as well as bacteria that live in close contact with eukaryotic cells, have developed many mechanisms to subvert their host cells and grow in intimate association with them. Many bacterial pathogens, such as Yersinia spp., Salmonella enterica, Pseudomonas aeroginosa, and Escherichia coli O157, use the type III secretion system as part of their pathogenesis determinants (14). Other bacteria such as Agrobacterium tumefaciens, Bordetella pertussis, and Legionella pneumophila use type IV secretion systems, which are functionally homologous to type III secretion systems but are evolutionarily related to bacterial conjugation systems, as opposed to the type III secretion systems, which are evolutionarily related to the bacterial flagellar basal body (9, 13).L. pneumophila, the causative agent of Legionnaires' disease, is a facultatively intracellular pathogen that is able to infect, multiply within, and kill human macrophages, as well as free-living amoebae (32, 48). Two regions of icm/dot genes that constitute the L. pneumophila icm/dot type IV secretion system have been discovered (reviewed in references 53 and 64). Region I contains 7 genes (icmV, -W, and -X and dotA, -B, -C, and -D) (3,6,39,63), and region II has been shown to contain 17 genes (icmT, -S, -R, -Q, -P, -O, -N, -M, -L, -K, -E, -G, -C, -D, -J, -B, and -F) (1,46,50,52,63). The icm/dot genes were shown to participate in m...

The DotL Protein, a Member of the TraG-Coupling Protein Family, Is Essential for Viability ofLegionella pneumophilaStrain Lp02

“…However, inactivation of three other dot/icm genes-icmF, dotU, and icmR-did suppress loss of dotL (Fig. 6), even though loss of these genes caused only a partial inhibition of growth in permissive hosts (12,53,54,62,68). These results indicate that, although inactivation of the majority of the dot/icm genes can suppress the lethality caused by loss of dotL, there is specificity to the suppression.…”

AttenuatedFrancisella novicidaTransposon Mutants Protect Mice against Wild-Type Challenge

“…6, in trans expression of the cloned dsbB gene provided nearly complete complementation of the virulence defect in three different cell types. Further analysis showed that the LD 50 was 60.25 CFU, which is comparable to the wild-type LD 50 (66.25 CFU).…”

“…Based on its in vitro growth defect, comparatively low LD 50 , relative lack of intracellular attenuation, and questionable dissemination patterns, we felt that the carB mutant was not a strong enough candidate to include in further development of a vaccine against tularemia, and therefore we did not attempt to complement the carB gene.…”

The bacterial type VI secretion machine: yet another player for protein transport across membranes

“…dotU and icmF mutants have similar intracellular growth phenotypes, i.e. partially defective in replication within macrophages (Sexton et al, 2004;Zusman et al, 2004), which suggested that the DotU and IcmF proteins worked together. Furthermore, the lack of IcmF resulted in a reduced level Fig.…”