Icm Genes Research Articles

Additive Effect on Intracellular Growth byLegionella pneumophilaIcm/Dot Proteins Containing a Lipobox Motif

Legionella pneumophila, the causative agent of Legionnaires' disease, utilizes a type IVB secretion system to subvert its host cells and grow intracellularly. This type IV secretion system is composed of 25 icm (or dot) genes that probably constitute parts of a secretion complex as well as more than 30 proteins that are translocated via this system into the host cells. Three of the Icm/Dot proteins (DotD, DotC, and IcmN) contain a lipobox motif at their N terminals and are predicted to be lipoproteins. Two of these lipoproteins (DotD and DotC) were found to be essential for intracellular growth in both HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii, while the third lipoprotein (IcmN) was found to be partially required for intracellular growth only in A. castellanii. Mutation analysis of the lipobox cysteine residue, which was shown previously to be indispensable for the lipobox function, indicated that both DotC and DotD are partially functional without this conserved residue. Cysteine mutations in both DotC and DotD or in DotC together with an icmN deletion or in DotD together with an icmN deletion were found to be additive, indicating that each of these lipoproteins performs its function independently from the others. Analysis of the transcriptional regulation of both the dotDC operon and the icmN gene revealed that both had higher levels of expression at stationary phase which were partially dependent on the LetA regulator. Our results indicate that the lipoproteins of the L. pneumophila icm (or dot) system are essential components of the secretion system and that they perform their functions independently.

Functional similarities between the icm/dot pathogenesis systems of Coxiella burnetii and Legionella pneumophila.

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular pathogen, whereas Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen. During infection of humans both of these pathogens multiply in alveolar macrophages inside a closed phagosome. L. pneumophila intracellular multiplication was shown to be dependent on the icm/dot system, which probably encodes a type IV-related translocation apparatus. Recently, genes homologous to all of the L. pneumophila icm/dot genes (besides icmR) were found in C. burnetii. To explore the similarities and differences between the icm/dot pathogenesis systems of these two pathogens, interspecies complementation analysis was performed. Nine C. burnetii icm homologous genes (icmT, icmS, icmQ, icmP, icmO, icmJ, icmB, icmW, and icmX) were cloned under regulation of the corresponding L. pneumophila icm genes and examined for the ability to complement L. pneumophila mutants with mutations in these genes. The C. burnetii icmS and icmW homologous genes were found to complement the corresponding L. pneumophila icm mutants to wild-type levels of intracellular growth in both HL-60-derived human macrophages and Acanthamoeba castellanii. In addition, the C. burnetii icmT homologous gene was found to completely complement an L. pneumophila insertion mutant for intracellular growth in HL-60-derived human macrophages, but it only partially complemented the same mutant for intracellular growth in A. castellanii. Moreover, as previously shown for L. pneumophila, the proteins encoded by the C. burnetii icmS and icmW homologous genes were found to interact with one another, and interspecies protein interaction was observed as well. Our results strongly indicate that the Icm/Dot pathogenesis systems of C. burnetii and L. pneumophila have common features.

The Legionella pneumophila GacA homolog (LetA) is involved in the regulation of icm virulence genes and is required for intracellular multiplication in Acanthamoeba castellanii

Legionella pneumophila, the causative agent of legionnaires' disease, is a broad-host-range facultative intracellular pathogen. Thus far, 24 genes ( icm/ dot genes) required for L. pneumophila intracellular growth, have been discovered. In this study, a deletion substitution was constructed in the L. pneumophila homolog of the gacA response regulator ( letA) and its involvement in L. pneumophila pathogenicity and icm/ dot gene expression was characterized. The letA mutant constructed had no intracellular growth defect when analyzed in HL-60 derived human macrophages, but it was found to be severely attenuated for intracellular growth in the protozoan host Acanthamoeba castellanii. The growth defect in amoebae was fully complemented by introducing the L. pneumophila letA gene on a plasmid. In addition, the LetA regulator was found to be involved in the expression of three icm genes ( icmT, icmP and icmR). The level of expression of the icmT::lacZ and icmR::lacZ fusions was found to be higher, while the level of expression of the icmP::lacZ fusion was found to be lower when analyzed in the letA mutant strain, in comparison to the wild-type strain. We concluded that LetA has a moderate effect on icm/ dot gene expression, but it probably plays a major role in the expression of L. pneumophila genes required for intracellular growth in protozoan hosts. A similar host specific phenotype was previously described for the RpoS sigma factor and the type II general secretion system of L. pneumophila.

Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth.

Legionella 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 two-hybrid 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.

Intracellular multiplication and human macrophage killing by Legionella pneumophila are inhibited by conjugal components of IncQ plasmid RSF1010.

Previously we have reported that Legionella pneumophila can mediate plasmid DNA transfer at a frequency of about 10(-3) transconjugants per donor and that this process is dependent on several icm genes. Here we characterize the icm-dependent conjugal ability of L. pneumophila and study its relationship to intracellular multiplication and host cell killing. We found that three icm genes and the RSF1010 mobA gene are completely required and that three icm genes and the RSF1010 mobC gene are partially required for conjugation. Conjugation occurred during lag phase and stopped when the cell number increased. Inhibition of transcription or translation in the donor had only a minor effect on conjugation frequency. These results suggest that stationary-phase bacteria contain a functional icm complex that can mediate conjugal DNA transfer and probably can initiate infection of human macrophages as well. We also found that a functional RSF1010 mobilization system inhibits intracellular multiplication and killing of human macrophages by L. pneumophila. The strongest inhibition was observed in icm insertion mutants complemented with wild-type icm genes on an RSF1010-derived plasmid. These results suggest that the conjugation substrate probably competes with the natural substrate of the L. pneumophila icm system for transfer outside the bacterial cell. We propose that the function of the L. pneumophila icm system is to transfer effector molecules to the host cell. These effector molecules may interact with components of the host cell that are involved in phagosome formation and fate.

Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila.

During infection, the Legionnaires' disease bacterium, Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism's ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes or dotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophila colocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icm and dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila-containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication.

Characterization of a new region required for macrophage killing by Legionella pneumophila

In a previous study, a collection of 55 Legionella pneumophila mutants defective for macrophage killing was isolated by transposon mutagenesis. In this study, nine of these mutants that belong to the same DNA hybridization group (group 3) were characterized. A wild-type DNA fragment that covers this DNA hybridization group was cloned and sequenced. This region was found to contain six new genes (designated icmT, icmS, icmR, icmQ, icmP, and icmO), five of which contain at least one transposon insertion. No transposon insertion was found in icmS. However, this gene was found to be required for macrophage killing, since a kanamycin resistance cassette introduced into icmS by gene replacement resulted in a mutant that was attenuated for macrophage killing. A plasmid containing the DNA fragment that covers this region complements all the mutants for macrophage killing, although various levels of complementation were observed for mutants in different genes. Complementation tests were also performed with plasmids containing one or two of these genes, as well as with plasmids containing nonpolar in-frame deletions. The results from these complementation tests indicated that all six genes located in this region are needed for macrophage killing and that they are probably arranged as two transcriptional units (icmTS and icmPO) and two genes (icmR and icmQ). A region upstream of the coding sequence of several icm genes may contain a potential promoter and/or regulatory site. Homology searches show that icmP and icmO bear significant homology to the trbA and trbC genes from the Salmonella R64 plasmid, respectively. The sequences of the other four genes do not show significant homology with any entries in sequence databases.