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. 2004 Oct;72(10):5972-82.
doi: 10.1128/IAI.72.10.5972-5982.2004.

IcmF and DotU are required for optimal effector translocation and trafficking of the Legionella pneumophila vacuole

Susan M VanRheenen  1 , Guillaume Duménil, Ralph R Isberg
Affiliations

IcmF and DotU are required for optimal effector translocation and trafficking of the Legionella pneumophila vacuole

Susan M VanRheenen et al. Infect Immun. 2004 Oct.

Abstract

The gram-negative bacterium Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease, characterized by bacterial replication within alveolar macrophages. Prior to intracellular replication, the vacuole harboring the bacterium must first escape trafficking to the host lysosome, a process that is dependent on the Dot/Icm type IV secretion system. To identify genes required for intracellular growth, bacterial mutants were isolated that were delayed in escape from the macrophage but which retain a minimally functional Dot/Icm machinery. The mutations were found in eight distinct genes, including three genes known to be required for optimal intracellular growth. Two of these genes, icmF and dotU, are located at one end of a cluster of genes that encode the type IV secretion system, yet both icmF and dotU lack orthologs in other type IV translocons. DotU protein is degraded in the early postexponential phase in wild-type L. pneumophila and at all growth phases in an icmF mutant. IcmF contains an extracytoplasmic domain(s) based on accessibility to a membrane-impermeant amine-reactive reagent. In the absence of either gene, L. pneumophila targets inappropriately to LAMP-1-positive compartments during macrophage infection, is defective in the formation of replicative vacuoles, and is impaired in the translocation of the effector protein SidC. Therefore, although IcmF and DotU do not appear to be part of the core type IV secretion system, these proteins are necessary for an efficiently functioning secretion apparatus.

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Figures

FIG. 1.
FIG. 1.
L. pneumophila strains bearing a deletion of either dotU or icmF are partially defective for growth in bone marrow-derived macrophages. Bone marrow-derived macrophages were infected at an MOI of 0.05 with each of the indicated strains for 2 h, after which the monolayer was washed to remove unbound bacteria. At each time point, the monolayer was lysed with 0.02% saponin, dilutions were plated onto CYET plates, and numbers of CFU were determined. The dot/icm+ strain Lp02 is the parent for both deletions as well as the dotA strain Lp03. All strains contained either a vector control (v; pJB908) or a complementing plasmid (picmF/pSV36 or pdotU/pSV38), as indicated. The experiment at each time point was completed in triplicate, and means and standard deviations are shown.
FIG. 2.
FIG. 2.
icmF and dotU mutants are defective for formation of replicative vacuoles. Bone marrow-derived macrophages were infected at an MOI of 1.0 for 1 h, after which they were washed and either fixed or incubated for an additional 13.5 h and then fixed. L. pneumophila were stained with antibody as described in the text (see Materials and Methods). The number of bacteria within each macrophage was assessed microscopically at each time point and assigned to one of the following four categories: 1 to 2 bacteria per macrophage, 3 to 6 bacteria per macrophage, 7 to 12 bacteria per macrophage, or more than 12 bacteria per macrophage. Approximately 100 infected macrophages were counted per coverslip (except for the dotA strain Lp03, for which 50 macrophages per coverslip were examined), and three coverslips were analyzed for each strain at each time point. Means and standard deviations are shown.
FIG. 3.
FIG. 3.
L. pneumophila strains lacking dotU or icmF are internalized into vacuoles that associate with the endocytic pathway. Bone marrow-derived macrophages were infected for 30 min at an MOI of 10 with each of the indicated strains, and then the cells were washed, fixed, and probed with antibodies against L. pneumophila and LAMP-1. The icmF1::miniTn10 (icmF1::Tn) strain bears a transposon mutation near the amino terminus of icmF. Each strain was analyzed in triplicate, and means and standard deviations are shown. v, vector.
FIG. 4.
FIG. 4.
Neither the icmF nor the dotU strain replicates in LAMP-1-positive compartments. Bone marrow-derived macrophages were infected for 75 min at an MOI of 1.0 with either the dot/icm+ strain Lp02 (filled bars), the dotA3 strain Lp03 (open bars), the ΔdotU strain (narrow-hatched bars), or the icmF1::miniTn10 (icmF1::Tn) strain (wide-hatched bars). Washed monolayers were then incubated for an additional 7 h prior to fixation and probing with anti-L. pneumophila and anti-LAMP-1 antibodies. Each strain contained the vector pJB908, and identical results were obtained with strains lacking this plasmid (data not shown). Three coverslips were analyzed per strain, and ∼50 vacuoles were scored per coverslip. For each strain, the percentage of phagosomes containing 1 to 2, 3 to 6, or 7 or more bacteria per phagosome (A) and the percentage of phagosomes staining positive for LAMP-1 in each category (B) were scored. Only singly infected macrophages were analyzed. Means and standard deviations are shown.
FIG. 5.
FIG. 5.
The presence of DotU in L. pneumophila lysates is dependent on IcmF and growth phase. (A) L. pneumophila strains were grown in AYET to either exponential phase (OD of 1.5 to 2.5; E) or postexponential phase (OD > 4.0; P), and equivalent amounts were resuspended in sample buffer and lysed by boiling. Proteins were resolved by SDS-PAGE, transferred to a membrane support, and probed with antibodies against DotU or IcmF. (B) L. pneumophila strains bearing the indicated plasmids (v, pJB908; picmF, pSV36) were grown to mid-exponential phase in AYE and processed as described for panel A. The migration of DotU and IcmF is shown on the left, and the migration of molecular mass markers is shown on the right for panel A. The IcmF antibody recognizes a second protein (marked with an asterisk) that is unrelated to IcmF.
FIG. 6.
FIG. 6.
IcmF and DotU each contain a putative transmembrane domain(s), and IcmF is accessible to a membrane-impermeant reagent in broth-grown L. pneumophila. Hydrophobicity plots of IcmF (A) and DotU (B) were generated with the TopPred program by using the Kyte-Doolittle scale and a window size of 29. The solid line indicates the hydrophobicity value, and the short-dashed and long-dashed lines represent the cutoff values for certain and putative transmembrane segments, respectively. (C) Wild-type L. pneumophila was grown to mid-logarithmic phase in AYE, and surface-exposed proteins were labeled with membrane-impermeant sulfo-NHS-biotin for 30 min. Labeled cells were then lysed by boiling, and biotinylated proteins were isolated on streptavidin-conjugated agarose beads. Equivalent amounts of the total extract (T) as well as supernatant (S) and bead (B) samples were resolved by SDS-PAGE and probed for the presence of IcmF, DotU, DotG, and ICDH with antibodies specific for each of these proteins. The migration of each protein is indicated on the left.
FIG. 7.
FIG. 7.
Translocation of effector protein SidC is reduced in strains with deletions of dotU or icmF. Bone marrow-derived macrophages were infected with the indicated strains for 1 h at an MOI of 2.0. Each strain expressed GFP from plasmid pAM239. After washing, monolayers were fixed and stained for external bacteria with an anti-L. pneumophila antibody prior to permeabilization and then stained with an anti-SidC antibody after permeabilization. (A to D) SidC staining of GFP-expressing L. pneumophila strains Lp02 (dot/icm+) (A), Lp03 (dotA3) (B), ΔdotU (C), and ΔicmF (D). GFP stains are shown in the left panels (GFP-L.p.), SidC stains are shown in the center panels (SidC), and merged images are shown in the right panels (merge). (E) Quantitation of SidC colocalization. For the experiment shown, three coverslips were examined per strain and approximately 100 vacuoles were characterized per coverslip. Means and standard deviations are indicated.
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