The Legionella pneumophila effector SidJ is required for efficient recruitment of endoplasmic reticulum proteins to the bacterial phagosome

Abstract

The virulence of Legionella pneumophila is dependent on the Dot/Icm type IV protein secretion system, which translocates effectors into infected cells. A large number of such translocated proteins have been identified, but few of these proteins are necessary for intracellular replication of the pathogen, making it difficult to correlate these genes with specific cell-biological events associated with L. pneumophila infection. We report here the identification and characterization of a family of two substrates, SidJ and SdjA, with distinctive phenotypes. In contrast to many Dot/Icm substrates, whose expression levels are elevated when bacteria are grown to postexponential phase, SidJ is produced at a constant rate during the entire bacterial growth cycle. Mutation in sidJ causes a significant growth defect in both macrophage and amoeba hosts, but an sdjA mutant is detectably defective only in protozoan hosts. However, in the amoeba host a mutant lacking both sidJ and sdjA does not display a more severe growth defect than the sidJ mutant. Despite its significant intracellular growth defect, the sidJ mutant is still able to effectively evade fusion with lysosomes. Importantly, recruitment of endoplasmic reticulum (ER) proteins by vacuoles containing the sidJ mutant was considerably delayed in both mammalian and amoeba cells. Our results suggest that SidJ modulates host cellular pathways, contributing to the trafficking or retention of ER-derived vesicles to L. pneumophila vacuoles.

FIG. 1.

sidJ and sdjA are closely linked to members of the sidE protein family. sidJ is situated between sdeB and sdeC and is separated from sdeC by an open reading frame (orf2) predicted to code for a protein of 301 amino acids (A), and sdjA is directly downstream of sdeD (B). A plasmid expressing SidJ almost completely complements the intracellular growth defect caused by the deletion of a chromosomal region spanning sdeC to sdeA (C). After challenge of murine macrophages with appropriate L. pneumophila strains, growth of bacteria was monitored by plating cell lysates at the indicated time points on bacteriological media. Similar results were obtained from more than three independent experiments performed in triplicate, and data shown were from one representative experiment. L. pneumophila strains used: Lp02, wild type (dot/icm⁺); Lp03, Lp02 (dotA); ZL015, Lp02 (ΔsdeC-ΔsdeA); ZL015C, ZL015 (pSidJ).

FIG. 2.

SidJ is translocated into host cells by the Dot/Icm system. Macrophages were infected at an MOI of 1 for 1 h with ΔsidC strains harboring plasmids expressing wild-type sidC (pSidC/pZL199) (A) or sidC lacking the region coding for the last 100 residues (psidCΔC100/pZL204) (B); fusions of the last 500 amino acids of SidJ to SidCΔC100 (psidCΔ100::sidJ500/pZL234) (C); or full-length sdjA (psidCΔC100::sdjA/pZL757) (D). L. pneumophila was labeled with an anti-L. pneumophila antibody and a secondary antibody conjugated to Texas red; SidC was labeled with an anti-SidC antibody and a secondary antibody linked to fluorescein isothiocyanate. Bar, 5 μm. Data were obtained by inspecting at least 100 vacuoles in each sample (E). Experiments were performed three times in triplicate, and similar results were obtained. Data shown are the means and standard deviations from one representative experiment. (F) Dot/Icm-dependent translocation of SidJ into host cells. U937 cells were infected with the indicated L. pneumophila strains at an MOI of 5.0 for 14 h, and infected cells were collected and extracted with 0.2% saponin as described in Materials and Methods. After being resolved by SDS-PAGE, proteins transferred to nitrocellulose membranes were detected with antibodies against SidJ and ICDH. WT (wild type), U937 cells infected with strain Lp02; dotA, U937 cells infected with Lp03; pSidJ, U937 cells infected with strain ZL101 (ΔsidJ) expressing sidJ from a high-copy-number plasmid (pZL223); -, uninfected U937 cells; WT-Bacteria, Lp02 grown to postexponential phase and lysed with SDS sample buffer. Experiments were repeated three times, and similar results were obtained. Statistical analyses were performed with the Student t test. ***, P < 0.0001.

FIG. 3.

Growth-phase-independent expression of sidJ and the expression of members of the sidE protein family in the sidJ mutant. (A) SidJ is produced by L. pneumophila constitutively during its growth cycle. Tested L. pneumophila strains were grown in AYE broth to the indicated OD₆₀₀, identical amounts of bacterial cells were lysed in SDS sample buffer, and cleared protein samples were separated by SDS-PAGE. After being transferred to membranes, proteins were probed with antibodies specific for SidJ and ICDH. WT (wild type), Lp02; dotA⁻, Lp03; ZL101, Lp02 (ΔsidJ); ZL102, Lp02 ΔsidJ/pSidJ. (B) Expression of sdeA, sdeB, and sdeC is not affected by the deletion of sidJ. Total proteins from equivalent amount of bacteria grown to postexponential phase were resolved by SDS-PAGE and probed with an anti-SdeC antibody (2). Bands representing individual proteins were indicated by arrows, and the cytosolic protein ICDH was used as a loading control. Relevant molecular mass standards are shown on the left (in kilodaltons).

FIG. 4.

sidJ and sdjA mutants display different growth defect phenotypes in host cells. L. pneumophila strains grown to postexponential phase were used to infect murine bone marrow macrophages (A) or D. discoideum (B and C) at an MOI of 0.05. Two hours after addition of bacteria, infection was synchronized by washing of the cells with medium. At the indicated time points, infected monolayers were lysed and the bacterial yield was determined by plating appropriate dilutions on CYE plates to obtain CFU. (A) Bone marrow macrophages were infected with Lp02 (wild type), Lp03 (dotA), ZL101 (Lp02 ΔsidJ), ZL102 (Lp02 ΔsidJ/pSidJ), or ZL51 (Lp02 ΔsdjA). (B) Growth of the wild type (Lp02), the sidJ mutant (ZL101), and the sidJ complementation strain (ZL102) in D. discoideum. (C) Growth of the sdjA mutant (ZL51) and complementation strain ZL103 (Lp02 ΔsdjA/pSdjA) in D. discoideum. The dotA mutant Lp03 was included in all experiments, and no growth was observed for this strain; instead, it was digested by the host efficiently, as described earlier (35). For clarity, the data on this mutant are not shown. Growth was determined by dividing CFU at a given time point by the input bacterial cell numbers. All experiments were performed in triplicate at least three times, and data shown are from one representative experiment. Statistical analyses were performed with the Student t test. *, P < 0.05; **, P < 0.0.001; ***, P < 0.0001.

FIG. 5.

The sidJ mutant forms more small vacuoles in bone marrow macrophages. Murine bone marrow macrophages attached to glass coverslips were infected with the indicated bacterial strains at an MOI of 0.5. After removal of extracellular bacteria by washing at 2 h after infection, samples were withdrawn and fixed at the indicated time points. Extracellular and intracellular bacteria were labeled with an anti-L. pneumophila antibody and different fluorescent dyes. The number of bacteria per vacuole in each strain was scored and categorized into small (1 to 2 bacteria), medium (3 to 10 bacteria), and large (more than 10 bacteria) vacuoles. Similar results were obtained in at least three independent experiments; data shown are averages of triplicate infections in which more than 100 vacuoles were scored per sample. Statistical analyses were performed with the Student t test. **, P < 0.0.001; ***, P < 0.0001.

FIG. 6.

SidJ is required for efficient acquisition of ER protein markers. For the recruitment of calnexin, macrophages were infected with the indicated bacterial strains at an MOI of 5 for 10 min or 1 h, and postnuclear supernatant containing LCVs was prepared and stained for calnexin (see Materials and Methods). Data shown are from infections using bone marrow macrophages (A) or differentiated U937 cells (B). For kinetic analysis of the recruitment of HDEL-GFP (C), D. discoideum strain AX4::HDEL::GFP was infected with the appropriate bacterial strains and samples withdrawn at the indicated times were processed for fluorescence microscopy analysis. Similar results were obtained in three independent experiments, and data shown are from one representative experiment performed in triplicate. At least 100 vacuoles were scored from each sample. Statistical analyses were performed with the Student t test. **, P < 0.001; ***, P < 0.0001.