Fig 1.
Genetic analysis of the SidE family and SidJ.
(A) sdeC-sdeA locus includes five genes (sdeC, lpg2154, sidJ, sdeB, and sdeA), whereas the sidE gene is located at a separate location. The SidE family consists of the related proteins SidE, SdeC, SdeB, and SdeA (shown with black arrows). (B-F) Replication of various L. pneumophila strains in A. castellanii was determined at the indicated time points post infection and expressed as fold growth. (B) Suppression of the growth defect of the CleanΔP170 mutant by expression of low amounts of SdeA. (C) Complementation of the ΔsidJ mutant by expression of SidJ. (D-F) Overexpression of SdeA does not inhibit the growth of the wild type strain Lp02 (D) or the CleanΔP170 mutant (E) but does inhibit the replication of the ΔsidJ mutant (F). (G) Overproduction of SdeA causes Legionella to traffic into the endocytic pathway of amoebae. A. castellanii pre-incubated with Texas Red Ovalbumin (TrOV), which labels their endocytic pathway and vacuole, were infected with Lp02, a dotA mutant (Lp03) and a ΔsidJ mutant containing vector (v) or a plasmid over-expressing SdeA (sdeA OP). The percent of bacteria that co-localized with TrOV was quantitated at 0, 1, and 3 hours post infection. Data are means ± SEM of three independent experiments. Approximately 100 bacteria were scored per condition and asterisks indicate statistical difference (P<0.05).
Fig 2.
SidJ suppresses SdeA toxicity in yeast and mammalian cells.
(A) Tenfold serial dilution of yeast strains expressing vector (v), Pgal-sidJ, Pgal-sdeA or Pcyc-sidJ were spotted and grown on selective plates containing glucose (repressing conditions) or galactose (inducing conditions). (B and C) Localization of SdeA and SidJ in HEK293 cells as observed by transfecting cells for 40-hours with mCherry, YFP, mCherry-SdeA and YFP-SidJ. Channels used to acquire images are indicated on the left of each row and merged images are shown in the bottom row. Arrow indicates a cell containing a dense mCherry-SdeA foci. Bar, 10 μm. (C) Scoring data of cells with dense SdeA loci observed in (B). (D) Shorter transfection time (20-hour) reveals mCherry-SdeA-induced Golgi fragmentation in HEK293 cells. Cells were fixed and stained with anti-giantin (Golgi marker). Bar, 10 μm. Arrows indicate Golgi. (E) Scoring data of Golgi fragmentation in (D). Data are means ± SEM of three independent experiments. Approximately 100 cells/condition were counted and asterisks indicate statistical difference (P<0.05).
Fig 3.
SidJ mediated the disappearance of SidE proteins from the LCV at later time points of infection.
(A) BMMs were infected with the wild-type strain Lp02, the T4SS-deficient strain Lp03 or the ΔsidJ mutant, fixed, and stained with antibodies specific for the SidE family, LidA, or SidM (green). DNA (L. pneumophila and host nuclei) was stained with propidium iodide (red). Representative images are shown for 1 hour or 7-hour infections. Cells were treated with MG132 (10 μM) for 15 min prior to infection as needed. (B-D) Co-localization of SidE, SidM, and LidA with the LCV were scored and recorded as a percentage. Percentage of co-localization is plotted over time for SidE proteins (B), SidM (C), and LidA (D) for the wild-type strain Lp02 (filled squares), Lp02 + MG132 (open triangles), and the ΔsidJ mutant (open squares). Approximately 75 LCVs were counted for each Dot/Icm substrate at each point. Two independent experiments were conducted and the data is representative of both.
Fig 4.
SidE proteins are not degraded during infection.
(A) Assay demonstrating secretion of Dot/Icm substrates SidE and SidJ. U937 cells were infected with wild-type Legionella, lysed by douncing, and post nuclear supernatant fractions (PNS) were prepared. Samples were processed in the absence (left column) or the presence of digitonin (right column). Fractions include total (T), organelles and LCV (O), cytoplasm (C), digitonin insoluble (I) and digitonin soluble (S), which contains secreted proteins. Fractions were analyzed by Western blot using antibodies specific for the indicated proteins. (B-E) U937 cells were infected with wild-type Legionella for 1 hour, washed, and the infection was allowed to proceed for the indicated times in the absence (WT) or presence of chloramphenicol (Cm) or MG132 (MG). The cells were processed as above including dounce lysis and digitonin treatment followed by centrifugation. The digitonin soluble data is shown for SidE (B), SidJ (C), LidA (D), and SidM (E) and is representative of three experiments.
Fig 5.
SidE proteins removed from the LCV associate with host organelles.
U937 cells were infected with wild-type Legionella for 1 hour or 3 hours, lysed by douncing, separated by sucrose gradient and analyzed by Western blot. (A) Western blots includes markers for various cell compartments including: actin (cytoplasm), LAMP-1 (endocytic compartments), PHB (mitochondria), GM130 (Golgi), calnexin (ER), and DotF (LCV). (B) Westerns for SidE proteins and SidJ at both 1 and 3 hour post infection (hpi). The box highlights the altered levels of SidE proteins in the LCV fraction as the infection progresses.
Fig 6.
In vitro assay for SidJ removal of SidE proteins from the LCV.
(A-D) BMMs were infected with a ΔsidJ mutant for the indicated time, lysed by gentle douncing, unbroken cells removed by centrifugation, and the PNS was incubated with 0.5 μM of purified SidJ, SidJ DD mutant or BSA. The reactions were stopped by addition of paraformaldehyde. The fixed cells were then stained with anti-SidE (A) or LidA (C) antibody. Representative images are shown with SidE and LidA in green and bacteria in blue. (B and D) Phagosomes were scored for co-localization with SidE proteins (B) or LidA (D). Removal of SidE proteins from the LCV is dependent on the concentration of SidJ (E) and the time of incubation (F). Data are means ± SEM of three independent experiments. Approximately 75 LCVs were counted for each reaction and asterisks indicate statistical difference among treatments in both B and D and in samples compared to the mock controls in both E and F, (P<0.05).
Fig 7.
Model showing SidJ-mediated removal of SidE proteins from the Legionella containing vacuole (LCV).
Shown is a time course for the initial hours of an infection by wild-type L. pneumophila and a ΔsidJ mutant. SidE proteins are indicated with a red letter E, SidJ protein with a purple letter J, a hypothetical protein necessary for retaining SidE proteins on the LCV with a blue letter X, the LCV membrane in green, L. pneumophila in maroon, the proteasome as a yellow Pac-man, and a host organelle in light blue. In a wild-type infection, SidE proteins localize to the LCV at early time points (1 hpi). At later time points, SidJ removes SidE proteins from the LCV either by localized degradation of SidE (top panel) and/or by degradation of a retention factor, thus leading to the relocalization of LCV-associated SidE proteins to a host organelle (middle panel). In a ΔsidJ infection, SidE proteins remain and accumulate on the LCV, which is detrimental for growth (lower panel).