LidA, a translocated substrate of the Legionella pneumophila type IV secretion system, interferes with the early secretory pathway - PubMed (original) (raw)

LidA, a translocated substrate of the Legionella pneumophila type IV secretion system, interferes with the early secretory pathway

Isabelle Derré et al. Infect Immun. 2005 Jul.

Abstract

Legionella pneumophila uses a type IV secretion system to deliver effector molecules into the host cell and establish its replication vacuole. In this study, we investigated the role of LidA, a translocated substrate associated with the surface of the L. pneumophila-containing vacuole. LidA is secreted into the host cell throughout the replication cycle of the bacteria and associates with compartments of the early secretory pathway. When overexpressed in mammalian cells or yeast, LidA interferes with the early secretory pathway, probably via a domain predicted to be rich in coiled-coil structure. Finally, during intracellular replication, the replication vacuoles are in close contact with the endoplasmic reticulum-Golgi intermediate compartment and the Golgi apparatus, suggesting a positive correlation between intracellular growth and association of the vacuole with compartments of the early secretory pathway. We propose that LidA is involved in the recruitment of early secretory vesicles to the L. pneumophila-containing vacuole and that the vacuole associates with the secretory pathway to facilitate this process.

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Figures

FIG. 1.

FIG. 1.

Membrane association of LidA after translocation into host cells. (A) L. pneumophila (WT) and an isogenic dotA mutant (dotA), both expressing GFP, were grown to postexponential phase and resuspended in PBS containing either 1% Triton X-100, 1% CHAPS, 1% OG, or 1% digitonin (see Materials and Methods). The detergent-soluble and -insoluble fractions were analyzed by immunoblotting using antibodies directed against LidA or GFP. (B) U937 cells were incubated for 1, 6, or 12 h with either L. pneumophila (WT) or the isogenic dotA mutant (dotA), both expressing GFP, prior to incubation for 20 min in PBS containing 1% digitonin. The detergent-soluble and -insoluble fractions were analyzed by immunoblotting using antibodies directed against LidA or GFP. Lp, wild-type L. pneumophila grown in bacteriological medium and directly resuspended in sample buffer; Uninf., uninfected. (C) U937 cells were incubated for 6 h with either L. pneumophila (WT) or the isogenic dotA mutant (dotA), both expressing GFP, prior to mechanical fractionation (see Materials and Methods). The cytosolic fraction was separated from the membrane fraction by ultracentrifugation, and the membrane fraction was subjected to digitonin extraction (see Materials and Methods). The cytosolic fraction (Cytoplasm) and the digitonin-soluble fraction (Membrane Dig. Sol.) were analyzed by immunoblotting using antibodies directed against LidA, calnexin, or RhoGDI.

FIG. 2.

FIG. 2.

The GFP-LidA fusion protein localizes to early secretory compartments and causes their disruption when overexpressed. (A and B) Immunofluorescence micrographs of COS1 cells expressing GFP-LidA at low (top row) or high (bottom row) levels and stained with antibodies directed against either the ERGIC marker, ERGIC53 (A), or the Golgi marker, GM130 (B). Left, GFPLidA; middle, ERGIC53/GM130; right, merge. Bars, 10 μm. (C and D) Quantification of ERGIC (C) or Golgi (D) disruption in either untransfected COS1 (Untransf.) or transfectants expressing either low levels (Low GFPLidA) or high levels (High GFPLidA) of GFPLidA. The cells were stained with anti-ERGIC53 to assay for ERGIC disruption and with anti-GM130, p115, or TGN46 to assay for disruption of the Golgi, the _cis_-Golgi, and the _trans_-Golgi, respectively. The values represent the mean ± standard deviation of three independent experiments.

FIG. 3.

FIG. 3.

The predicted coiled-coil domain of LidA targets the protein to early secretory compartments and causes ERGIC/Golgi disruption. (A) Schematic representation of truncated LidA polypeptides that were fused to GFP. The extent of each deletion is shown on the left. The coiled-coil-rich domain is indicated in grey. Dark-grey circles indicate the predicted coiled-coil structures, and dark- and light-grey squares indicate the repeats (D1 to D4), with the square color indicating regions of highest sequence similarity (see the text). The cellular localization of each construct and its ability to disrupt the ERGIC or the Golgi apparatus or to affect L. pneumophila intracellular growth is indicated on the right. +, present; −, absent; ND, not determined. (B) Immunofluorescence micrographs of COS1 cells expressing truncated versions of LidA: amino acids 1 to 190 (top) or amino acids 191 to 549 (bottom) fused to the GFP and stained with antibodies directed against the Golgi marker, GM130. Left, GFP-LidA; middle, GM130; right, merge. Bars, 10 μm.

FIG. 4.

FIG. 4.

Delay in CPY maturation in yeast cells expressing LidA. (A) Yeast strains harboring chromosomal copies of lidA under the control of the galactose-inducible promoter (A6, A2, and A7) or containing a vector control (v) grown in YPGal were analyzed by immunoblotting using antibody directed against LidA. The strains were adjusted to equivalent numbers of 0.1 _A_600 units. (B) Tenfold serial dilutions of different yeast strains containing lidA under the control of a galactose-inducible promoter (A6, A2, and A7) or containing a vector control (v) were spotted onto YPD (Glucose) or YPGal (Galactose) plates and incubated at 30°C for 3 days. (C) Yeast cells containing a vector control (v) or a chromosomal copy of lidA under the control of a galactose-inducible promoter (A2) were grown at 30°C in glucose (Glc) or galactose (Gal) to an _A_600 of 1. The cells were pulse-labeled with [35S]Met/Cys for 5 min and chased for 0 min (0 min) or 30 min (30 min) (see Materials and Methods). Immunoprecipitations of CPY were performed, immunoprecipitates were subjected to SDS-PAGE, and relative abundance was determined as described in Materials and Methods. (D) Yeast cells containing a chromosomal copy of lidA under the control of a galactose-inducible promoter (A2) were grown at 30°C in galactose to an _A_600 of 1 and were processed as described for panel C, except that samples were collected 0, 30, and 120 min after the beginning of the chase. (E) Quantification of CPY maturation. The percentages of CPY precursor, corresponding to the experiment shown in panel C, were calculated (see Materials and Methods) for both strains (v and A2) grown in glucose (+ Glc) or galactose (+ Gal) after 0 min or 30 min of chase. (F) Quantification of CPY maturation corresponding to the experiment shown in panel D.

FIG. 5.

FIG. 5.

Yeast cells expressing LidA accumulate vesicles and multimembranous compartments. Yeast cells containing a vector control (A and B) or a chromosomal copy of lidA under the control of a galactose-inducible promoter (C and D, A6; E, F, G, and H, A2) were grown at 30°C in galactose to an _A_600 of 1 and processed for electron microscopy. The panels on the right represent enlargements of a section of the pictures presented in the panels on the left. Arrowheads and arrows indicate accumulation of vesicles and multimembranous compartments, respectively. N, nucleus; Va, vacuole; m, mitochondria. Bars, 1 μm (right panels) and 0.2 μm (left panels).

FIG. 6.

FIG. 6.

The L. pneumophila replication vacuole is in close contact with early secretory compartments. (A and B) Immunofluorescence of COS1 cells incubated for 14 h with wild-type L. pneumophila and stained for the ERGIC (A; anti-ERGIC53) or the Golgi apparatus (B; anti-GM130). (Top rows) L. pneumophila is shown in green, and the ERGIC or the Golgi apparatus in red. (Bottom rows) Merged and phase images. N, nucleus. Bar, 10 μm. (C) Quantification of the association of the replication vacuoles with the Golgi apparatus. Displayed are the numbers (Nbr) of cells for which the vacuoles are in association with the Golgi apparatus and that have the indicated numbers of bacteria per vacuole. The values represent the means plus standard deviations of three independent experiments.

FIG. 7.

FIG. 7.

Schematic representation of LidA-mediated ER-derived vesicle recruitment to the _L. pneumophila_-containing vacuole. Upon L. pneumophila uptake by the host cell, LidA (stars) is injected into the host cell cytoplasm via the type IV secretion system (double lines). The protein then associates with the phagosomal membrane and with compartments of the secretory pathway, such as the ERGIC. In this model, LidA interacts with a host protein(s) of the secretory pathway to facilitate the recruitment of ER-derived vesicles to the L. pneumophila (Lp)-containing vacuole. This may be facilitated by the migration of the phagosome in the vicinity of the ERGIC and the Golgi apparatus.

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