E3 ubiquitin ligase activity and targeting of BAT3 by multiple Legionella pneumophila translocated substrates

Abstract

The intracellular bacterial pathogen Legionella pneumophila modulates a number of host processes during intracellular growth, including the eukaryotic ubiquitination machinery, which dictates the stability, activity, and/or localization of a large number of proteins. A number of L. pneumophila proteins contain eukaryotic-like motifs typically associated with ubiquitination. Central among these is a family of five F-box-domain-containing proteins of Legionella pneumophila. Each of these five proteins is translocated to the host cytosol by the Dot/Icm type IV protein translocation system during infection. We show that three of these proteins, LegU1, LegAU13, and LicA, interact with components of the host ubiquitination machinery in vivo. In addition, LegU1 and LegAU13 are integrated into functional Skp-Cullin-F-box (SCF) complexes that confer E3 ubiquitin ligase activity. LegU1 specifically interacts with and can direct the ubiquitination of the host chaperone protein BAT3. In a screen for additional L. pneumophila proteins that associate with LegU1 in mammalian cells, we identified the bacterial protein Lpg2160. We demonstrate that Lpg2160 also associates with BAT3 independently of LegU1. We show that Lpg2160 is a translocated substrate of the Dot/Icm system and contains a C-terminal translocation signal. We propose a model in which LegU1 and Lpg2160 may function redundantly or in concert to modulate BAT3 activity during the course of infection.

FIG. 1.

L. pneumophila contains several Dot/Icm translocated substrates with F-box domains. (A) Structure of the SCF complex. An F-box-domain-containing protein associates with SKP1. SKP1 and RBX1 associate with CUL1, which serves as a scaffold for complex formation. (B) Five proteins contain predicted F-box motifs in the Legionella pneumophila strain Philadelphia-1 genome. aa, amino acids; Ank, ankyrin repeat domain; RCC1, regulation of chromatin condensation domain. (C) Each of the L. pneumophila F-box proteins is translocated into the host cytosol in a Dot/Icm-dependent manner. U937 cells were challenged with wild-type or translocation-deficient dotA bacteria expressing CyaA (negative control) or CyaA::LegU1, CyaA::LegAU13, CyaA::LicA, CyaA::PpgA, or Cya::Lpg2525. After 1 h, host cells were lysed and cAMP levels were quantified. The data represented are the averages of three replicates ± standard errors of the means.

FIG. 2.

Regulation of L. pneumophila F-box transcription in different phases of growth. The majority of the F-box proteins are transcribed at a low level and are not upregulated during post-exponential growth. Wild-type L. pneumophila was grown in broth until it reached early exponential phase (EE; A₆₀₀ = 0.9), mid-exponential phase (ME; A₆₀₀ = 2.0), or post-exponential phase (PE; A₆₀₀ = 3.7) growth. Total RNA was purified from each and reverse transcribed, and individual levels of each transcript were measured using quantitative real-time RT-PCR. The levels of each transcript were normalized to the level of an endogenous control locus, 16S rRNA, the transcript level of which was set equal to 100,000 on this scale. For comparison, the transcript level of the translocated substrate, SidC, was also measured in each of these samples.

FIG. 3.

A subset of L. pneumophila F-box proteins associate with SCF components in human cells. (A) Mammalian SCF components associate with a subset of L. pneumophila F-box proteins. HEK-293T cells were transfected with 3XFLAG-tagged L. pneumophila F-box proteins, and lysates were subjected to immunoprecipitation with anti-FLAG resin. The immunoprecipitates were analyzed by Western blot analysis using antibodies to endogenous human SKP1, CUL1, and ARP2, a negative control for nonspecific enrichment. Lanes IN, inputs, normalized to 1% of the amount of the immunoprecipitate (IP) fraction loaded on the gel (asterisks, predicted size of each 3XFLAG-tagged F-box protein). (B) Plasmids encoding 3XFLAG-tagged LegU1 and LegAU13 were cotransfected with a plasmid encoding HA-tagged ubiquitin in HEK-293T cells and subjected to anti-FLAG immunoprecipitation. Western blot analysis was performed using anti-HA antibody in order to reveal ubiquitinated species. After the membrane was stripped, it was probed with antibody directed against the FLAG epitope. (C) The association between SCF components and LegU1 and LegAU13 depends on their F-box domains. HEK-293T cells were transfected with plasmids encoding 3XFLAG-tagged LegU1 and LegAU13 with and without intact F-box domains. Cell lysates from these transfections were immunoprecipitated with anti-FLAG resin and analyzed by Western blotting using the noted antibodies (see Materials and Methods). Lanes S/N, unbound supernatant. Inputs and unbound supernatants were normalized to 1% of the immunoprecipitate fraction (lanes IP) loaded on the gel.

FIG. 4.

LegU1 and LegAU13 associate with E3 ubiquitin ligase activity, as measured through an in vitro ubiquitination assay. (A) The E2 enzymes UBCH5A and UBCH5C support the robust generation of high-molecular-weight ubiquitinated species by LegU1 and LegAU13 in vitro. Ubiquitination reactions were performed in vitro (see Materials and Methods) using a panel of E2 enzymes, as noted above each displayed blot. Reactions were analyzed by Western blot analysis using HRP-conjugated streptavidin to detect biotin-ubiquitinated species and comparison of the results to those for a vector-only negative control. Western blot analysis was also performed using anti-mouse IgG antibody to identify any differences in the amount of SCF-bound resin aliquoted into each reaction mixture. (B) The F-box domains of LegU1 and LegAU13 are required for in vitro ubiquitination. In vitro ubiquitination reactions were performed in complete reaction mixtures that included the UBCH5A E2 enzyme, using either 3XFLAG-tagged LegU1, LegAU13, LegU1ΔF-box,or LegAU13ΔF-box or a vector-only control.

FIG. 5.

LegU1 associates with the host protein BAT3 and can direct its ubiquitination. (A) Among the five Philadelphia-1 F-box proteins, only LegU1 associates with BAT3. HEK-293T cells were transfected with plasmids encoding 3XFLAG-tagged LegU1, LicA, LegAU13, PpgA, or Lpg2525. Cell lysates from these transfections were immunoprecipitated with anti-FLAG resin and analyzed by Western blotting using anti-BAT3 antibody or anti-VCP as a control for nonspecific enrichment. Lanes IN, inputs; lanes IP, immunoprecipitates. (B) LegU1 associates with BAT3 independently of the F-box domain. HEK-293T cells were transfected with 3XFLAG-tagged LegU1, LegU1ΔF-box, or the 3XFLAG vector alone. After anti-FLAG immunoprecipitation, samples were subjected to Western blot analysis using anti-BAT3 antibody and anti-TUBA4A antibody as controls for nonspecific enrichment. (C) Immunoprecipitation of LegU1 by BAT3. HEK-293T cells were transfected with either Myc-tagged BAT3 and the 3XFLAG vector, 3XFLAG-tagged LegU1 and the Myc vector, or Myc-tagged BAT3 together with 3XFLAG-tagged LegU1. Lysates from these cells were subjected to anti-Myc immunoprecipitation, and immunoblot analysis was performed using anti-FLAG antibody. Transfection combinations are noted above the displayed blot (asterisk, predicted size of 3XFLAG-tagged LegU1). For panels A to C, each input fraction was normalized to 1% of the immunoprecipitate fraction loaded on the gel. (D) LegU1 directs the polyubiquitination of BAT3 in vitro in a manner dependent on the F-box domain. Resin harboring 3XFLAG-tagged LegU1 or LegU1ΔF-box was prepared by anti-FLAG immunoprecipitation from HEK-293T cells transfected with each translocated substrate. Aliquots of this resin were used for in vitro ubiquitination experiments (see Materials and Methods) in a complete reaction mixture with wild-type ubiquitin [Ub(WT)], E1 enzyme, and an E2 enzyme, UBCH5A. Using other aliquots of the same resin, parallel reactions were performed using a reaction mixture lacking the E1 enzyme or containing only an isoform of ubiquitin (NoK) that lacks all lysine residues and that is incapable of extending polyubiquitin chains. Western blot analysis using anti-BAT3 antibody was used to detect any endogenous BAT3 protein that coimmunoprecipitated with complexes and was subjected to these reactions.

FIG. 6.

A second L. pneumophila translocated substrate, Lpg2160, associates with BAT3 and LegU1 in mammalian cells. (A) The L. pneumophila protein Lpg2160, but not its close paralog, Lpg2638, associates with LegU1 independently of the F-box domain. HEK-293T cells were cotransfected with plasmids encoding the 3XFLAG vector, 3XFLAG::LegU1, or 3XFLAG::LegU1ΔF-box, along with GFP-tagged Lpg2160 or Lpg2638. Cell lysates were subjected to anti-FLAG immunoprecipitation, followed by anti-GFP immunoblot analysis. Lanes IN, inputs; lanes IP, immunoprecipitates. (B) Lpg2160 associates with BAT3 independently of LegU1. HEK-293T cells were transfected with either the HA::mYFP vector or HA::mYFP-tagged Lpg2160, and cell lysates were subjected to immunoprecipitation with anti-HA resin. Western blot analysis was performed using anti-BAT3. In addition, anti-HA antibodies were used to detect ectopically expressed HA::mYFP-tagged proteins (asterisk), and anti-TUBA4A was used as a control for nonspecific enrichment. For panels A and B, each input fraction was normalized to 1% of the immunoprecipitate fraction loaded on the gel. (C) Lpg2160 does not serve as a target of ubiquitination by LegU1 in vitro, despite its coassociation. In vitro ubiquitination reactions were performed as described in the text using resin containing 3XFLAG or 3XFLAG::LegU1 (see Materials and Methods). Aliquots of resin were incubated in either the complete reaction mixture, reaction mixtures lacking the E1 enzyme, or reaction mixtures using a mutant form of ubiquitin (NoK) incapable of forming polyubiquitin chains. After the reactions were stopped, samples were analyzed by Western blotting using anti-HA or anti-BAT3 antibodies. (D) Lpg2160 is translocated into the host cytosol in a Dot/Icm-dependent manner. U937 cells were challenged with wild-type or translocation-deficient dotA bacteria expressing CyaA (negative control) or CyaA fused to full-length Lpg2160(1-482), Lpg2160(1-451), or Lpg2160(450-482). After 1 h, host cells were lysed and cAMP levels were quantified. The data represented are the averages of three replicates ± standard errors of the means.

FIG. 7.

LegU1, LegAU13, and Lpg2160 are dispensable for intracellular growth in both primary mouse macrophages and amoebae. Intracellular growth of L. pneumophila within A/J mouse primary bone marrow-derived macrophages (A) and the amoeba Acanthamoeba castellanii (B). Host cells were challenged with the indicated L. pneumophila strains: the wild type; ΔlegU1, ΔlegAU13, Δlpg2160, ΔlegU1, and Δlpg2160 strains; and a dotA mutant. The number of viable bacteria at each time point was determined by lysing host cells and plating dilutions of bacteria onto CYET plates. The data were plotted as the averages of three experiments per strain per time point. Error bars represent the standard errors of the means of these measurements.

FIG. 8.

Composition and potential role of each L. pneumophila F-box-associated complex in mammalian cells. On the basis of the results of our experiments, we propose a model in which LegU1 and LegAU13 form intact SCF complexes with E3 ligase activity. SCF^LegU1 directs the ubiquitination of the host protein BAT3, which may result in a reduced ER stress response or suppression of host apoptotic pathways. Another L. pneumophila translocated substrate, Lpg2160, also associates with the LegU1-BAT3 complex but does not get ubiquitinated. SCF^LegAU13 is an E3 ubiquitin ligase complex with unknown target affinity. In addition to being subject to autoubiquitination, we hypothesize that LegAU13 directs the ubiquitination of host and/or bacterial proteins during intracellular replication. The F-box protein LicA associates with SKP1 but does not associate with CUL1. The consequences of this interaction are unknown, but we propose that LicA-SKP1 complexes may not direct the ubiquitination of target proteins.