Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of <i>Mycobacterium tuberculosis</i>

<div><p>The success of <i>Mycobacterium tuberculosis</i> (Mtb) as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-γ) is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1) promotes IFN-γ-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli <i>in vitro</i>. In IFN-γ activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-γ. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.</p></div

UBQLN1 binds Mtb <i>in vitro</i>.

<p>(A) Mtb-UBQLN1 binding assay. Mtb were incubated with HEK293 cell lysate (B, C) or recombinant protein (D) (input) and washed 5 times to remove unbound proteins. The pellet contains Mtb and associated host proteins (elution). (B) Mtb were incubated with lysate from HEK293 cells transfected with vector control, myc-UBQLN1, GFP, or V5-parkin. WB analysis of the input (I), 5^th wash (W5), and elution (E), probed with indicated antibodies. Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet. (C) Mtb were incubated with lysate from HEK293 cells transfected with myc-UBQLN1-ΔUBA, myc-UBQLN1-ΔUBL, and myc-UBQLN1-UBA. WB containing the input, 5^th wash, and elution were incubated with anti-myc antibody. The predicted sizes of myc-UBQLN1-ΔUBL, myc-UBQLN1-ΔUBA, and myc-UBQLN1-UBA are 55 kD, 50 kD, and 6 kD, respectively. (D) Mtb were incubated with recombinant GST-UBQLN1, GST-UBQLN1-ΔUBL, or GST-UBQLN1-ΔUBA. WB containing the input and elution was probed with an anti-GST antibody.</p

MUPs bind ubiquilins.

<p>(A) Domain structure of human UBQLN1 (Interpro: <a href="http://www.ebi.ac.uk/interpro/" target="_blank">http://www.ebi.ac.uk/interpro/</a>). (B) Gal4 DNA-binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins (murine) in Y2H. (C) HEK293 cells transfected with indicated MUP-V5, myc-UBQLN1 (human), or empty vector were immunoprecipitated (IP) with anti-myc or anti-UBQLN1 antibodies. Western blot (WB) of IP and lysate were probed as indicated. (D-E) WB of input lysate and IP from HEK293 cells expressing myc-UBQLN1 and Rv1926-V5 (D) or Rv1566-V5 (E). IP was performed using antibodies directed against myc, V5, or isotype control (IgG).</p

UBQLN1 localizes to Mtb, restricts its growth, and promotes the ability of macrophages to active CD4+ T cell.

<p>(A) BMDMs were infected with GFP-expressing wt Mtb or Δ<i>esxA</i> for 24h and immunostained with UBQLN1 antibody. IFN-<b>γ</b> was added 1d prior to infection as indicated. (B) Quantification of results from <i>A</i> from 3 independent experiments with at least 100 bacteria analyzed per experiment; ***<i>P</i>< 0.001, Fisher’s exact test, comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples. (C) Cellular lysate from BMDMs that were treated with IFN-<b>γ</b> as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2. (D) BMDMs were transfected with siRNAs targeting UBQLN1 or a non-targeting control (siCON) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2. (C-D) Actin served as a loading control. (E) BMDMs were infected with Mtb live/dead strain and treated with AnTc 24h prior to fixation. (F) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live/dead Mtb strain. IFN-<b>γ</b> was added 1d prior to infection if indicated. %Viable bacteria were quantified 24 and 48 hpi. Data are from 2 independent experiments. *<i>P</i><0.05; ***<i>P</i><0.001, Fisher’s exact test, comparing the proportion of viable and non-viable bacteria in indicated samples. (G) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN-<b>γ</b> one day prior to infection with H37Rv or Δ<i>esxA</i>. CFU were enumerated 4 and 96 hpi. *<i>P</i>< 0.05, unpaired Student’s <i>t</i>-test. Data are representative of three independent experiments. (H) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb (H37Rv) (I) UBQLN1-silenced BMDMs and controls were infected with H37Rv or Δ<i>esxA</i>. (H-I) 24 hpi infected BMDMs were co-cultured with P25TCR-Tg CD4+ T cells, and IFN-<b>γ</b> was measured by ELISA. Data is representative of 3 independent experiments. *<i>P</i><0.05, **<i>P</i><0.005, unpaired Student’s <i>t</i>-test. (B, F, Γ, H) Results are mean +/- SEM; ns, not significant.</p

UBQLN1 co-localizes with Mtb that are cleared through autophagy.

<p>(A) BMDMs treated with IFN-<b>γ</b> and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and either ubiquitin (using the FK2 antibody) or p62. GFP-LC3 BMDMs infected with DsRed-Mtb were immunostained for UBQLN1. (B) Quantification of those Mtb that co-localize with at least one of the cellular markers shown in A. More than 375 total bacteria were counted in two independent experiments, and the number co-localizing with at least one marker is indicated as “total” under each bar. (C) Wt macrophages (Atg16L1^flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1^flox/flox Lyz-cre; Cre+) were activated with IFN-<b>γ</b> or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **<i>P</i><0.005, ***<i>P</i><0.0001, Fisher’s exact test (in which the proportions of viable to non-viable Mtb was compared). (D) Atg16L1^flox/flox Cre+ and Cre- BMDMs treated with IFN-<b>γ</b> and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and p62. The number of Mtb co-localizating with the cellular markers is shown. (C-D) At least 100 bacteria were analyzed in three independent experiments. (E) Images from IFN-<b>γ</b> treated BMDMs of indicated genotype infected with GFP-Mtb and immunostained with UBQLN1 and p62. Arrows indicates p62+ UBQLN1+ Mtb.</p

UBQLN1 promotes ubiquitin, p62, and LC3 recruitment to Mtb.

<p>(A-C) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA. Images show IFN-<b>γ</b> treated macrophages infected with GFP-Mtb (A-B) or DsRed-Mtb (C) for 24h. Cells were immunostained for ubiquitin (Ub) with the FK2 antibody (A) or p62 (B), which are shown in red. (C) GFP-LC3 BMDMs were infected with DsRed-Mtb. LC3 is shown in green, whereas Mtb are red. (A-C) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN-<b>γ</b> as indicated. Results are mean +/- SEM from two independent experiments, with at least 100 bacteria analyzed per experiment. ***<i>P</i><0.001, Fisher’s exact test (in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN-<b>γ</b>).</p

UBQLN1 acts in parallel to parkin.

<p>(A) WT and <i>Park2</i>^-/- BMDMs were pretreated with IFN-<b>γ</b>, infected with GFP-Mtb for 24h, and immunostained with the FK2 antibody. Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments. (B) <i>Park2</i>^<i>-/-</i> BMDMs pretreated or not treated with IFN-<b>γ</b> were infected with GFP-Mtb for 24h and immunostained with UBQLN1 specific antibody; Arrows indicate Mtb co-localized with UBQLN1. ns- not significant. (C) <i>Park2</i>^<i>-/-</i> BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection, treated with IFN-<b>γ,</b> and infected with GFP-Mtb for 24h prior to immunostaining for ubiquitin (FK2 antibody). Ubiquitin (FK2+) bacteria were quantified from two independent experiments. (A, C) **<i>P</i><0.01, Fisher’s exact test (comparing the proportion of FK2 positive bacteria in indicated samples). Results are mean +/- SEM. A minimum of 100 Mtb were counted in each experiment.</p