Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection

Abstract

Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

Figure 1

AGS3 stable cell lines exhibit increased proteolysis without an alteration in autophagic induction. (A,B,C) Immunoblot of cell lysates prepared from PMA-differentiated AGS3^lo and AGS3^hi THP-1 cells (A), or from AGS3^lo and AGS3^hi HeLa cells (B), or from Bafilomycin A1-treated AGS3^lo and AGS3^hi THP-1 cells (C) to examine the expression of the indicated proteins. The results obtained from three experiments are shown below and presented as % of the THP-1 AGS3^lo (A) and HeLa AGS3^lo (B). The LC3-II and p62 levels were normalized to the actin level in the corresponding lysates. (D) qRT-PCR of RNA prepared from AGS3^lo and AGS3^hi THP-1 cell lines for the indicated genes.

Figure 2

Lysosome levels are regulated by AGS3 expression. (A,B) Immunoblots of the indicated proteins in lysates from AGS3^lo and AGS3^hi stable THP-1 (A) and in AGS3^lo and AGS3^hi stable HeLa cells (B). Lysosome marker/actin band intensity ratios were normalized and the results shown as percentage of AGS3^lo cells in the graphs to the right of the immunoblots. (C) Immunoblots of the indicated proteins in THP-1 stable cells expressing GFP, AGS3^lo THP-1 cells, or AGS3^hi THP-1 cells. (D) Confocal images of lysosomal vesicles in an AGS3^lo HeLa cell (marked by asterisks) and AGS3^hi (GFP positive) HeLa cell immunostained for LAMP-1 (right panel). The lysosomal mass was quantified by counting LAMP-1 positive vesicles in 50-70 cells of each cell type. The graph is shown to the right of the images. Scale bar: 10 μm. (E) Confocal images of an AGS3^lo cell (** in the upper panel, visible in lower panel) and an AGS3^hi THP-1 cell incubated with DQ-BSA overnight. Scale bar: 10 μm. (F) Immunoblot of indicated protein levels in cell lysates from HeLa cells transiently expressing AGS3 for 60 hours. Lysosome marker/actin band intensity ratios were normalized and shown as percentage of the control transfected cells. (G,H) Immunoblots of the indicated proteins in lysates from AGS3^hi HeLa cells transfected with scrambled (ctrl) or GFP siRNAs (20nM), or HeLa cells transfected with scrambled or AGS3 siRNA (20 nM) for 72 hours. The results were normalized to actin levels and shown as a percentage of HeLa AGS3^hi (G, right panel) or HeLa cells transfected with control siRNAs (H, right panel).

Figure 3

AGS3 levels alter lysosomal biogenesis by regulating the nuclear translocation of TFEB. (A) Immunoblot of the indicated proteins in cell lysates from THP-1 cells, AGS3^lo and AGS3^hi stable THP-1 cells; and from BMDM derived from WT and Gpsm1^−/− mice. In some instances the BMDM were stimulated with LPS overnight (100 ng/ml). Normalized AGS3 and LAMP-1 levels are shown as a percentage of non-stimulated (left and middle) or WT (right). (B) Immunoblot of TFEB and actin expression in cell lysates prepared from the indicated cells. The LPS treated cells were from overnight stimulation. (C) qRT-PCR of RNA prepared from AGS3^lo and AGS3^hi THP-1 cell lines for the indicated genes. (D) Confocal images of HeLa cells expressing TFEB-GFP and AGS3-RFP or only TFEB-GFP. The location of the AGS3^lo cell in the middle panel is marked by asterisks. Scale bar: 10 μm. (E) Quantification of TFEB nuclear localization in HeLa cells as in part D. Localization assessed in 500 cells. (F) Immunoblots of the indicated proteins in cytosolic and nuclear fractions isolated from HeLa cells transfected with vector or AGS3 expression plasmids overnight or treated with a GSK3β inhibitor (VIII).

Figure 4

MyD88- and TRIF-dependent signaling pathways involved in AGS3 and LAMP-1 upregulation in macrophages. (A) Representative immunoblots of cell lysates prepared from WT, MyD88, and TRIF deficient immortalized bone marrow macrophages (iBMDMs) to examine AGS3 and LAMP-1 expression. The results of three separate experiments are shown below each of the immunoblots. The data is shown as a percentage increase versus the non-stimulated cells. The cells were exposed to LPS (100 ng/ml) overnight prior to cell lysis and immunoblotting. (B) Immunoblot of cell lysates from iBMDMs exposed overnight to S. aureus, E. coli bioparticles at MOI of 2.0, or not, to examine LAMP-1 and AGS3 expression. Quantification of three separate experiments is shown to the right.

Figure 5

AGS3 regulates mTOR complex via the AKT/TSC2/mTOR pathway. (A-D) Immunoblots of the indicated proteins in cell lysates from AGS3^lo and AGS3^hi THP-1 cells (A), control or HeLa cells transiently expressing AGS3 for 72 hours (B), HeLa cells treated with control or siRNA targeting AGS3 expression for 72 hours (C), or BMDM from WT or Gpsm1^−/− mice stimulated with LPS overnight (100 ng/ml). Shown below each immunoblot is the summation of results from at least 3 experiments normalized to actin and presented as a percentage of AGS3^lo, % of control, % of control, or % of WT as indicated.

Figure 6

High levels of AGS3 reduce Gβ expression and inhibit chemokine receptor signaling. (A) Immunoblot of cell lysates prepared from AGS3^lo and AGS3^hi THP-1 cells for the expression of Gα_i and Gβ. The % reduction in Gβ1 and Gβ2 expression compared to the AGS3^lo cells are shown below the immunoblot. (B) Trace of the intracellular calcium levels following exposure of AGS3^hi and AGS3^lo cells to different concentrations of CXCL12. (C) Flow cytometry to examine CXCR4 expression on AGS3^lo and AGS3^hi THP-1 cells. The geometric means are shown to the right of the flow cytometry plot.

Figure 7

AGS3-induced lysosomal enrichment mediates resistance against antibiotic resistant bacteria. (A) Confocal microscopy images of THP-1 AGS3^lo and AGS3^hi cells infected with DsRed-tagged B. cenocepacia J2315 strain at MOI of 1.0 for 24 hours. The location of the AGS3^lo cell is indicated with asterisks in the GFP panel. Scale bars: 10 μm. (B) The percentage of THP-1 AGS3^lo and AGS3^hi cells harboring ≥15 bacteria per cytosol 24 hours post-infection. (C) Quantification of CFUs from WT and Gpsm1^−/− BMDM infected with B. cenocepacia J2315 at an MOI 1.0 at 24 hours. Results were normalized to WT cells. The results obtained from three separate infection experiments are shown for each specific bacteria. (D) Confocal microscopy images of THP-1 AGS3^lo and AGS3^hi cells infected with B. cenocepacia J2315 at an MOI of 1.0 at 24 hours post-infection. The cells were immunostained with B. cenocepacia and LAMP-2 specific antibodies. The location of the AGS3^lo cell is indicated with asterisks in the GFP panel. Scale bars: 10 μm. (E) Quantification of the co-localization rate between B. cenocepacia J2315 and LAMP-2 (F) Kinetic analysis of CFU counts upon Mycobacterium tuberculosis infection. THP-1 AGS3^lo and AGS3^hi cells were infected with M. tuberculosis at MOI 0.5 for 4 hours. The cells were lysed at various time points post infection and the lysates were plated on 7H11 media for CFU counts. The results are from three independent experiments performed in triplicate. (G) Brightfield microscopy images of THP-1 AGS3^lo and AGS3^hi cells infected with MRSA252 at MOI of 4:1 for 6 hours. Scale bar: 400 μm. (H,I) Quantification of LDH release from THP-1 AGS3^lo and AGS3^hi cells (H) and from WT and Gpsm1^−/− BMDM (I) 6 hours post-infection with MRSA252 or USA300 clone LAC at MOIs of 6:1 and 30:1, respectively. Control cells were incubated with a 2% Triton-X solution to determine 100% LDH release. The amounts of LDH release following bacterial infections are shown as a percentage of the Triton-X triggered release. The results obtained from three separate infection experiments are shown for each specific bacteria.