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. 2016 May 24;15(8):1728-42.
doi: 10.1016/j.celrep.2016.04.052. Epub 2016 May 12.

An Evolutionarily Conserved PLC-PKD-TFEB Pathway for Host Defense

Mehran Najibi  1 , Sid Ahmed Labed  1 , Orane Visvikis  1 , Javier Elbio Irazoqui  2
Affiliations

An Evolutionarily Conserved PLC-PKD-TFEB Pathway for Host Defense

Mehran Najibi et al. Cell Rep. .

Abstract

The mechanisms that tightly control the transcription of host defense genes have not been fully elucidated. We previously identified TFEB as a transcription factor important for host defense, but the mechanisms that regulate TFEB during infection remained unknown. Here, we used C. elegans to discover a pathway that activates TFEB during infection. Gene dkf-1, which encodes a homolog of protein kinase D (PKD), was required for TFEB activation in nematodes infected with Staphylococcus aureus. Conversely, pharmacological activation of PKD was sufficient to activate TFEB. Furthermore, phospholipase C (PLC) gene plc-1 was also required for TFEB activation, downstream of Gαq homolog egl-30 and upstream of dkf-1. Using reverse and chemical genetics, we discovered a similar PLC-PKD-TFEB axis in Salmonella-infected mouse macrophages. In addition, PKCα was required in macrophages. These observations reveal a previously unknown host defense signaling pathway, which has been conserved across one billion years of evolution.

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Figures

Figure 1
Figure 1. DKF-1/PKD is necessary and sufficient for HLH-30/TFEB activation
(A) HLH-30::GFP animals were reared on E. coli carrying empty vector (EV), dkf-1 RNAi, or dkf-2 RNAi, and subsequently fed with E. coli OP50 (top row) or infected with S. aureus (middle row). Shown are representative epifluorescence micrographs. Hatched boxes indicate areas enlarged in detail (Bottom Row). (B) Quantitative analysis. Data are mean ± SEM (two biological replicates, n ≥ 50 per condition). *** p ≤ 0.001 (two-sample t test). (C) Survival of wild type and hlh-30 mutant animals reared on E. coli carrying dkf-1 RNAi or empty vector control prior to infection with S. aureus. *** p ≤ 0.001 (Log-Rank test). (D) Animals were treated with dkf-1 RNAi as in (A) and subsequently incubated with 1 μg/ml PMA for 30 min. Shown are representative epifluorescence micrographs (middle row). Hatched boxes indicate areas enlarged in detail (bottom row). Top row shows animals treated with vehicle. (E) Quantitative analysis. Data are mean ± SEM (two biological replicates, n ≥ 50 per condition). ** p ≤ 0.01 (two-sample t test). (F) qRT-PCR of ilys-2 in wild type or hlh-30 mutants. Animals were incubated with 1 μg/ml PMA for 8 h. Results are normalized to control wild type animals. Data are mean ± SEM (three biological replicates, three technical replicates, n ≥ 3,000 per condition). (G) qRT-PCR of ilys-2 in worms reared on E. coli carrying empty vector control or dkf-1 RNAi. Animals were incubated with 1 μg/ml PMA for 8 h. Results are normalized to empty vector control. * p ≤ 0.05 (two-sample t test). (H) HLH-30::GFP animals were treated with kb-NB142-70 or Bisindolylmaleimide IV, and subsequently fed with E. coli OP50 (top row) or infected with S. aureus (middle row). Shown are representative epifluorescence micrographs. Hatched boxes indicate areas enlarged in detail (Bottom Row). (I) Quantitative analysis. Data are mean ± SEM (two biological replicates, n ≥ 50 per condition). ** p ≤ 0.01 (two-sample t test). (J) HLH-30::GFP animals were reared on E. coli carrying empty vector (EV), pkc-1, pkc-2, or tpa-1 RNAi, and subsequently infected with S. aureus (top row). Shown are representative epifluorescence micrographs. Hatched boxes indicate areas enlarged in detail (bottom row). (K) Quantitative analysis. Data are mean ± SEM (three biological replicates, n ≥ 50 per condition). See also Figure S1.
Figure 2
Figure 2. A Gαq-PLCε-PKD pathway controls TFEB in C. elegans
(A) HLH-30::GFP animals were reared on E. coli carrying empty vector or egl-30 RNAi, and subsequently infected with S. aureus. Shown are representative epifluorescence micrographs. Hatched boxes indicate areas enlarged in detail. EV, empty vector control RNAi. (B) Quantitative analysis. Data are mean ± SEM (two biological replicates, n ≥ 50 per condition). *** p ≤ 0.001 (two-sample t test). (C) Survival of wild type and egl-30 mutant animals infected with S. aureus. *** p ≤ 0.001 (Log-Rank test). (D) HLH-30::GFP animals were reared on E. coli carrying empty vector or plc-1, plc-2, plc-3, plc-4, or egl-8 RNAi, and subsequently infected with S. aureus. Shown are representative epifluorescence micrographs. (E) Quantitative analysis. Data are mean ± SEM (two biological replicates, n ≥ 50 per condition). *** p ≤ 0.001 (two-sample t test). (F) Animals were treated with dkf-1, plc-1, or egl-30 RNAi and subsequently incubated with 1 μg/ml PMA for 30 min. Shown are representative epifluorescence micrographs (top row). Hatched boxes indicate areas enlarged in detail (bottom row). (G) Quantitative analysis. Data are mean ± SEM (two biological replicates, n ≥ 50 per condition). *** p ≤ 0.001 (two-sample t test). (H) Proposed hypothetical model for HLH-30 regulation by infection.
Figure 3
Figure 3. PKD1 and PKCα/γ are necessary for activation of TFEB by infection
TFEB-GFP RAW264.7 cells were preincubated with PKC and PKD inhibitors for 1 h previous to infection with S. enterica (MOI = 100) for 2 h. Shown are representative images from one replicate, and quantification of three biological replicates of three technical replicates each. (A) DMSO control. (A′) detail. (B) S. enterica SL1344. (B′) detail. (C) 10 μM kb-NB142-70 (PKD inhibitor). (C′) detail. (D) 5 μM Gö 6983 (pan-PKC inhibitor). (D′) detail. (E) 1 mM HBDDE (selective inhibitor of PKCα and PKCγ). (E′) detail. (F) 10 μM LY333531 (selective inhibitor of PKCβ1 and PKCβ2). (F′) detail. (G) Percentage of cells with nuclear translocation was measured with Gen5 analysis software. (H) GFP intensity in nucleus compared to cytoplasm (N/C ratio) was measured using CellProfiler. See Methods for details. ** p ≤ 0.01, *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). Scale bars = 100 μm. (I–M′) TFEB-Flag RAW264.7 cells were infected with Salmonella after shRNA treatment. Shown are anti-FLAG immunofluorescence micrographs. Scale bars = 100 μm. (I) scrambled shRNA control with PBS. (I′) detail. (J) scrambled shRNA control with S. enterica SL1344. (J′) detail. (K) PKD1 shRNA with S. enterica SL1344. (K′) detail. (L) PKD2 shRNA with S. enterica SL1344. (L′) detail. (M) PKD3 shRNA with S. enterica SL1344. (M′) detail. (N) Percentage of cells with nuclear translocation. (O) GFP intensity in nucleus compared to cytoplasm (N/C ratio). ** p ≤ 0.01, *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). (P) Anti- PKD1, PKD2, PKD3, and β actin immunoblots of lysates from sh-PKD1, sh-PKD2, sh-PKD3, and scrambled control cells. (Q) Quantitative analysis of PKD1 immunoblot, normalized to β actin loading control. See also the Figure S2.
Figure 4
Figure 4. Activation of PKC or PKD is sufficient for TFEB activation
TFEB-GFP RAW264.7 cells were preincubated with inhibitors for 1 h previous to addition of 100 ng/ml PMA for 30 min. Shown are representative images from one replicate, and quantification of three biological replicates of three technical replicates each. (A) DMSO control. (A′) detail. (B) DMSO plus PMA. (B′) detail. (C) 10 μM kb-NB142-70 (specific PKD inhibitor). (C′) detail. (D) 5 μM CRT0066101 (specific PKD inhibitor). (D′) detail. (E) 1 mM HBDDE (selective inhibitor of PKCα and PKCγ). (E′) detail. (F) 10 μM LY333531 (PKCβ1 and PKCβ2 inhibitor). (F′) detail. Scale bars = 100 μm. (G) Percentage of cells with nuclear translocation was measured with Gen5 analysis software. (H) GFP intensity in nucleus compared to cytoplasm (N/C ratio) was measured using CellProfiler. Please see Methods for more detail. ** p ≤ 0.01, *** p ≤ 0.001 (One-way ANOVA followed by Tukey’s post-hoc test). (I) Images from immunoblot following addition of 100 ng/ml PMA, primary antibodies are indicated on the left. (J) Quantitative analysis of TFEB immunoblot, normalized to β actin loading control. See also the Figure S3.
Figure 5
Figure 5. PKD and PKCα are quickly activated after infection
(A–N) RAW264.7 cells were infected with S. enterica SL1344 (MOI = 100) for 0 (control), 10, 20, 30, 60, and 120 min, lysed, and subjected to immunoblot analysis. Shown are representative results from three biological replicates. (A) Images from immunoblots. Primary antibodies are indicated on the left. (B–J) Quantitative analysis, normalized to β actin loading control. (K) Images from immunoblots after Salmonella infection plus 10 μM kb-NB142-70 (specific PKD inhibitor). Primary antibodies are indicated on the left. (L) Quantitative analysis, normalized to β actin loading control.
Figure 6
Figure 6. Salmonella enterica must be alive to activate the PKD-TFEB pathway in macrophages
(A) Anti-phospho-PKD immunoblot. RAW264.7 cells were incubated with live or dead S. enterica SL1344 (MOI = 100) for 0 (control), 10, 20, 30, 60, and 120 min, lysed, and subjected to immunoblot analysis. (B,C) Quantitative analysis, normalized to β actin loading control. (D–G) TFEB-GFP RAW264.7 cells were incubated with live or dead S. enterica (MOI = 100) for 2 h. For heat killed condition, bacteria were heated to 75 °C for 1 h and 100% killing was confirmed by culture for 48 h on LB-streptomycin agar at 37°C. For antibiotic-killed bacteria, gentamicin (100 μg/ml) was added to washed bacteria in PBS for 2 h and 100% killing was confirmed by culture for 48 h on LB-streptomycin agar at 37°C. Shown are representative images from one replicate, and quantification of three biological replicates of three technical replicates each. (D) PBS control. (E) Live S. enterica SL1344. (F) Heat-killed S. enterica. (G) Antibiotic-killed S. enterica. (H) Percentage of cells with nuclear translocation was measured with Gen5 analysis software. (I) GFP intensity in nucleus compared to cytoplasm (N/C ratio) was measured using CellProfiler. ** p ≤ 0.01, *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test).
Figure 7
Figure 7. PC-PLC activity is required for TFEB activation by infection
TFEB-GFP RAW264.7 cells were preincubated with PLC inhibitors for 1 h prior to infection with S. enterica (MOI = 100) for 2 h. Shown are representative images from one replicate, and quantification of three biological replicates of three technical replicates each. Scale bars = 100 μm. (A) DMSO control. (A′) detail. (B) S. enterica SL1344. (B′) detail. (C) 50 μM tricyclodecan-9-yl-xanthogenate (D609), which inhibits phosphatidylcholine-specific phospholipase C (PC-PLC). (C′) detail. (D) 50 μM U-73122, which inhibits phosphoinositide-specific phospholipase C (PI-PLC). (D′) detail. (E) 10 μM VU0359595, which inhibits phospholipase D1 (PLD1). (E′) detail. (F) 10 μM CAY10594, which inhibits phospholipase D2 (PLD2). (F′) detail. (G) 10 μM FIPI, which inhibits PLD1 and PLD2. (G′) detail. (H) 10 μM halopemide, which inhibits PLD1 and PLD2. (H′) detail. (I) Percentage of cells with nuclear translocation was measured with Gen5 analysis software. (J) GFP intensity in nucleus compared to cytoplasm (N/C ratio) was measured using CellProfiler. ** p ≤ 0.01, *** p ≤ 0.001 (One-way ANOVA followed by Tukey’s post-hoc test). (K,L) RAW264.7 cells were incubated with 50 μM D609 for 1 hour and then infected with S. enterica SL1344 (MOI = 100) for 0 (control), 10, 20, 30, 60, and 120 min, lysed, and subjected to immunoblot analysis. Shown are representative results from three biological replicates. (K) Images from immunoblots. Primary antibodies are indicated on the left. (L) Quantitative analysis, normalized to β actin loading control. (M) Proposed genetic pathways for signal transduction and activation of TFEB in C. elegans and mammals by infection. * denotes mammalian steps proposed by analogy with C. elegans.
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