Autophagosomes induced by a bacterial Beclin 1 binding protein facilitate obligatory intracellular infection

Abstract

Autophagy, a cytoplasmic catabolic process, plays a critical role in defense against intracellular infection. In turn, evasion or inhibition of autophagy has emerged as an important virulence factor for intracellular pathogens. However, Anaplasma phagocytophilum, the obligatory intracellular bacterium that causes human granulocytic anaplasmosis, replicates in the membrane-bound compartment resembling early autophagosome. Here, we found that Anaplasma translocated substrate 1 (Ats-1), a type IV secretion effector, binds Beclin 1, a subunit of the class III PI3K and Atg14L, and it nucleates autophagosomes with markers of omegasomes, double FYVE-containing protein 1, Atg14L, and LC3. Ats-1 autophagy induction did not activate the starvation signaling pathway of mammalian target of rapamycin. These autophagy proteins were also localized to the Anaplasma inclusion. Ectopically expressed Ats-1 targeted the Anaplasma inclusions and enhanced infection, whereas host cytoplasmic delivery of anti-Ats-1 or Beclin 1 depletion by siRNA suppressed the infection; beclin 1 heterozygous-deficient mice were resistant to Anaplasma infection. Furthermore, Anaplasma growth arrest by the class III PI3K inhibitor 3-methyladenine was alleviated by essential amino acid supplementation. Thus, Anaplasma actively induces autophagy by secreting Ats-1 that hijacks the Beclin 1-Atg14L autophagy initiation pathway likely to acquire host nutrients for its growth.

Fig. 1.

Ats-1 binds Beclin 1 and triggers vesicle formation. (A–C) Co-IP assay for protein interaction between Ats-1 (without a tag) and HA-Beclin 1 (A), Ats-1(90–250)-GFP and HA-Beclin 1(1–272) (B), and Ats-1(218–376)-GFP and HA-Beclin 1(1–272) (C). Cotransfected HEK293 cells were immunoprecipitated (IP) with preimmune IgG or anti (α)–Ats-1. Precipitates were immunoblotted (IB) with anti-HA and anti–Ats-1. IgG, rabbit IgG heavy chain. (D) Ats-1–GFP forms clusters of vesicles of ∼0.5–1 μm in diameter in transfected RF/6A cells. (E) Localization of HA-Beclin 1 to vesicles containing tag-less Ats-1 in cotransfected RF/6A cells. (F) Ats-1–GFP vesicles are distinct from mitochondria labeled with anticytochrome c. (Scale bars: D–F, 10 μm.) Insets show magnified boxed areas.

Fig. 2.

Ats-1 nucleates autophagosomes involving ER autophagy proteins. (A and B) Co-IP assay for protein interaction between Ats-1–GFP and HA-Atg14L (A) or Myc-UVRAG (B). Cotransfected HEK293 cells were immunoprecipitated (IP) with preimmune IgG or anti (α)–Ats-1. Precipitates were immunoblotted (IB) with anti–Ats-1 and anti-HA or anti-Myc. (C) The diffuse pattern of Ats-1(∆N17)-GFP in transfected RF/6A cells. (D and E) Co-IP assay for protein interaction between HA-Atg14L, and Ats-1(∆N17)-GFP (D) or Cox8A-Ats-1(∆N17)-GFP (E). Cotransfected HEK293 cells were IP with preimmune IgG or α–Ats-1. Precipitates were IB with anti–Ats-1 and anti-HA. (F) Localization of Cox8A-Ats-1(∆N17)-GFP (green) with mitochondria labeled with anti–Mn-Sod (red). The boxed area is magnified on the right. (G–I) Localization of HA-Atg14L (G), HA-DFCP1 (H), and HA-LC3 (I) to Ats-1–GFP vesicles. Boxed areas are magnified on the right. (Scale bars: C, E, and G–I, 10 μm.) (J) Correlative light-electron microscopy for RF/6A cells expressing Ats-1–GFP or GFP alone. (i and iv) Fluorescence micrographs of cells expressing Ats-1–GFP and GFP, respectively. (Scale bars: 10 μm.) (ii and v) TEM of the fluorescent cells in i and iv. (Scale bars: 10 μm.) (iii and vi) Magnified boxed area in ii and v. Yellow pseudocolored areas denote autophagosomes. (Scale bars: 1.0 μm.) (K) Phosphorylated mTOR level in Ats-1–GFP-expressing cells. RF/6A cells transfected with plasmid encoding GFP or Ats-1–GFP were subjected to Western blot analysis using antibodies against actin, mTOR, and phosphorylated mTOR (p-mTOR). Rapamycin-treated RF/6A cells were used as positive control. The values under the bands show the relative ratios of band intensities of p-mTOR to actin, with the ratio in the GFP lane set as one.

Fig. 3.

Ats-1 localizes to Ap inclusions. (A) Ats-1–GFP targets Ap inclusions. Live Ats-1–GFP-expressing RF/6A cells were infected with mCherry-Ap. Bacterial inclusions surrounded by Ats-1–GFP in two cells are shown. (Scale bars: 10 μm.) (B) Localization of native Ats-1 on the cytoplasmic side of Ap inclusions. The plasma membrane of infected cells was selectively permeabilized with SLO and labeled with anti–Ats-1 (Ats-1/SLO) and anti-P44 (P44/SLO). After the first round of staining, all cell membranes were permeabilized with saponin, and cells were stained again with anti-P44 (P44/saponin). Secondary antibodies with distinct fluorochromes were used for P44 labeling before (red) and after (blue and gray pseudocolor) saponin treatment. Contours of the HL-60 cell are marked with white dashed lines. Ap, Ap inclusions. N, nucleus. (Scale bar: 5 μm.) (C) Immuno-EM for Ats-1 in infected cells. Yellow pseudocolored area, Ap inclusion. The arrow indicates double membranes. (Scale bar: 0.5 μm.)

Fig. 4.

Autophagy proteins localize to Ap inclusions. (A) Ap-infected cells at 2 d postinfection (p.i.). Ap morulae/inclusions are indicated with arrows in Diff-Quik staining. N, nucleus. (Scale bar: 5 μm.) (B) Ap-infected RF/6A cells expressing GFP, HA-Atg14L, HA-DFCP1, or GFP-LC3 were immunostained with anti-Ap and anti-HA at 2 d p.i. N, nucleus. Boxed areas are magnified on the right. (Scale bars: 10 μm.) Arrowheads indicate the presence of GFP-LC3 inside of Ap inclusions. (C) Immunogold labeling for GFP-LC3 in Ap-infected GFP-LC3–expressing RF/6A cells. Yellow pseudocolored area, Ap inclusion. An arrowhead indicates the GFP-LC3 inside of Ap inclusion. An asterisk indicates Ap. (D) Selected sequential video images of a single inclusion containing an mCherry-Ap (red) in live RF/6A cells transfected with plasmid encoding GFP-LC3 (green). GFP-LC3 puncta inside of Ap inclusion are indicated with arrowheads. (Scale bar, 4 μm.) (E) An autophagosome (arrow) is closely apposed to the Ap (asterisk) inclusion membrane in HL-60 cells. (F) An Ap inclusion containing autophagic bodies (arrowheads). Note that the content of autophagic vesicles is similar to the host cell cytoplasm. (C, E, and F) TEM. (Scale bars: 0.5 μm.) (G) Phosphorylated mTOR level in Ap-infected HL-60 cells harvested at 4, 12, 24, 48, and 72 h p.i. Western blot analysis using antibodies against α-tubulin, mTOR, and p-mTOR. HL-60, uninfected HL-60 cells. Rapamycin-treated HL-60 cells were used as positive control. The values under the bands show the relative ratios of band intensities of p-mTOR to tubulin, with the ratio in HL-60 lane set as one.

Fig. 5.

Cellular fractionation of Ap-infected cells. Immunoblotting of fractions (1–12) of Ap-infected HL-60 cells (I) and uninfected HL-60 cells (U) collected after OptiPrep density gradient centrifugation using anti–Ats-1 and various organelle markers: anti-LC3 (autophagosome), anticalnexin (ER), anti–LAMP-2 (lysosomes and late endosomes), anti-EEA1 (early endosomes), and anti-P44 (Ap inclusions).

Fig. 6.

Ats-1 promotes Ap infection. Ap infection (A and B) and autophagy induction (B) in HL-60 cells 2 d after cytoplasmic delivery of anti–Ats-1 or preimmune IgG by the Chariot system. (A) Percent infected cells. Data are shown as the mean ± SD from three independent experiments. *Significantly different (P < 0.01). (B) Immunoblotting using anti-P44, antitubulin, and anti-LC3. The values under the bands show the relative ratios of band intensities, with the ratios of those band intensities from preimmune IgG control set as one. (C) Ap infection in RF/6A cells transfected with plasmid encoding GFP or Ats-1–GFP [0.5 d posttransfection (p.t.), 3 d p.i.]. Immunoblot analysis using anti-P44 and antiactin. The values under the bands show the relative ratios of band intensities, with the ratios of those band intensities from GFP set as one. The expressions of GFP and Ats-1–GFP in transfected cells are shown at the bottom.

Fig. 7.

Beclin 1 depletion impairs Ap infection, and amino acid supplementation overrides 3-MA inhibition of Ap replication. (A) Immunoblotting of lysates of Ap-infected RF/6A cells transfected with beclin 1 siRNA or scrambled siRNA (2 d p.t., 3 d p.i.) using anti-P44, antiactin, and anti-Beclin 1. The values under the bands show the relative ratios of band intensities, with the ratios of those band intensities from scrambled siRNA set as one. (B) The dot plot of Ap load in the blood from beclin 1^+/− and WT mice at 5 d p.i. Quantitative PCR of Ap 16S rDNA normalized to mouse G3PDH DNA. *Significantly different (P < 0.05). (C) Ap-infected cells: untreated (Control), or treated with amino acids supplementation (AA), 3-MA (3-MA), or 3-MA and amino acids (3-MA + AA). Diff-Quik staining. (Scale bar: 10 μm.) (D) Quantitative PCR of Ap 16S rDNA normalized to human G3PDH DNA. *Significantly different (P < 0.05) by ANOVA. (E and F) The effect of MG-132 on Ap growth. Diff-Quik–stained images of Ap in solvent control or MG-132–treated HL-60 cells (E) and the percentage of Ap infected HL-60 cells on 3 d p.i. (F). (Scale bar: 10 μm.) (G–I) The effect of DFP on Ap growth. Host cell-free Ap pretreated with solvent control or DFP was used to infect HL-60 cells (G). HL-60 cells were pretreated with solvent or DFP followed by washing and infection with Ap (H). HL-60 cells were infected with Ap followed by the treatment with solvent or DFP at 1 d p.i. (I). The relative ratios of Ap/cell were shown, with the ratios from control set as one. *Significantly different (P < 0.01).

Fig. 8.

Proposed model of autophagosome nucleation by Ats-1 in Ap-infected cells. Ats-1 is translocated from Ap to the cytoplasm of infected cells by the type IV secretion system. A portion of Ats-1 interacts with the host autophagosome initiation complex (Atg14L-Beclin 1-Vps34), stimulating the formation of omegasomes in ER. Another portion of Ats-1 targets mitochondria, where it exerts antiapoptotic activity. The omegasome is marked with DFCP1. N terminus of Ats-1 is required for Atg14L recruiting and thus, autophagosome formation, but it is cleaved off when Ats-1 is imported into mitochondria. The isolation membrane elongates to envelop the cytoplasmic content into the double-membrane vacuole, the autophagosome, which is decorated with LC3. Ats-1 autophagosomes are recruited to Ap inclusion, and the outer membrane fuses with the Ap inclusion membrane, resulting in the release of the autophagic body-like content into the Ap inclusion.