p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy pathways

Abstract

Autophagy is an important mechanism of innate immune defense. We have recently shown that autophagy components are recruited with septins, a new and increasingly characterized cytoskeleton component, to intracytosolic Shigella that have started to polymerize actin. On the other hand, intracytosolic Listeria avoids autophagy recognition by expressing ActA, a bacterial effector required for actin polymerization. Here, we exploit Shigella and Listeria as intracytosolic tools to characterize different pathways of selective autophagy. We show that the ubiquitin-binding adaptor proteins p62 and NDP52 target Shigella to an autophagy pathway dependent upon septin and actin. In contrast, p62 or NDP52 targets the Listeria ActA mutant to an autophagy pathway independent of septin or actin. TNF-α, a host cytokine produced upon bacterial infection, stimulates p62-mediated autophagic activity and restricts the survival of Shigella and the Listeria ActA mutant. These data provide a new molecular framework to understand the emerging complexity of autophagy and its ability to achieve specific clearance of intracytosolic bacteria.

FIGURE 1.

NDP52 recruitment to Shigella. A, HeLa cells were infected with S. flexneri for 4 h 40 min, fixed for fluorescent light microscopy, and stained with antibodies to NDP52 and ubiquitin (FK2). Scale bar, 1 μm. Similar images were obtained labeling for FK1. B, HeLa cells were transfected with SEPT6-GFP, infected with S. flexneri for 4 h 40 min, fixed for fluorescent light microscopy, and stained with antibodies to NDP52. Scale bar, 1 μm. Similar images were obtained for cells transfected with SEPT2 or SEPT9 fluorescent constructs. C, HeLa cells were treated with control (CTRL), p62, SEPT2, or SEPT9 siRNA. Whole cell lysates of siRNA-treated cells were immunoblotted for GAPDH, p62, SEPT2, or SEPT9 to show the efficiency of p62, SEPT2, or SEPT9 depletion (top). After 4 h 4 0min of infection with S. flexneri, cells were fixed and labeled for quantitative microscopy. Graphs represent the mean percent ± S.E. (error bars) of Shigella with NDP52 recruitment from three independent experiments per treatment. p values, Student's t test. D, HeLa cells were transfected with SEPT6-GFP, infected with S. flexneri for 4 h 40 min, fixed for fluorescent light microscopy, and stained with antibodies to p62 and NDP52. Scale bar, 1 μm. Similar images were obtained for cells transfected with SEPT2 or SEPT9 fluorescent constructs. E, HeLa cells were treated with control (CTRL), p62, or NDP52 siRNA. Whole cell lysates of siRNA-treated cells were immunoblotted for GAPDH, p62, or NDP52 to show the efficiency of p62 or NDP52 depletion (top). After 4 h 40 min of infection with S. flexneri, cells were fixed and labeled for quantitative microscopy. Graphs represent the mean percent ± S.E. of Shigella with p62 or NDP52 recruitment from three independent experiments per treatment. p values, Student's t test.

FIGURE 2.

NDP52 is required for p62-mediated autophagic activity and vice versa. HeLa cells were treated with control (CTRL), p62, or NDP52 siRNA, treated or not with bafilomycin, and immunoblotted for GAPDH, p62, or NDP52. Representative blots from three independent experiments are shown.

FIGURE 3.

NDP52 and p62 are recruited independently to Listeria. A, HeLa cells were infected with L. monocytogenes for 3 h and fixed and labeled for quantitative microscopy. Graphs represent the mean percent ± S.E. (error bars) of Listeria EGD or EGDΔactA having recruited ubiquitin (Ub), p62, or NDP52 from three independent experiments per strain. p values, Student's t test. B, HeLa cells were infected with L. monocytogenes EGDΔactA for 3 h, fixed for fluorescent light microscopy, and stained with antibodies to p62 and NDP52. White arrowheads point to representative bacteria shown in inset. Scale bar, 1 μm. C, HeLa cells were treated with control (CTRL), p62, NDP52, SEPT2, or SEPT9 siRNA, infected with L. monocytogenes EGDΔactA for 3 h and fixed and labeled for quantitative microscopy. Graphs represent the mean percent ± S.E. of Listeria with p62 or NDP52 recruitment from three independent experiments per treatment. p values, Student's t test.

FIGURE 4.

NDP52 and p62 recruitment to Shigella depends on actin and Shigella IcsB. A, HeLa cells were infected with S. flexneri, treated with dimethyl sulfoxide (DMSO) or cytochalasin D (CytD), and after 4 h 40 min were fixed and labeled for quantitative microscopy. Graphs represent the mean percent ± S.E. (error bars) (of Shigella having recruited ubiquitin (Ub), p62, or NDP52 from three independent experiments per treatment. p values, Student's t test. B, HeLa cells were infected with S. flexneri M90TΔicsB for 4 h 40 min, fixed for fluorescent light microscopy, and stained with antibodies to p62 and NDP52. Scale bar, 1 μm. C, HeLa cells were infected with S. flexneri for 4 h 40 min and fixed and labeled for quantitative microscopy. Graphs represent the mean percent ± S.E. of Shigella M90T or M90TΔicsB having recruited ubiquitin, p62, or NDP52 from three independent experiments per strain. p values, Student's t test.

FIGURE 5.

Autophagic activity is stimulated by TNF-α. A, untreated or TNF-α-treated HeLa cells were infected with S. flexneri for 4 h 40 min and fixed and labeled for quantitative microscopy. Graphs represent the mean percent ± S.E. (error bars) of Shigella having recruited p62 or NDP52 from three independent experiments per treatment. p values, Student's t test. B, gentamicin survival assays for S. flexneri were performed in HeLa cells treated or not TNF-α. Graphs represent the relative number (i.e. normalized to −TNF-α cells) of colony-forming units (CFU) counted from cells after 4 h 40 min. On the graph −TNF-α is figuratively presented as 1, and data represent the mean ± S.E. from n ≥ 3 experiments. p values, Student's t test. C, gentamicin survival assays for L. monocytogenes EGDΔactA were performed in HeLa cells treated or not with TNF-α. Graphs represent the relative number (i.e. normalized to −TNF-α cells) of colony-forming units counted from cells after 4 h 40 min. On the graph −TNF-α is figuratively presented as 1, and data represent the mean ± S.E. from n ≥ 3 experiments. p values, Student's t test. D, untreated or TNF-α-treated HeLa cells were treated or not with bafilomycin and immunoblotted for GAPDH, p62, NDP52, or LC3-II. Representative blots from three independent experiments are shown. A representative blot showing the ratio of LC3-I:II for TNF-α-treated cells in the presence and absence of bafilomycin is provided as supplemental Fig. S1A. E, p62-GFP reporter cells were treated or not with TNF-α. Cells were treated for 24 h with tetracycline (i.e. On), and after 16 h in the absence of tetracycline (i.e. Off), cells were fixed and labeled for flow cytometry to measure levels of p62-GFP. Values provided are the mean ± S.E. from three triplicate experiments. One representative analysis comparing untreated and TNF-α-treated cells of three is shown, and the others are shown as supplemental Fig. S1B. F, p62 and NDP52 mRNA expression following TNF-α stimulation are shown. p62 and NDP52 cDNA expression was determined by real-time PCR in HeLa cells following 0, 12, or 24 h of stimulation with TNF-α. Data are represented as relative -fold change (ddCt units), and the housekeeping gene used for normalization was GUS. Values provided are mean ± S.E. from n ≥ 3 independent experiments per treatment.