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p62 and NDP52 Proteins Target Intracytosolic Shigella and Listeria to Different Autophagy Pathways

p62 and NDP52 Proteins Target Intracytosolic Shigella and Listeria to Different Autophagy Pathways THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 30, pp. 26987–26995, July 29, 2011 © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. p62 and NDP52 Proteins Target Intracytosolic Shigella and □ S Listeria to Different Autophagy Pathways Received for publication, January 21, 2011, and in revised form, May 22, 2011 Published, JBC Papers in Press, June 6, 2011, DOI 10.1074/jbc.M111.223610 ‡§¶1  ‡§¶ ‡‡ ‡‡ Serge Mostowy , Vanessa Sancho-Shimizu **,Me´lanie Anne Hamon , Roxane Simeone , Roland Brosch , §§ ‡§¶2 Terje Johansen , and Pascale Cossart ‡ § From the Institut Pasteur, Unite´ des Interactions Bacte´ries-Cellules, Paris F-75015, France, INSERM U604, Paris F-75015, France, Institut National de la Recherche Agronomique (INRA), USC2020, Paris F-75015, France, the Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U980, Necker Medical School, Paris 75015 France, the **University Paris Descartes, ‡‡ Paris 75015, France, the Institut Pasteur, Unite´ Postulante Pathoge´nomique Mycobacte´rienne Inte´gre´e, Paris F-75015, France, §§ and the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway Autophagy is an important mechanism of innate immune binding proteins in selective autophagy, p62 (sequestosome 1; defense. We have recently shown that autophagy components SQSTM1) has emerged as the prototype autophagy receptor are recruited with septins, a new and increasingly characterized (7). p62 is an LC3 interaction partner in vivo and is constantly cytoskeleton component, to intracytosolic Shigella that have degraded by autophagy, establishing it as a useful marker for started to polymerize actin. On the other hand, intracytosolic autophagic vesicle turnover (8). NDP52 has more recently Listeria avoids autophagy recognition by expressing ActA, a emerged as another autophagy receptor and shares with p62 bacterial effector required for actin polymerization. Here, we the ability to bind LC3 and ubiquitinated cargo simultaneously exploit Shigella and Listeria as intracytosolic tools to character- (9). The respective roles of p62 and NDP52 are not understood. ize different pathways of selective autophagy. We show that the Whether these individual autophagy receptors recognize dif- ubiquitin-binding adaptor proteins p62 and NDP52 target Shi- ferent ubiquitinated proteins and/or perform independent gella to an autophagy pathway dependent upon septin and actin. functions in cells may be critical for the complete understand- In contrast, p62 or NDP52 targets the Listeria ActA mutant to ing of autophagy and its therapeutic potential. an autophagy pathway independent of septin or actin. TNF-,a Recent evidence has implicated the cytoskeleton as a critical host cytokine produced upon bacterial infection, stimulates mediator of selective autophagy. We have shown that septins, a p62-mediated autophagic activity and restricts the survival of novel component of the cytoskeleton (10), are recruited with Shigella and the Listeria ActA mutant. These data provide a new autophagy proteins to “cage” Shigella flexneri in the cytosol of molecular framework to understand the emerging complexity of infected cells and restrict bacterial dissemination (11). These autophagy and its ability to achieve specific clearance of intra- results suggest an interdependence between the two evolution- cytosolic bacteria. arily conserved processes of septin assembly and autophagy: when septins are absent, autophagy markers accumulate less, and vice versa. However, in the case of Listeria monocytogenes, Autophagy is an evolutionarily conserved catabolic pathway a bacterial pathogen that also escapes to the cytosol, no efficient that allows eukaryotes to degrade and recycle intracellular septin caging has been observed (11). Listeria has been reported components by sequestering proteins and organelles in special- to avoid autophagic recognition by expressing ActA, a bacterial ized double-membrane vesicles named autophagosomes (1–3). effector required for actin tail motility (12, 13). Therefore Lis- Although autophagosomes can sequester cytosolic material teria evades both septin caging and autophagy via its surface nonspecifically, e.g. as a response to starvation, there is increas- expression of ActA (11). Given these fundamental differences ing evidence for selective autophagic degradation of various between Shigella and Listeria, it is clear that in-depth investi- cellular structures, including protein aggregates, mitochondria, gation of these two bacteria will help to describe precisely the and microbes (4, 5). The mechanism of selective autophagy is coordination among actin, septin, and selective autophagy. not well understood, yet the role of ubiquitin in this process is The role of p62 and/or NDP52 in selective autophagy of Sal- critical (5, 6). By simultaneous binding to both ubiquitin and monella enterica serovar Typhimurium (S. typhimurium) has the autophagosome-associated ubiquitin-like proteins (i.e. recently been characterized (9, 14, 15). It has been proposed LC3/GABARAP proteins) autophagy receptors can mediate that p62 and NDP52 act independently to drive efficient bacte- docking of ubiquitinated cargo to the autophagosome, thereby rial autophagy of S. typhimurium within Salmonella-contain- ensuring their selective degradation (5, 6). Of the ubiquitin- ing vacuoles (15). To assess the role of p62 and NDP52 in the autophagy of bacterial pathogens that escape to the cytosol and □ S The on-line version of this article (available at http://www.jbc.org) contains to determine the role of the actin/septin cytoskeleton in this supplemental Figs. S1–S4. process, we examined these components in the case of Shigella To whom correspondence may be addressed. Tel.: 33-1-40-61-32-69; Fax: 33-1-45-68-87-06; E-mail: [email protected]. International research scholar from the Howard Hughes Medical Institute and recipient of European Research Council Advanced Grant Award The abbreviations used are: NDP52, 52-kDa nuclear dot protein; CFP, 233348. To whom correspondence may be addressed. Tel.: 33-1-40-61-30- cyan fluorescent protein; IF, immunofluorescence; LC3, light chain 3; 32; Fax: 33-1-45-68-87-06; E-mail: [email protected]. SEPT, Septin. This is an Open Access article under the CC BY license. JULY 29, 2011• VOLUME 286 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 26987 Autophagy of Shigella versus Listeria and Listeria and reveal that these different intracytosolic bac- Listeria was added to host cells at a multiplicity of infection of teria are targeted to different pathways of selective autophagy. 100. Bacteria and cells were centrifuged at 1000 g for 1 min at 21 °C and, then incubated at 37 °C and 10% CO for 1 h, washed EXPERIMENTAL PROCEDURES with minimum essential medium, and incubated with fresh Bacteria and Cell Culture—L. monocytogenes EGD (BUG gentamicin-containing complete medium (10 g/ml) for an 600) and EGDactA (BUG 2140) were grown overnight at 37 °C additional 2 h, after which they were washed with 1 PBS and in brain heart infusion medium (Difco Laboratories), diluted fixed and processed for IF. Shigella was added to cells at an 15  in fresh brain heart infusion medium, and cultured until multiplicity of infection of 100 (for quantification analyses), or A  0.8. S. flexneri M90T (BUG 2505), M90TicsA (BUG 400l of growth (A  0.6) was diluted in minimum essen- 600 nm 600 nm 1791), and M90TicsB were cultured overnight in trypticase tial medium and added directly to cells (for imaging analyses). soy, diluted 80  in fresh trypticase soy, and cultured until Bacteria and cells were centrifuged at 700 g for 10 min at 21 °C A  0.6. Mycobacterium marinum M-DsRed were cul- and then placed at 37 °C and 10% CO for 30 min, washed with 600 nm 2 tured at 30 °C in Middlebrook 7H9 (BD Biosciences) supple- minimum essential medium, and incubated with fresh gentam- g/ml) for 4 h, after mented with 0.2% glycerol, 0.05% Tween 80, and 10% ADC icin-containing complete medium (50 Enrichment (Fisher Scientific), diluted 48 h prior to infection in which they were washed with 1 PBS and fixed and processed fresh medium, and cultured until A  0.6. for IF. For infection of cells with M. marinum,2  10 RAW 600 nm HeLa (American Type Culture Collection (ATCC) CCL-2) macrophages or mouse embryonic fibroblasts were plated onto cells were cultured in minimum essential medium plus Glu- glass coverslips in 6-well plates and used for experiments 48 h taMAX (Invitrogen) supplemented with 1 mM sodium pyruvate later. M. marinum was washed twice in 1 PBS and passaged (Invitrogen), 0.1 mM nonessential amino acid solution (Invitro- through a 26-gauge needle. Bacteria were added to cells in gen), and 10% fetal calf serum (FCS). p62-GFP cells were cul- medium without FCS at an multiplicity of infection of 10 tured in DMEM plus GlutaMAX supplemented with 15 g/ml (RAW) or 100 (mouse embryonic fibroblasts). Bacteria and hygromycin, 200 g/ml blasticidin, and 10% FCS (16). Cells cells were centrifuged at 700 g for 5 min at 21 °C and then were grown at 37 °C and 10% CO . RAW 264.7 cells (ATCC placed for2hat32 °Cand5%CO . Infected cells were then 2 2 TIB-71) were cultured in DMEM plus GlutaMAX supple- washed with 1 PBS and incubated with 200 g/ml amikacin M glutamine (Invitrogen), 1 mM sodium pyru- for2hat32 °Cand5%CO mented with 2 m . After this time, macrophages were vate, and 10% FCS. Cells were grown at 37 °C and 10% CO . washed with 1 PBS and incubated in complete medium at Mouse embryonic fibroblasts from C57BL/6 mice were main- 32 °C and 5% CO for 20 or 44 h. After a total of 24 or 48 h, tained in DMEM containing 10% FCS. infected cells were washed with 1 PBS and fixed and pro- Antibodies—Rabbit polyclonal antibodies used were anti- cessed for IF. SEPT2 (R170) and anti-SEPT9 (R69) (11), anti-p62 (Clini- Images were acquired on a fluorescence inverted microscope science, PM045), anti-NDP52 (AbCam, ab68588), and anti- Axiovert 200 M (Carl Zeiss MicroImaging, Inc.) equipped with LC3/Atg8 (Novus Biologicals, NB100-2331). The anti-NBR1 a cooled digital charge-coupled device camera (Cool SNAPHQ, (Abnova, H00004077-A01) used was a mouse polyclonal anti- Photometrics) driven by Metamorph Imaging System software body. Mouse monoclonal antibodies used were anti-GAPDH (Universal Imaging Corp). For three-dimensional representa- (AbCam, 6C5), FK2 (Enzo Life Sciences, PW8810), FK1 (Enzo tion, quantitative microscopy (i.e. counting autophagy receptor Life Sciences, PW8805), and anti-p62 lck ligand (BD Biosci- recruitment) was performed using Z-stack image series of ences, 610832). Secondary antibodies used were Cy5- (Jackson infected cells, counting 250–1000 bacteria/experiment. Images ImmunoResearch Laboratories), Alexa Fluor 488-, or Alexa were processed using ImageJ. Where mentioned, IF micros- Fluor 546-conjugated goat anti-rabbit or goat anti-mouse copy was performed with SEPT2-YFP (BUG 2444), SEPT6-GFP (Molecular Probes). F-actin was labeled with Alexa Fluor 488-, (BUG 2445), SEPT9-CFP (BUG 2309), SEPT9-tdTomato (BUG 546-, or 647-phalloidin (Molecular Probes). 2723), or GFP-LC3 (BUG 3046) (11). Cells were transfected For immunoblotting, total cellular extracts were blotted with with jetPEI (PolyPlus Transfection). the above-mentioned antibodies followed by peroxidase-con- siRNA, Pharmacological Inhibitors, and Cytokine Treatment— jugated goat anti-mouse or anti-rabbit antibodies (Biosys Lab- 0.8 10 HeLa cells were plated in 6-well plates and transfected oratories). GAPDH was used throughout as a loading control. the following day using Oligofectamine (Invitrogen). Control Proteins were run on 8, 10, or 14% acrylamide gels. siRNA (AM4635) as well as predesigned siRNA for SEPT2 Infections and Microscopy—1–1.5  10 HeLa cells were (14709), SEPT9 (18228), NDP52 (s19994), and NBR1 (s8381) plated on glass coverslips in 6-well plates (Techno Plastic Prod- were all from Ambion. siRNA sequences for p62 were taken from Refs. 11, 17. Cells were tested 72 h after siRNA ucts) and used for experiments 48 h later. Cells on coverslips were fixed for 15 min in 4% paraformaldehyde and then washed transfection. with 1 PBS and processed for immunofluorescence (IF). After For experiments involving pharmacological inhibitors, HeLa 10 min of incubation in 50 mM ammonium chloride, cells were cells were infected and treated for 30 min prior to fixation with permeabilized for 4 min with 0.1% Triton X-100 and then incu- dimethyl sulfoxide or cytochalasin D (5 M). Cytochalasin D bated in 1 PBS. Incubation with primary or secondary anti- was suspended in dimethyl sulfoxide and handled as suggested bodies was performed in 1 PBS. Vectashield hard set mount- by the manufacturer (Sigma). To monitor autophagic flux, cells ing medium with DAPI (Vector Laboratories) or mounting were treated with bafilomycin A1 (Sigma, B1793) for 12 h (160 medium for IF (Interchim) was used. nM). For experiments involving TNF- (R&D Systems, 210- 26988 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 30 •JULY 29, 2011 Autophagy of Shigella versus Listeria TA), cells were treated with 20 ng/ml TNF- for 12, 20, or 24 h. co-localization of NDP52 with ubiquitinated proteins Treatment was continued throughout infection. For experi- around Shigella (Fig. 1A) and showed that 84  3% of Shigel- ments involving IL-1 (R&D Systems, 201-LB) or IFN- la-septin cages recruited NDP52 (means  S.E. from n  3 (Imukin, Roche Applied Biosciences), cells were treated with 20 experiments) (Fig. 1B). To determine whether NDP52 ng/ml or 5000 units/ml, respectively, for 12 or 24 h. recruitment is dependent upon septins, we used siRNA to Gentamicin Survival Assays—Gentamicin survival assays deplete cells of SEPT2 or SEPT9 and evaluated NDP52 were adapted from Refs. 18, 19. Cells treated with or without recruitment to Shigella in these siRNA-treated cells. In both TNF- were incubated with Shigella or Listeria as detailed above. cases, NDP52 recruitment was significantly reduced (3.8 Cells were washed and then lysed with distilled H O. The number 0.4-fold or 3.0  0.6-fold, respectively) (Fig. 1C), concordant with the view that septins play a role in the recruitment of of viable bacteria released from the cells was assessed by plating on Luria Bertani (LB) or brain heart infusion agar plates. autophagic markers (11). Flow Cytometry—Gene expression for p62-GFP cells was We then asked whether p62 and NDP52 were recruited to induced (i.e. “On”) by adding 1 g/ml tetracycline (Sigma, the same Shigella-septin cage. Indeed, 83  2% of Shigella- T7660) to the culture medium for 24 h (16). After these 24 h, septin cages recruited both p62 and NDP52 (mean  S.E. from gene expression was turned off (i.e. “Off”) by washing cells twice n  3 experiments) (Fig. 1D). To determine whether p62 and with DMEM and incubating with fresh tetracycline-free com- NDP52 have independent roles at the Shigella-septin cage, we plete medium for 16 h (16). After these times, treated p62-GFP used siRNA to deplete cells of p62 or NDP52 and evaluated cells were washed in 1 PBS, detached with 0.05% EDTA-tryp- adaptor recruitment to Shigella in these siRNA-treated cells. sin, and permeabilized/fixed using BD Cytofix/Cytoperm Fixa- Strikingly, p62 or NDP52 recruitment was significantly reduced tion/Permeabilization kit (BD Biosciences, 554714). Samples in either case (31.1  5.1-fold or 2.4  0.3-fold in p62-depleted were analyzed using a FACSCalibur instrument (BD Biosci- cells, respectively, or 6.6  2.3-fold or 32.6  14.6-fold in ences). Dead cells were excluded on the basis of forward and NDP52-depleted cells, respectively) (Fig. 1E). These results side scatter, and a minimum of 10,000 events were acquired per strongly suggest an interdependent relationship between p62 sample. Results were analyzed using FlowJo software. and NDP52 to target Shigella toward autophagy. Immunoprecipitation Studies—HeLa cells (uninfected) NDP52 Regulates p62-mediated Autophagic Activity and treated or not with bafilomycin, or HeLa cells infected with Vice Versa—p62 is constantly degraded by autophagy and has Shigella or the Listeria ActA mutant in the absence and pres- thus been established as a marker for autophagic activity, i.e. ence of TNF-, were lysed in Igepal buffer (20 mM Tris, pH 8.0, autophagic flux (8). To address the role of NDP52 in autophagic 1% (v/v) Igepal CA-630 (Sigma), 150 mM NaCl, 10% (v/v) glyc- activity, we examined the steady-state levels of p62 in the erol, and protease inhibitors mixture). p62, NDP52, SEPT2, or absence and presence of bafilomycin (an inhibitor of autopha- SEPT9 was immunoprecipitated from 800 g of the total pro- gosome-lysosome fusion) in control cells or in cells depleted of tein extracts by the addition of 1 g of anti-p62, anti-NDP52, NDP52. Levels of p62 were significantly reduced in cells anti-SEPT2, or anti-SEPT9 antibody and 40 l of a 50% slurry depleted for NDP52 in the absence (2.1  0.4-fold) and pres- suspension of protein A-Sepharose beads (Amersham Biosci- ence (1.5 0.1-fold) of bafilomycin (Fig. 2), suggesting that p62 ences). Samples were analyzed by SDS-PAGE and immunoblotted and NDP52 are interdependent to promote autophagy. We with anti-p62, anti-NDP52, anti-SEPT2, or anti-SEPT9 antibody. next examined steady-state levels of NDP52. Levels of NDP52 HRP-conjugated goat anti-mouse or anti-rabbit secondary anti- accumulated in control cells treated with bafilomycin (3.9 bodies were subsequently used. Protein input was evaluated by 0.5-fold), indicating that NDP52, like p62, can be used as a probing blots of cell lysates prior to the immunoprecipitation step marker of autophagic vesicle turnover (Fig. 2). In cells depleted using antibodies specific to p62, NDP52, SEPT2, or SEPT9. for p62, levels of NDP52 were significantly reduced in the Gene Expression Studies—Cells were treated with TNF-, absence (1.5  0.1-fold) and presence (1.2  0.1-fold) of bafi- IL-1, or IFN- for 0, 12, or 24 h. After this time RNA was lomycin (Fig. 2). Thus, p62 can significantly regulate NDP52 extracted using an RNeasy mini-kit (Qiagen) and converted to activity and vice versa. cDNA using the High Capacity RNA-to-cDNA mastermix p62 and NDP52 Are Recruited Independently to Listeria— (Applied Biosystems 4375575). Gene expression probes Different pathogens have evolved different ways to escape (Applied Biosystems) included GUS (4326320E), p62 autophagy (20). In the case of L. monocytogenes, these bacteria (Hs00177654_m1), and NDP52 (Hs00414663_m1). Gene avoid ubiquitination, p62 recognition, and septin caging by expression was evaluated using the ddCt method, and levels of expressing the surface protein ActA, a bacterial effector p62 and NDP52 were normalized to GUS. required to polymerize actin (11, 12). We addressed the role of NDP52 and showed that L. monocytogenes EGDactA, but not RESULTS the wild-type strain EGD, is recognized by ubiquitin, p62, and The Recruitment of p62 and NDP52 to Shigella Is NDP52 (Fig. 3A). 95  2% of EGDactA recruiting p62 also Interdependent—We have reported previously that p62 is recruited NDP52 (mean S.E. from n 3 experiments), show- recruited to intracytosolic Shigella entrapped by septin cages, ing that p62 and NDP52 were recruited to the same bacteria and the recruitment of p62 is dependent upon SEPT2 and (Fig. 3B). SEPT9 (11). NDP52 shares with p62 the ability to bind ubiq- We used siRNA to deplete cells of p62, NDP52, SEPT2, or uitin-coated S. typhimurium (9, 14, 15). We thus addressed SEPT9 and evaluated adaptor recruitment to EGDactA in the recruitment of NDP52 to Shigella. We first observed the these siRNA-treated cells. Unlike what was observed for Shi- JULY 29, 2011• VOLUME 286 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 26989 Autophagy of Shigella versus Listeria FIGURE 1. NDP52 recruitment to Shigella. A, HeLa cells were infected with S. flexneri for4h40 min, fixed for fluorescent light microscopy, and stained with antibodies to NDP52 and ubiquitin (FK2). Scale bar,1m. Similar images were obtained labeling for FK1. B, HeLa cells were transfected with SEPT6-GFP, infected with S. flexneri for4h40 min, fixed for fluorescent light microscopy, and stained with antibodies to NDP52. Scale bar,1 m. 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). After4h4 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 for4h40 min, fixed for fluorescent light microscopy, and stained with antibodies to p62 and NDP52. Scale bar,1 m. 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). After4h40minof 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. gella (Fig. 1E), NDP52 was recruited to bacteria in p62-depleted be recruited independently of each other, and independently of cells and vice versa (Fig. 3C). Septin depletion did not affect septins, during autophagy of Listeria. autophagy receptor recruitment to EGDactA (Fig. 3C), in Autophagy Receptor Recruitment Is Dependent on Shigella agreement with our previous work showing no septin caging of Effectors IcsA and IcsB—As an interdependence between p62 these bacteria (11). These data reveal that p62 and NDP52 can and NDP52 was clearly identified in cells infected with Shigella 26990 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 30 •JULY 29, 2011 Autophagy of Shigella versus Listeria (Fig. 1E), we analyzed more precisely the signals that recruit p62 (11), and NDP52 (Fig. 1C) to wild-type bacteria. Therefore, and NDP52. Shigella avoids autophagy via the bacterial effector accumulation of ubiquitinated proteins and the targeting of protein IcsB which prevents the binding of Atg5, a protein crit- Shigella to autophagy by p62 and NDP52 are dependent on ical for autophagosome maturation (21), to IcsA (22). We thus actin and septin. addressed the role of IcsA and IcsB in the recruitment of p62 It has been shown previously that the IcsB mutant is more and NDP52. efficiently targeted to autophagy (22, 23) and is more efficiently S. flexneri M90TicsA, i.e. an isogenic mutant strain unable entrapped in septin cages (11). Interestingly, S. flexneri to polymerize actin or recruit septin cages in the cytosol of cells M90TicsB recruited significantly more ubiquitin, p62, and (11), failed to recruit ubiquitin, p62, and NDP52 (data not NDP52 than did wild-type bacteria (1.8  0.1-fold, 1.9  0.2- shown). In agreement with this result, the inhibition of actin fold, and 1.9  0.2-fold, respectively) (Fig. 4, B and C). Taken polymerization by cytochalasin D significantly reduced the together, these data highlight the role of IcsB in preventing recruitment of ubiquitin, p62, and NDP52 to wild-type bacteria ubiquitin protein recruitment/formation and the recruitment (3.1  0.5-fold, 2.3  0.6-fold, and 2.2  0.5-fold, respectively) of p62 and NDP52 and confirm the critical role of actin and (Fig. 4A). Moreover, SEPT2 or SEPT9 depletion similarly septin in this process. reduced the recruitment of ubiquitin (data not shown), p62 p62-mediated Autophagic Activity Is Stimulated by TNF-— We next considered host factors that control the recruitment of -Bafilomycin +Bafilomycin autophagy receptors during Shigella infection. TNF- is a CTRL p62 NDP52 CTRL p62 NDP52 pleiotropic cytokine that orchestrates a wide range of biological siRNA siRNA siRNA siRNA siRNA siRNA functions, including host defense against pathogens (24), and is prominently induced upon Shigella infection (25). TNF- is GAPDH also known to stimulate septin caging (11). We therefore tested p62 and NDP52 recruitment to Shigella in TNF--treated cells. p62 In agreement with the increase observed for septin caging, p62 and NDP52 recruitment to Shigella significantly increased NDP52 (1.9 0.2-fold and 1.9 0.2-fold, respectively) upon treatment with TNF- (Fig. 5A). In the case of EGDactA, TNF- did not FIGURE 2. NDP52 is required for p62-mediated autophagic activity and vice versa. HeLa cells were treated with control (CTRL), p62, or NDP52 siRNA, affect the recruitment of p62 and NDP52 (data not shown), in treated or not with bafilomycin, and immunoblotted for GAPDH, p62, or agreement with our previous work showing no septin caging of NDP52. Representative blots from three independent experiments are these bacteria (11). 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 EGDactA 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 EGDactA 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,1m. C, HeLa cells were treated with control (CTRL), p62, NDP52, SEPT2, or SEPT9 siRNA, infected with L. monocytogenes EGDactA 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. JULY 29, 2011• VOLUME 286 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 26991 Autophagy of Shigella versus Listeria 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 after4h40min 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 M90TicsB for4h40 min, fixed for fluorescent light microscopy, and stained with antibodies to p62 and NDP52. Scale bar,1 m. 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 M90TicsB having recruited ubiquitin, p62, or NDP52 from three independent experiments per strain. p values, Student’s t test. To determine the impact of increased autophagy receptor p62 and NDP52 gene expression in response to TNF- over recruitment on the fate of Shigella, we performed bacterial sur- time. p62 gene expression progressively increased in the pres- vival assays in cells treated or not with TNF-. TNF- treat- ence of TNF- (1.8 0.9-fold at 12 h and 4.0 1.9-fold at 24 h), ment resulted in 28.0 4.1% reduced bacterial counts (Fig. 5B). unlike NDP52 gene expression (1.2 0.4-fold at 12 h and 1.6 Interestingly, similar results were obtained using EGDactA, 0.3-fold at 24 h) (Fig. 5F). These data, together with results i.e. TNF- treatment resulted in 36.3  2.2% reduced bacterial obtained from the autophagic flux assay (Fig. 5D) and the counts (Fig. 5C), raising the possibility that TNF- treatment is induced expression of p62-GFP (Fig. 5E), highlight TNF- as a a general stimulant of autophagic activity, not excluding, how- potent stimulant of p62-mediated autophagic activity. ever, that other events could lead to Listeria reduced counts. DISCUSSION To test whether the impact of TNF- on autophagic activity could be extended to uninfected cells, we used the autophagic Autophagy has recently emerged as an important mecha- flux assay (8) on cells treated or not with TNF-. Strikingly, nism for controlling intracellular pathogens (26, 27). A variety levels of p62 and LC3-II were significantly increased in cells of different bacteria, including mycobacteria, Salmonella, Shi- treated with TNF- in the absence (p62 by 2.3  0.3-fold and gella, and Listeria, are recognized by autophagy (9, 11, 12, 14, LC3-II by 2.4  0.5-fold) and presence (p62 by 1.4  0.2-fold 22, 28), yet the specific signals that mediate recognition of intra- and LC3-II by 2.0  0.4-fold) of bafilomycin (Fig. 5D and sup- cellular pathogens by the autophagy machinery have not been plemental Fig. S1A). To confirm this, we applied a novel defined thus far. A role for ubiquitin in this process has become reporter cell system based on induced expression of p62-GFP, increasingly recognized (5, 6), and it is currently thought that where the degradation of GFP can be followed after promoter ubiquitin associated with intracellular pathogens promotes tar- shutoff by flow cytometry (16). In accordance with the geted autophagosome formation and pathogen destruction. autophagic flux assay (Fig. 5D), TNF- induced significantly Here, we use Shigella and Listeria to characterize different more GFP before (1.5  0.0-fold) and after (1.5  0.1-fold) pathways of selective autophagy. We show that the ubiquitin- promoter shutoff than did untreated cells (Fig. 5E and supple- binding adaptor proteins p62 and NDP52 targeted Shigella to mental Fig. S1B). an autophagy pathway dependent upon septin and actin. In Interestingly, TNF- treatment did not increase levels of contrast, p62 or NDP52 targeted the Listeria ActA mutant to an NDP52 in the absence and presence of bafilomycin (Fig. 5D), autophagy pathway independent of septin or actin. Taken suggesting that TNF- may particularly stimulate the together, these results reveal that p62 and NDP52 mediate dif- autophagic activity of p62. To test this, we measured levels of ferent pathways of selective autophagy for Shigella and Listeria 26992 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 30 •JULY 29, 2011 Autophagy of Shigella versus Listeria FIGURE 5. Autophagic activity is stimulated by TNF-. A, untreated or TNF--treated HeLa cells were infected with S. flexneri for4h40minand 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-. Graphs represent the relative number (i.e. normalized to TNF- cells) of colony-forming units (CFU) counted from cells after4h40 min. On the graph TNF- 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 EGDactA were performed in HeLa cells treated or not with TNF-. Graphs represent the relative number (i.e. normalized to TNF- cells) of colony-forming units counted from cells after4h40 min. On the graph TNF- 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--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--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-. 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--treated cells of three is shown, and the others are shown as supplemental Fig. S1B. F, p62 and NDP52 mRNA expression following TNF- 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-. 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. and provide novel insight into the mechanisms by which adap- SEPT9. However, very few direct interaction partners have tor proteins target bacteria to autophagy. been identified for septins (19, 29), possibly because septin- The molecular events underlying the autophagy of Shigella binding partners interact with specific conformations of higher have not been completely elucidated, although a number of order septin assemblies and not with individual septins or sep- contributing factors are herein identified. Given the interde- tin complexes (30). pendence between p62 and NDP52 observed for Shigella (Fig. Remarkably, autophagy activation for Listeria is actin- and 1E), there could be a single mechanism for autophagy activation septin-independent. Together with the finding that p62 and in this pathway, and the concentration of autophagic compo- NDP52 are not interdependent for autophagy of Listeria (Fig. nents to the substrates by septin cages enable autophagy to 3C), these results demonstrate that Listeria induces a different achieve specific and efficient clearance of bacteria. The mech- autophagy process than Shigella and reinforces the view that anisms by which septins modulate properties of the autophagy different bacterial pathogens invoke different pathways of receptors are not yet clear. Immunoprecipitation experiments on cells using antibodies for SEPT2/SEPT9 or p62/NDP52 did not reveal a direct interaction between p62/NDP52 and SEPT2/ S. Mostowy and P. Cossart, unpublished observations. JULY 29, 2011• VOLUME 286 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 26993 Autophagy of Shigella versus Listeria selective autophagy. In particular, by expressing ActA to may detect ubiquitin chains of different linkage types, recruit polymerize actin, Listeria prevents ubiquitin protein recruit- distinct signaling molecules, and/or determine the specific ment/formation, autophagy, and septin caging (11, 12, 31). On autophagy pathway (5, 6, 9). Alternatively, through their bind- the other hand, by expressing IcsA to polymerize actin, Shigella ing to LC3 or different LC3 proteins (38), different adaptors allows ubiquitin protein recruitment/formation, autophagy, may help recruit membrane, potentially from different sources and septin caging (11, 22, 31). We suggest that autophagosomes (39, 40), to generate autophagosomes around bacteria. acting on Shigella and Listeria are intrinsically distinct, and the Although the exact relationship between actin polymerization differential requirement of the actin cytoskeleton could reflect and septin assembly in regulating autophagy is not yet clear, we the diverse nature of the two autophagic pathways. In the hypothesize that septins are required to scaffold ubiquitinated future, a detailed comparative survey of the composition of Shi- proteins and autophagy receptors around actin-polymerizing gella- versus Listeria-induced autophagosomes would be cru- substrates for clearance by autophagy. It is thus of great interest cial to elucidate the molecular and biochemical basis that estab- to study the respective role of different autophagy receptors, lishes features unique to these autophagic pathways. actin polymerization, and septin assembly in the different path- Investigation of infection by other intracytosolic bacteria will ways of selective autophagy. also help to describe precisely the different autophagy mecha- Acknowledgments—We thank D. Judith and members of the P. nisms. In this respect, preliminary data show that M. marinum, Cossart and M. Lecuit laboratories for helpful discussions. which is similar to Shigella in that it also recruits WASP family proteins for actin tail polymerization (32), promotes septin cage formation (11) and is recognized by autophagy markers (sup- REFERENCES plemental Fig. S2). 1. Yang, Z., and Klionsky, D. J. (2010) Nat. Cell Biol. 12, 814–822 To examine whether physiological stimuli induce selective 2. Levine, B., and Kroemer, G. 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p62 and NDP52 Proteins Target Intracytosolic Shigella and Listeria to Different Autophagy Pathways

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American Society for Biochemistry and Molecular Biology
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Copyright © 2011 Elsevier Inc.
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0021-9258
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10.1074/jbc.m111.223610
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 30, pp. 26987–26995, July 29, 2011 © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. p62 and NDP52 Proteins Target Intracytosolic Shigella and □ S Listeria to Different Autophagy Pathways Received for publication, January 21, 2011, and in revised form, May 22, 2011 Published, JBC Papers in Press, June 6, 2011, DOI 10.1074/jbc.M111.223610 ‡§¶1  ‡§¶ ‡‡ ‡‡ Serge Mostowy , Vanessa Sancho-Shimizu **,Me´lanie Anne Hamon , Roxane Simeone , Roland Brosch , §§ ‡§¶2 Terje Johansen , and Pascale Cossart ‡ § From the Institut Pasteur, Unite´ des Interactions Bacte´ries-Cellules, Paris F-75015, France, INSERM U604, Paris F-75015, France, Institut National de la Recherche Agronomique (INRA), USC2020, Paris F-75015, France, the Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U980, Necker Medical School, Paris 75015 France, the **University Paris Descartes, ‡‡ Paris 75015, France, the Institut Pasteur, Unite´ Postulante Pathoge´nomique Mycobacte´rienne Inte´gre´e, Paris F-75015, France, §§ and the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway Autophagy is an important mechanism of innate immune binding proteins in selective autophagy, p62 (sequestosome 1; defense. We have recently shown that autophagy components SQSTM1) has emerged as the prototype autophagy receptor are recruited with septins, a new and increasingly characterized (7). p62 is an LC3 interaction partner in vivo and is constantly cytoskeleton component, to intracytosolic Shigella that have degraded by autophagy, establishing it as a useful marker for started to polymerize actin. On the other hand, intracytosolic autophagic vesicle turnover (8). NDP52 has more recently Listeria avoids autophagy recognition by expressing ActA, a emerged as another autophagy receptor and shares with p62 bacterial effector required for actin polymerization. Here, we the ability to bind LC3 and ubiquitinated cargo simultaneously exploit Shigella and Listeria as intracytosolic tools to character- (9). The respective roles of p62 and NDP52 are not understood. ize different pathways of selective autophagy. We show that the Whether these individual autophagy receptors recognize dif- ubiquitin-binding adaptor proteins p62 and NDP52 target Shi- ferent ubiquitinated proteins and/or perform independent gella to an autophagy pathway dependent upon septin and actin. functions in cells may be critical for the complete understand- In contrast, p62 or NDP52 targets the Listeria ActA mutant to ing of autophagy and its therapeutic potential. an autophagy pathway independent of septin or actin. TNF-,a Recent evidence has implicated the cytoskeleton as a critical host cytokine produced upon bacterial infection, stimulates mediator of selective autophagy. We have shown that septins, a p62-mediated autophagic activity and restricts the survival of novel component of the cytoskeleton (10), are recruited with Shigella and the Listeria ActA mutant. These data provide a new autophagy proteins to “cage” Shigella flexneri in the cytosol of molecular framework to understand the emerging complexity of infected cells and restrict bacterial dissemination (11). These autophagy and its ability to achieve specific clearance of intra- results suggest an interdependence between the two evolution- cytosolic bacteria. arily conserved processes of septin assembly and autophagy: when septins are absent, autophagy markers accumulate less, and vice versa. However, in the case of Listeria monocytogenes, Autophagy is an evolutionarily conserved catabolic pathway a bacterial pathogen that also escapes to the cytosol, no efficient that allows eukaryotes to degrade and recycle intracellular septin caging has been observed (11). Listeria has been reported components by sequestering proteins and organelles in special- to avoid autophagic recognition by expressing ActA, a bacterial ized double-membrane vesicles named autophagosomes (1–3). effector required for actin tail motility (12, 13). Therefore Lis- Although autophagosomes can sequester cytosolic material teria evades both septin caging and autophagy via its surface nonspecifically, e.g. as a response to starvation, there is increas- expression of ActA (11). Given these fundamental differences ing evidence for selective autophagic degradation of various between Shigella and Listeria, it is clear that in-depth investi- cellular structures, including protein aggregates, mitochondria, gation of these two bacteria will help to describe precisely the and microbes (4, 5). The mechanism of selective autophagy is coordination among actin, septin, and selective autophagy. not well understood, yet the role of ubiquitin in this process is The role of p62 and/or NDP52 in selective autophagy of Sal- critical (5, 6). By simultaneous binding to both ubiquitin and monella enterica serovar Typhimurium (S. typhimurium) has the autophagosome-associated ubiquitin-like proteins (i.e. recently been characterized (9, 14, 15). It has been proposed LC3/GABARAP proteins) autophagy receptors can mediate that p62 and NDP52 act independently to drive efficient bacte- docking of ubiquitinated cargo to the autophagosome, thereby rial autophagy of S. typhimurium within Salmonella-contain- ensuring their selective degradation (5, 6). Of the ubiquitin- ing vacuoles (15). To assess the role of p62 and NDP52 in the autophagy of bacterial pathogens that escape to the cytosol and □ S The on-line version of this article (available at http://www.jbc.org) contains to determine the role of the actin/septin cytoskeleton in this supplemental Figs. S1–S4. process, we examined these components in the case of Shigella To whom correspondence may be addressed. Tel.: 33-1-40-61-32-69; Fax: 33-1-45-68-87-06; E-mail: [email protected]. International research scholar from the Howard Hughes Medical Institute and recipient of European Research Council Advanced Grant Award The abbreviations used are: NDP52, 52-kDa nuclear dot protein; CFP, 233348. To whom correspondence may be addressed. Tel.: 33-1-40-61-30- cyan fluorescent protein; IF, immunofluorescence; LC3, light chain 3; 32; Fax: 33-1-45-68-87-06; E-mail: [email protected]. SEPT, Septin. This is an Open Access article under the CC BY license. JULY 29, 2011• VOLUME 286 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 26987 Autophagy of Shigella versus Listeria and Listeria and reveal that these different intracytosolic bac- Listeria was added to host cells at a multiplicity of infection of teria are targeted to different pathways of selective autophagy. 100. Bacteria and cells were centrifuged at 1000 g for 1 min at 21 °C and, then incubated at 37 °C and 10% CO for 1 h, washed EXPERIMENTAL PROCEDURES with minimum essential medium, and incubated with fresh Bacteria and Cell Culture—L. monocytogenes EGD (BUG gentamicin-containing complete medium (10 g/ml) for an 600) and EGDactA (BUG 2140) were grown overnight at 37 °C additional 2 h, after which they were washed with 1 PBS and in brain heart infusion medium (Difco Laboratories), diluted fixed and processed for IF. Shigella was added to cells at an 15  in fresh brain heart infusion medium, and cultured until multiplicity of infection of 100 (for quantification analyses), or A  0.8. S. flexneri M90T (BUG 2505), M90TicsA (BUG 400l of growth (A  0.6) was diluted in minimum essen- 600 nm 600 nm 1791), and M90TicsB were cultured overnight in trypticase tial medium and added directly to cells (for imaging analyses). soy, diluted 80  in fresh trypticase soy, and cultured until Bacteria and cells were centrifuged at 700 g for 10 min at 21 °C A  0.6. Mycobacterium marinum M-DsRed were cul- and then placed at 37 °C and 10% CO for 30 min, washed with 600 nm 2 tured at 30 °C in Middlebrook 7H9 (BD Biosciences) supple- minimum essential medium, and incubated with fresh gentam- g/ml) for 4 h, after mented with 0.2% glycerol, 0.05% Tween 80, and 10% ADC icin-containing complete medium (50 Enrichment (Fisher Scientific), diluted 48 h prior to infection in which they were washed with 1 PBS and fixed and processed fresh medium, and cultured until A  0.6. for IF. For infection of cells with M. marinum,2  10 RAW 600 nm HeLa (American Type Culture Collection (ATCC) CCL-2) macrophages or mouse embryonic fibroblasts were plated onto cells were cultured in minimum essential medium plus Glu- glass coverslips in 6-well plates and used for experiments 48 h taMAX (Invitrogen) supplemented with 1 mM sodium pyruvate later. M. marinum was washed twice in 1 PBS and passaged (Invitrogen), 0.1 mM nonessential amino acid solution (Invitro- through a 26-gauge needle. Bacteria were added to cells in gen), and 10% fetal calf serum (FCS). p62-GFP cells were cul- medium without FCS at an multiplicity of infection of 10 tured in DMEM plus GlutaMAX supplemented with 15 g/ml (RAW) or 100 (mouse embryonic fibroblasts). Bacteria and hygromycin, 200 g/ml blasticidin, and 10% FCS (16). Cells cells were centrifuged at 700 g for 5 min at 21 °C and then were grown at 37 °C and 10% CO . RAW 264.7 cells (ATCC placed for2hat32 °Cand5%CO . Infected cells were then 2 2 TIB-71) were cultured in DMEM plus GlutaMAX supple- washed with 1 PBS and incubated with 200 g/ml amikacin M glutamine (Invitrogen), 1 mM sodium pyru- for2hat32 °Cand5%CO mented with 2 m . After this time, macrophages were vate, and 10% FCS. Cells were grown at 37 °C and 10% CO . washed with 1 PBS and incubated in complete medium at Mouse embryonic fibroblasts from C57BL/6 mice were main- 32 °C and 5% CO for 20 or 44 h. After a total of 24 or 48 h, tained in DMEM containing 10% FCS. infected cells were washed with 1 PBS and fixed and pro- Antibodies—Rabbit polyclonal antibodies used were anti- cessed for IF. SEPT2 (R170) and anti-SEPT9 (R69) (11), anti-p62 (Clini- Images were acquired on a fluorescence inverted microscope science, PM045), anti-NDP52 (AbCam, ab68588), and anti- Axiovert 200 M (Carl Zeiss MicroImaging, Inc.) equipped with LC3/Atg8 (Novus Biologicals, NB100-2331). The anti-NBR1 a cooled digital charge-coupled device camera (Cool SNAPHQ, (Abnova, H00004077-A01) used was a mouse polyclonal anti- Photometrics) driven by Metamorph Imaging System software body. Mouse monoclonal antibodies used were anti-GAPDH (Universal Imaging Corp). For three-dimensional representa- (AbCam, 6C5), FK2 (Enzo Life Sciences, PW8810), FK1 (Enzo tion, quantitative microscopy (i.e. counting autophagy receptor Life Sciences, PW8805), and anti-p62 lck ligand (BD Biosci- recruitment) was performed using Z-stack image series of ences, 610832). Secondary antibodies used were Cy5- (Jackson infected cells, counting 250–1000 bacteria/experiment. Images ImmunoResearch Laboratories), Alexa Fluor 488-, or Alexa were processed using ImageJ. Where mentioned, IF micros- Fluor 546-conjugated goat anti-rabbit or goat anti-mouse copy was performed with SEPT2-YFP (BUG 2444), SEPT6-GFP (Molecular Probes). F-actin was labeled with Alexa Fluor 488-, (BUG 2445), SEPT9-CFP (BUG 2309), SEPT9-tdTomato (BUG 546-, or 647-phalloidin (Molecular Probes). 2723), or GFP-LC3 (BUG 3046) (11). Cells were transfected For immunoblotting, total cellular extracts were blotted with with jetPEI (PolyPlus Transfection). the above-mentioned antibodies followed by peroxidase-con- siRNA, Pharmacological Inhibitors, and Cytokine Treatment— jugated goat anti-mouse or anti-rabbit antibodies (Biosys Lab- 0.8 10 HeLa cells were plated in 6-well plates and transfected oratories). GAPDH was used throughout as a loading control. the following day using Oligofectamine (Invitrogen). Control Proteins were run on 8, 10, or 14% acrylamide gels. siRNA (AM4635) as well as predesigned siRNA for SEPT2 Infections and Microscopy—1–1.5  10 HeLa cells were (14709), SEPT9 (18228), NDP52 (s19994), and NBR1 (s8381) plated on glass coverslips in 6-well plates (Techno Plastic Prod- were all from Ambion. siRNA sequences for p62 were taken from Refs. 11, 17. Cells were tested 72 h after siRNA ucts) and used for experiments 48 h later. Cells on coverslips were fixed for 15 min in 4% paraformaldehyde and then washed transfection. with 1 PBS and processed for immunofluorescence (IF). After For experiments involving pharmacological inhibitors, HeLa 10 min of incubation in 50 mM ammonium chloride, cells were cells were infected and treated for 30 min prior to fixation with permeabilized for 4 min with 0.1% Triton X-100 and then incu- dimethyl sulfoxide or cytochalasin D (5 M). Cytochalasin D bated in 1 PBS. Incubation with primary or secondary anti- was suspended in dimethyl sulfoxide and handled as suggested bodies was performed in 1 PBS. Vectashield hard set mount- by the manufacturer (Sigma). To monitor autophagic flux, cells ing medium with DAPI (Vector Laboratories) or mounting were treated with bafilomycin A1 (Sigma, B1793) for 12 h (160 medium for IF (Interchim) was used. nM). For experiments involving TNF- (R&D Systems, 210- 26988 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 30 •JULY 29, 2011 Autophagy of Shigella versus Listeria TA), cells were treated with 20 ng/ml TNF- for 12, 20, or 24 h. co-localization of NDP52 with ubiquitinated proteins Treatment was continued throughout infection. For experi- around Shigella (Fig. 1A) and showed that 84  3% of Shigel- ments involving IL-1 (R&D Systems, 201-LB) or IFN- la-septin cages recruited NDP52 (means  S.E. from n  3 (Imukin, Roche Applied Biosciences), cells were treated with 20 experiments) (Fig. 1B). To determine whether NDP52 ng/ml or 5000 units/ml, respectively, for 12 or 24 h. recruitment is dependent upon septins, we used siRNA to Gentamicin Survival Assays—Gentamicin survival assays deplete cells of SEPT2 or SEPT9 and evaluated NDP52 were adapted from Refs. 18, 19. Cells treated with or without recruitment to Shigella in these siRNA-treated cells. In both TNF- were incubated with Shigella or Listeria as detailed above. cases, NDP52 recruitment was significantly reduced (3.8 Cells were washed and then lysed with distilled H O. The number 0.4-fold or 3.0  0.6-fold, respectively) (Fig. 1C), concordant with the view that septins play a role in the recruitment of of viable bacteria released from the cells was assessed by plating on Luria Bertani (LB) or brain heart infusion agar plates. autophagic markers (11). Flow Cytometry—Gene expression for p62-GFP cells was We then asked whether p62 and NDP52 were recruited to induced (i.e. “On”) by adding 1 g/ml tetracycline (Sigma, the same Shigella-septin cage. Indeed, 83  2% of Shigella- T7660) to the culture medium for 24 h (16). After these 24 h, septin cages recruited both p62 and NDP52 (mean  S.E. from gene expression was turned off (i.e. “Off”) by washing cells twice n  3 experiments) (Fig. 1D). To determine whether p62 and with DMEM and incubating with fresh tetracycline-free com- NDP52 have independent roles at the Shigella-septin cage, we plete medium for 16 h (16). After these times, treated p62-GFP used siRNA to deplete cells of p62 or NDP52 and evaluated cells were washed in 1 PBS, detached with 0.05% EDTA-tryp- adaptor recruitment to Shigella in these siRNA-treated cells. sin, and permeabilized/fixed using BD Cytofix/Cytoperm Fixa- Strikingly, p62 or NDP52 recruitment was significantly reduced tion/Permeabilization kit (BD Biosciences, 554714). Samples in either case (31.1  5.1-fold or 2.4  0.3-fold in p62-depleted were analyzed using a FACSCalibur instrument (BD Biosci- cells, respectively, or 6.6  2.3-fold or 32.6  14.6-fold in ences). Dead cells were excluded on the basis of forward and NDP52-depleted cells, respectively) (Fig. 1E). These results side scatter, and a minimum of 10,000 events were acquired per strongly suggest an interdependent relationship between p62 sample. Results were analyzed using FlowJo software. and NDP52 to target Shigella toward autophagy. Immunoprecipitation Studies—HeLa cells (uninfected) NDP52 Regulates p62-mediated Autophagic Activity and treated or not with bafilomycin, or HeLa cells infected with Vice Versa—p62 is constantly degraded by autophagy and has Shigella or the Listeria ActA mutant in the absence and pres- thus been established as a marker for autophagic activity, i.e. ence of TNF-, were lysed in Igepal buffer (20 mM Tris, pH 8.0, autophagic flux (8). To address the role of NDP52 in autophagic 1% (v/v) Igepal CA-630 (Sigma), 150 mM NaCl, 10% (v/v) glyc- activity, we examined the steady-state levels of p62 in the erol, and protease inhibitors mixture). p62, NDP52, SEPT2, or absence and presence of bafilomycin (an inhibitor of autopha- SEPT9 was immunoprecipitated from 800 g of the total pro- gosome-lysosome fusion) in control cells or in cells depleted of tein extracts by the addition of 1 g of anti-p62, anti-NDP52, NDP52. Levels of p62 were significantly reduced in cells anti-SEPT2, or anti-SEPT9 antibody and 40 l of a 50% slurry depleted for NDP52 in the absence (2.1  0.4-fold) and pres- suspension of protein A-Sepharose beads (Amersham Biosci- ence (1.5 0.1-fold) of bafilomycin (Fig. 2), suggesting that p62 ences). Samples were analyzed by SDS-PAGE and immunoblotted and NDP52 are interdependent to promote autophagy. We with anti-p62, anti-NDP52, anti-SEPT2, or anti-SEPT9 antibody. next examined steady-state levels of NDP52. Levels of NDP52 HRP-conjugated goat anti-mouse or anti-rabbit secondary anti- accumulated in control cells treated with bafilomycin (3.9 bodies were subsequently used. Protein input was evaluated by 0.5-fold), indicating that NDP52, like p62, can be used as a probing blots of cell lysates prior to the immunoprecipitation step marker of autophagic vesicle turnover (Fig. 2). In cells depleted using antibodies specific to p62, NDP52, SEPT2, or SEPT9. for p62, levels of NDP52 were significantly reduced in the Gene Expression Studies—Cells were treated with TNF-, absence (1.5  0.1-fold) and presence (1.2  0.1-fold) of bafi- IL-1, or IFN- for 0, 12, or 24 h. After this time RNA was lomycin (Fig. 2). Thus, p62 can significantly regulate NDP52 extracted using an RNeasy mini-kit (Qiagen) and converted to activity and vice versa. cDNA using the High Capacity RNA-to-cDNA mastermix p62 and NDP52 Are Recruited Independently to Listeria— (Applied Biosystems 4375575). Gene expression probes Different pathogens have evolved different ways to escape (Applied Biosystems) included GUS (4326320E), p62 autophagy (20). In the case of L. monocytogenes, these bacteria (Hs00177654_m1), and NDP52 (Hs00414663_m1). Gene avoid ubiquitination, p62 recognition, and septin caging by expression was evaluated using the ddCt method, and levels of expressing the surface protein ActA, a bacterial effector p62 and NDP52 were normalized to GUS. required to polymerize actin (11, 12). We addressed the role of NDP52 and showed that L. monocytogenes EGDactA, but not RESULTS the wild-type strain EGD, is recognized by ubiquitin, p62, and The Recruitment of p62 and NDP52 to Shigella Is NDP52 (Fig. 3A). 95  2% of EGDactA recruiting p62 also Interdependent—We have reported previously that p62 is recruited NDP52 (mean S.E. from n 3 experiments), show- recruited to intracytosolic Shigella entrapped by septin cages, ing that p62 and NDP52 were recruited to the same bacteria and the recruitment of p62 is dependent upon SEPT2 and (Fig. 3B). SEPT9 (11). NDP52 shares with p62 the ability to bind ubiq- We used siRNA to deplete cells of p62, NDP52, SEPT2, or uitin-coated S. typhimurium (9, 14, 15). We thus addressed SEPT9 and evaluated adaptor recruitment to EGDactA in the recruitment of NDP52 to Shigella. We first observed the these siRNA-treated cells. Unlike what was observed for Shi- JULY 29, 2011• VOLUME 286 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 26989 Autophagy of Shigella versus Listeria FIGURE 1. NDP52 recruitment to Shigella. A, HeLa cells were infected with S. flexneri for4h40 min, fixed for fluorescent light microscopy, and stained with antibodies to NDP52 and ubiquitin (FK2). Scale bar,1m. Similar images were obtained labeling for FK1. B, HeLa cells were transfected with SEPT6-GFP, infected with S. flexneri for4h40 min, fixed for fluorescent light microscopy, and stained with antibodies to NDP52. Scale bar,1 m. 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). After4h4 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 for4h40 min, fixed for fluorescent light microscopy, and stained with antibodies to p62 and NDP52. Scale bar,1 m. 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). After4h40minof 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. gella (Fig. 1E), NDP52 was recruited to bacteria in p62-depleted be recruited independently of each other, and independently of cells and vice versa (Fig. 3C). Septin depletion did not affect septins, during autophagy of Listeria. autophagy receptor recruitment to EGDactA (Fig. 3C), in Autophagy Receptor Recruitment Is Dependent on Shigella agreement with our previous work showing no septin caging of Effectors IcsA and IcsB—As an interdependence between p62 these bacteria (11). These data reveal that p62 and NDP52 can and NDP52 was clearly identified in cells infected with Shigella 26990 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 30 •JULY 29, 2011 Autophagy of Shigella versus Listeria (Fig. 1E), we analyzed more precisely the signals that recruit p62 (11), and NDP52 (Fig. 1C) to wild-type bacteria. Therefore, and NDP52. Shigella avoids autophagy via the bacterial effector accumulation of ubiquitinated proteins and the targeting of protein IcsB which prevents the binding of Atg5, a protein crit- Shigella to autophagy by p62 and NDP52 are dependent on ical for autophagosome maturation (21), to IcsA (22). We thus actin and septin. addressed the role of IcsA and IcsB in the recruitment of p62 It has been shown previously that the IcsB mutant is more and NDP52. efficiently targeted to autophagy (22, 23) and is more efficiently S. flexneri M90TicsA, i.e. an isogenic mutant strain unable entrapped in septin cages (11). Interestingly, S. flexneri to polymerize actin or recruit septin cages in the cytosol of cells M90TicsB recruited significantly more ubiquitin, p62, and (11), failed to recruit ubiquitin, p62, and NDP52 (data not NDP52 than did wild-type bacteria (1.8  0.1-fold, 1.9  0.2- shown). In agreement with this result, the inhibition of actin fold, and 1.9  0.2-fold, respectively) (Fig. 4, B and C). Taken polymerization by cytochalasin D significantly reduced the together, these data highlight the role of IcsB in preventing recruitment of ubiquitin, p62, and NDP52 to wild-type bacteria ubiquitin protein recruitment/formation and the recruitment (3.1  0.5-fold, 2.3  0.6-fold, and 2.2  0.5-fold, respectively) of p62 and NDP52 and confirm the critical role of actin and (Fig. 4A). Moreover, SEPT2 or SEPT9 depletion similarly septin in this process. reduced the recruitment of ubiquitin (data not shown), p62 p62-mediated Autophagic Activity Is Stimulated by TNF-— We next considered host factors that control the recruitment of -Bafilomycin +Bafilomycin autophagy receptors during Shigella infection. TNF- is a CTRL p62 NDP52 CTRL p62 NDP52 pleiotropic cytokine that orchestrates a wide range of biological siRNA siRNA siRNA siRNA siRNA siRNA functions, including host defense against pathogens (24), and is prominently induced upon Shigella infection (25). TNF- is GAPDH also known to stimulate septin caging (11). We therefore tested p62 and NDP52 recruitment to Shigella in TNF--treated cells. p62 In agreement with the increase observed for septin caging, p62 and NDP52 recruitment to Shigella significantly increased NDP52 (1.9 0.2-fold and 1.9 0.2-fold, respectively) upon treatment with TNF- (Fig. 5A). In the case of EGDactA, TNF- did not FIGURE 2. NDP52 is required for p62-mediated autophagic activity and vice versa. HeLa cells were treated with control (CTRL), p62, or NDP52 siRNA, affect the recruitment of p62 and NDP52 (data not shown), in treated or not with bafilomycin, and immunoblotted for GAPDH, p62, or agreement with our previous work showing no septin caging of NDP52. Representative blots from three independent experiments are these bacteria (11). 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 EGDactA 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 EGDactA 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,1m. C, HeLa cells were treated with control (CTRL), p62, NDP52, SEPT2, or SEPT9 siRNA, infected with L. monocytogenes EGDactA 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. JULY 29, 2011• VOLUME 286 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 26991 Autophagy of Shigella versus Listeria 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 after4h40min 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 M90TicsB for4h40 min, fixed for fluorescent light microscopy, and stained with antibodies to p62 and NDP52. Scale bar,1 m. 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 M90TicsB having recruited ubiquitin, p62, or NDP52 from three independent experiments per strain. p values, Student’s t test. To determine the impact of increased autophagy receptor p62 and NDP52 gene expression in response to TNF- over recruitment on the fate of Shigella, we performed bacterial sur- time. p62 gene expression progressively increased in the pres- vival assays in cells treated or not with TNF-. TNF- treat- ence of TNF- (1.8 0.9-fold at 12 h and 4.0 1.9-fold at 24 h), ment resulted in 28.0 4.1% reduced bacterial counts (Fig. 5B). unlike NDP52 gene expression (1.2 0.4-fold at 12 h and 1.6 Interestingly, similar results were obtained using EGDactA, 0.3-fold at 24 h) (Fig. 5F). These data, together with results i.e. TNF- treatment resulted in 36.3  2.2% reduced bacterial obtained from the autophagic flux assay (Fig. 5D) and the counts (Fig. 5C), raising the possibility that TNF- treatment is induced expression of p62-GFP (Fig. 5E), highlight TNF- as a a general stimulant of autophagic activity, not excluding, how- potent stimulant of p62-mediated autophagic activity. ever, that other events could lead to Listeria reduced counts. DISCUSSION To test whether the impact of TNF- on autophagic activity could be extended to uninfected cells, we used the autophagic Autophagy has recently emerged as an important mecha- flux assay (8) on cells treated or not with TNF-. Strikingly, nism for controlling intracellular pathogens (26, 27). A variety levels of p62 and LC3-II were significantly increased in cells of different bacteria, including mycobacteria, Salmonella, Shi- treated with TNF- in the absence (p62 by 2.3  0.3-fold and gella, and Listeria, are recognized by autophagy (9, 11, 12, 14, LC3-II by 2.4  0.5-fold) and presence (p62 by 1.4  0.2-fold 22, 28), yet the specific signals that mediate recognition of intra- and LC3-II by 2.0  0.4-fold) of bafilomycin (Fig. 5D and sup- cellular pathogens by the autophagy machinery have not been plemental Fig. S1A). To confirm this, we applied a novel defined thus far. A role for ubiquitin in this process has become reporter cell system based on induced expression of p62-GFP, increasingly recognized (5, 6), and it is currently thought that where the degradation of GFP can be followed after promoter ubiquitin associated with intracellular pathogens promotes tar- shutoff by flow cytometry (16). In accordance with the geted autophagosome formation and pathogen destruction. autophagic flux assay (Fig. 5D), TNF- induced significantly Here, we use Shigella and Listeria to characterize different more GFP before (1.5  0.0-fold) and after (1.5  0.1-fold) pathways of selective autophagy. We show that the ubiquitin- promoter shutoff than did untreated cells (Fig. 5E and supple- binding adaptor proteins p62 and NDP52 targeted Shigella to mental Fig. S1B). an autophagy pathway dependent upon septin and actin. In Interestingly, TNF- treatment did not increase levels of contrast, p62 or NDP52 targeted the Listeria ActA mutant to an NDP52 in the absence and presence of bafilomycin (Fig. 5D), autophagy pathway independent of septin or actin. Taken suggesting that TNF- may particularly stimulate the together, these results reveal that p62 and NDP52 mediate dif- autophagic activity of p62. To test this, we measured levels of ferent pathways of selective autophagy for Shigella and Listeria 26992 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 30 •JULY 29, 2011 Autophagy of Shigella versus Listeria FIGURE 5. Autophagic activity is stimulated by TNF-. A, untreated or TNF--treated HeLa cells were infected with S. flexneri for4h40minand 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-. Graphs represent the relative number (i.e. normalized to TNF- cells) of colony-forming units (CFU) counted from cells after4h40 min. On the graph TNF- 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 EGDactA were performed in HeLa cells treated or not with TNF-. Graphs represent the relative number (i.e. normalized to TNF- cells) of colony-forming units counted from cells after4h40 min. On the graph TNF- 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--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--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-. 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--treated cells of three is shown, and the others are shown as supplemental Fig. S1B. F, p62 and NDP52 mRNA expression following TNF- 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-. 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. and provide novel insight into the mechanisms by which adap- SEPT9. However, very few direct interaction partners have tor proteins target bacteria to autophagy. been identified for septins (19, 29), possibly because septin- The molecular events underlying the autophagy of Shigella binding partners interact with specific conformations of higher have not been completely elucidated, although a number of order septin assemblies and not with individual septins or sep- contributing factors are herein identified. Given the interde- tin complexes (30). pendence between p62 and NDP52 observed for Shigella (Fig. Remarkably, autophagy activation for Listeria is actin- and 1E), there could be a single mechanism for autophagy activation septin-independent. Together with the finding that p62 and in this pathway, and the concentration of autophagic compo- NDP52 are not interdependent for autophagy of Listeria (Fig. nents to the substrates by septin cages enable autophagy to 3C), these results demonstrate that Listeria induces a different achieve specific and efficient clearance of bacteria. The mech- autophagy process than Shigella and reinforces the view that anisms by which septins modulate properties of the autophagy different bacterial pathogens invoke different pathways of receptors are not yet clear. Immunoprecipitation experiments on cells using antibodies for SEPT2/SEPT9 or p62/NDP52 did not reveal a direct interaction between p62/NDP52 and SEPT2/ S. Mostowy and P. Cossart, unpublished observations. JULY 29, 2011• VOLUME 286 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 26993 Autophagy of Shigella versus Listeria selective autophagy. In particular, by expressing ActA to may detect ubiquitin chains of different linkage types, recruit polymerize actin, Listeria prevents ubiquitin protein recruit- distinct signaling molecules, and/or determine the specific ment/formation, autophagy, and septin caging (11, 12, 31). On autophagy pathway (5, 6, 9). Alternatively, through their bind- the other hand, by expressing IcsA to polymerize actin, Shigella ing to LC3 or different LC3 proteins (38), different adaptors allows ubiquitin protein recruitment/formation, autophagy, may help recruit membrane, potentially from different sources and septin caging (11, 22, 31). We suggest that autophagosomes (39, 40), to generate autophagosomes around bacteria. acting on Shigella and Listeria are intrinsically distinct, and the Although the exact relationship between actin polymerization differential requirement of the actin cytoskeleton could reflect and septin assembly in regulating autophagy is not yet clear, we the diverse nature of the two autophagic pathways. In the hypothesize that septins are required to scaffold ubiquitinated future, a detailed comparative survey of the composition of Shi- proteins and autophagy receptors around actin-polymerizing gella- versus Listeria-induced autophagosomes would be cru- substrates for clearance by autophagy. It is thus of great interest cial to elucidate the molecular and biochemical basis that estab- to study the respective role of different autophagy receptors, lishes features unique to these autophagic pathways. actin polymerization, and septin assembly in the different path- Investigation of infection by other intracytosolic bacteria will ways of selective autophagy. also help to describe precisely the different autophagy mecha- Acknowledgments—We thank D. Judith and members of the P. nisms. In this respect, preliminary data show that M. marinum, Cossart and M. Lecuit laboratories for helpful discussions. which is similar to Shigella in that it also recruits WASP family proteins for actin tail polymerization (32), promotes septin cage formation (11) and is recognized by autophagy markers (sup- REFERENCES plemental Fig. S2). 1. Yang, Z., and Klionsky, D. J. (2010) Nat. Cell Biol. 12, 814–822 To examine whether physiological stimuli induce selective 2. Levine, B., and Kroemer, G. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Jul 29, 2011

Keywords: Autophagy; Bacteria; Cytoskeleton; Tumor Necrosis Factor (TNF); Ubiquitination; Listeria; Septin; Shigella

References