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• W2034167623 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. 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. Autophagy is an evolutionarily conserved catabolic pathway that allows eukaryotes to degrade and recycle intracellular components by sequestering proteins and organelles in specialized double-membrane vesicles named autophagosomes (1Yang Z. Klionsky D.J. Nat. Cell Biol. 2010; 12: 814-822Crossref PubMed Scopus (1636) Google Scholar, 2Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5542) Google Scholar, 3Xie Z. Klionsky D.J. Nat. Cell Biol. 2007; 9: 1102-1109Crossref PubMed Scopus (1712) Google Scholar). Although autophagosomes can sequester cytosolic material nonspecifically, e.g. as a response to starvation, there is increasing evidence for selective autophagic degradation of various cellular structures, including protein aggregates, mitochondria, and microbes (4Kraft C. Peter M. Hofmann K. Nat. Cell Biol. 2010; 12: 836-841Crossref PubMed Scopus (508) Google Scholar, 5Johansen T. Lamark T. Autophagy. 2011; 7: 279-296Crossref PubMed Scopus (1290) Google Scholar). The mechanism of selective autophagy is not well understood, yet the role of ubiquitin in this process is critical (5Johansen T. Lamark T. Autophagy. 2011; 7: 279-296Crossref PubMed Scopus (1290) Google Scholar, 6Kirkin V. McEwan D.G. Novak I. Dikic I. Mol. Cell. 2009; 34: 259-269Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar). By simultaneous binding to both ubiquitin and the autophagosome-associated ubiquitin-like proteins (i.e. LC3/GABARAP proteins) autophagy receptors can mediate docking of ubiquitinated cargo to the autophagosome, thereby ensuring their selective degradation (5Johansen T. Lamark T. Autophagy. 2011; 7: 279-296Crossref PubMed Scopus (1290) Google Scholar, 6Kirkin V. McEwan D.G. Novak I. Dikic I. Mol. Cell. 2009; 34: 259-269Abstract Full Text Full Text PDF PubMed Scopus (990) Google Scholar). Of the ubiquitin-binding proteins in selective autophagy, p62 (sequestosome 1; SQSTM1) has emerged as the prototype autophagy receptor (7Pankiv S. Clausen T.H. Lamark T. Brech A. Bruun J.A. Outzen H. Øvervatn A. Bjørkøy G. Johansen T. J. Biol. Chem. 2007; 282: 24131-24145Abstract Full Text Full Text PDF PubMed Scopus (3307) Google Scholar). p62 is an LC3 interaction partner in vivo and is constantly degraded by autophagy, establishing it as a useful marker for autophagic vesicle turnover (8Mizushima N. Yoshimori T. Levine B. Cell. 2010; 140: 313-326Abstract Full Text Full Text PDF PubMed Scopus (3532) Google Scholar). NDP52 3The abbreviations used are: NDP5252-kDa nuclear dot proteinCFPcyan fluorescent proteinIFimmunofluorescenceLC3light chain 3SEPTSeptin. has more recently emerged as another autophagy receptor and shares with p62 the ability to bind LC3 and ubiquitinated cargo simultaneously (9Thurston T.L. Ryzhakov G. Bloor S. von Muhlinen N. Randow F. Nat. Immunol. 2009; 10: 1215-1221Crossref PubMed Scopus (657) Google Scholar). The respective roles of p62 and NDP52 are not understood. Whether these individual autophagy receptors recognize different ubiquitinated proteins and/or perform independent functions in cells may be critical for the complete understanding of autophagy and its therapeutic potential. 52-kDa nuclear dot protein cyan fluorescent protein immunofluorescence light chain 3 Septin. Recent evidence has implicated the cytoskeleton as a critical mediator of selective autophagy. We have shown that septins, a novel component of the cytoskeleton (10Weirich C.S. Erzberger J.P. Barral Y. Nat. Rev. Mol. Cell Biol. 2008; 9: 478-489Crossref PubMed Scopus (256) Google Scholar), are recruited with autophagy proteins to “cage” Shigella flexneri in the cytosol of infected cells and restrict bacterial dissemination (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). These results suggest an interdependence between the two evolutionarily 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, a bacterial pathogen that also escapes to the cytosol, no efficient septin caging has been observed (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Listeria has been reported to avoid autophagic recognition by expressing ActA, a bacterial effector required for actin tail motility (12Yoshikawa Y. Ogawa M. Hain T. Yoshida M. Fukumatsu M. Kim M. Mimuro H. Nakagawa I. Yanagawa T. Ishii T. Kakizuka A. Sztul E. Chakraborty T. Sasakawa C. Nat. Cell Biol. 2009; 11: 1233-1240Crossref PubMed Scopus (345) Google Scholar, 13Ogawa M. Yoshikawa Y. Mimuro H. Hain T. Chakraborty T. Sasakawa C. Autophagy. 2011; 7: 310-314Crossref PubMed Scopus (34) Google Scholar). Therefore Listeria evades both septin caging and autophagy via its surface expression of ActA (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Given these fundamental differences between Shigella and Listeria, it is clear that in-depth investigation of these two bacteria will help to describe precisely the coordination among actin, septin, and selective autophagy. The role of p62 and/or NDP52 in selective autophagy of Salmonella enterica serovar Typhimurium (S. typhimurium) has recently been characterized (9Thurston T.L. Ryzhakov G. Bloor S. von Muhlinen N. Randow F. Nat. Immunol. 2009; 10: 1215-1221Crossref PubMed Scopus (657) Google Scholar, 14Zheng Y.T. Shahnazari S. Brech A. Lamark T. Johansen T. Brumell J.H. J. Immunol. 2009; 183: 5909-5916Crossref PubMed Scopus (438) Google Scholar, 15Cemma M. Kim P.K. Brumell J.H. Autophagy. 2011; 7: 341-345Crossref PubMed Scopus (158) Google Scholar). It has been proposed that p62 and NDP52 act independently to drive efficient bacterial autophagy of S. typhimurium within Salmonella-containing vacuoles (15Cemma M. Kim P.K. Brumell J.H. Autophagy. 2011; 7: 341-345Crossref PubMed Scopus (158) Google Scholar). To assess the role of p62 and NDP52 in the autophagy of bacterial pathogens that escape to the cytosol and to determine the role of the actin/septin cytoskeleton in this process, we examined these components in the case of Shigella and Listeria and reveal that these different intracytosolic bacteria are targeted to different pathways of selective autophagy. L. monocytogenes EGD (BUG 600) and EGDΔactA (BUG 2140) were grown overnight at 37 °C in brain heart infusion medium (Difco Laboratories), diluted 15 × in fresh brain heart infusion medium, and cultured until A600 nm = 0.8. S. flexneri M90T (BUG 2505), M90TΔicsA (BUG 1791), and M90TΔicsB were cultured overnight in trypticase soy, diluted 80 × in fresh trypticase soy, and cultured until A600 nm = 0.6. Mycobacterium marinum M-DsRed were cultured at 30 °C in Middlebrook 7H9 (BD Biosciences) supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% ADC Enrichment (Fisher Scientific), diluted 48 h prior to infection in fresh medium, and cultured until A600 nm = 0.6. HeLa (American Type Culture Collection (ATCC) CCL-2) cells were cultured in minimum essential medium plus GlutaMAX (Invitrogen) supplemented with 1 mm sodium pyruvate (Invitrogen), 0.1 mm nonessential amino acid solution (Invitrogen), and 10% fetal calf serum (FCS). p62-GFP cells were cultured in DMEM plus GlutaMAX supplemented with 15 μg/ml hygromycin, 200 μg/ml blasticidin, and 10% FCS (16Larsen K.B. Lamark T. Øvervatn A. Harneshaug I. Johansen T. Bjørkøy G. Autophagy. 2010; 6: 784-793Crossref PubMed Scopus (121) Google Scholar). Cells were grown at 37 °C and 10% CO2. RAW 264.7 cells (ATCC TIB-71) were cultured in DMEM plus GlutaMAX supplemented with 2 mm glutamine (Invitrogen), 1 mm sodium pyruvate, and 10% FCS. Cells were grown at 37 °C and 10% CO2. Mouse embryonic fibroblasts from C57BL/6 mice were maintained in DMEM containing 10% FCS. Rabbit polyclonal antibodies used were anti-SEPT2 (R170) and anti-SEPT9 (R69) (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), anti-p62 (Cliniscience, PM045), anti-NDP52 (AbCam, ab68588), and anti-LC3/Atg8 (Novus Biologicals, NB100-2331). The anti-NBR1 (Abnova, H00004077-A01) used was a mouse polyclonal antibody. Mouse monoclonal antibodies used were anti-GAPDH (AbCam, 6C5), FK2 (Enzo Life Sciences, PW8810), FK1 (Enzo Life Sciences, PW8805), and anti-p62 lck ligand (BD Biosciences, 610832). Secondary antibodies used were Cy5- (Jackson ImmunoResearch Laboratories), Alexa Fluor 488-, or Alexa Fluor 546-conjugated goat anti-rabbit or goat anti-mouse (Molecular Probes). F-actin was labeled with Alexa Fluor 488-, 546-, or 647-phalloidin (Molecular Probes). For immunoblotting, total cellular extracts were blotted with the above-mentioned antibodies followed by peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Biosys Laboratories). GAPDH was used throughout as a loading control. Proteins were run on 8, 10, or 14% acrylamide gels. 1–1.5 × 105 HeLa cells were plated on glass coverslips in 6-well plates (Techno Plastic Products) and used for experiments 48 h later. Cells on coverslips were fixed for 15 min in 4% paraformaldehyde and then washed with 1× PBS and processed for immunofluorescence (IF). After 10 min of incubation in 50 mm ammonium chloride, cells were permeabilized for 4 min with 0.1% Triton X-100 and then incubated in 1× PBS. Incubation with primary or secondary antibodies was performed in 1× PBS. Vectashield hard set mounting medium with DAPI (Vector Laboratories) or mounting medium for IF (Interchim) was used. Listeria was added to host cells at a multiplicity of infection of 100. Bacteria and cells were centrifuged at 1000× g for 1 min at 21 °C and, then incubated at 37 °C and 10% CO2 for 1 h, washed with minimum essential medium, and incubated with fresh gentamicin-containing complete medium (10 μg/ml) for an additional 2 h, after which they were washed with 1× PBS and fixed and processed for IF. Shigella was added to cells at an multiplicity of infection of 100 (for quantification analyses), or 400 μl of growth (A600 nm = 0.6) was diluted in minimum essential medium and added directly to cells (for imaging analyses). Bacteria and cells were centrifuged at 700× g for 10 min at 21 °C and then placed at 37 °C and 10% CO2 for 30 min, washed with minimum essential medium, and incubated with fresh gentamicin-containing complete medium (50 μg/ml) for 4 h, after which they were washed with 1× PBS and fixed and processed for IF. For infection of cells with M. marinum, 2 × 105 RAW macrophages or mouse embryonic fibroblasts were plated onto glass coverslips in 6-well plates and used for experiments 48 h later. M. marinum was washed twice in 1× PBS and passaged through a 26-gauge needle. Bacteria were added to cells in medium without FCS at an multiplicity of infection of 10 (RAW) or 100 (mouse embryonic fibroblasts). Bacteria and cells were centrifuged at 700× g for 5 min at 21 °C and then placed for 2 h at 32 °C and 5% CO2. Infected cells were then washed with 1× PBS and incubated with 200 μg/ml amikacin for 2 h at 32 °C and 5% CO2. After this time, macrophages were washed with 1× PBS and incubated in complete medium at 32 °C and 5% CO2 for 20 or 44 h. After a total of 24 or 48 h, infected cells were washed with 1× PBS and fixed and processed for IF. Images were acquired on a fluorescence inverted microscope Axiovert 200 M (Carl Zeiss MicroImaging, Inc.) equipped with a cooled digital charge-coupled device camera (Cool SNAPHQ, Photometrics) driven by Metamorph Imaging System software (Universal Imaging Corp). For three-dimensional representation, quantitative microscopy (i.e. counting autophagy receptor recruitment) was performed using Z-stack image series of infected cells, counting 250–1000 bacteria/experiment. Images were processed using ImageJ. Where mentioned, IF microscopy was performed with SEPT2-YFP (BUG 2444), SEPT6-GFP (BUG 2445), SEPT9-CFP (BUG 2309), SEPT9-tdTomato (BUG 2723), or GFP-LC3 (BUG 3046) (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Cells were transfected with jetPEI (PolyPlus Transfection). 0.8 × 105 HeLa cells were plated in 6-well plates and transfected the following day using Oligofectamine (Invitrogen). Control siRNA (AM4635) as well as predesigned siRNA for SEPT2 (14709), SEPT9 (18228), NDP52 (s19994), and NBR1 (s8381) were all from Ambion. siRNA sequences for p62 were taken from Refs. 11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 17Pohl C. Jentsch S. Nat. Cell Biol. 2009; 11: 65-70Crossref PubMed Scopus (157) Google Scholar. Cells were tested 72 h after siRNA transfection. For experiments involving pharmacological inhibitors, HeLa cells were infected and treated for 30 min prior to fixation with dimethyl sulfoxide or cytochalasin D (5 μm). Cytochalasin D was suspended in dimethyl sulfoxide and handled as suggested by the manufacturer (Sigma). To monitor autophagic flux, cells were treated with bafilomycin A1 (Sigma, B1793) for 12 h (160 nm). For experiments involving TNF-α (R&D Systems, 210-TA), cells were treated with 20 ng/ml TNF-α for 12, 20, or 24 h. Treatment was continued throughout infection. For experiments involving IL-1β (R&D Systems, 201-LB) or IFN-γ (Imukin, Roche Applied Biosciences), cells were treated with 20 ng/ml or 5000 units/ml, respectively, for 12 or 24 h. Gentamicin survival assays were adapted from Refs. 18Mostowy S. Danckaert A. Tham T.N. Machu C. Guadagnini S. Pizarro-Cerdá J. Cossart P. J. Biol. Chem. 2009; 284: 11613-11621Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 19Mostowy S. Nam Tham T. Danckaert A. Guadagnini S. Boisson-Dupuis S. Pizarro-Cerdá J. Cossart P. PLoS ONE. 2009; 4: e4196Crossref PubMed Scopus (71) Google Scholar. Cells treated with or without TNF-α were incubated with Shigella or Listeria as detailed above. Cells were washed and then lysed with distilled H2O. The number of viable bacteria released from the cells was assessed by plating on Luria Bertani (LB) or brain heart infusion agar plates. Gene expression for p62-GFP cells was induced (i.e. “On”) by adding 1 μg/ml tetracycline (Sigma, T7660) to the culture medium for 24 h (16Larsen K.B. Lamark T. Øvervatn A. Harneshaug I. Johansen T. Bjørkøy G. Autophagy. 2010; 6: 784-793Crossref PubMed Scopus (121) Google Scholar). After these 24 h, gene expression was turned off (i.e. “Off”) by washing cells twice with DMEM and incubating with fresh tetracycline-free complete medium for 16 h (16Larsen K.B. Lamark T. Øvervatn A. Harneshaug I. Johansen T. Bjørkøy G. Autophagy. 2010; 6: 784-793Crossref PubMed Scopus (121) Google Scholar). After these times, treated p62-GFP cells were washed in 1× PBS, detached with 0.05% EDTA-trypsin, and permeabilized/fixed using BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences, 554714). Samples were analyzed using a FACSCalibur instrument (BD Biosciences). Dead cells were excluded on the basis of forward and side scatter, and a minimum of 10,000 events were acquired per sample. Results were analyzed using FlowJo software. HeLa cells (uninfected) treated or not with bafilomycin, or HeLa cells infected with Shigella or the Listeria ActA mutant in the absence and presence of TNF-α, were lysed in Igepal buffer (20 mm Tris, pH 8.0, 1% (v/v) Igepal CA-630 (Sigma), 150 mm NaCl, 10% (v/v) glycerol, and protease inhibitors mixture). p62, NDP52, SEPT2, or SEPT9 was immunoprecipitated from 800 μg of the total protein extracts by the addition of 1 μg of anti-p62, anti-NDP52, anti-SEPT2, or anti-SEPT9 antibody and 40 μl of a 50% slurry suspension of protein A-Sepharose beads (Amersham Biosciences). Samples were analyzed by SDS-PAGE and immunoblotted with anti-p62, anti-NDP52, anti-SEPT2, or anti-SEPT9 antibody. HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies were subsequently used. Protein input was evaluated by probing blots of cell lysates prior to the immunoprecipitation step using antibodies specific to p62, NDP52, SEPT2, or SEPT9. Cells were treated with TNF-α, IL-1β, or IFN-γ for 0, 12, or 24 h. After this time RNA was extracted using an RNeasy mini-kit (Qiagen) and converted to cDNA using the High Capacity RNA-to-cDNA mastermix (Applied Biosystems 4375575). Gene expression probes (Applied Biosystems) included GUS (4326320E), p62 (Hs00177654_m1), and NDP52 (Hs00414663_m1). Gene expression was evaluated using the ddCt method, and levels of p62 and NDP52 were normalized to GUS. We have reported previously that p62 is recruited to intracytosolic Shigella entrapped by septin cages, and the recruitment of p62 is dependent upon SEPT2 and SEPT9 (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). NDP52 shares with p62 the ability to bind ubiquitin-coated S. typhimurium (9Thurston T.L. Ryzhakov G. Bloor S. von Muhlinen N. Randow F. Nat. Immunol. 2009; 10: 1215-1221Crossref PubMed Scopus (657) Google Scholar, 14Zheng Y.T. Shahnazari S. Brech A. Lamark T. Johansen T. Brumell J.H. J. Immunol. 2009; 183: 5909-5916Crossref PubMed Scopus (438) Google Scholar, 15Cemma M. Kim P.K. Brumell J.H. Autophagy. 2011; 7: 341-345Crossref PubMed Scopus (158) Google Scholar). We thus addressed the recruitment of NDP52 to Shigella. We first observed the co-localization of NDP52 with ubiquitinated proteins around Shigella (Fig. 1A) and showed that 84 ± 3% of Shigella-septin cages recruited NDP52 (means ± S.E. from n = 3 experiments) (Fig. 1B). To determine whether NDP52 recruitment is dependent upon septins, we used siRNA to deplete cells of SEPT2 or SEPT9 and evaluated NDP52 recruitment to Shigella in these siRNA-treated cells. In both cases, NDP52 recruitment was significantly reduced (3.8 ± 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 autophagic markers (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). We then asked whether p62 and NDP52 were recruited to the same Shigella-septin cage. Indeed, 83 ± 2% of Shigella-septin cages recruited both p62 and NDP52 (mean ± S.E. from n = 3 experiments) (Fig. 1D). To determine whether p62 and NDP52 have independent roles at the Shigella-septin cage, we used siRNA to deplete cells of p62 or NDP52 and evaluated adaptor recruitment to Shigella in these siRNA-treated cells. Strikingly, p62 or NDP52 recruitment was significantly reduced in either case (31.1 ± 5.1-fold or 2.4 ± 0.3-fold in p62-depleted cells, respectively, or 6.6 ± 2.3-fold or 32.6 ± 14.6-fold in NDP52-depleted cells, respectively) (Fig. 1E). These results strongly suggest an interdependent relationship between p62 and NDP52 to target Shigella toward autophagy. p62 is constantly degraded by autophagy and has thus been established as a marker for autophagic activity, i.e. autophagic flux (8Mizushima N. Yoshimori T. Levine B. Cell. 2010; 140: 313-326Abstract Full Text Full Text PDF PubMed Scopus (3532) Google Scholar). To address the role of NDP52 in autophagic activity, we examined the steady-state levels of p62 in the absence and presence of bafilomycin (an inhibitor of autophagosome-lysosome fusion) in control cells or in cells depleted of NDP52. Levels of p62 were significantly reduced in cells depleted for NDP52 in the absence (2.1 ± 0.4-fold) and presence (1.5 ± 0.1-fold) of bafilomycin (Fig. 2), suggesting that p62 and NDP52 are interdependent to promote autophagy. We next examined steady-state levels of NDP52. Levels of NDP52 accumulated in control cells treated with bafilomycin (3.9 ± 0.5-fold), indicating that NDP52, like p62, can be used as a marker of autophagic vesicle turnover (Fig. 2). In cells depleted for p62, levels of NDP52 were significantly reduced in the absence (1.5 ± 0.1-fold) and presence (1.2 ± 0.1-fold) of bafilomycin (Fig. 2). Thus, p62 can significantly regulate NDP52 activity and vice versa. Different pathogens have evolved different ways to escape autophagy (20Mostowy S. Cossart P. Locht C. Simonet M. Bacterial Pathogenesis. Horizon Scientific Press, Rowan House, Hethersett, Norwich, U.K2012: 241-258Google Scholar). In the case of L. monocytogenes, these bacteria avoid ubiquitination, p62 recognition, and septin caging by expressing the surface protein ActA, a bacterial effector required to polymerize actin (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 12Yoshikawa Y. Ogawa M. Hain T. Yoshida M. Fukumatsu M. Kim M. Mimuro H. Nakagawa I. Yanagawa T. Ishii T. Kakizuka A. Sztul E. Chakraborty T. Sasakawa C. Nat. Cell Biol. 2009; 11: 1233-1240Crossref PubMed Scopus (345) Google Scholar). We addressed the role of NDP52 and showed that L. monocytogenes EGDΔactA, but not the wild-type strain EGD, is recognized by ubiquitin, p62, and NDP52 (Fig. 3A). 95 ± 2% of EGDΔactA recruiting p62 also recruited NDP52 (mean ± S.E. from n = 3 experiments), showing that p62 and NDP52 were recruited to the same bacteria (Fig. 3B). We used siRNA to deplete cells of p62, NDP52, SEPT2, or SEPT9 and evaluated adaptor recruitment to EGDΔactA in these siRNA-treated cells. Unlike what was observed for Shigella (Fig. 1E), NDP52 was recruited to bacteria in p62-depleted cells and vice versa (Fig. 3C). Septin depletion did not affect autophagy receptor recruitment to EGDΔactA (Fig. 3C), in agreement with our previous work showing no septin caging of these bacteria (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). These data reveal that p62 and NDP52 can be recruited independently of each other, and independently of septins, during autophagy of Listeria. As an interdependence between p62 and NDP52 was clearly identified in cells infected with Shigella (Fig. 1E), we analyzed more precisely the signals that recruit p62 and NDP52. Shigella avoids autophagy via the bacterial effector protein IcsB which prevents the binding of Atg5, a protein critical for autophagosome maturation (21Mizushima N. Sugita H. Yoshimori T. Ohsumi Y. J. Biol. Chem. 1998; 273: 33889-33892Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar), to IcsA (22Ogawa M. Yoshimori T. Suzuki T. Sagara H. Mizushima N. Sasakawa C. Science. 2005; 307: 727-731Crossref PubMed Scopus (703) Google Scholar). We thus addressed the role of IcsA and IcsB in the recruitment of p62 and NDP52. S. flexneri M90TΔicsA, i.e. an isogenic mutant strain unable to polymerize actin or recruit septin cages in the cytosol of cells (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), failed to recruit ubiquitin, p62, and NDP52 (data not shown). In agreement with this result, the inhibition of actin polymerization by cytochalasin D significantly reduced the recruitment of ubiquitin, p62, and NDP52 to wild-type bacteria (3.1 ± 0.5-fold, 2.3 ± 0.6-fold, and 2.2 ± 0.5-fold, respectively) (Fig. 4A). Moreover, SEPT2 or SEPT9 depletion similarly reduced the recruitment of ubiquitin (data not shown), p62 (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), and NDP52 (Fig. 1C) to wild-type bacteria. Therefore, accumulation of ubiquitinated proteins and the targeting of Shigella to autophagy by p62 and NDP52 are dependent on actin and septin. It has been shown previously that the IcsB mutant is more efficiently targeted to autophagy (22Ogawa M. Yoshimori T. Suzuki T. Sagara H. Mizushima N. Sasakawa C. Science. 2005; 307: 727-731Crossref PubMed Scopus (703) Google Scholar, 23Travassos L.H. Carneiro L.A. Ramjeet M. Hussey S. Kim Y.G. Magalhães J.G. Yuan L. Soares F. Chea E. Le Bourhis L. Boneca I.G. Allaoui A. Jones N.L. Nuñez G. Girardin S.E. Philpott D.J. Nat. Immunol. 2010; 11: 55-62Crossref PubMed Scopus (1025) Google Scholar) and is more efficiently entrapped in septin cages (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Interestingly, S. flexneri M90TΔicsB recruited significantly more ubiquitin, p62, and NDP52 than did wild-type bacteria (1.8 ± 0.1-fold, 1.9 ± 0.2-fold, and 1.9 ± 0.2-fold, respectively) (Fig. 4, B and C). Taken together, these data highlight the role of IcsB in preventing ubiquitin protein recruitment/formation and the recruitment of p62 and NDP52 and confirm the critical role of actin and septin in this process. We next considered host factors that control the recruitment of autophagy receptors during Shigella infection. TNF-α is a pleiotropic cytokine that orchestrates a wide range of biological functions, including host defense against pathogens (24Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3007) Google Scholar), and is prominently induced upon Shigella infection (25Perdomo O.J. Cavaillon J.M. Huerre M. Ohayon H. Gounon P. Sansonetti P.J. J. Exp. Med. 1994; 180: 1307-1319Crossref PubMed Scopus (251) Google Scholar). TNF-α is also known to stimulate septin caging (11Mostowy S. Bonazzi M. Hamon M.A. Tham T.N. Mallet A. Lelek M. Gouin E. Demangel C. Brosch R. Zimmer C. Sartori A. Kinoshita M. Lecuit M. Cossart P. Cell Host Microbe. 2010; 8: 433-444Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). We therefore tested p62 and NDP52 recruitment to Shigella in TNF-α-treated cells. In agreement with the increase observed for septin caging, p62 and NDP52 recruitment to Shigella significantly increased (1.9 ± 0.2-fold and 1.9 ± 0.2-fold, respectively) upon treatment with TNF-α (Fig. 5A). In the cas" @default.
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