FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan
2021; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.15252/embr.202152584
ISSN1469-3178
AutoresAkihiro Yamada, Miyako Hikichi, Takashi Nozawa, Ichirô Nakagawa,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle13 September 2021free access Source DataTransparent process FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan Akihiro Yamada Akihiro Yamada orcid.org/0000-0002-0996-3343 Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Miyako Hikichi Miyako Hikichi Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Takashi Nozawa Takashi Nozawa orcid.org/0000-0002-1822-5553 Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Ichiro Nakagawa Corresponding Author Ichiro Nakagawa [email protected] orcid.org/0000-0001-6552-1702 Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Akihiro Yamada Akihiro Yamada orcid.org/0000-0002-0996-3343 Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Miyako Hikichi Miyako Hikichi Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Takashi Nozawa Takashi Nozawa orcid.org/0000-0002-1822-5553 Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Ichiro Nakagawa Corresponding Author Ichiro Nakagawa [email protected] orcid.org/0000-0001-6552-1702 Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Author Information Akihiro Yamada1, Miyako Hikichi1, Takashi Nozawa1 and Ichiro Nakagawa *,1 1Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan *Corresponding author. Tel: +81 75 753 4448; E-mail: [email protected] EMBO Reports (2021)22:e52584https://doi.org/10.15252/embr.202152584 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Xenophagy, also known as antibacterial selective autophagy, degrades invading bacterial pathogens such as group A Streptococcus (GAS) to defend cells. Although invading bacteria are known to be marked with ubiquitin and selectively targeted by xenophagy, how ubiquitin ligases recognize invading bacteria is poorly understood. Here, we show that FBXO2, a glycoprotein-specific receptor for substrate in the SKP1/CUL1/F-box protein (SCF) ubiquitin ligase complex, mediates recognition of GlcNAc side chains of the GAS surface carbohydrate structure and promotes ubiquitin-mediated xenophagy against GAS. FBXO2 targets cytosolic GAS through its sugar-binding motif and GlcNAc expression on the GAS surface. FBXO2 knockout resulted in decreased ubiquitin accumulation on intracellular GAS and xenophagic degradation of bacteria. Furthermore, SCF components such as SKP1, CUL1, and ROC1 are required for ubiquitin-mediated xenophagy against GAS. Thus, SCFFBXO2 recognizes GlcNAc residues of GAS surface carbohydrates and functions in ubiquitination during xenophagy. Synopsis During xenophagy invading bacteria are marked with ubiquitin, which is then selectively targeted by autophagic membranes. FBXO2 directly recognizes bacterial surface glycan and facilitates SCF complex-mediated ubiquitination to eliminate bacteria by xenophay. The GlcNAc side chain of the bacterial surface glycan is recognized by FBXO2, a glycoprotein-specific substrate receptor of the SCF ubiquitin ligase complex. FBXO2 targets invading bacteria via its sugar-binding motif. FBXO2 and SCF complex components are required for ubiquitin accumulation on intracellular Group A Streptococcus and the cellular defense by xenophagy. Introduction Autophagy is a membrane trafficking process in which double-membrane spherical structures known as autophagosomes deliver cytosolic contents to lysosomes for degradation. Autophagy not only degrades its components but also selectively targets invading microbes and plays a key role in innate immune defense. Autophagy against microbes is named xenophagy and has been reported to degrade various intracellular bacterial pathogens such as Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, and group A Streptococcus (GAS) (Levine & Deretic, 2007; Campoy & Colombo, 2009). Intracellular bacteria are marked with ubiquitin and ubiquitin-binding autophagy receptors such as p62 and NDP52 and are recruited for linking to LC3 on autophagosomes (Boyle & Randow, 2013). Ubiquitin is also involved in recruiting ATG16L1 for xenophagy (Fujita et al, 2013). Although ubiquitin ligases such as LRSAM1, Parkin, Smurf1, Nedd4-1, and RNF213 have been identified as crucial factors in xenophagy against various bacterial pathogens (Huett et al, 2012; Manzanillo et al, 2013; Franco et al, 2017; Ogawa et al, 2018; Otten et al, 2021), how these ligases selectively recognize invading pathogens is poorly understood. Group A Streptococcus, also known as Streptococcus pyogenes, is a group of gram-positive bacteria that cause a variety of illnesses, including pharyngitis, impetigo, and severe systemic infections such as necrotizing fasciitis and streptococcal toxic shock syndrome (Cole et al, 2011). GAS invades non-phagocytic human cells via endocytosis and escapes from endosomes into the cytosol through the activity of streptolysin O (SLO), a pore-forming toxin secreted by GAS. GAS exposed to the cytoplasm is selectively targeted by xenophagy (Nakagawa et al, 2004). Some GAS strains avoid xenophagic degradation by secreting toxins (Barnett et al, 2013; O'Seaghdha & Wessels, 2013; Toh et al, 2020). We previously reported that GAS invasion through SLO triggers the recruitment of ubiquitin-binding autophagy receptors to ubiquitin-marked GAS through TBC1D9 and RAB35 (Minowa-Nozawa et al, 2017; Nozawa et al, 2020), suggesting that ubiquitination is essential for xenophagy against GAS infection. However, the substrate(s) and ligase(s) responsible for ubiquitin-coating GAS have not been identified. In addition to ubiquitin-mediated recognition, glycan binding proteins such as galectin-3 and galectin-8 have been reported to direct xenophagy by binding host glycans exposed on damaged endosomes and lysosomes. Galectin-8, a cytosolic β-galactoside-binding lectin, activates xenophagy by recruiting the autophagy receptor NDP52 during Salmonella Typhimurium infection (Thurston et al, 2012). Galectin-3 recruits TRIM16, a RING-type ubiquitin ligase, to add K63-linked ubiquitin to damaged endomembranes (Chauhan et al, 2016). In addition, although not involved in xenophagy, the SCFFBXO27 ubiquitin ligase complex has been reported to ubiquitinate lysosomal glycoprotein exposed following membrane damage for lysophagy (Yoshida et al, 2017). Therefore, recent advances have revealed that glycan recognition is important for xenophagy and lysophagy. The recognized glycan substrate is thought to be a host membrane component. However, despite the abundance of glycans and glycoproteins on the bacteria surface, whether bacterial glycans are also recognized in xenophagy is unknown. Group A carbohydrate (GAC) is a bacterial surface rhamnose polysaccharide that constitutes half of the cell wall of GAS, is essential for bacterial growth, and contributes to infection ability (McCarty, 1952). The N-acetylglucosamine (GlcNAc) side chain, present in GAC, is a virulence factor (Henningham et al, 2018). Recent studies showed that mutants defective in GlcNAc side chain addition improve intracellular viability by increasing the sensitivity to neutrophils and serum killing, attenuating virulence, and suppressing host immune responses (van Sorge et al, 2014). However, it is unclear how the GAS surface carbohydrate structure and its GlcNAc side chains contribute to the intracellular dynamics of GAS in host cells. In this study, we investigated the involvement of GlcNAc residues of GAS surface carbohydrate and FBXO2, a glycoprotein-specific F-box protein, in xenophagy. Our findings reveal that the bacterial sugar chain is among the markers of xenophagy recognition, providing insight into the pathway linking bacterial surface components and ubiquitin ligase in xenophagy. Results GAC side chain GlcNAc is involved in xenophagy of GAS Although recent studies showed that ubiquitin-binding autophagy receptors are required for the xenophagy of GAS (Ito et al, 2013; Minowa-Nozawa et al, 2017; Lin et al, 2019), whether ubiquitin itself directly mediates xenophagy is not clear. To investigate the necessity of ubiquitination in xenophagy during GAS infection, we used the ubiquitin-activating enzyme (E1)-specific inhibitor PYR41, which blocks ubiquitination in cells (Yang et al, 2007). Human epithelial cells (HeLa cells) were treated with PYR41 and infected with GAS. The recruitment of galectin-3 was not affected by PYR41 treatment (Fig EV1A and B), suggesting that PYR41 does not inhibit GAS invasion into the host cytosol. In control (non-PYR41-treated) cells, intracellular GAS was coated with ubiquitin, whereas ubiquitin-positive GAS was rarely observed in PYR41-treated cells (Fig EV1A and B). Additionally, PYR41 treatment significantly decreased LC3-positive GAS (Fig EV1A and B). Because LC3 recruitment to bacteria is a hallmark of xenophagy induction (Xu et al, 2019), these results suggest that ubiquitination is required for the xenophagy of GAS. Click here to expand this figure. Figure EV1. Ubiquitination is critical for xenophagy of GAS A, B. GAS xenophagy depends on ubiquitination. WT HeLa cells treated with PYR41 (E1 inhibitor) were infected with WT GAS for 4 h, fixed, and immunostained for anti-galectin-3 (Gal3), LC3, or ubiquitin (magenta). Cellular and bacterial DNAs were stained with DAPI (cyan); scale bar, 10 μm. (A) Representative confocal images and (B) percentages of Gal3, ubiquitin, or LC3 localized to GAS. C. Bacterial invasion rates into host cells (CFU recovered at 2 h post-infection/CFU at 1 h post-infection) which were normalized to that in wild-type cells. D. Intracellular bacterial survival rates (CFU recovered at 6 h post-infection/CFU at 2 h post-infection) which were normalized to that in wild-type cells. E. HeLa cells were infected with GAS for 3 h, and immunostained for ubiquitin and galectin-3. Scale bar, 10 μm. Data information: Data are shown as the mean ± SEM of more than three independent experiments. Asterisks indicate significant differences (**P > 0.01) as determined by Student's t-test. Download figure Download PowerPoint Next, to investigate whether ubiquitination is required for xenophagic degradation of GAS, we examined the intracellular survival of GAS in PYR41-treated cells. Although the invasion efficiency of GAS was unchanged (Fig EV1C), the survival of bacteria at 6 h after infection was significantly increased upon PYR41 treatment (Fig EV1D). Taken together, these results suggest that ubiquitination is required for xenophagy against GAS. We next observed the localization of galectin-3 and ubiquitin in GAS-infected cells and found that some ubiquitin-coated GAS was galectin-3-negative (Fig EV1E). Because galectin-3 targets damaged endosomal membranes (Paz et al, 2010), structures other than damaged endosomal membranes may be targeted by the ubiquitination system during GAS infection. The GAS surface harbors the GAC comprised of a polyrhamnose backbone with GlcNAc side chains. Twelve GAC genes (gacA–gacL) are involved in GAC biosynthesis and transport (Rush et al, 2017). The first three genes, gacA–gacC, are responsible for polyrhamnose backbone biosynthesis and are essential for bacterial viability, and gacI–gacK are required for the addition of GlcNAc side chains to the polyrhamnose backbone (Fig 1A). To examine the involvement of GAC components in xenophagy, we constructed GAC deletion mutants of GAS. We obtained six gac mutants of ΔgacG–gacL, but not of ΔgacA–gacF, which are thought to be critical for core rhamnose backbone construction. To determine whether these mutants were deficient in GlcNAc expression on the GAS surface, we treated GAS mutants with succinylated wheat germ agglutinin (sWGA), a lectin that specifically binds GlcNAc, and measured the fluorescence intensity of sWGA attached to each strain (Nagata & Burger, 1974). As expected, binding of sWGA to ΔgacI was not observed, and the deletion of gacJ and gacK significantly decreased sWGA binding to GAS (Fig 1B). These data are consistent with the results of a previous study (van Sorge et al, 2014). We then examined whether targeting of these GAC mutants by xenophagy was reduced. We found that LC3-positive GAS was significantly reduced in ΔgacI-, ΔgacJ-, and ΔgacK-infected cells than in wild-type GAS-infected cells (Fig 1C and D). In addition, although ΔgacL harbored GlcNAc on the cell surface, targeting LC3 to ΔgacL significantly decreased (Fig 1B and D). As GacL is required to transfer GlcNAc from GlcNAc-phosphate-undecaprenol to polyrhamnose, GlcNAc bound to the rhamnose backbone may be involved in xenophagy induction. Figure 1. Screening of gac deletion mutant A. Schematic representation of the group A carbohydrate (GAC) gene cluster. Biogenesis of GAC is obtained with 12 gac operons. Genes from gacA to gacC are responsible for rhamnose backbone biogenesis, and those from gacI to gacL are involved in GlcNAc side chain addition. B. Expression levels of GlcNAc in wild-type (WT) GAS, ΔgacG, H, I, J, K, and L were measured as the sWGA fluorescence intensity (normalized to WT GAS). C, D. HeLa cells expressing mCherry-LC3 (magenta) were infected with WT GAS and the indicated gac deletion mutant for 2 h at an MOI of 100 and then stained with DAPI (cyan); scale bar, 10 μm. (C) Representative confocal images are shown at 2 h post-infection, and (D) percentages of GcAV-positive cells were quantified. Data information: Data represent the mean ± SEM of (B) nine and (D) four independent experiments. Asterisks indicate significant differences (**P > 0.01) as determined by Student's t-test. Download figure Download PowerPoint GlcNAc residues are involved in recognition by the host ubiquitination system To validate the involvement of GlcNAc of GAS in xenophagy, we complemented gacI to ΔgacI because deletion of gacI most profoundly decreased GlcNAc expression on GAS. The recovery of GlcNAc expression was confirmed by the sWGA binding assay (Fig 2A). To confirm that gacI was involved in xenophagy induction, we examined LC3 lipidation levels during infection. The levels of the lipidated form of LC3 (LC3-II) during ΔgacI infection were significantly lower than those during GAS wild-type and ΔgacI::gacI infection (Fig 2B and C), suggesting that GlcNAc is involved in xenophagy induction during GAS infection. We next investigated which step upstream of LC3 recruitment (endocytosed internalization of GAS, cytosolic escape via SLO, or recognition of GAS by the ubiquitination machinery) was affected by gacI deletion. The invasion efficiency of ΔgacI GAS cells was comparable to that of wild-type GAS (Fig EV2A), and recruitment of galectin-3 was unchanged (Fig EV2B and C), demonstrating that GlcNAc was not associated with bacterial invasion into the host cytosol. We found that deletion of gacI resulted in decreased ubiquitin-coated GAS (Fig 2D and E). To validate whether GlcNAc residues are involved in ubiquitination during GAS infection, we isolated bacteria from infected cells through affinity purification using anti-lipoteichoic acid antibodies. Similar numbers of wild-type and ΔgacI GAS were isolated from host cells at 4 h after infection (Fig EV2D). We then detected conjugated ubiquitin by immunoblotting and found that isolated wild-type GAS carried multiple ubiquitinated molecules with 100–200 and 17 kDa (Fig 2F). In contrast, the size of the bands from ΔgacI GAS was 140–200 kDa (Fig 2F), indicating that GlcNAc residues on the GAS surface are involved in ubiquitination of bacteria in infected cells. Figure 2. GlcNAc affects ubiquitination of GAS A. Expression levels of GlcNAc in WT GAS, ΔgacI, and ΔgacI::gacI were measured as the sWGA fluorescence intensity (normalized to WT GAS). B, C. Accumulation of LC3-II. (B) Representative Western blot of LC3-II during non-infection (NI), WT GAS, ΔgacI, and ΔgacI::gacI. (C) LC3-II intensity normalized to NI. D, E. Recruitment of ubiquitin in GAS-infected cells. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta). Cellular and bacterial DNAs were stained with DAPI (cyan). (D) Representative confocal images and (E) percentage of cells with ubiquitin-positive GAS. Scale bar, 10 μm. F. Immunoblot of the indicated GAS isolated from host cells. Lysate of GAS-infected HeLa cells was precipitated with anti-lipoteichoic acid antibody to isolate GAS, and the immunoprecipitates were analyzed by immunoblotting. G, H. Localization of ubiquitin in sWGA-treated GAS. HeLa WT was infected with the indicated GAS strains masked with sWGA for 4 h, fixed, and immunostained for ubiquitin (magenta). (G) Representative confocal images and (H) percentages of cell with ubiquitin-positive GAS. Scale bar, 10 μm. I, J. Recruitment of p62 to gacI mutants. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for p62 (magenta). Cellular and bacterial DNAs were stained with DAPI (cyan). (I) Representative confocal images and (J) percentage of cells with p62-positive GAS. Scale bar, 10 μm. K. Bacterial viability rates of GAS mutants in cells (CFU recovered at 6 h post-infection/CFU at 2 h post-infection). Data information: Data are shown as the mean ± SEM of (A) nine and (C, E, H, J, and K) three independent experiments. Asterisks indicate significant differences (*P > 0.05, **P > 0.01) as determined by Student's t-test. Source data are available online for this figure. Source Data for Figure 2 [embr202152584-sup-0002-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. GacI is not essential for GAS invasion into host cytosol A. Percentage of invasion rate (CFU recovered at 2 h post-infection/CFU at 1 h post-infection). Values were normalized to that of wild-type GAS. B, C. Localization of Gal3 in GAS-infected cells. (B) HeLa cells expressing mCherry-Gal3 (magenta) were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 2 h at an MOI of 100 and stained with DAPI (cyan); scale bar, 10 μm. (C) Percentages of Gal3-positive cells. D. Numbers of wild-type and ΔgacI GAS (CFU/ml). HeLa cells were infected with WT GAS or ΔgacI GAS for 4 h and isolated from infected cells by affinity purification. Isolated bacteria were incubated on THY plates, and the number of isolated bacteria was determined. E, F. HeLa cells were infected with sWGA-treated GAS and immunostained for Gal3. Representative images (E) and quantification of cells with Gal3-positive GAS (F). Data information: Data are shown as the mean ± SEM of three independent experiments. NS indicates not significant determined by Student's t-test. Download figure Download PowerPoint To confirm the involvement of GlcNAc residues in ubiquitin-mediated recognition in xenophagy, we pre-treated GAS with sWGA to mask GlcNAc residues on the bacterial surface and infected cells with sWGA-coated GAS. Pre-treatment of GAS with sWGA did not inhibit the recruitment of galectin-3 to GAS (Fig EV2E and F) but significantly decreased ubiquitin-positive GAS in wild-type and ΔgacI::gacI-infected cells (Fig 2G and H). In contrast, no significant reduction in ubiquitin-positive GAS was observed after sWGA treatment in ΔgacI-infected cells (Fig 2G and H). Taken together, these results demonstrate that GlcNAc residues are involved in bacterial recognition by the ubiquitination system for xenophagy during GAS infection. As ubiquitin-coated GAS is recognized by ubiquitin-binding autophagy receptors such as p62, we next examined the recruitment of p62 to invading GAS. Deletion of gacI abolished the recruitment of p62, whereas complementation of gacI restored the p62 recruitment to GAS (Fig 2I and J). Because targeting of ΔgacI GAS by ubiquitin, p62, and LC3 was decreased, we next examined whether this mutant avoided xenophagic degradation. The survival rate of ΔgacI GAS was significantly higher than that of the wild-type and ΔgacI::gacI GAS strains (Fig 2K). Therefore, GlcNAc is involved in recognition by host ubiquitin-mediated xenophagy. GlcNAc residues induce K48- and K63-ubiquitin coating during GAS infection K48-, K63-, and M1-linked polyubiquitination is reportedly crucial for xenophagy (Ito et al, 2013; Noad et al, 2017; Ogawa et al, 2018). K48-ubiquitin signals coated each bacterium, whereas K63-ubiquitin signals localized around aggregated bacteria (Fig EV3A). In contrast, minimal M1-linked linear ubiquitin signals were detected around GAS (Fig EV3A). This observation is consistent with the localization of ubiquitin during Streptococcus pneumoniae (Ogawa et al, 2018). To determine the types of polyubiquitin chains induced via GAS surface GlcNAc residues, we observed chain-specific ubiquitin signals on gacI mutants. Confocal microscopic images revealed that the recruitment of both K48 ubiquitin and K63 ubiquitin during ΔgacI infection was reduced compared with that during wild-type and ΔgacI::gacI infection (Fig EV3B and C). Therefore, GlcNAc on the GAS surface is involved in inducing both K48 ubiquitination and K63 ubiquitination during xenophagy of GAS. Click here to expand this figure. Figure EV3. Recruitment of K48- and K63-ubiquitin chain to GAS A. HeLa cells were infected with GAS for 4 h and immunostained for K48 ubiquitin, K63 ubiquitin, and M1 ubiquitin (green). Cellular and bacterial DNAs were stained with DAPI (magenta); scale bar, 2 μm. B, C. Localization of K48- or K63 ubiquitin in GAS-infected cells. HeLa cells were infected with the indicated GAS strains for 4 h; scale bar, 10 μm. Representative confocal images at 4 h of WT GAS, ΔgacI, and ΔgacI::gacI (B) and quantification of cells with K48 or K63 ubiquitin-positive GAS (C). Data information: Data are shown as the mean ± SEM of three independent experiments. Asterisks indicate significant differences (**P > 0.01) as determined by Student's t-test. Download figure Download PowerPoint GAS is targeted by FBXO2 through GlcNAc residues Because GlcNAc residues on the GAS surface carbohydrate were suggested to be involved in ubiquitin-mediated recognition during GAS infection, we focused on glycoprotein-specific F-box proteins (FBXO2, FBXO6, and FBXO27), which are components of the SCF ubiquitin ligase complex (Yoshida et al, 2002). We determined the subcellular localization of EmGFP-tagged FBXO2, FBXO6, and FBXO27 during GAS infection. EmGFP-FBXO2 and EmGRP-FBXO6 were recruited to intracellular GAS, whereas EmGFP-FBXO27 was not (Fig 3A). More than 50 and 30% of infected cells exhibited FBXO2- and FBXO6-positive GAS, respectively (Fig 3B). Confocal microscopy images show that FBXO2 and FBXO6 were co-localized with LC3, and some GAS-associated FBXO2 signals were LC3-negative (Fig 3C, arrowhead). Furthermore, signal-intensity plots of FBXO2 and LC3 revealed that the signal peaks of FBXO2 were inside the LC3-positive circles (Fig 3C), suggesting that FBXO2 targets bacteria rather than LC3 vacuoles. We also examined the kinetics of the recruitment of FBXO2 and LC3 to GAS and found that FBXO2 was recruited to GAS more frequently than LC3 at 2 and 4 h after infection (Fig 3D). To determine whether FBXO2 targeted cytosolic GAS, we observed the localization of FBXO2 during WT or Δslo GAS infection. As shown in Fig EV4, EV5, galectin-3, LC3, and FBXO2 were not recruited to Δslo GAS, suggesting that FBXO2 translocation occurred proximal to invading bacteria upon cytosolic escape through SLO during GAS infection. Because LC3 recruitment to GAS was ubiquitination-dependent and decreased after infection with a GlcNAc deletion mutant, we examined the recruitment of FBXO2 to ΔgacI GAS. Recruitment of FBXO2 to GAS decreased after gacI deletion (Fig 3E and F), suggesting that GlcNAc is involved in targeting FBXO2 to GAS. Taken together, these results suggest that invading GAS is targeted by FBXO2 through GAS surface GlcNAc residues. Figure 3. FBXO2 targets GAS and is involved in xenophagy A, B. Co-localization of GFP-FBXO2 and mCherry-LC3 in GAS-infected cells. HeLa cells transfected with the indicated EmGFP-FBXO (green) and mCherry-LC3 (magenta) were infected with WT GAS for 2 h; scale bar, 10 μm. Representative confocal images (A) and (B) percentages of FBXO-positive cells. C. Enlarged image of GAS-associated FBXO2 of (A). Graphs show the signal intensities of EmGFP-FBXO2 and mCherry-LC3 measured along the arrows in the merged image. Green and red arrows indicate the signal peaks observed in EmGFP-FBXO2 and mCherry-LC3, respectively. White arrow indicates bacteria that are targeted by FBXO2 but not by LC3. Scale bar, 10 μm. D. Time course of FBXO2 and LC3 recruitment during GAS infection. E, F. Localization of EmGFP-FBXO2 in GAS-infected cells. HeLa cells transfected with GFP-FBXO2 (green) were infected with the indicated GAS strains for 2 or 4 h at an MOI of 100; scale bar, 10 μm. Representative confocal images at 4 h of WT GAS, ΔgacI, and ΔgacI::gacI (E) and (F) percentages of FBXO2-positive cells. Data information: Data are shown as the mean ± SEM of (B–D, F; n > 200 cells per condition) three independent experiments. Asterisks indicate significant differences (**P > 0.01) as determined by Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. GAS induces FBXO2 recruitment in a SLO-dependent manner A, B. Secretion of SLO required for FBXO2 recruitment to GAS. WT HeLa cells transfected with GFP-FBXO2 (green) and mCherry-Gal3 (red) were infected with WT GAS or GASΔslo for 2 h; scale bar, 10 μm. (A) Representative confocal images and (B) percentages of FBXO2 or Gal3 localized to GAS were quantified. C, D. Secretion of SLO required for FBXO2 and xenophagy recruitment to GAS. WT HeLa cells transfected with GFP-FBXO2 (green) and mCherry-LC3 (red) were infected with WT GAS and GAS Δslo for 2 h at an MOI of 100; scale bar, 10 μm. (C) Representative confocal images and (D) percentages of LC3 localized to GAS. E. Western blotting of FBXO2 in FBXO-KO HeLa cells. F. Representative images of mCherry-LC3 in FBXO2-KO cells complemented with indicated FBXO2 constructs. Data information: Data are shown as the mean ± SEM of three independent experiments. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Involvement of FBXO6 in ubiquitination-mediated xenophagy of GAS A. Immunoblots of cells knocked down expression of FBXO2 or FBXO6. B, C. Recruitment of ubiquitin and LC3 to intracellular GAS in the indicated knockdown cells. Cells were transfected with the indicated siRNA and infected with GAS for 4 h. Cells were immunostained for ubiquitin (FK2: magenta) or LC3 (magenta); scale bar, 10 μm. Representative images and quantification of cells with ubiquitin- and LC3-positive GAS. Data are shown as the mean ± SEM of three independent experiments. Asterisks indicate significant differences (*P > 0.05; **P > 0.01) as determined by Student's t-test. D. Immunoblots of cells with knocked down expression of SKP1, CUL1, and ROC1. E. Lysates from HeLa cells were infected with WT GAS for 2 h, or non-infected (NI) was immunoprecipitated with an anti-FLAG or anti-FBXO2 antibody and protein A agarose. Immunoprecipitates were analyzed by immunoblotting. Source data are available online for this figure. Download figure Download PowerPoint FBXO2 targets intracellular GAS through sugar-binding motifs FBXO2 consists of four distinct domains: the PEST domain (residues 1–54), F-box domain (FBD; residues 55–95), linker domain (residues 96–124), and sugar-binding domain (SBD; residues 125–297). FBD binds to SKP1 and SBD binds to the innermost chitobiose in high-mannose N-glycans, but mutations Y279 and W280 in SBD prevent this binding (Mizushima et al, 2004). To investigate the translocation mechanism of FBXO2 to GAS in more detail, we constructed a series of deletion and substitution mutants of FBXO2 (Fig 4A). FBXO2 ΔFBD was localized to GAS, whereas the FBXO2 Y279A, W280A, and Y279A/W280A mutants showed cytosol and nuclear localization and were not recruited to GAS (Fig 4B and C), demonstrating that the sugar-binding motif is critical for targeting of FBXO2 to invading GAS. Figure 4. FBXO2 targets intracellular GAS through sugar-binding motifs A. Schematic representation of wild-type FBXO2 and domain deletion mutants of FBXO2. Numbers above the constructs represent the amino acid positions of FBXO2. PEST (residues 1–54) and F-box domains (residues 55–95), linker sequence (residues 96–124), and sugar-binding domain (residues 125–297) are indicated by P, FBD, linker, and SBD, respectively. B, C. HeLa cells transfected the indicated GFP-FBXO2 mutants (green) were infected with WT GAS for 4 h; scale bar, 10 μm. Representative confocal images (B) and percentages of each FBXO2 mutants localized to GAS (C). D. EmGFP intensities bound to GAS cells in vitro. GAS cells were in
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