Cellular Microbiology (2014) 16(12), 1806–1821

doi:10.1111/cmi.12329 First published online 30 August 2014

Rab17-mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion Bijaya Haobam,1† Takashi Nozawa,1,2*† Atsuko Minowa-Nozawa,1,2 Misako Tanaka,1 Seiichiro Oda,1 Takayasu Watanabe,1 Chihiro Aikawa,1,2 Fumito Maruyama1,2 and Ichiro Nakagawa1,2 1 Section of Bacterial Pathogenesis, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. 2 Department of Microbiology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. Summary Autophagy plays a crucial role in host defence by facilitating the degradation of invading bacteria such as Group A Streptococcus (GAS). GAScontaining autophagosome-like vacuoles (GcAVs) form when GAS-targeting autophagic membranes entrap invading bacteria. However, the membrane origin and the precise molecular mechanism that underlies GcAV formation remain unclear. In this study, we found that Rab17 mediates the supply of membrane from recycling endosomes (REs) to GcAVs. We showed that GcAVs contain the RE marker transferrin receptor (TfR). Colocalization analyses demonstrated that Rab17 colocalized effectively with GcAV. Rab17 and TfR were visible as punctate structures attached to GcAVs and the Rab17-positive dots were recruited to the GAScapturing membrane. Overexpression of Rab17 increased the TfR-positive GcAV content, whereas expression of the dominant-negative Rab17 form (Rab17 N132I) caused a decrease, thereby suggesting the involvement of Rab17 in RE–GcAV fusion. The efficiency of GcAV formation was lower

Received 28 January, 2014; revised 4 June, 2014; accepted 25 June, 2014. *For correspondence. E-mail [email protected], [email protected]; Tel. (+81) 3 5408 5457; Fax (+81) 3 5408 5457. † These authors contributed equally to this work.

in Rab17 N132I-overexpressing cells. Furthermore, knockdown of Rabex-5, the upstream activator of Rab17, reduced the GcAV formation efficiency. These results suggest that Rab17 and Rab17mediated REs are involved in GcAV formation. This newly identified function of Rab17 in supplying membrane from REs to GcAVs demonstrates that RE functions as a primary membrane source during antibacterial autophagy.

Introduction Autophagy refers to the catabolic homeostatic process whereby cells degrade their own components for recycling purposes. During this process, a small portion of the cytoplasm is surrounded by a double-membrane organelle, the autophagosome. The autophagosome then fuses with the lysosome to form the single-layered autolysosome, in which cellular contents are ultimately degraded (Ohsumi, 2001; Wang and Klionsky, 2003; Yoshimori, 2004; Mizushima et al., 2008). This mechanism of degradation can be used selectively for several purposes, including the removal of misfolded proteins or damaged organelles. Intracellular invading pathogens, such as Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Shigella flexneri and Streptococcus pyogenes (Group A Streptococcus, GAS), can also be targeted selectively by autophagy (Levine et al., 2011). GAS is a common pathogen that causes a variety of acute infections including pharyngitis, skin infections, acute rheumatic fever and life-threatening necrotizing fasciitis (Cunningham, 2000). GAS bacteria are destroyed inside GAS-containing autophagosome-like vacuoles (GcAVs) following their invasion of non-phagocytic cells (Nakagawa et al., 2004). The GAS comprise chainforming cocci where the diameter of each coccus is approximately 1 μm, thus the GAS-surrounding autophagic vacuoles need to be bigger than the canonical autophagosomes, which have a diameter of 0.3–0.9 μm. Yamaguchi et al. (2009) showed that homotypic fusion of the initial GcAV (isolation membrane) leads to the formation of a large GcAV, which can be 10 times larger than the canonical autophagosome. Our previous study also suggested that Rab9A increases the size of GcAVs via

© 2014 John Wiley & Sons Ltd

cellular microbiology

Role of recycling endosomes in GcAV formation homotypic fusion between closed GcAVs, which leads to more efficient bacterial eradication (Nozawa et al., 2012). Overall, these reports suggest that alternative and/or additional membrane trafficking pathways are involved in the supply of membrane for autophagosome formation during GAS infection. The source of the autophagosomal membrane and its mechanism of formation are unresolved questions in autophagy research, thus they remain poorly understood. Recent advances in this area have shown that many organelles, such as the plasma membrane, trans-Golgi network, endoplasmic reticulum (ER) and mitochondria, might act as potential membrane sources in mammalian cells (Young et al., 2006; Axe et al., 2008; Hayashi-Nishino et al., 2009; Hailey et al., 2010; Ravikumar et al., 2010; Tooze and Yoshimori, 2010). ER–mitochondria contact sites are known to be the source of autophagosome formation in mammalian cells (Hamasaki et al., 2013). Recently, recycling endosomes (REs) were proposed as potential membrane sources for autophagosomes. REs contain the early acting autophagy proteins Atg9 and ULK1, and they can be incorporated into newly forming autophagosomes. Rab11, an RE-localized Rab GTPase, is required for RE-mediated autophagosome formation (Longatti et al., 2012). However, the Rab proteins involved in GcAV formation differ substantially from those in starvation-induced autophagosomes, and the Rab proteins recruited to GcAVs from REs, ER, and mitochondria have not been identified (Nozawa et al., 2012). Therefore, the membrane source and the mechanism that regulates the supply of membrane for GcAV formation remain unclear, and it is unknown whether GcAVs are derived from the same membrane source as that used to produce starvationinduced autophagosomes. In the present study, we found that GcAVs contain RE marker protein transferrin receptor (TfR). Rab17, a protein resident in the RE, localizes to GcAVs and is required for the fusion of GcAVs and REs. Rab17 is also involved in GcAV formation. Thus, we propose that RE is an important membrane source and that the fusion of RE during the formation of GcAV involves Rab17, but not Rab11.

Results Identifying the membrane sources for GcAV formation To identify the membrane sources that contribute to the growing GcAV, we transfected HeLa cells with mCherry– LC3 (a red fluorescent protein fused to an autophagic membrane marker), before infecting them with GAS and observing the localization of GcAV using various organelle membrane markers. We examined the localization of mCherry–LC3 with TfR, calnexin (an ER marker), © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

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TOMM20 (a mitochondrial marker), and GM130 (a cisGolgi apparatus marker) at 2 and 4 h after infection. These time points were considered to represent the early and late stages of GcAV formation respectively (Nozawa et al., 2012). As shown in Fig. 1A and B, about 30% of the GcAVs colocalized with the TfR at 2 and 4 h, whereas the calnexin, TOMM20, and GM130 signals did not overlap significantly with GcAVs. These results suggest that RE membranes are used for GcAV formation. Involvement of Rab11 in GcAV formation Previous studies have indicated that starvation-induced autophagosomes contain the TfR and the RE-resident Rab GTPase protein Rab11, which is required for autophagosome formation via REs (Longatti et al., 2012). Therefore, to examine the involvement of Rab11 in GcAV formation, we observed the localization of emerald-green fluorescent protein (EmGFP)–Rab11 during GAS infection. EmGFP–Rab11 clearly colocalized with LC3-positive puncta in starvation conditions (Fig. 2A). However, EmGFP–Rab11 was not associated with GcAV at every time point examined (Fig. 2A). In addition, EmGFP– Rab11 did not colocalize with LC3-positive puncta during GAS infection. These results are consistent with previous reports (Fader et al., 2008; Nozawa et al., 2012). Next, we investigated the effects of Rab11 knockdown on GcAV formation using a microRNA–RNA interference (miR–RNAi) system, and we used Western blotting (Fig. 2B) to confirm the effect of the knockdown on Rab11 expression. Knockdown of Rab11 significantly decreased the number of autophagosomes in starved cells (Fig. 2C and D), but the efficiency of GcAV formation was not affected (Fig. 2E and F). In addition, the ratio of TfRpositive GcAVs was not affected in Rab11-knockdown cells compared with control cells (Fig. 2G), suggesting that Rab11 is not required for fusion between GcAVs and REs. Taken together, these data suggest that Rab11 is involved in starvation-induced autophagy but not in GASinduced autophagy. Colocalization of Rab17 with GcAVs Both starvation-induced and GAS-induced autophagosomes contain the TfR, but Rab11 is a specific regulator of starvation-induced autophagy. The specific type of autophagy (i.e. starvation-induced versus GASinduced) primarily dictates the type of Rab protein that is involved in autophagosome formation (Nozawa et al., 2012). Therefore, we hypothesized that other RE-resident Rab proteins might regulate fusion between GcAVs and REs. To identify the specific Rab proteins involved in GcAV–RE fusion, we examined the localization of three RE-resident Rab proteins (Rab4, Rab17 and Rab22A) in

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Fig. 1. TfR localizes to the GcAV. A. GcAVs contain the TfR. HeLa cells that transiently expressed mCherry–LC3 were infected with GAS at an moi of 100 for 4 h, fixed, and stained with anti-TfR (RE marker), anti-calnexin (ER marker), anti-TOMM20 (mitochondria marker), or anti-GM130 (cis-Golgi marker) antibody. DNA was stained with DAPI. Representative confocal z-slices are shown, with a higher magnification in the boxed area. Bars, 10 μm (white) and 2 μm (yellow). B. The percentages of GcAVs that colocalized with each indicated marker were determined by confocal microscopy. The colocalization efficiency was calculated as the percentage relative to the total number of GcAVs. The data shown represent the results for > 50 GcAVs and each percentage represents the mean ± SD from three independent experiments. © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

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Fig. 2. RE-resident Rab11 is involved in starvation-induced autophagy but not in GAS-induced autophagy. A. EmGFP–Rab11 colocalized with mCherry–LC3 in starvation conditions but not in cells infected with GAS. HeLa cells that transiently expressed mCherry–LC3 and EmGFP–Rab11 were cultured in starvation medium for 1 h or infected with GAS for 4 h. B. HeLa cells were transfected with miR vectors for the control and Rab11. At 48 h after transfection, the expression of Rab11 was analysed by Western blotting using anti-Rab11 antibody. C. Knockdown of Rab11 inhibited starvation-induced autophagosome formation. HeLa cells that stably expressed EmGFP-LC3 were transfected with each miR vector. At 48 h after transfection the cells were cultured with starvation medium for 1 h or infected with GAS for 4 h. DNA was stained with DAPI. D. Quantification of the number of LC3 puncta per cell in the indicated knockdown cells. The data represent the mean ± SD based on 10 images. *P < 0.05. E. Effect of Rab11 knockdown on GcAV formation. HeLa cells that transiently expressed EmGFP–LC3 were infected with GAS for 4 h, fixed, and stained with DAPI. Bars, 10 μm. F. Quantification of the percentage of GcAV-positive cells. The data represent the mean ± SD based on three independent experiments. G. Knockdown of Rab11 did not affect the percentage of TfR-positive GcAVs. Quantification of the percentage of TfR-positive GcAVs. HeLa cells that transiently expressed mCherry–LC3 were infected with GAS for 4 h, fixed, and stained with anti-TfR. The colocalization efficiency was calculated as the percentage relative to the total number of GcAVs. The data shown represent results for > 50 GcAVs and each percentage represents the mean ± SD based on three independent experiments. © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

1810 B. Haobam et al. GAS-infected cells. All of these Rab proteins localized clearly to TfR-positive REs in uninfected cells (Fig. S1). As shown in Fig. 3A and B, only EmGFP–Rab17 colocalized frequently with GcAV at 2 and 4 h after infection. We also examined the time-course of EmGFP– Rab17 colocalization with GcAV from 1 to 5 h after infection and found that the percentages of EmGFP– Rab17-positive GcAVs at all time points tested were > 60% (Fig. 3C), where these colocalization efficiencies were higher than those of TfR-positive GcAVs. Rab17 is involved in the regulation of receptor-mediated transcytosis, apical recycling, and melanosome trafficking (Hunziker and Peters, 1998; Zacchi et al., 1998; Beaumont et al., 2011). To our knowledge, this is the first study to find an association between Rab17 and the LC3positive compartment. Recruitment of Rab17 to the GAS-capturing isolation membrane with TfR-positive REs We hypothesized that Rab17 and the TfR might be supplied from REs to GcAVs. In uninfected cells, EmGFP– Rab17 colocalized with the TfR but not with LC3. In contrast, in GAS-infected cells, EmGFP–Rab17 localized with the TfR to GAS-containing LC3-positive structures (Fig.4A). In addition, mCherry–LC3 puncta colocalized partially with EmGFP–Rab17 and the TfR (Fig. 4A). To examine whether Rab17 is recruited to growing LC3positive vacuoles, we performed time-lapse imaging of live cells during GAS infection. As shown in Fig. 4B, an LC3-positive membrane initially contained a single Rab17-dotted structure, but the number of Rab17 signals on the LC3-positive membrane increased gradually with time, and the final closed LC3-positive vacuole harboured at least five Rab17 signals. These results suggest that Rab17 and the TfR are recruited to autophagic vacuoles during GAS infection. Interestingly, the EmGFP–Rab17 signal did not overlap completely with mCherry–LC3, but instead was represented by several dotted structures associated with GcAVs (arrowheads in Fig. 4A magnified images). These GcAV-associated EmGFP–Rab17positive dots colocalized clearly with the TfR. We also investigated whether this structure was a representative image and found that all the GcAVs we observed possessed EmGFP–Rab17- and TfR-positive dots (Fig. S2). Therefore, we propose a new model of Rab17-mediated GcAV formation where Rab17-positive REs fuse with GcAVs. LC3 can localize to unclosed isolation membranes, closed double-membrane autophagosomes, and lysosome-fused autolysosomes, thus GcAV may also be classified with these three types of autophagic structures. To identify the structures to which Rab17 is recruited, we examined the localization of Atg5, LC3, LAMP1 and

cathepsin D. Previously, we demonstrated that Atg5 localizes specifically to the isolation membrane and can be used as an isolation membrane marker protein during GAS infection (Nozawa et al., 2012). LAMP1 is a lysosome marker, thus LAMP1-positive GcAVs represent autolysosomes. Cathepsin D is a lysosomal protease that can also be used as a lysosome marker. At 2 h post infection, EmGFP–Rab17 localized to the GAS-targeting Atg5-positive membrane (Fig. 4C), and at 4 h post infection, LAMP1- or cathepsin D-positive GcAV also colocalized with EmGFP–Rab17 (Figs 4D and S3). Rab17-positive dot-like structures were observed on the GcAVs, even in isolation membranes and autolysosomes (arrowheads in Fig. 4C and D magnified images). The observation that the LAMP1- or cathepsin D-positive dots on GcAVs did not overlap with the Rab17-positive dots indicates that the Rab17-positive dot-like structures are not recruited to GcAVs via endosome/lysosome fusion. These results suggest that Rab17 and Rab17-positive REs are recruited to the isolation membrane, thus they localize to both autophagosomes and autolysosomes.

Requirement of Rab17 during fusion between REs and GcAVs To examine whether Rab17 is involved in RE–GcAV fusion, we attempted to knock down Rab17 using a miR– RNAi system. However, none of the designed miR–RNAi vectors decreased the expression of Rab17 in an effective manner. Next, we overexpressed Rab17 wild type (WT), Rab17 N132I (dominant negative, GDP-bound form of Rab17) and Rab17 Q77L (constitutively active, GTPbound form of Rab17), and quantified the percentage of TfR-positive GcAVs. The percentages of TfR-positive GcAVs were higher in Rab17 WT and Rab17 Q77Loverexpressing cells compared with the control cells (Fig. 5A and B). By contrast, the percentage was significantly lower following overexpression of Rab17 N132I. Therefore, we propose that Rab17 is involved in the fusion of GcAVs and REs.

Requirement of Rab17 for GcAV formation and GAS destruction Next, to identify the roles of REs in GcAV formation, we investigated the effects of Rab17 WT, Rab17 N132I and Rab17 Q77L overexpression on GcAV formation. Rab17 and Rab17 Q77L overexpression significantly increased the percentage of GcAV-harbouring cells compared with the control cells (Fig.6A and B). By contrast, Rab17 N132I overexpression resulted in lower percentages at both 2 and 4 h post infection, thereby suggesting that Rab17 is involved in GcAV formation. © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

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© 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

1812 B. Haobam et al. Fig. 4. Rab17 localized at the GAS-targeting isolation membrane and autophagosome. A. EmGFP–Rab17 and the TfR colocalized with LC3 in GAS-infected cells. HeLa cells that transiently expressed EmGFP–Rab17 and mCherry–LC3 were infected with GAS at an moi of 100 for 4 h and fixed. Cells were stained with anti-TfR antibody. Bars, 10 μm (white) and 2 μm (yellow). B. EmGFP–Rab17 puncta were recruited to GcAVs. HeLa cells that transiently expressed EmGFP–Rab17 and mCherry–LC3 were infected with GAS for 2 h and images were captured by confocal microscopy. C. EmGFP–Rab17 colocalized with the GAS-capturing isolation membrane. HeLa cells that transiently expressed FLAG–Atg5 and EmGFP–Rab17 were infected with GAS for 2 h, fixed, and stained with anti-FLAG antibody. Bars, 10 μm. D. EmGFP–Rab17 localized to LAMP1-positive GcAVs. HeLa cells that transiently expressed EmGFP–Rab17 and mCherry–LC3 were infected with GAS for 4 h, fixed, and stained with anti-LAMP1 antibody. Bars, 10 μm. Yellow arrowheads, LAMP1-positive dot-like structures. White arrowheads, Rab17-positive dot-like structures.

GcAVs can efficiently destroy invading GAS, and this process is facilitated by lysosomal proteases (Nakagawa et al., 2004). If Rab17 and Rab17-mediated REs are required for GcAV formation, Rab17 N132I overexpression should increase the intracellular survival of GAS. Therefore, we examined the invasion efficiency of GAS and the number of GAS that survived at 6 h after infection. As expected, although the efficiency of GAS invasion of cells was not affected by Rab17 N132I overexpression (Fig. 6C), the number of surviving GAS was significantly higher at 6 h (Fig. 6D) compared with that in the control cells. This observation indicates that the bactericidal efficiency was significantly reduced by Rab17 dysfunction. Furthermore, the bactericidal efficiency was increased in Rab17- or Rab17 Q77L-overexpressing cells. These data suggest that GTP-bound Rab17 is important for GcAV formation and the subsequent destruction of GAS. We also suggest that the recruitment of REs into growing GcAVs is required for GcAV formation.

Involvement of Rabex-5 in GcAV formation Rabex-5 is known to be a guanine nucleotide exchange factor (GEF) for Rab5 (Horiuchi et al., 1997), and it was recently shown to have a GEF activity with Rab17, which regulates the localization of Rab17 (Yoshimura et al., 2010; Mori et al., 2013). Thus, we hypothesized that Rabex-5 might also be involved in the formation of GcAV. To test this hypothesis, we examined the localization of EmGFP-Rabex-5 in GAS-infected HeLa cells. EmGFPRabex-5 colocalized with TfR on GcAVs and the percentages of EmGFP-Rabex-5-positive GcAV were > 80% at both 2 and 4 h post infection (Fig. 7A and B). In addition, the ratio of TfR-positive GcAVs was increased by Rabex-5 overexpression (Fig. 7C). Next, we knocked down the expression of Rabex-5 to confirm the involvement of Rabex-5 in GcAV formation and GcAV–RE fusion, and confirmed the effect of the knockdown by Western blotting (Fig. 7D). As expected, the efficiency of GcAV formation and the ratio of TfR-positive GcAVs were reduced significantly by the knockdown of Rabex-5 (Fig. 7E and F). These results demonstrate that Rabex-5 is involved in RE–GcAV fusion and GcAV formation.

Involvement of Rab17 in starvation-induced autophagosome formation To determine whether Rab17 regulates starvationinduced autophagy, we examined the colocalization of EmGFP-Rab17 with mCherry-LC3 in starvation conditions, where the majority of the LC3 punctate structures did not colocalize with EmGFP-Rab17 (Fig. 8A). Next, we examined the effects of the overexpression of Rab17 WT or NI on starvation-induced autophagosome formation. Overexpression of Rab17 WT or NI did not affect the number of LC3-positive puncta formed in starvation conditions (Fig. 8B and C). Moreover, we also found that EmGFP-Rabex-5 did not colocalize with LC3 puncta (Fig. 8D). These results suggest that Rab17 is not required for starvation-induced autophagosome formation.

Discussion In the present study, we demonstrated that Rab17 mediates the supply of membrane from REs during GcAV formation (Fig. 9). Rab17 as well as Rabex-5 were visible in GcAVs as Rab17- and Rabex-5-positive structures that colocalized clearly with the TfR. Rab17-positive structures were present on the GAS-capturing isolation membrane, thereby indicating that Rab17-positive REs were recruited to the growing GcAVs. Overexpression analyses showed that Rab17 is involved in RE–GcAV fusion, GcAV formation, and bacterial destruction. Therefore, we propose that Rab17-mediated REs are required for GcAV formation and are an important membrane source (Fig. 9). Various membrane sources have been suggested to supply lipids for starvation-induced autophagosome formation, but the details of their mechanisms have remained unclear. In the present study, we found that the GcAV is also TfR positive (similar to starvation-induced autophagy), which suggests that the RE is a common membrane source. It is not clear whether the continuous supply of membrane from the RE pathway is sufficient to support large autophagosome formation. However, our microscopic analyses demonstrated that the recruitment of many RE-like structures to for the formations of autophagosomes supports this possibility. Interestingly, Rab17, Rabex-5 and TfR signals occur simultaneously as © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

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dotted structures on the GAS-containing autophagic vacuoles (Figs 4A and 7A). The GcAV formation process involves a complex membrane network system, thus it is important to characterize the ‘hot spots’ to facilitate studies of the dynamic mechanical behaviour of vacuoles. A previous study of SNARE proteins on GcAV showed that some SNAREs (VAMP8 and Vtilb) localize to the GcAV, where they are involved in GcAV–lysosome fusion (Furuta et al., 2010), and the microscopic images obtained in that study illustrated the dotted structure of mCherry–VAMP8 or –Vtilb on GcAVs. Therefore, the Rab17/TfR-positive dotted structures may also be related to these or other SNAREs. We detected no colocalization of GcAV with the ER, mitochondria, or Golgi markers; however, we cannot exclude the possibility that these organelles are potential membrane donors for GcAV formation. For example, calnexin is a membrane-bound chaperone in the ER that has been used as an ER marker, but starvation-induced autophagosomes do not contain calnexin. Hamasaki et al. (2013) showed that autophagosomes form at the ER–mitochondria contact site where the ER-resident SNARE protein syntaxin 17 is involved in autophagosome biogenesis, as well as demonstrating that syntaxin 17 is required for GcAV formation. These results indicate that the source of autophagosome formation for GcAVs is the same as that in starvationinduced autophagy. TfR–ULK1-positive REs are incorporated into the autophagosomes formed during starvation (Longatti et al., 2012). Recently, Puri et al. (2013) showed that mAtg9 vesicles are trafficked from the plasma membrane to the RE. Atg16L1 also localizes to the RE and participates in vesicle fusion with mAtg9-positive vesicles

at that site. Thus, the RE is likely to be the site where the initial Atg proteins assemble. It is not known whether these core Atg proteins are required for GcAV formation, but REs may play similar roles in GcAV formation, as found in canonical autophagy. The delivery of the RE to the autophagosome requires Rab11 in starvation conditions. However, our data indicate that Rab11 is not involved in GcAV formation or RE–GcAV fusion. Further microscopic analysis revealed that Rab17 and its activator Rabex-5 colocalized with GcAVs. EmGFP–Rab17 or EmGFP–Rabex-5 signals were visible as dot structures that localized on GcAVs. These Rab17/ Rabex-5-positive dots contained TfR, suggesting that Rab17-TfR-positive REs are recruited to and fused with GcAVs. Expression of the dominant-negative form of Rab17 decreased the number of GcAVs and TfR-positive GcAVs. In contrast, the expression of Rab17 WT and the constitutively active form of Rab17 increased the abundance of GcAVs and TfR-positive GcAVs. This is the first time the role of Rab17 in autophagy has been demonstrated, although many Rab proteins have been implicated in both canonical and antibacterial autophagy (Chua et al., 2011). Rab17 is involved mainly in epithelial cell-specific polarized trafficking (Hunziker and Peters, 1998; Zacchi et al., 1998), and GAS are internalized through the apical membrane. This specific Rab17mediated pathway that defends against GAS invasion might be a product of evolutionary pressure. In addition to Rab23, Rab17 was recruited during the formation of GcAV and it may be involved in GcAV formation (Nozawa et al., 2012). Rab17 and Rab23 are known to function during the biogenesis of primary cilia, which are sensory structures © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

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Fig. 6. Rab17 is required for GcAV formation and the degradation of GAS. A. Confocal microscopic images of GcAVs in EmGFP-, EmGFP–Rab17 NI- and EmGFP–Rab17 QL-overexpressing cells. HeLa cells that expressed mCherry–LC3 and EmGFP, EmGFP–Rab17 NI, or EmGFP–Rab17 QL were infected with GAS at an moi of 100 for 4 h and fixed. DNA was stained by DAPI. Arrowheads, GcAV. Bars, 10 μm. B. Effects of the overexpression of Rab17, Rab17 NI and Rab17 QL on the GcAV formation efficiency. The percentage of GcAV-positive cells was calculated as the ratio of GcAV-positive cells relative to GAS-infected cells in confocal microscopic images. The data shown represent the results for > 200 infected cells and each percentage represents the mean ± SD based on three independent experiments. **P < 0.01, *P < 0.05. C. Overexpression of Rab17 WT, Rab17 NI or Rab17 QL did not affect GAS invasion. Cells were infected with GAS and the viable intracellular bacteria were counted in triplicate. The results represent the ratio of intracellular live GAS at 2 h after infection relative to intracellular and adherent GAS at 1 h. D. Effects of the overexpression of Rab17 WT, Rab17 NI and Rab17 QL on the number of intracellular surviving GAS. The number of intracellular surviving GAS at 6 h after infection was determined by a bacterial viability assay. The data shown represent the mean ± SE based on three independent experiments. *P < 0.05. © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

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Fig. 7. Rabex-5 is involved in RE–GcAV fusion and GcAV formation. A. Confocal microscopic images of GcAVs with EmGFP–Rabex-5. HeLa cells that expressed mCherry–LC3 and EmGFP–Rabex-5 were infected with GAS at an moi of 100 for 4 h, fixed, and stained with anti-TfR antibody. DNA was stained by DAPI. Bars, 10 μm. B. Colocalization efficiencies of Rabex-5 with GcAVs. HeLa cells that expressed mCherry–LC3 and EmGFP–Rabex-5 were infected with GAS for 1 h and incubated further with antibiotics. The colocalization efficiencies of GcAVs and EmGFP–Rabex-5 were calculated as percentages relative to the total numbers of GcAVs. The data shown represent the results for > 40 GcAVs and each percentage represents the mean value ± SD based on three independent experiments. C. Quantification of the percentage of TfR-positive GcAVs. HeLa cells transfected with EmGFP or EmGFP–Rabex-5 were infected with GAS for 4 h and fixed. The ratio of TfR-positive GcAV was calculated as the percentage relative to the total number of GcAVs in confocal microscopic images. The data shown represent the results for > 50 GcAVs and each percentage represents the mean ± SD based on three independent experiments. **P < 0.01. D. Effects of the knockdown of Rabex-5 expression. HeLa cells were transfected with EmGFP–Rabex-5 expression vectors and miR vectors as the control, and Rabex-5. At 48 h after transfection, Rabex-5 expression was analysed by Western blotting using anti-GFP antibodies. E. Effect of Rabex-5 knockdown on the formation efficiency of GcAVs. HeLa cells transfected with EmGFP–LC3 and miR-knockdown vectors as the control, or Rabex-5 were infected with GAS at an moi of 100 for 4 h and fixed. The percentage of GcAV-positive cell formation was calculated as the ratio of GcAV-positive cells relative to GAS-infected cells in confocal microscopic images. The data shown represent the results for > 200 infected cells and each percentage represents the mean ± SD based on three independent experiments. *P < 0.05. F. Quantification of the percentage of TfR-positive GcAVs. The colocalization efficiency was calculated as the percentage relative to the total number of GcAVs. The data shown represent the results for > 50 GcAVs and each percentage represents the mean ± SD based on three independent experiments. *P < 0.05.

involved in morphogen signalling on the cell surface (Yoshimura et al., 2007). During ciliogenesis, these Rab proteins cooperate and regulate the microtubule dynamics in primary cilia formation. Thus, it would be interesting to study the functional cooperativity of Rab17 and Rab23, as well as the relationship between Rab-mediated cytoskeleton dynamics and GcAV formation. Our data also indicate that the delivery of REs to GcAVs requires the active (GTP-bound) form of Rab17. The dominant-negative GDP-bound form of Rab17 inhibited RE–GcAV fusion and GcAV formation, and abolished their localization to GcAVs. The nucleotide cycle is coupled to membrane attachment and release, thus the localization of the GEF and GTPase activating protein (GAP) may determine the correct targeting of Rab proteins. Recently, it was reported that several Rab GAPs

interact directly with the LC3 and GABARAP families (Itoh et al., 2011; Popovic et al., 2012). However, over half of them do not colocalize with starvation-induced autophagosomes. These observations suggest that a series of Rab GAPs that bind to LC3 may provide the scaffold for appropriate Rab proteins in different types of autophagy. Rabex-5, which is also known as Rab5–GEF, was recently identified as a GEF for Rab17 and a major determinant of the specific membrane targeting of Rab5 (Blümer et al., 2013; Mori et al., 2013). In the present study, we showed that Rabex-7 also localizes to GcAVs where it regulates GcAV formation. Rab5 localizes to endosomes and functions in the GAS invasion step (Sakurai et al., 2010), thus Rabex-5 may be involved in both the invasion and autophagy processes during GAS infection. © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

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Fig. 8. Rab17 and Rabex-5 are dispensable for starvation-induced autophagosome formation. A. Confocal microscopic images of mCherry–LC3 puncta with EmGFP–Rab17 in starvation conditions. HeLa cells that expressed mCherry–LC3 and EmGFP–Rab17 were cultured in starvation medium for 2 h and fixed. Bars, 10 μm. B. Confocal microscopic images of mCherry–LC3 puncta in EmGFP–Rab17 NI-expressing cells. HeLa cells that expressed mCherry–LC3 and EmGFP–Rab17 NI were cultured in starvation medium for 2 h and fixed. Bars, 10 μm. C. Effect of overexpression of the Rab17 inactive mutant on starvation-induced autophagosome formation. HeLa cells transfected with EmGFP, EmGFP–Rab17, or EmGFP–Rab17 NI were incubated in starvation medium. The number of LC3 dots was quantified in confocal microscopic images. The data shown represent the results for > 10 images and each percentage represents the mean value ± SD based on three independent experiments. D. Confocal microscopic images of mCherry–LC3 puncta with EmGFP–Rabex-5 in starvation conditions. HeLa cells that expressed mCherry–LC3 and EmGFP–Rabex-5 were cultured in starvation medium for 2 h and fixed. Bars, 10 μm.

It is still unclear why Rab17, but not Rab11, is involved in RE recruitment to autophagosome formation during GAS infection. For many years, the RE has been considered to be a compartment that recycles internalized cargoes to the plasma membrane, but its involvement in a more complex set of intracellular pathways is increasingly accepted (Hsu and Prekeris, 2010). The RE pathway is involved in exocytic and retrograde transport, as well as in degradation pathways (Matsui et al., 2011). It has also been suggested to serve as a sorting hub for multiple membrane trafficking pathways (Taguchi, 2013). In the present study, although Rab11 colocalized frequently with Rab17 in GAS-infected cells, the colocalization was not complete and some puncta exhibited only one of the two signals (data not shown). Thus, Rab17-positive, but not © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

Rab11-positive, REs may be involved specifically in GcAV formation, although their physiological role is unknown. Alternatively, Rab11 may be inhibited by an unknown factor and Rab17 could function during GAS infection. Invasive pathogenic bacteria secrete various virulence factors that disrupt or manipulate host membrane traffic, and these activities can affect a number of host–cell endocytic trafficking proteins that are located in intracellular compartments (Mallo et al., 2008; Bakowski et al., 2010; Alix et al., 2011; Ham et al., 2011). A recent study suggested that the NADase, one of the proteins secreted by GAS, inhibits autophagosome–lysosome fusion (O’Seaghdha and Wessels, 2013). However, there have been no previous investigations of the molecular mechanisms that underlie the inhibition of

1818 B. Haobam et al.

Fig. 9. Proposed model of Rab17-mediated RE recruitment during GcAV formation. Model showing the involvement of Rab17-positive REs in GcAV formation. Rab17 and Rabex-5 are involved in the fusion of REs during the formation of GcAVs and the recruitment of REs is required for GcAV formation.

lysosomal fusion or studies of other virulence factors that affect host membrane traffic. Further details of the molecular interaction between GAS and host proteins are needed to understand the involvement of specific Rab proteins. In summary, we demonstrated that Rab17 is important for the supply of membrane from REs to nascent GcAVs. Thus, the Rab17-mediated recruitment of REs is crucial for the autophagic defence against invading GAS. This is the first study to demonstrate that REs are a source of membrane for GcAV formation during GAS autophagy. Experimental procedures GAS strain GAS strain JRS4 (M6+ F1+) was grown in Todd–Hewitt broth (BD Diagnostic Systems; Sparks, MD) supplemented with 0.2% yeast extract, as described previously (Nakagawa et al., 2004).

Cell cultures HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum and 50 μg ml−1 gentamicin (Nacalai Tesque) in a 5% CO2 incubator at 37°C. To induce starvation, cells were washed with phosphate-buffered saline (PBS) and incubated in Hanks’ balanced salt solution (Difco) (starvation medium) for 1 h.

Plasmids The plasmids used for transient expression were constructed with Gateway cloning technology (Invitrogen). The cDNA for

human Rab protein was amplified using the polymerase chain reaction (PCR) from human peripheral blood mononuclear cell cDNA with the following primer pairs: Rab17-F, 5′-CACCATGGCACAGGCACACAGGACCCCCCAGCCCAG-3′ and Rab17-R, 5′-GCACCTAGTGGGCGCAGCATTTGGCCT GCCTCGCGG-3′; Rab22A-F, 5′-CACCATGGCGCTGAGGG AGCTCAAAGTGTGTCTGCT-3′ and Rab22A-R, 5′-TCAGCA GCAGCTCCGCTTTGGCTCTGAAGGCTGTCT-3′; Rabex-5-F, 5′-CACCATGAGCCTTAAGTCTGAACGCCGAGGAATTCA-3′; and Rabex-5-R, 5′-TCATCCTGCATAAACTTGAGGTTGCAG TGGTGGAGG-3′. The PCR products were cloned into a pENTR/ D-TOPO vector using a pENTR Directional TOPO cloning kit (Invitrogen) and subcloned into pcDNA6.2/N-EmGFP-DEST. The Rab17 dominant-negative mutant (Rab17 N132I) and constitutively active mutant (Rab17 Q77L) were constructed by introducing point mutations using a PrimeSTAR Mutagenesis Basal Kit (Takara) and the following primer pairs: Rab17 NI-forward, 5′-GTGGGCATCAAGACGGACCTCAGCCAG-3′ and Rab17 NI-reverse, 5′-CGTCTTGATGCCCACCAGCATCACCAG-3′; Rab17 QL-forward, 5′-GCTGGCCTGGAGAAGTACCACA GCGTC-3′; and Rab17 QL-reverse, 5′-CTTCTCCAGG CCAGCTGTGTCCCAGAT-3′. pBABEpuro–GFP–LC3 (plasmid 22405), which was generated by Jayanta Debnath (Fung et al., 2008), was purchased from Addgene. A BLOCK-iT Pol II miR– RNAi expression vector kit (Invitrogen) was used to knock down Rab11 and Rabex-5 expression. We designated the targeting sequences for Rab11 as 5′-GGAGCTGTAGGTGCCTTATTG-3′ (GenBank Accession No. NM_001206836) and for Rabex-5 as 5′-CGAGAAGATAATGGATCAGAT-3′ (GenBank Accession No. NM_001287062). The miRNA double-strand sequences were ligated to pcDNA-6.2-GW/miR (Invitrogen), according to the supplier’s instructions. pcDNA6.2-GW/miR-neg (Invitrogen) was used as the miRNA control. These plasmids were transfected into HeLa cells as described below.

Transfection of plasmids and Western blotting Plasmids were transfected using polyethylenimine (Polysciences) and incubated for 48 h until they were used in the experimental assays. Western blotting was performed as described previously (Nakagawa et al., 2001). The HeLa cells transfected with plasmids were lysed in Triton lysis buffer [1% Triton X-100, 150 mM NaCl, 50 mM Hepes (pH 7.4), and protease inhibitor cocktail (Nacalai Tesque)]. The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Pall Corporation). The immunoblot analysis was performed using the antibodies indicated and visualized with Chemi-Lumi One Super (Nacalai Tesque).

Retroviral infections and generation of stable cell lines Plat-E cells (kindly provided by T. Kitamura, The University of Tokyo) were transiently transfected with pBABEpuro–GFP–LC3 using FuGENE HD reagent (Roche Applied Science). The cells were cultured for 48 h and the growth medium that contained the retrovirus was collected. HeLa cells were incubated with the virus-containing medium. Uninfected cells were removed by selection with 2 μg ml−1 puromycin (Invitrogen). © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

Role of recycling endosomes in GcAV formation Antibodies The following antibodies were used: mouse anti-TfR/CD71 (Santa Cruz Biotechnology, sc-53059), mouse anti-calnexin (BD Transduction Laboratories, 610524), rabbit anti-TOMM20 (Abcam, ab78547) and anti-GM130 (BD Transduction Laboratories, 610822), rabbit anti-Rab11 (Cell Signaling Technology, 3539), mouse anti-FLAG M2 (Sigma-Aldrich), mouse anti-LAMP1 (Santa Cruz Biotechnology, H4A3), mouse anti-β-actin (13E5) (Cell Signaling Technology, 4970), and mouse anti-GFP (Nacalai Tesque). The secondary antibodies used for Western blotting were horseradish peroxidase-conjugated anti-mouse or antirabbit IgG (Jackson ImmunoResearch Laboratories). The fluorescent secondary antibodies used for immunofluorescence were Alexa Fluor 488-conjugated goat anti-rabbit or anti-mouse IgG, Alexa Fluor 594-conjugated goat anti-rabbit or anti-mouse IgG, or Alexa Fluor 660-conjugated goat anti-rabbit or antimouse IgG (Molecular Probes/Invitrogen).

GAS infection For GAS infection, HeLa cells (4 × 104 cells per well) were cultured in 24-well culture plates on 12-mm-diameter glass coverslips. At 48 h after transfection, the cells were infected with harvested bacteria at a multiplicity of infection (moi) of 1:100 without antibiotics. After 1 h, the medium was changed to 10% fetal calf serum/DMEM with antibiotics (100 μg ml−1 gentamicin and 100 U ml−1 penicillin G) to kill any extracellular bacteria. The cells were then cultured until the time points indicated. The cells were placed on a glass-bottomed dish to facilitate time-lapse imaging.

Fluorescence microscopy For confocal microscopy, the cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton in PBS for 10 min, blocked with blocking solution (5% skim milk, 2.5% goat serum, and 2.5% donkey serum in 0.1% gelatin-containing PBS) for 1 h, and incubated with primary antibodies for 1 h. After washing, the cells were incubated with secondary antibodies for 30 min. After further washes, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Nacalai Tesque) in PBS to detect bacterial and cellular DNA. Images were acquired using a confocal laser microscope (FV1000; Olympus) and captured with FluoView software (Olympus).

Statistical analysis Colocalization and GcAV formation were quantified by direct visualization via confocal microscopy. Unless indicated otherwise, at least 50 GcAVs or 200 GAS-infected cells were counted per condition for each experiment. At least three independent experiments were performed for each trial. Unless indicated otherwise, the results represent the mean ± standard deviation (SD) and P-values were calculated using a two-tailed Student’s t-test.

Acknowledgements This research was supported in part by Grant-in-Aid for Scientific Research (25115708, 25293370, 25305013, 70294113, © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

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24790413), the funding program for Next Generation WorldLeading Researchers (LS041) (I.N.), a Sasakawa Scientific Research Grant (T.N.) from The Japan Science, and Daiichi Sankyo Foundation of Life Science (I.N.) and the Japanese Ministry of Education Global Center of Excellence (GCOE) Program ’International Research Center for Molecular Science in Tooth and Bone Diseases’.

Competing interests statement The authors declare no competing interests.

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1806–1821

Role of recycling endosomes in GcAV formation Fig. S1. Subcellular localizations of Rab4A, Rab17, and Rab22A. Rab4A, Rab17, and Rab22A localized to REs. HeLa cells that transiently expressed EmGFP–Rab4A, EmGFP– Rab17, or mCherry–Rab22A were immunostained to identify the TfR. Bars, 10 μm. Fig. S2. Representative images of GcAVs with EmGFP–Rab17 and TfR. Confocal microscopic images of GcAVs immunostained with EmGFP–Rab17 and to show the TfR. HeLa cells that transiently expressed EmGFP–Rab17 were infected with GAS

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for 4 h, fixed, and immunostained to identify the TfR. Bars (yellow), 2 μm. Fig. S3. EmGFP–Rab17 localized to cathepsin D-positive GcAVs. HeLa cells that transiently expressed EmGFP– Rab17 and mCherry–LC3 were infected with GAS for 4 h, fixed, and stained with anti-cathepsin D antibody. Bars, 10 μm. Yellow arrowhead, cathepsin D-positive dot-like structures. White arrowheads, Rab17-positive dot-like structures.

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