Sep 14, 2011 - None of the other kinesins tested had a significant effect on ..... Photoshop and assembled in Adobe Illustrator. Live cell .... C.C., Jiang, X., et al.
Cellular Microbiology (2011) 13(11), 1812–1823
doi:10.1111/j.1462-5822.2011.01663.x First published online 14 September 2011
Salmonella exploits Arl8B-directed kinesin activity to promote endosome tubulation and cell-to-cell transfer Natalia A. Kaniuk,1 Veronica Canadien,1 Richard D. Bagshaw,2 Malina Bakowski,1,7 Virginie Braun,1 Marija Landekic,1 Shuvadeep Mitra,1 Ju Huang,1 Won Do Heo,3 Tobias Meyer,4 Laurence Pelletier,2,7 Helene Andrews-Polymenis,5 Michael McClelland,6 Tony Pawson,2,7 Sergio Grinstein1,8,9 and John H. Brumell1,7,9* 1 Cell Biology Program, Hospital for Sick Children, Toronto, ON, Canada, M5G 1X8. 2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada, M5G 1X5. 3 Department of Biological Sciences and KI for the BioCentury, KAIST, Daejeon 305-701, Korea. 4 Chemical and Systems Biology, Stanford University Medical School, Stanford, CA 94305, USA. 5 Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, 77843-1114, TX, USA. 6 Department of Pathology and Laboratory Medicine, University of California, Irvine, CA 92697-4800, USA. Departments of 7Molecular Genetics, 8Biochemistry and 9 Institute of Medical Science, University of Toronto, Toronto, ON, Canada, M5S 1A8. Summary The facultative intracellular pathogen Salmonella enterica serovar Typhimurium establishes a replicative niche, the Salmonella-containing vacuole (SCV), in host cells. Here we demonstrate that these bacteria exploit the function of Arl8B, an Arf family GTPase, during infection. Following infection, Arl8B localized to SCVs and to tubulated endosomes that extended along microtubules in the host cell cytoplasm. Arl8B+ tubules partially colocalized with LAMP1 and SCAMP3. Formation of LAMP1+ tubules (the Salmonella-induced filaments phenotype; SIFs) required Arl8B expression. SIFs formation is known to require the activity of kinesin-1. Here we find that Arl8B is required for kinesin-1 recruitment to SCVs. We have previously shown that SCVs undergo
Received 3 February, 2011; revised 1 August, 2011; accepted 2 August, 2011. *For correspondence. E-mail john.brumell@ sickkids.ca; Tel. (+1) 416 813 7654 ext. 3555; Fax (+1) 416 813 5028.
centrifugal movement to the cell periphery at 24 h post infection and undergo cell-to-cell transfer to infect neighbouring cells, and that both phenotypes require kinesin-1 activity. Here we demonstrate that Arl8B is required for migration of the SCV to the cell periphery 24 h after infection and for cell-to-cell transfer of bacteria to neighbouring cells. These results reveal a novel host factor co-opted by S. Typhimurium to manipulate the host endocytic pathway and to promote the spread of infection within a host.
Introduction Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular bacterial pathogen that can cause disease in a variety of hosts (Haraga et al., 2008). After invading host cells, these bacteria occupy a membrane-bound compartment named the Salmonellacontaining vacuole (SCV) that interacts selectively with the host cell endocytic pathway (Bakowski et al., 2008). The SCV rapidly matures and acquires a subset of late endocytic markers, including lysosomal-associated membrane protein 1 (LAMP1), which serves to identify the SCV after 60 min of infection (Garcia-del Portillo et al., 1993a; Steele-Mortimer et al., 1999). Despite the presence of LAMP1 on SCVs, these compartments undergo limited fusion with lysosomes, allowing bacteria to avoid degradation (Ishibashi and Arai, 1990; Buchmeier and Heffron, 1991; Rathman et al., 1997; Hashim et al., 2000). Intracellular replication of S. Typhimurium is optimal from 4 to 14 h post infection (p.i.) (Steele-Mortimer, 2008). At this time, mature SCVs are localized to the juxtanuclear region and associate closely with the transGolgi network (Salcedo and Holden, 2003). At this stage, long LAMP1+ membrane tubules known as Salmonellainduced filaments (SIFs) extend from SCVs towards the cell periphery (Garcia-del Portillo et al., 1993b). SIFs formation requires the actions of bacterial virulence proteins (effectors), which are translocated into host cells via type 3 secretion systems (T3SS) encoded on Salmonella pathogenicity islands (SPI)-1 and -2 (Bakowski et al., 2008). Bacterial mutants that do not promote SIF formation are attenuated for virulence (Hensel et al., 1998; Beuzon et al., 2000) suggesting SIFs are important for
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cellular microbiology
Arl8B in Salmonella infection 1813 pathogenesis. Tubulation of secretory carrier membrane protein 3 (SCAMP3)-containing compartments has also been observed at this time, and may represent a disruption in trafficking from post-Golgi compartments (Mota et al., 2009). At later stages of infection (14–24 h p.i.), SCVs undergo centrifugal movement, and are localized to the cell periphery (Szeto et al., 2009). Centrifugal SCV movement is associated with cell-to-cell transfer of bacteria in vitro (Szeto et al., 2009). Cell-to-cell transfer of bacteria has also been described in a mouse model of S. Typhimurium infection (Sheppard et al., 2003). The mechanisms that regulate centrifugal SCV movement and cell-to-cell transfer of bacteria remain unclear. In eukaryotic cells, intracellular trafficking is regulated by the co-ordinated actions of members of the Arf and Rab families (Behnia and Munro, 2005; Burguete et al., 2008). Late endocytic compartments undergo bidirectional movement along microtubules, and this movement is required for membrane repair (Idone et al., 2008), phagosome maturation (Harrison et al., 2003) and antigen presentation (Sanjuan et al., 2009). Rab7 is thought to play a major role in controlling movement of late endocytic compartments via the recruitment of RILP (Rab7-interacting lysosomal protein) and the dynein/dynactin complex (Harrison et al., 2004; Progida et al., 2007). Recently, Arl8B (also known as Arl10C, Gie1) was implicated in regulating movement of late endocytic compartments (Bagshaw et al., 2006; Hofmann and Munro, 2006). Microtubule-dependent redistribution of LAMP1+ compartments towards the cell periphery occurs in Arl8B-overexpressing cells (Hofmann and Munro, 2006), implicating this GTPase in the regulation of microtubule motor activity. However, the mechanism by which Arl8B impacts on the movement of late endocytic compartments is unknown. We examined the mechanisms that regulate intracellular trafficking of S. Typhimurium and discovered a novel association of Arl8B with SCVs. We show that Arl8B controls the positioning of late endocytic compartments and mediates kinesin-1 recruitment to SCVs. Furthermore we show that S. Typhimurium exploits Arl8B-directed kinesin activity to promote endosome tubulation and cell-to-cell transfer of bacteria. Collectively, these studies reveal an important role for Arl8B in Salmonella pathogenesis and point to this GTPase as a key host factor that is exploited by an intracellular pathogen.
Results Arl8B associates with SCVs during their maturation Salmonella can modulate the function of specific Rab GTPases to control the fate of the SCV (Harrison et al., 2004; Smith et al., 2007; Mallo et al., 2008; Bakowski et al., 2010) but infection-induced alterations of other © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 1812–1823
Fig. 1. Arl8B is associated with Salmonella-containing vacuoles during maturation. A and B. HeLa cells transfected with Arl8B-GFP were infected for 3 h with wild-type SL1344 (A) or DinvA/Inv S. Typhimurium (B). The inner panels represent a higher magnification of the boxed areas. Scale bar, 10 mm. C. The percentage of Arl8B-GFP+ bacteria in (A) and (B) was determined by fluorescence microscopy. Data represent the mean ⫾ standard deviation for at least three independent experiments. N.D., not determined at this time point.
Ras superfamily GTPases has not been described. We performed a screen for GTPases associated with maturing SCVs during Salmonella infection of HeLa cells. The collection of CFP-tagged GTPases used for our initial screen was previously described (Heo et al., 2006). We found that Arl8B associated with the SCV as early as 10 min p.i. (Fig. 1A and C). Association of Arl8B-GFP with SCVs increased with time, with kinetics similar to late endosome markers that are normally acquired by the SCV, including Niemann-Pick type C1 and lysobisphosphatidic acid (Brumell et al., 2002; Smith et al., 2007). In parallel, we infected cells with a SPI-1 T3SSdeficient (DinvA/Inv) mutant of S. Typhimurium that traffics to lysosomes and is degraded (Smith et al., 2007). Arl8B also associated with phagosomes containing DinvA/Inv bacteria (Fig. 1B and C). Therefore, Arl8B localizes to both the SCV and model phagosomes during bacterial infection.
1814 N. A. Kaniuk et al. Arl8B is not required for endocytic trafficking to lysosomes Arl8B is a member of the ADP-ribosylation factor (Arf) family of GTPases that play various roles in endosomal traffic (Chavrier and Goud, 1999; Burd et al., 2004). It is ubiquitously expressed in mammalian cells, including HeLa cells. Arl8B was previously observed to localize to late endocytic compartments, including lysosomes (Bagshaw et al., 2006; Hofmann and Munro, 2006). We tested the role of Arl8B in endocytic trafficking to lysosomes using DQ-BSA, an indicator of proteolytic activity (Bakowski et al., 2010). Cells were treated with control siRNA or siRNA directed against either Arl8B or Rab5 isoforms (A, B, C). Effective knock-down of Arl8B and Rab5 was confirmed by Western blotting (Fig. S1A). Cells were then loaded with DQ-BSA, which becomes fluorescent after cleavage in lysosomes. Expression of Arl8B was not required for delivery of endocytic cargo to lysosomes, while expression of Rab5 isoforms was indispensable (Fig. S1B and C). Therefore Arl8B does not play an essential role in endocytic trafficking of DQ-BSA to lysosomes.
Arl8B controls positioning of late endocytic compartments We found that overexpression of Arl8B-GFP caused redistribution of LAMP1+ compartments to the cell periphery (Fig. S2B, compare with Fig. S2A), consistent with previous observations (Bagshaw et al., 2006; Hofmann and Munro, 2006). Conversely, we observed that knock-down of Arl8B expression by siRNA caused perinuclear clustering of LAMP1+ compartments (Fig. S2D and E). Knockdown of SKIP (SifA and Kinesin interacting protein), a kinesin-1 binding and activating protein, was previously shown to induce perinuclear clustering of LAMP1+ compartments (Jackson et al., 2008) and served as a positive control. Thus, Arl8B promotes outward movement of late endocytic compartments.
Arl8B is required for the formation of SIFs Salmonella has the ability to promote the tubulation of different endosomal compartments during infection of host cells (Leone and Meresse, 2011). This includes formation of LAMP1+ tubules known as SIFs (Garcia-del Portillo et al., 1993b). Maximal SIF induction occurs at 8–10 h p.i. and effectors of the bacterial SPI-2 T3SS are required for SIF formation (Brumell et al., 2002; Haraga and Miller, 2003; Steele-Mortimer, 2008). SIFs are dynamic structures that emanate from Golgi-localized bacterial microcolonies, and extend outward to the cell periphery along microtubules (Brumell et al., 2002). We
Fig. 2. Arl8B is required for the formation of Salmonella-induced endosome tubules. A. HeLa cells were transfected with Arl8B-GFP and infected with wild-type S. Typhimurium for 10 h. Cells were then fixed and stained for LAMP1. Arrow indicates Arl8B+ tubule that is LAMP1-. Scale bar, 10 mm. B. HeLa cells were transfected with Arl8B-GFP and infected with wild-type S. Typhimurium for 10 or 24 h. The percentage of infected cells displaying LAMP1+ tubules (SIFs, black) or Arl8B+ tubules (grey) was determined by fluorescence microscopy. Data represent the mean ⫾ standard deviation for at least three independent experiments. C. HeLa cells were treated with control or Arl8B siRNA and then infected with wild-type S. Typhimurium for 10 h. The percentage of infected cells that displayed LAMP1+ tubules (SIFs) was determined by fluorescence microscopy. Data represent the mean ⫾ standard deviation for at least three independent experiments. Asterisks indicate significantly different from control siRNA-treated cells (P < 0.01), as determined by two-tailed, unpaired Student’s t-test.
found that Arl8B-GFP colocalized with SIFs in HeLa cells at 10 h p.i. (Fig. 2A). Tubular structures containing Arl8B but not LAMP1 were also found in infected cells (Fig. 2A, arrows, Fig. S3, arrowheads) suggesting a more extensive Arl8B+ tubular network, although it must be kept in mind that Arl8B-GFP is overexpressed under these experimental conditions. Arl8B-GFP+ tubules were also observed in MDCK cells, COS-7 cells and Henle cells at 10 h p.i. (Fig. S4). In HeLa cells, both Arl8B+ tubules and SIFs were largely absent at 24 h p.i. (Fig. 2B), suggesting they may be regulated in a similar manner. Formation of Arl8B+ tubules required the SPI-2 T3SS and the effectors SseG and SifA (Fig. S5A and B). It is noteworthy that these effectors also play a role in formation of SIFs (Brumell et al., 2002; Salcedo and Holden, 2003; Mota et al., 2009). Other effectors tested did not have a significant role in Arl8B+ tubule formation (Fig. S5B). Treatment of cells with Arl8B siRNA decreased SIF formation significantly (Fig. 2C). Conversely, overexpression of Arl8BGFP relative to endogenous Arl8B (Fig. S6A) increased © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 1812–1823
Arl8B in Salmonella infection 1815 SIF formation significantly (Fig. S6B). We conclude that Arl8B plays a significant role in the SIFs phenotype, consistent with the role of this GTPase in outward-directed movement of late endocytic compartments. Recently, a SCAMP3-containing tubular network was characterized in Salmonella-infected cells (Mota et al., 2009). SCAMP3+ tubules partially overlap with SIFs, but also comprise distinct tubules that are LAMP1- (Mota et al., 2009). SCAMP3 is normally localized to the transGolgi network, and Salmonella-induced SCAMP3+ tubule formation requires ER-to-Golgi transport (Mota et al., 2009). We found that Arl8B colocalized with SCAMP3+ tubules in infected cells (Fig. S7A, arrows). However, when cells were treated with Arl8B siRNA, there was no obvious change in SCAMP3+ tubule formation (Fig. S7B). Therefore, Arl8B is not required for the formation of SCAMP3+ tubules, indicating that its role may be limited to modulation of LAMP1+ endocytic compartments and not post-Golgi transport. Kinesins are required for Arl8B+ tubule formation Arl8B+ tubules were associated with microtubules during S. Typhimurium infection (Fig. S8A) and were no longer observed when cells were treated with the microtubule disrupting agent nocodazole (Fig. S8B). SIFs formation was previously shown to require kinesin-1 activity (Guignot et al., 2004; Harrison et al., 2004). Therefore we asked whether kinesins play a role in the formation of Arl8B+ tubules. We screened 30 kinesins using endoribonuclease-prepared siRNA (esiRNA) to identify those required for SIFs and Arl8B+ tubule formation. Knock-down of Kif5B (heavy chain for kinesin-1) inhibited formation of Arl8B+ tubules (Fig. S8C). KifC1 depletion also impaired both tubulation phenotypes, consistent with its known association with Kif5B (Nath et al., 2007). Kif11 and Kif24 knock-down also decreased Arl8B+ tubules and SIFs (Fig. S8C). Rab7 esiRNA inhibited SIFs formation, as expected (Brumell et al., 2002) and also inhibited Arl8B+ tubule formation. None of the other kinesins tested had a significant effect on SIFs or on Arl8B+ tubule formation (data not shown). Knock-down of Kif5B, KifC1 and Kif11 led to perinuclear clustering of LAMP1+ compartments, a phenotype similar to that of cells treated with Arl8B siRNA (Fig. S9A and B). Thus, multiple kinesins are involved in both late endocytic compartment positioning and infection-driven tubule formation, two phenotypes regulated by Arl8B. Arl8B plays a role in kinesin-1 accumulation on SCVs of sifA-mutant bacteria We wished to determine if Arl8B plays a role in kinesin recruitment to SCVs. However, endogenous levels of kinesins are difficult to detect via immunofluorescence. There© 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 1812–1823
fore we took advantage of mutant bacteria lacking the SPI-2 T3SS effector SifA. In previous studies, SifA was shown to bind SKIP (Boucrot et al., 2005) and promote peripheral movement of LAMP1+ vesicles and tubules during S. Typhimurium infection (Dumont et al., 2010). In the absence of SifA, SCVs accumulate kinesin-1 after 6 h p.i. (Boucrot et al., 2005). We asked whether Arl8B is involved in this accumulation of kinesin-1 on DsifA SCVs. When cells were infected with wild-type bacteria, no kinesin-1 accumulation was observed on SCVs (identified by LAMP1 staining; Fig. 3A and E). In contrast, DsifA SCVs accumulated kinesin-1, as previously described (Henry et al., 2006) (Fig. 3C and E). However, knock-down of Arl8B impaired kinesin-1 recruitment to wild-type and DsifA SCVs (Fig. 3B, D and E). Together, these results show that Arl8B is required for the recruitment of kinesin-1 to SCVs. Arl8B is required for peripheral SCV displacement at 24 h p.i. Salmonella-containing vacuoles are localized to the perinuclear region (particularly the Golgi) between 8 and 14 h p.i., the time when endosome tubulation is maximal (Salcedo and Holden, 2003; Boucrot et al., 2005; Szeto et al., 2009). However, centrifugal displacement of SCVs towards the host cell periphery is observed at 14–24 h p.i. when endosome tubulation is minimal (Szeto et al., 2009). SCV displacement to the cell periphery requires the SPI-2 T3SS, an intact microtubule network and kinesin-1 activity (Szeto et al., 2009). Since Arl8B regulates positioning of late endocytic compartments (Fig. S2), we investigated the role of Arl8B in peripheral displacement of SCVs. As expected, SCVs in cells treated with control siRNA were located near the nucleus at 10 h p.i. and redistributed to the cell periphery at 24 h p.i. (Fig. 4A–C). In contrast, when cells were treated with Arl8B siRNA, SCVs remained in a position close to the nucleus at 24 h p.i. (Fig. 4B and C). Therefore, Arl8B is required for SCV migration to the cell periphery at late times p.i., after endosome tubulation has ceased. Arl8B is required for cell-to-cell transfer of S. Typhimurium Since we previously demonstrated that S. Typhimurium can undergo cell-to-cell transfer to promote infection of neighbouring cells in vitro (Szeto et al., 2009), we asked if Arl8B plays a role in this process. In order to study this question we used a cell-to-cell transfer assay previously used to study Listeria monocytogenes (Alberti-Segui et al., 2007). Briefly, we examined the ability of intracellular S. Typhimurium to infect a newly introduced population of fluorescently labelled host cells. Bacteria were used to infect cells treated with control and Arl8B siRNA, then
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Fig. 3. Arl8B mediates kinesin recruitment to the SCVs of sifA-deficient bacteria. A–D. HeLa cells were treated with control (A and C) or Arl8B (B and D) siRNA and then infected with wild-type S. Typhimurium (A and B) or an isogenic DsifA mutant (C and D) for 10 h. Cells were fixed and co-stained for LAMP1 (red) and Kinesin-1 heavy chain (green) and then with DAPI (white) to label bacteria and the nucleus. Arrows in (A) and (B) indicate wild-type bacteria in LAMP1+ SCVs that do not colocalize with Kinesin-1. Arrow in (C) shows kinesin-1 accumulation on the SCV of DsifA mutant bacteria. Arrow in (D) shows SCV of DsifA mutant bacteria that does not accumulate Kinesin-1. Scale bar, 10 mm. E. The percentage of infected cells in (A)–(D) that displayed Kinesin-1 accumulation on LAMP-1+ SCVs was determined by fluorescence microscopy. Data represent the mean ⫾ standard deviation for at least three independent experiments. The asterisk indicates significant difference from control siRNA-treated cells (P < 0.001), as determined by two-tailed, unpaired Student’s t-test. Scale bar, 10 mm.
after 2 h p.i., uninfected cells fluorescently labelled with CellTracker Blue CMAC were seeded onto the previously infected cells. A cell-impermeant antibiotic (Gentamicin) was maintained in the medium to kill extracellular bacteria. Cells were examined at 24 h p.i. for LAMP1+ bacteria residing within CellTracker Blue-labelled cells as evidence of cell-to-cell infection. In control siRNA-treated cells, bacteria were observed to infect 20% of CellTracker Bluelabelled cells (Fig. 5A and B). However, Arl8B siRNAtreated cells displayed greatly reduced cell-to-cell transfer of bacteria (Fig. 5A and B). Therefore, Arl8B plays a role in cell-to-cell transfer of S. Typhimurium. Discussion Previous studies have focused on the role of Rab GTPases in intracellular trafficking by pathogenic bacteria
(Brumell and Scidmore, 2007). However, little is known about the role of members of the Arf family of small GTPases. In this study, we demonstrate that Arl8B is associated with SCVs during their maturation. Arl8B is also associated with phagosomes containing DinvA/Inv bacteria, indicating that it may play an important role in endocytic trafficking that is exploited by S. Typhimurium. However, our data suggest that Arl8B does not play a critical role in trafficking to lysosomes (Fig. S1). Our findings are in contrast to those of Katada and colleagues who found that the Arl8B homologue in Caenorhabditis elegans is required for endocytic trafficking to lysosomes in this organism (Nakae et al., 2010). It is possible that the sensitivity of our DQ-BSA assay is not sufficient to detect subtle defects in endocytic trafficking under Arl8B knockdown conditions, or that mammalian cells have other factors that can compensate for trafficking to lysosomes in © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 1812–1823
Arl8B in Salmonella infection 1817 Fig. 4. Arl8B is required for peripheral SCV displacement at 24 h post infection. A and B. HeLa cells were treated with control or Arl8B siRNA and then infected with wild-type S. Typhimurium for either 10 (A) or 24 h (B). Cells were then fixed and immunostained for bacteria (red) and LAMP1 (green), and then stained with DAPI (blue) to label bacteria and the host cell nucleus. Dotted lines demarcate the edges of the cells in fluorescence images (left panels), which can also be visualized in accompanying DIC images (right panels). Arrowheads indicate LAMP1+ SCVs. C. The distance of SCVs to the closest nuclear edge was determined by fluorescence microscopy in (A) and (B). Data represent the mean ⫾ standard deviation for at least three independent experiments. Asterisk indicates significant difference (P < 0.05) from control siRNA-treated cells as determined by two-tailed, unpaired Student’s t-test. Scale bar, 10 mm.
the absence of Arl8B. With regard to the latter possibility, mammalian cells express a closely related Arf family GTPase, Arl8A, for which very little is known. Arl8A may act redundantly for endocytic delivery to lysosomes, a possibility that will require further study. However, our experiments indicate that it cannot compensate for loss of Arl8B expression in the phenotypes studied during S. Typhimurium infection (SIFs, SCV positioning, cell-to-cell transfer). Previous studies have implicated Rab5, Rab7 © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 1812–1823
and Rab9 in endocytic trafficking events at the SCV (Brumell et al., 2001a; Smith et al., 2007; Jackson et al., 2008; Mallo et al., 2008). Whether Arl8B acts in conjunction with these or other members of the Rab GTPase family to co-ordinate endocytic trafficking remains to be determined. We observed that during S. Typhimurium infection (6–10 h p.i.), Arl8B is located on endosomal tubules that radiate from the SCV. We also show that the bacteria exploit Arl8B towards endosome tubulation, including the formation of LAMP1+ SIFs (Fig. 2). Arl8B+ tubules that did not colocalize with LAMP1 were also observed, indicating that endosome tubulations containing this GTPase are more extensive than the SIFs network. However, it must be kept in mind that overexpression of Arl8B may lead to enhanced detection relative to the signal for LAMP1, or non-specific targeting to other compartments. Effectors of the SPI-2 T3SS, namely SifA and SseG, were found to be required for the Arl8B+ tubules phenotype, although it is not clear how these effectors contribute to their formation. Formation of SCAMP3+ tubules did not require Arl8B expression (Fig. S7). However, these structures require distinct bacterial and host factors for their formation and are thought to accumulate membrane from a different intracellular source compared with SIFs formation (Mota et al., 2009). Therefore, it is not surprising that Arl8B plays a role specific to SIFs. The function of endosome tubulation during S. Typhimurium infection is not clear, although it is appreciated that their formation and dynamics are regulated by many virulence factors (Watson and Holden, 2010; Schroeder et al., 2011). In addition to regulating SIFs formation, we show that Arl8B is required for centrifugal movement of SCVs to the cell periphery at late (14–24 h p.i.) stages of infection. These data are consistent with the role of Arl8B in controlling plus end-directed movement of late endosome/ lysosome movement along microtubules (Bagshaw et al., 2006; Hofmann and Munro, 2006), although the mechanisms that control dynamic SCV positioning, which are
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Fig. 5. Arl8B is required for cell-to-cell transfer of S. Typhimurium. (A and B) HeLa cells were treated with control or Arl8B siRNA and then infected with wild-type S. Typhimurium for 2 h. Cell cultures were then overlaid with uninfected HeLa cells labelled with CellTracker Blue CMAC (blue) and cultured an additional 22 h in the presence of Gentamicin. Cells were then fixed and immunostained for Salmonella (red) and LAMP1 (green). LAMP1+ SCVs appearing in CellTracker Blue-labelled cells were used as indicators of cell-to-cell transfer. Dotted lines demarcate the edges of the cells in fluorescence images (left panels), which can also be visualized in accompanying DIC images (right panels). The percentage of CellTracker Blue-labelled cells that contained LAMP1+ SCVs at 24 h p.i. was determined by fluorescence microscopy in (B). Data represent the mean ⫾ standard deviation for at least three independent experiments. Asterisk indicates significant difference (P < 0.05) from control siRNA-treated cells as determined by two-tailed, unpaired Student’s t-test. Scale bar, 10 mm.
localized to the perinuclear Golgi apparatus (8–10 h p.i.) and then subsequently to the cell periphery (14–24 h p.i.), remain unclear. Our previous studies indicate that co-ordinated changes in the expression/stability of T3SS effectors play an important role, but how these effectors impact on Arl8B remains to be determined (Szeto et al., 2009). Our data are consistent with a model whereby Arl8B promotes the activity of kinesins on late endocytic compartments. Indeed, we show that Arl8B is required for
kinesin-1 recruitment to SCVs, which bear many markers of late endocytic compartments (Fig. 3). In addition to kinesin-1, KifC1 and Kif11 were also found to regulate positioning of late endocytic compartments (Fig. S4), suggesting that other kinesins may also be recruited to SCVs via Arl8B to promote endosome tubulations and SCV movement to the cell periphery. We previously demonstrated that S. Typhimurium can undergo cell-to-cell transfer during infection of HeLa cells in a microtubule and kinesin-1-dependent manner (Szeto et al., 2009). Our current study identifies Arl8B as a novel host factor required for cell-to-cell transfer by these bacteria. We found that Arl8B expression is not required for establishment of an intracellular replicative niche by S. Typhimurium (Fig. S10). This further underlines the importance of bacterial exploitation of Arl8B function for cell-to-cell transfer. The role of Arl8B in cell-to-cell transfer within a host remains to be determined once suitable reagents (e.g. Arl8B knockout mice) become available. In summary, our studies reveal Arl8B to be a novel host factor exploited by S. Typhimurium to manipulate the endocytic system of host cells during infection. Since Arl8B does not play an essential role in endocytic trafficking to lysosomes in mammalian cells (Fig. S1), it may represent a suitable therapeutic target in the host cell to prevent cell-to-cell transfer by S. Typhimurium. It is also possible that other vacuole-adapted pathogens exploit Arl8B function during infection of host cells. Further studies of Arl8B function and its manipulation by intracellular pathogens are warranted. Experimental procedures Bacterial strains and plasmids Salmonella enterica serovar Typhimurium strains used in this work were wild-type SL1344 (Hoiseth and Stocker, 1981), wildtype CS401 (Miao and Miller, 1999) and a derivate of wild-type ATCC14028 (HA431) (Santiviago et al., 2009) and their derivatives as follows. Strains derived from wild-type CS401 were: DsifA (MBO213) (Datsenko and Wanner, 2000). Strains derived from HA431 (ATCC14028) were: DsseG, DsifA, DsopD2, DpipB2, DsseJ and DssaT (Santiviago et al., 2009). DinvA/pRI203 (DinvA/ Inv) 14028S (Steele-Mortimer et al., 2002) was also used in this study. The DinvA mutant is defective for SPI-1 T3SS activity and cannot undergo normal invasion of host cells. Expression of the Invasin protein from Yersinia pseudotuberculosis allows the DinvA/Inv strain to enter the host cell via binding to b1-integrins, and the bacteria are subsequently degraded in lysosomes (Smith et al., 2007). The cDNA for Arl8B (NM_018184) was PCR-amplified from a cDNA library of a human fibroblast cell line using Arl8B-specific primers without encoding a stop codon. The cDNA was cloned into the mammalian expression vector pcDNA3.1/CT-GFP (Invitrogen) in frame with the GFP sequence to produce a C-terminal tagged Arl8B expression vector (Arl8B-GFP). All constructs were confirmed by DNA sequencing before use. © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 1812–1823
Arl8B in Salmonella infection 1819 Cell culture and transfection HeLa, MDCK, Henle and COS-7 cells were obtained from ATCC. Cells were maintained in growth medium [DMEM (HyClone) supplemented with 10% FBS (Wisent)] at 37°C in 5% CO2. Cultures were used between passage 5 and 20. HeLa cells were seeded at 5 ¥ 104 cells per well in 24-well tissue culture plates containing coverslips 16–24 h before use. Late-log bacterial cultures were used for infecting HeLa cells as outlined previously (Szeto et al., 2009). Briefly, bacteria were pelleted at 10 000 g for 2 min and resuspended in PBS. The innoculum was diluted and added to HeLa cells at 37°C for 10 min. The cells were then washed extensively with PBS and growth medium containing 100 mg ml-1 gentamicin was added until 2 h p.i., at which point the cells were washed and growth medium containing 10 mg ml-1 gentamicin was added for the duration of the experiment. Invasion studies using S. Typhimurium DinvA/Inv were performed as described previously (Smith et al., 2007). Nocodazole was used at 2 mg ml-1 and tetracycline at 20 mg ml-1 where indicated. For transfection of HeLa cells, Genejuice (Novagen) or Lipofectamine RNAiMAX (Invitrogen) transfection reagents were used according to manufacturer’s instructions. Cells were transfected with plasmids encoding GFP or Arl8B-GFP at 2 h p.i. with S. Typhimurium strains.
RNA interference Arl8B-directed and SKIP-directed siRNA used in this work were obtained from Thermo Scientific Dharmacon (siGENOME SMARTpool) and transfected using Lipofectamine RNAiMAX (Invitrogen) at a final concentration of 100 nM and allowed to proceed for 48 h. The control siRNA was siCONTROL NonTargeting siRNA #2 (Dharmacon). esiRNA duplexes for luciferase (control), Rab7, Kif5B, KifC1, Kif24 and Kif11 were generated using gene-specific primer pairs in Table S1, as previously described (Kittler et al., 2007). All esiRNA duplexes were transfected into HeLa cells using Lipofectamine RNAiMAX (Invitrogen) at a final concentration of 100 ng ml-1 and allowed to proceed for 48 h.
microscope with a 63¥ oil immersion objective and a heated stage assembly [Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu Back-Thinned EM-CCD camera, spinning disk confocal scan head and Volocity 3.7.0 acquisition software (Improvision)]. Images were processed using Volocity software to generate a time-lapse threedimensional z-stack reconstruction.
Antibodies and reagents Rabbit polyclonal antibodies to S. Typhimurium O antiserum were obtained from Difco Laboratories (Kansas City, MP) and used at 1:50 for immunofluorescence studies. Mouse anti-human LAMP1 antibody (clone H4A3), used at 1:20 dilution, was developed by J. Thomas August and obtained from the Developmental Studies Hybridoma Bank under the auspices of the NICHD, maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA. In order to better visualize transfected GFP constructs, mouse anti-GFP antibody was used at a dilution of 1:200 (Invitrogen). Rabbit anti-SCAMP3 (VA226) was used at a dilution of 1:200 (Guo et al., 2002). Rabbit anti-kinesin heavy chain (uKHC) was used at a dilution of 1:20 (Santa Cruz). Mouse anti-b-tubulin was used at a dilution of 1:200 (Sigma). Secondary antibodies used for immunofluorescence were all Alexa conjugated and from Molecular Probes. To stain host cell nuclei in fixed cells, coverslips were immersed in 2 mg ml-1 DAPI or 10 min prior to mounting on glass slides. For Western blotting, rabbit antiArl8B was used at a dilution of 1:100 (ProteinTech); rabbit anti-Rab5 was used at a dilution of 1:500 (Santa Cruz); mouse anti-b-tubulin was used at a dilution of 1:2000 (Sigma).
Intracellular positioning of SCVs and cell-to-cell bacterial transfer The intracellular position of SCVs was determined by measuring the distance of LAMP1+ SCVs to the nearest edge of the host cell nucleus (labelled by DAPI staining) in fixed HeLa cells as previously described (Szeto et al., 2009). Cell-to-cell bacterial transfer assays were performed as previously described (Szeto et al., 2009).
Immunofluorescence microscopy Cells were fixed with 2.5% paraformaldehyde in PBS for 10 min at 37°C. Fixed cells were immunostained as previously described (Brumell et al., 2001b). Immunostaining before permeabilization was used to differentiate between intracellular and extracellular bacteria (Smith et al., 2007). Coverslips were mounted onto glass slides using DakoCytomation fluorescent mounting medium. Images were acquired using an inverted Leica DMIRE2 fluorescent microscope equipped with a Hamamatsu ORCA-ER camera and OpenLab 3.1.7 software. Images were imported into Adobe Photoshop and assembled in Adobe Illustrator.
DQ-Red-BSA assay HeLa cells were plated at 6 ¥ 104 cells ml-1 in eight-well chambered coverglass (Thermo Fisher). After transfection (plasmid or siRNA), cells were incubated with DQ-Red-BSA (Invitrogen) at 0.25 mg ml-1 in growth medium for 1 h then washed with GM for 4 h. The medium was then replaced by RPMI and images were acquired in live cells by fluorescence microscopy. Quantification of the intensity per mm of the DQ-Red-BSA signal was measured using Volocity 5.0. Results are expressed as percentage of control.
Acknowledgements Live cell imaging Cells were grown on 2.5 cm coverslips, co-transfected 12–16 h before invasion with Arl8B-GFP and LAMP1-RFP constructs, and then infected with SL1344 bacteria. At 8 h p.i. culture medium was switched to RPMI-1640 and time-lapse 0.5 mm confocal z-stacks of the cells were imaged using a Quorum spinning disk © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 1812–1823
John H. Brumell, PhD, holds an Investigators in Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. Infrastructure for the Brumell laboratory was provided by a New Opportunities Fund from the Canadian Foundation for Innovation and the Ontario Innovation Trust. N.A.K. was supported by a postdoctoral fellowship from CAG/CIHR/Axcan Pharma adminis-
1820 N. A. Kaniuk et al. tered by the Canadian Association of Gastroenterology and a Postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. R.D.B. was supported by a CIHR fellowship. We thank Andrea Tagliaferro for help with esiRNA preparation. Work in the Brumell lab was supported by the Canadian Institutes of Health Research (MOP62890) and work in the Pelletier lab was supported by NSERC (RGPIN355644–2008), NCIC (019562) and HFSP (CDA0044/200). Work in the Grinstein lab was supported by CIHR grant MOP7075. S.G. is the current holder of the Pitblado Chair in Cell Biology at The Hospital for Sick Children. L.P. holds a Canada Research Chair in Centrosome Biogenesis and Function. H.A.-P and M.M. are supported by NIH Grants R21AI083964, NIH 1R01AI083646, NIH 1R56AI077645, NIH R01 AI075093, and USDA Grant AFRI CSREES 2009-03579. W.D.H. is supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008127012011), Rural Development Administration, Republic of Korea. We are grateful to Drs J.D. Castle and D. Holden for providing reagents used in this study. We also thank Michael Woodside and Paul Paroutis for help with confocal microscopy.
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Supporting information Additional Supporting Information may be found in the online version of this article:
1822 N. A. Kaniuk et al. Fig. S1. Arl8B expression is not required for endocytic trafficking to lysosomes. A. HeLa cells were transfected with control siRNA (siCTL), siRNA against Arl8B (siArl8B) or siRNA against Rab5A, B and C (siRab5). The efficiency of the knock-down was analysed by Western immunoblotting. B. HeLa cells were treated with the indicated siRNA for 48 h. Cells were then washed and incubated with DQ-Red-BSA at 0.25 mg ml-1 for 1 h, then washed and incubated with growth medium for 4 h. The medium was replaced by RPMI and images were acquired from live cells by fluorescence microscopy. Dotted lines demarcate the edges of the cells. Scale bar, 10 mm. C. The intensity of the DQ-Red-BSA signal was quantified using Volocity software. Data represent the mean ⫾ standard deviation for at least three independent experiments. Asterisk indicates significant difference (P < 0.01) from control siRNA-treated cells as determined by two-tailed, unpaired Student’s t-test. Fig. S2. Arl8B controls positioning of late endocytic compartments. A and B. HeLa cells expressing GFP (A) and Arl8B-GFP (B) were fixed and labelled with an antibody to LAMP1 and then stained for DAPI to label the nucleus. Dotted lines demarcate the edges of the cells. Arrowheads in (B) indicate LAMP1+ compartments located at the cell periphery. Scale bar, 10 mm. C. Western blot showing effective knock-down of Arl8B expression by siRNA treatment (upper panel). b-Tubulin blot as loading control (lower panel). D. Cells were treated with the indicated siRNA and stained with anti-LAMP1 antibody (green) and then DAPI to label the nucleus (blue). Dotted lines demarcate the edges of the cells in fluorescence images (left panels), which can also be visualized in accompanying DIC images (right panels). Scale bar, 10 mm. E. The percentage of cells displaying clustering of LAMP1+ compartments in (D) was determined by fluorescence microscopy. Data represent the mean ⫾ standard deviation for at least three independent experiments. Asterisks indicate significantly different from control siRNA (P < 0.01), as determined by two-tailed, unpaired Student’s t-test. Fig. S3. Arl8B-GFP is recruited to Salmonella-induced filaments. HeLa cells co-transfected with Arl8B-GFP and LAMP1RFP and infected for 8 h with wild-type S. Typhimurium. Cells were then examined by spinning disk microscopy with a heated stage assembly. Three-dimensional z-stack reconstructions are shown. The elapsed time after the start of imaging is indicated. Peripheral clusters of Arl8B-GFP and LAMP1-RFP positive membrane are indicated with arrows and the arrowheads indicate dynamic Arl8B+ tubules that were observed with and without coincident LAMP1-RFP. Fig. S4. Arl8B+ tubules are present in multiple cell types during Salmonella infection. MDCK (Madin Darby canine kidney), COS-7 (monkey kidney fibroblasts) and Henle 407 (human intestinal epithelial) cells were transfected with Arl8B-GFP and then infected with wild-type S. Typhimurium for 10 h. Cells were then fixed and stained for GFP (green) or bacteria (red). Arrowheads indicate Arl8B+ tubules in infected cells. Scale bar, 10 mm. Fig. S5. Formation of Arl8B+ tubules requires the SPI-2 T3SS and the effectors SseG and SifA. (A and B) HeLa cells were transfected with Arl8B-GFP (green) and then infected with either wild-type (HA431) S. Typhimurium or isogenic mutants deficient in T3SS effectors for 10 h. A strain deficient in secretion by the SPI-2 T3SS (DssaT) was also examined. Cells were then fixed
and stained with anti-Salmonella antibody (blue). The percentage of infected cells that displayed Arl8B+ tubules was determined by fluorescence microscopy in (B). Data represent the mean ⫾ standard deviation for at least three independent experiments. Asterisks indicate significantly different from control siRNA-treated cells (P < 0.00001), as determined by two-tailed, unpaired Student’s t-test. Scale bar, 10 mm. Fig. S6. Overexpression of Arl8B increases Salmonella-induced filaments (SIFs) formation. A. HeLa cells were transfected with either GFP or Arl8B-GFP, or indicated siRNA. Cell lysates were then subjected to western immunoblotting with antibodies to Arl8B or tubulin (loading control), as indicated. Mobilities of overexpressed Arl8B-GFP or endogenous Arl8B are indicated. Knock-down of Arl8B confirms mobility of endogenous protein. B. HeLa cells were transfected with either GFP or Arl8B-GFP and then infected with wild-type S. Typhimurium for 10 h. The percentage of infected cells (expressing GFP construct) that displayed LAMP1+ tubules (SIFs) was determined by fluorescence microscopy. Asterisks indicate significantly different from GFP transfected cells (P < 0.01), as determined by two-tailed, unpaired Student’s t-test. Fig. S7. Arl8B colocalizes with SCAMP3+ tubules in infected cells but is not required for their formation. A. HeLa cells were transfected with Arl8B-GFP and then infected with wild-type S. Typhimurium for 10 h. Cells were then fixed and stained with anti-SCAMP3 antibody. Arrows indicates SCAMP3+ tubules that colocalize with Arl8B. Scale bar, 10 mm. B. HeLa cells were treated with control or Arl8B siRNA and then infected with wild-type S. Typhimurium for 10 h. The percentage of infected cells that displayed SCAMP3+ tubules was determined by fluorescence microscopy. Data represent the mean ⫾ standard deviation for at least three independent experiments. Fig. S8. Kinesins are required for Arl8B+ tubule formation. A. HeLa cells were transfected with Arl8B-GFP and infected with wild-type S. Typhimurium for 10 h. Cells were then fixed and stained with anti-b-tubulin antibody and then DAPI to label nucleus and bacteria (blue in merge). The inner panels represent a higher magnification of the boxed areas. Arrowhead indicates Arl8B+ tubule associating with microtubule. Scale bar, 10 mm. B. HeLa cells were transfected with Arl8B-GFP and infected with wild-type S. Typhimurium for 10 h in the presence of nocodazole. Cells were then fixed and stained with anti-LAMP1 antibody and then DAPI to label nucleus and bacteria (blue in merge). The inner panels represent a higher magnification of the boxed areas. Arrowhead indicates Arl8B+ vesicles that colocalize with LAMP1. Scale bar, 10 mm. C. HeLa cells were treated with the indicated siRNA and then transfected with Arl8B-GFP. Cells were then infected with wildtype S. Typhimurium for 10 h. The percentage of infected cells that displayed Arl8B+ tubules (black) or LAMP1+ tubules (SIFs, grey) was determined by fluorescence microscopy. Data represent the mean ⫾ standard deviation for at least three independent experiments. Asterisks indicate significantly different from control siRNA-treated cells (P < 0.05), as determined by twotailed, unpaired Student’s t-test. Fig. S9. Kinesins regulate positioning of late endocytic compartments. (A and B) HeLa cells were treated with the indicated esiRNA for 48 h, then fixed, and stained with anti-LAMP1 antibody (green) and DAPI (blue). Dotted lines demarcate the edges © 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 1812–1823
Arl8B in Salmonella infection 1823 of the cells in fluorescence images (left panels), which can also be visualized in accompanying DIC images (right panels). Scale bar, 10 mm. The distance of SCVs to the closest nuclear edge was determined by fluorescence microscopy in (B). Data represent the mean ⫾ standard deviation for at least three independent experiments. Asterisk indicates significant difference (P < 0.01) from control siRNA-treated cells as determined by two-tailed, unpaired Student’s t-test. Fig. S10. Arl8B expression is not required for establishment of an intracellular replicative niche by S. Typhimurium. HeLa cells were treated with either control or Arl8B siRNA for 48 h. Cells were then infected with S. Typhimurium. At the indicated times post infection (1 h and 8 h), cells were fixed and immunostained with a polyclonal antibody recognizing the bacteria. Intracellular
© 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 1812–1823
bacterial numbers were enumerated (100 infected cells for each condition). The percentage of infected cells with different bacterial numbers per cell (1–5, 6–10, 11–15, 16–20, > 20) were quantified and grouped into five categories. Shown are the means ⫾ SEM from three independent experiments. Table S1. Gene-specific primers used to generate endoribonuclease prepared siRNA for this study. T7 promoter sequences were used for sequencing and are indicated in bold.
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