Since the original observation that S. typhimurium induces phagosome tubulation, elucidation of the contributing factors and their molecular mechanisms has been occult. Here, we show that two SPI2 effector proteins, SseJ and SifA, cooperatively induce endosomal membrane tubulation (ET), which, like Salmonella-induced phagosome tubulation, required microtubules and SKIP. Furthermore, SseJ was found to bind RhoA and to induce ET with the GTP-bound form. The structure of the SifA-SKIP PHD complex demonstrated that the N-terminus of SifA binds SKIP, while the C-terminus folds similar to the SPI1 GEF SopE. In addition, SifA was shown to interact with the GDP-bound form of RhoA, as would be expected for a GEF. These results suggest that both SseJ and SifA interact in a protein complex with SKIP and RhoA family GTPases as a mechanism to promote phagosome tubulation.
Our observation that expression of SseJ and SifA recapitulated phagosome tubulation was surprising since previous reports suggested that SseJ is not required for phagosome tubulation (Birmingham et al., 2005
) and that SifA expressed alone, or with the SPI2 effector SopD2, induces LAMP1-positive filamentous structures (Brumell et al., 2001a
; Jiang et al., 2004
). However, we only rarely observed ET on expression of SifA alone or with SopD2, using a variety of different cell lines (data not shown). These differences could reflect experimental techniques since others have measured SifA-induced phenotypes using LAMP1 staining. Assaying specifically for SseJ-localized compartments revealed an increase in SifA-induced ET, indicating that even if SifA alone can induce ET at low frequency, the activity is increased in the presence of SseJ. In the absence of SseJ, other S. typhimurium
effectors with redundant membrane altering activities likely contribute to phagosome tubulation in vivo. A redundant-effector scenario is not without precedent as effectors in other TTSS, including those encoded on SPI1 required for S. typhimurium
invasion, have been demonstrated to have overlapping and/or redundant functions (Staskawicz et al., 2001
). For example, deletion of at least three SPI1 effectors, including the GEF SopE, is required for strains to exhibit reduced bacterial invasion and ruffling phenotypes (Zhou et al., 2001
), while exogenous expression of a single effector in mammalian cells will produce membrane ruffling.
Previous work on WxxxE-containing bacterial effector proteins indicated that they mimicked the activities of different Rho GTPases (Alto et al., 2006
). Expression of Map, IpgB1, and IpgB2, produced the classic GTPase-specific actin cytoskeleton effects of filopodia, lamellipodia, and stress fibers, respectively, which allowed characterization of their activities. Although SifA-expressing cells did not exhibit the actin cytoskeleton phenotype of any activated GTPase, including the stress fibers characteristic of RhoA, our work provides evidence that a property of SifA is to stimulate RhoA-family GTPase signaling pathways on the phagosome membrane, as constitutively active RhoA, RhoB, and RhoC were able to substitute for SifA in cooperating with SseJ to induce ET. Moreover, the SifA structure revealed that SifA contains a C-terminal domain that resembles the GEF SopE. This structural observation is consistent with the result that SifA can interact with the GDP-bound form of RhoA, as would be expected for a GEF. Though preliminary attempts to detect SifA GEF activity for RhoA were unsuccessful (data not shown) it is plausible that additional proteins could be essential for SifA GEF activity or that RhoA may not be the SifA substrate. The native substrate of SifA could be RhoB or RhoC, since they are also competent to induce ET with SseJ, and like mammalian GEFs, SifA could be GTPase specific. RhoB is an attractive candidate for the specific activity of SifA since it localizes to trafficking vesicles in mammalian cells (Adamson et al., 1992
). We attempted to identify which GTPase participates in ET by screening for reduced ET in the presence of siRNA targeting each RhoA family GTPase, and found that ET was reduced when RhoABC or RhoC alone were depleted (data not shown). However, since these proteins are important for cell cycle and their depletion can result in major alterations to the cytoskeleton, this method could have indirect effects that may not directly relate to mechanisms of ET induction.
Interestingly, SifA contains a stretch of residues (243–257) that is unique among WxxxE effectors (Alto et al., 2006
). In the structure of SifA, this region appears to be stabilized by W197 of the WxxxE motif, and is analogous to the catalytic loop of SopE that interacts with Cdc42 (Buchwald et al., 2002
), suggesting that the same region of SifA may also be involved in interactions with RhoA family GTPases. Consistent with this idea, mutating W197 and E201 reduced the ET-inducing activity of SifA (). Therefore, though it is unknown whether SifA functions to bind or activate GTPases, we tentatively conclude that rather than functioning independently of small GTPases, as originally postulated for the WxxxE family, SifA interacts with GDP-bound RhoA family GTPases as a mechanism to manipulate host cell processes. In addition, it is possible that SifA binds and/or activates RhoA as a mechanism to modulate the activity of SseJ, since it also binds RhoA.
Importantly, this study also demonstrated the structural basis of the interaction of SifA with the kinesin-binding protein SKIP and provided additional evidence that the SifA-SKIP complex is essential to ET. The crystal structure of the PH domain of SKIP with SifA provided fine detail of the interaction between SKIP and the amino-terminal domain of SifA, and mutants generated based on the interface provided additional evidence that the interaction of SKIP with SifA is essential to ET. Thus, SKIP likely facilitates ET by linking SifA protein complexes to the microtubule network.
Our results indicate that at least four proteins are required to induce ET. This leads to a working model for the mechanism of membrane tubulation, as depicted in . Lipidated SifA localizes to the membrane and binds SKIP via its N-terminus, which could serve to link SifA and the membrane to the microtubule network by binding to kinesin. Moreover, this interaction could also provide the direction and stability for membrane tubulation. SifA could also bind membrane associated GDP-bound RhoA (or RhoB or RhoC) via its C-terminus, and possibly activate it through GEF activity. Since SseJ also interacts with RhoA, RhoA may link SseJ and SifA on the membrane, and possibly other mammalian binding partners interact with the SKIP/SifA/RhoA/SseJ complex through additional direct protein-protein interactions. Each of the proteins in the complex appears to be required for membrane tubulation to occur, and its possible that their interaction influences the specific activity of each other.
Precedent for the analysis of membrane tubulation has been established by analyzing cultured cells treated with Brefeldin A (BFA), which causes tubulation of Golgi membranes (Lippincott-Schwartz et al., 1991
). BFA inactivates the secretory GTPase Arf, causing Golgi compartments to elongate and fuse with the ER, creating tubular structures (Nebenfuhr et al., 2002
). Interestingly, BFA-tubulation is blocked when cytoplasmic phospholipases are inhibited (de Figueiredo et al., 2001
), suggesting that phospholipases are required for BFA-tubulation, and this may be similar to the requirement of SseJ activity for ET. Additional proteins may also participate in ET, such as BAR domain-containing proteins, which induce membrane tubulation in vitro by sensing and maintaining membrane curvature (McMahon and Gallop, 2005
). Regardless of the specific mechanism, our work indicates that SifA and SseJ recruit protein complexes that link GTPase activity and membrane alteration with movement along microtubules. This is consistent with live videomicroscopy of S. typhimurium
infected HeLa cells, which indicates that LAMP1-positive phagosome tubules demonstrate dynamic and directional motility (http://faculty.washington.edu/merza/sifdynamics/
, courtesy of Alex Merz and Maggie So).
One of the most fascinating, yet perplexing, questions regarding S. typhimurium
pathogenesis is what is the function of phagosome tubulation with respect to intracellular replication and virulence in animals? Perhaps directional phagosome movement in infected cells is necessary for nutrient acquisition. Or, the bacteria could be attempting to move in a directional manner, such as through polarized epithelia, to the plasma membrane, or to accomplish cell-to-cell spread. Tubular endo/lysosomal structures containing MHC class II antigen have been shown to form in dendritic cells in response to exposure to bacterial LPS and capsule (Stephen et al., 2007
; Vyas et al., 2007
), suggesting that ET may be a host mechanism for movement of bacterial products that has been co-opted by salmonellae. The relevance of dendritic cell membrane tubulation to MHC presentation remains to be established, however it is interesting to note that S. typhimurium
inhibits MHC class II presentation in a SifA-dependent manner (Mitchell et al., 2004
). Although the exact function of phagosome tubulation is currently undefined, the discovery reported here that SifA and SseJ can interact with host GTPases and promote manipulation of host membranes should allow such questions to be better addressed, and should provide a bounty of information about bacterial mechanisms that promote intracellular replication and mammalian proteins involved in vesicular traffic.