L. pneumophila is a pathogen that targets and modifies host Rab GTPases to ultimately manipulate vesicular trafficking for its own benefit. The organism employs multiple effector proteins that act sequentially to achieve Rab1 recruitment and manipulation of the GTPase at the LCV. Of the four effectors shown to influence Rab1 dynamics on the vacuole (SidM, LidA, SidD, and LepB), LidA's role in Rab1 manipulation is the least defined.
LidA's early presence on the LCV seems to coincide with its role in boosting SidM-driven recruitment of Rab1 to the LCV (31
). However, it is not clear how LidA is involved in this process. One possibility is that LidA does not directly recruit Rab1 but, rather, that it interferes with its inactivation and removal in order to prolong the presence of active Rab1 on the LCV. We found that LidA inhibited LepB- or TBC1D20-stimulated GTP hydrolysis by forming a complex with Rab1 (A and B). LidA's inhibitory effect on Rab1 inactivation is most likely due to its close interaction with active Rab1, which probably sterically hinders GAP access to the nucleotide binding pocket of active Rab1. LidA binds to Rab1 with unparalleled affinity among known Rab effectors, including GAP proteins (41
). This affinity may increase the stability of the LidA-Rab1 complex to such an extent that GAP proteins could not compete for access to Rab1 (41
). Thus, LidA binding locks GTP-loaded Rab1 in the active conformation, thereby extending its presence on the LCV surface by preventing its GDI-mediated extraction from the membrane. This conclusion is further supported by the fact that EDTA, a small molecule with a molecular weight of only 292 Da, was unable to cause extraction of GTP or GDP from Rab1 in the presence of LidA (C and D), suggesting that LidA binds Rab1 close to its guanine nucleotide binding pocket. Another bacterial effector that acts similarly is EspG, a type III effector protein of the enterohemorrhagic Escherichia coli
O157:H7. EspG forms a complex with ARF6 and blocks its inactivation by GAPs (42
). Though examples of cellular ligands exist that were found to decelerate GTP hydrolysis by binding to their target GTPase (22
), pathogenic bacteria seem to use this mechanism in a more forceful manner to precisely control signaling events regulated by small GTPases without interference from the host.
We reasoned that LidA might keep Rab1 in an active conformation in order to present it to downstream effectors involved in vesicle tethering and fusion. However, our results so far do not support this idea, since LidA had an inhibitory rather than a stimulating effect on Rab1 interaction with GM130, giantin, and p115 (E). The possibility that other cellular downstream effectors might be able to interact with Rab1 even in the presence of LidA cannot be excluded. Alternatively, we speculate that while LidA interferes with binding of host downstream effectors of Rab1, it may facilitate the interaction of this GTPase with L. pneumophila
effectors that mimic the function of host Rab1 ligands. The inhibition of Rab1 binding to its downstream ligands by LidA is reminiscent of the inhibitory effect of SidM-catalyzed AMPylation of Rab1 which also blocks the interaction of the GTPase with at least one of its cellular binding partners, MICAL-3 (33
). L. pneumophila
may use this strategy to restrict Rab1 interaction to a selected set of ligands, thereby allowing the organism to efficiently compete with host proteins on the Golgi compartment for recruitment of secretory vesicles.
Our studies revealed that LidA interfered with Rab1 AMPylation and de-AMPylation in vitro
, raising the question about its effect on these posttranslational modifications during infection. Given that Rab1 recruitment to LCVs is dependent on SidM, it is most likely that LidA primarily interacts with Rab1 after it has been AMPylated by SidM. Consistent with this, LidA is the only known protein that interacts with Rab1 in its AMPylated (35
) or phosphocholinated form (E). The inhibitory effect of LidA on the de-AMPylation (and dephosphocholination) of Rab1 is corroborated by the recently released structure of LidA in complex with another host GTPase, Rab8 (41
). The serine and tyrosine residues of Rab8 that are equivalent to the ones phosphocholinated or AMPylated in Rab1 are buried within the complex with LidA and may not be easily accessible to SidD (or Lem3), respectively. It should also be noted that activation of Rab1 by SidM precedes AMPylation and, in vitro
, it seems to take place at a higher rate than AMPylation. Due to SidM's low binding affinity for GTP-bound Rab1, we could envision an alternative role for LidA in capturing those Rab1 molecules on the LCV surface that have been activated by SidM but escaped AMPylation, thereby protecting them from GAP-stimulated inactivation. Overall L. pneumophila
appears to delay the inactivation of Rab1 in several ways that, at a first glance, may seem redundant but that are necessary to efficiently antagonize host cell processes that may otherwise interfere with Rab1-mediated binding of ER-derived vesicles to the LCV.
The concept of functional redundancy explains why interference with individual factors or pathways within living cell systems does not always result in a detectable phenotype. The emerging consensus is that L. pneumophila
infection displays a high degree of functional redundancy which makes it challenging to detect growth phenotypes when individual or even several effector proteins or host cell pathways are disrupted (15
). For instance, an L. pneumophila
strain lacking SidM is unable to recruit host cell Rab1, yet this mutant is as proficient as wild-type bacteria in intracellular survival and replication vacuole formation (31
). In fact, none of the known L. pneumophila
effector proteins that modulate the activity of Rab1 are required for virulence. This suggests that L. pneumophila
targets multiple vesicle-trafficking routes simultaneously to reroute membrane material to the LCV. Consistent with this, we found that LCVs containing L. pneumophila
mutants showed extensive colocalization with host cell vesicles (), presumably because deletion of sidM
, alone or together, affected only the recruitment of Rab1-dependent vesicles to the LCV, leaving intact other pathways that provided material for the vacuolar transformation process. Thus, it will be interesting to identify the source(s) of the Rab1-independent membrane material and to determine which bacterial and host factors are involved in diverting those vesicles to the LCV.
LidA has characteristics of tethering factors, proteins that mediate docking of transport vesicles with the membrane of the destination compartments. Since LidA is exclusively membrane-associated within infected cells (14
) but lacks an obvious transmembrane domain, it is most likely that LidA is a “peripheral” membrane protein that is recruited to the cytosolic surface of the LCV or to surrounding vesicles by binding either Rab1 or the phosphoinositide PI(3)P (8
). These features may allow LidA to also bind membranes elsewhere in the cell on which this GTPase-lipid combination is found. Several effectors, including SidM, have been shown to bind PI(4)P and use this interaction for attachment to the vacuolar membrane. We found that, unlike SidM, LidA does not have separate regions for binding Rab1 and PI(3)P; instead, the same central coiled-coil region binds both Rab1 and the phosphoinositide, while the N- and C-terminal regions do not bind either (C to E). PI(3)P is commonly found on early endosomal membranes, and intracellular pathogens can interfere with its metabolism to avoid phagosome maturation. SapM from Mycobacterium tuberculosis
is a PI(3)P phosphatase (49
), and it avoids phagosome maturation by preventing the PI(3)P-dependent recruitment of lysosomal enzymes and vacuolar ATPase (28
). No enzymatic activities have been described for LidA, but given its affinity for PI(3)P, it could either interfere with its metabolism or simply mask its presence on the vacuole to occlude potential binding sites for the early endosomal marker EEA1. The presence of PI(3)P on ER subdomains involved in autophagosome formation (3
) suggests that additional host cell membrane compartments enriched in this phosphoinositide may be targeted by LidA.
We propose a model in which LidA promotes docking of host vesicles with the LCV early during infection (). In one scenario, LidA could localize to the LCV membrane, where it keeps Rab1 recruited by SidM in an active conformation by blocking inactivation through GAP-stimulated GTP hydrolysis. The LidA-Rab1 complex could act as an adaptor for vesicular tethering and fusion proteins, thereby bridging the membrane of the LCV with that of secretory transport vesicles surrounding the LCV. Alternatively, upon translocation, LidA might localize to the surface of surrounding vesicles through interaction with phosphoinositides and mediate docking of the vesicles with the LCV via interaction with AMPylated Rab1 and/or other L. pneumophila
effectors present on the LCV. The former model would explain how LidA enhances Rab1 accumulation on LCVs (31
), whereas the latter model would be consistent with the fact that LidA is the only known protein capable of binding Rab1 in its AMPylated form (33
Fig 6 Model(s) of LidA-mediated attachment of ER-derived vesicles to the LCV. (A) LidA translocated to the cytosol of infected host cells localizes to the LCV membrane by binding AMPylated Rab1 and/or phosphoinosides present on the vacuolar membrane. LidA keeps (more ...)
In order to fully understand the function of LidA during L. pneumophila
infection, several questions remain to be answered. It is currently unclear what protein or signal causes the high-affinity LidA-Rab1 complex to dissociate in order to allow SidD-mediated de-AMPylation and subsequent inactivation of Rab1. Also, a function for the N- and C-terminal domain of LidA has yet to be determined, and the identity of other bacterial or host proteins or pathways likely to be targeted by LidA is currently unclear. Though mammalian Rab6 and Rab8 were found to bind LidA (31
), the role of these interactions for L. pneumophila
virulence has not been elucidated yet. Addressing these questions is essential to obtain a clear perspective on LidA's role during infection of a host cell.