The biogenesis of the unusual Legionella
-containing vacuole (LCV) and the consequences of L. pneumophila
infection for the host cell were examined in several presentations. After uptake by macrophages, L. pneumophila
avoids endosome fusion and actively intercepts vesicle trafficking in the secretory pathway of the infected cell. The critical role of the Dot/Icm type IV secretion system (T4SS) in LCV biogenesis and intracellular replication of L. pneumophila
continues to stimulate immense research interest in mechanisms of substrate secretion and translocation and in the cell biology of Dot/Icm effectors. Joseph Vogel (Washington University, St Louis, USA) presented evidence that some Dot/Icm effectors contain two C-terminal secretion signals and proposed that these two distinct sequences provide temporal regulation of effector translocation (unpublished data). Both Youssef Abu Kwaik (University of Louisville, Buffalo, USA) and Mariella Lomma and Carmen Buchrieser (Pasteur Institute, Paris, France) presented new work on the importance of the host cell ubiquitination pathway for LCV biogenesis. Several Dot/Icm effectors translocated into infected cells carry F-box domains, which in eukaryotic cells mediate the interaction of a protein substrate with the host cell ubiquitination machinery, the SCF (Skp1/Cullin1/F-box) ubiquitin ligase complex. Both groups found that the SCF machinery is needed for LCV biogenesis and L. pneumophila
replication, and that the Dot/Icm F-box protein AnkB/Lpp2082 mediates an interaction with Skp1 and induces the ubiquitination of proteins around the LCV (Price et al., 2009
; unpublished results). The Buchrieser lab further found that Lpp2082 is needed for virulence in mice and that Lpp2082 induces the de-ubiquitination of a focal adhesion protein ParvB, which was important for L. pneumophila
replication (unpublished results).
Further developments in LCV biogenesis were presented by Zhao Qing Luo (Purdue University, Indiana, USA). Despite the fact that the LCV largely avoids interaction with endosomes and maintains a neutral pH, several components of the vacuolar ATPase, usually a late endosomal marker, are known to be associated with the LCV (see below). To examine the recruitment and function of the v-ATPase on the LCV, the Luo lab screened for Dot/Icm proteins that are toxic to yeast at neutral pH. They identified SidK, which interacts directly with the v-ATPase and inhibits ATP hydrolysis, although the vacuolar pH does not change in cells infected with a ΔsidK, mutant suggesting that other effectors are involved in pH maintenance (unpublished results).
Hubert Hilbi (University of Zürich, Zürich, Switzerland) provided an update on L. pneumophila
effector proteins that bind to phosphoinositide (PI) lipids, which are pivotal regulators of eukaryotic signal transduction and membrane dynamics. The PI-binding effectors anchor via
mono-phosphorylated PIs to the LCV membrane, where they recruit ER vesicles (SidC), function as guanine nucleotide exchange factor (GEF) for the Rab1 GTPase (SidM), or interact with the PI phosphatase OCRL1 (LpnE) (Weber et al., 2009
New Dot/Icm effectors were introduced by Elizabeth Hartland (University of Melbourne, Melbourne, Australia). In collaboration with Trevor Lithgow (Monash University, Melbourne, Australia), the Hartland lab showed that L. pneumophila
produces a protein with all the hallmarks of a mitochondrial carrier protein (unpublished data). Since mitochondrial carrier proteins are believed to be exclusive to eukaryotes and critical to the evolution of mitochondria from prokaryotes, the function of this Dot/Icm effector in L. pneumophila
-host interactions might turn out to be very interesting. E. Hartland also presented an update on the CD39 family of ecto-nucleoside triphosphate diphosphohydrolases (NTPDase) and showed that Lpg1905 from L. pneumophila
is a structural and functional mimic of mammalian NTPDases (Vivian et al
., 2009). This member of the L. pneumophila
eukaryotic proteins hydrolyses both ATP and GTP, and this activity is important for L. pneumophila
replication within the LCV and for mouse lung infection (Sansom et al., 2008
The issue of effector redundancy was tackled by Tamara O’Connor and Ralph Isberg (Tufts University, Boston, USA). Their previous work showed that an L. pneumophila
mutant lacking 46 Dot/Icm substrates did not exhibit any replication defect and that LCV biogenesis depends on intercepting vesicles trafficking in at least two pathways. L. pneumophila
replication is severely affected in cells with reduced levels of both Sec22 and Bet5, (Dorer et al., 2006
). The Isberg lab used RNAi to silence one trafficking pathway while screening for mutants defective for replication. The identification of one essential L. pneumophila
gene in each pathway led to the construction of double mutants that showed a replication defect in wild type amoebae, though not in macrophages (unpublished data). Nevertheless, this approach lends substantial weight to the argument that the LCV interacts with vesicles from different sources and that subsets of effector proteins act on specific trafficking pathways.
Craig Roy (Yale University, New Haven, USA) presented investigations into the spatial and temporal regulation of Dot/Icm effector function. The first Dot/Icm effector, RalF, which was identified by the Roy lab, has a eukaryotic Sec7 domain that functions as a guanine exchange factor (GEF) for the host GTPase, Arf1 (Nagai et al., 2002
). The C-terminus of RalF contains a 20 amino acid translocation signal that is connected to a novel structural region that appears to cap the active site of the GEF and mediate subcellular localization of the effector (Amor et al., 2005
). Other effectors, DrrA/SidM, Lpg2603 and Lpg1101, were shown to have a conserved C-terminus that mediates localization of GFP fusion proteins to the host plasma membrane when the gene fusions are expressed ectopically in mammalian cells. Conserved amino acid substitutions at invariant residues in this region abolished plasma membrane localization of these effectors, suggesting that this region is involved in localizing these effectors to the plasma membrane-derived vacuole in which L. pneumophila
initially resides (unpublished data). Thus, in addition to contributing to Dot/Icm-mediated translocation into host cells, the C-terminus of these proteins might determine subcellular localization and spatial regulation of effector protein function.
Dot/Icm dependent MAPK signalling is induced upon infection of macrophages (Shin et al., 2008
), although the consequences of MAPK activation for L. pneumophila
replication are unknown. Ralf Isberg (Tufts University, Boston, USA) reported that L. pneumophila
inhibits the equivalent of the MAPK pathway in amoebae by inducing expression of DupA, a tyrosine kinase/dual specificity phosphatase that is likely a negative regulator of MAPK signalling (Li et al., 2009
). (Li et al., 2009
). Ralf Isberg also reported that the Sel1-repeat protein EnhC is required to maintain cell wall integrity and that ΔenhC
mutants leak peptidoglycan by-products during macrophage infection, thereby stimulating Nod1 (unpublished data). This phenomenon explains why ΔenhC
mutants exhibit retarded growth 24 h after infection in macrophages but not amoebae, which lack Nod1 (Liu et al., 2008
). EnhC interacts with Lpg0663, a soluble lytic transglycosylase (Slt) involved in peptidoglycan synthesis and inhibits its activity (unpublished data). In other ongoing work, the Isberg lab is using serial passage over multiple generations to test the idea that L. pneumophila
growth in amoebae has simultaneously selected for growth in macrophages. Interestingly, the results so far suggest that optimal growth in amoebae does not confer optimal growth in macrophages (unpublished data).