Summary Intracellular pathogens manipulate host organelles to support replication within cells. For Legionella pneumophila, the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated replication compartment. We show here that the bacterial Sde proteins target host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule rearrangements as well as alterations in Rtn4 associated with the replication compartment. These rearrangements are triggered via Sde-promoted ubiquitin transfer to Rtn4, occurring almost immediately after bacterial uptake. Ubiquitin transfer requires two sequential enzymatic activities from a single Sde polypeptide: an ADP-ribosyltransferase and a nucleotidase/phosphohydrolase. The ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase, resulting in either transfer of ubiquitin to Rtn4, or phosphoribosylation of ubiquitin in the absence of a ubiquitination target. Therefore, a single bacterial protein drives a multistep biochemical pathway to control ubiquitination and tubular ER function independently of the host ubiquitin machinery.

SummaryLegionella pneumophila possesses a large arsenal of type IV translocated substrates. Over 100 such proteins have been identified, but the functions of most are unknown. Previous studies have demonstrated that L. pneumophila activates NF-kB, a master transcriptional regulator of the mammalian innate immune response. Activation of NF-kB is dependent on the Legionella Icm/Dot type IV protein translocation system, consistent with the possibility that translocated bacterial proteins contribute to this response. To test this hypothesis, an expression library of 159 known and putative translocated substrates was created to evaluate whether ectopic production of a single L. pneumophila protein could activate NF-kB in mammalian cells. Expression of two of these proteins, LnaB (Legionella NF-kB activator B) and LegK1, resulted in~150-fold induction of NF-kB activity in HEK293T cells, levels similar to the strong induction that occurs with ectopic expression of the known activator Nod1. LnaB is a substrate of the Icm/Dot system, and in the absence of this protein, a partial reduction of NF-kB activation in host cells occurs after challenge by post-exponential phase bacteria. These data indicate that LnaB is an Icm/Dot substrate that contributes to NF-kB activation during L. pneumophila infection in host cells.

Intracellular growth of Legionella pneumophila occurs in a replication vacuole constructed by host proteins that regulate vesicular traffic from the host endoplasmic reticulum (ER). This process is promoted by a combination of approximately 300 Icm/Dot translocated substrates (IDTS). One of these proteins, Ceg9, was previously identified in a screen for L. pneumophila IDTS that manipulate secretory traffic when overexpressed in yeast. Using ectopic expression of Ceg9 in mammalian cells, we demonstrate that Ceg9 interacts with isoforms of host reticulon 4 (Rtn4), a protein that regulates ER tubule formation. Binding occurs under conditions that prevent association with other known reticulon binding proteins, arguing that Ceg9 binding is stable. A tripartite complex was demonstrated among Rtn4, Ceg9, and atlastin 1, a previously characterized reticulon interacting partner. The binding of Ceg9 to Rtn4 was not due to bridging by atlastin 1 but resulted from the two interacting partners binding independently to reticulon. When Ceg9 is ectopically expressed in mammalian cells, it shows a localization pattern that is indistinguishable from that of Rtn4, perhaps due to interactions between and similar structural features of the two proteins. Consistent with Rtn4 playing a role in the formation of the Legionella-containing vacuole, it was recruited to almost 50% of the vacuoles within 20 min postinfection. Our studies suggest that L. pneumophila proteins interact with ER tubules at an early stage of replication vacuole formation. I ntravacuolar growth of intracellular pathogens involves intimate interaction with specific subsets of host cell membrane compartments (1). In the case of Chlamydia trachomatis, the association of the replication vacuole with the trans-Golgi and the endoplasmic reticulum (ER) network is a key step during the establishment of an intracellular niche (2-5). Similarly, vacuoles harboring Brucella, Salmonella, and Mycobacterium species also interface with defined membrane compartments that aid the growth of these bacteria in host cells (6-10). Failure to establish association with the appropriate organelles can have dire consequences, including loss of vacuolar membrane integrity, activation of inflammatory host cell death, and trafficking of the microorganism into degradative compartments (3,6,11,12).Legionella pneumophila is one such intravacuolar pathogen that targets specific intracellular compartments during its replication cycle (13-16). The bacterium is a pathogen of amoebae and humans. In humans, L. pneumophila's primary manifestation of virulence is Legionnaires' disease, a potentially lethal pneumonia resulting from growth of the bacteria within alveolar macrophages (17, 18). Biogenesis of the microbial replication vacuole is evolutionarily conserved from amoebae to eukaryotic cells (16,19), with the bacterium growing in an ER-encompassed compartment called the Legionella-containing vacuole (LCV) (13,15,20). The connection between the LCV and ER dynamics is well established, since the prop...

Successful pathogens have evolved to evade innate immune recognition of microbial molecules by pattern recognition receptors (PRR), which control microbial growth in host tissues. Upon Legionella pneumophila infection of macrophages, the cytosolic PRR Nod1 recognizes anhydro-disaccharide-tetrapeptide (anhDSTP) generated by soluble lytic transglycosylase (SltL), the predominant bacterial peptidoglycan degrading enzyme, to activate NF-κB-dependent innate immune responses. We show that L. pneumophila periplasmic protein EnhC, which is uniquely required for bacterial replication within macrophages, interferes with SltL to lower anhDSTP production. L. pneumophila mutant strains lacking EnhC (ΔenhC) increase Nod1-dependent NF-κB activation in host cells, while reducing SltL activity in a ΔenhC strain restores intracellular bacterial growth. Further, L. pneumophila ΔenhC is specifically rescued in Nod1- but not Nod2-deficient macrophages, arguing that EnhC facilitates evasion from Nod1 recognition. These results indicate that a bacterial pathogen regulates peptidoglycan degradation to control the production of PRR ligands and evade innate immune recognition.