A Single Legionella Effector Catalyzes a Multistep Ubiquitination Pathway to Rearrange Tubular Endoplasmic Reticulum for Replication.

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.

Endoplasmic Reticulum Tubule Protein Reticulon 4 Associates with the Legionella pneumophila Vacuole and with Translocated Substrate Ceg9.

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.

The Legionella pneumophila EnhC protein interferes with immunostimulatory muramyl peptide production to evade innate immunity.

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.

Control of host cell phosphorylation by legionella pneumophila.

Phosphorylation is one of the most frequent modifications in intracellular signaling and is implicated in many processes ranging from transcriptional control to signal transduction in innate immunity. Many pathogens modulate host cell phosphorylation pathways to promote growth and establish an infectious disease. The intracellular pathogen Legionella pneumophila targets and exploits the host phosphorylation system throughout the infection cycle as part of its strategy to establish an environment beneficial for replication. Key to this manipulation is the L. pneumophila Icm/Dot type IV secretion system, which translocates bacterial proteins into the host cytosol that can act directly on phosphorylation cascades. This review will focus on the different stages of L. pneumophila infection, in which host kinases and phosphatases contribute to infection of the host cell and promote intracellular survival of the pathogen. This includes the involvement of phosphatidylinositol 3-kinases during phagocytosis as well as the role of phosphoinositide metabolism during the establishment of the replication vacuole. Furthermore, L. pneumophila infection modulates the NF-κB and mitogen-activated protein kinase pathways, two signaling pathways that are central to the host innate immune response and involved in regulation of host cell survival. Therefore, L. pneumophila infection manipulates host cell signal transduction by phosphorylation at multiple levels.

LnaB: a Legionella pneumophila activator of NF-kappaB.

Legionella 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-kappaB, a master transcriptional regulator of the mammalian innate immune response. Activation of NF-kappaB 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-kappaB in mammalian cells. Expression of two of these proteins, LnaB (Legionella NF-kappaB activator B) and LegK1, resulted in approximately 150-fold induction of NF-kappaB 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-kappaB 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-kappaB activation during L. pneumophila infection in host cells.