LnaB: a Legionella pneumophila activator of NF-κB

Abstract

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.

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages

“…A second facet of innate immunity that controls cytokine transcription and is triggered by L. pneumophila infection in murine and human cells is signaling through NF-B (32,33,(35)(36)(37)(38)(39). Thus, we assessed levels of I B␣, the inhibitor of NF-B that keeps the transcription factor sequestered in the cytoplasm of the cell.…”

Legionella pneumophila-derived outer membrane vesicles promote bacterial replication in macrophages

“…Pro-inflammatory cytokines and anti-apoptotic proteins are equally induced after L. pneumophila infection and are both dependent on p65 translocation into the nucleus [35,42], which we observed after short time of OMV incubation. L. pneumophila itself can establish a phosphorylation of IκBα at ser-52 and ser-36 by LnaB and LegK1 which mimic eukaryotic serine/threonine kinases; this again leads to an IKK independent and robust induction of apoptosis antagonist genes as well as pro-inflammatory genes [54,55]. As L. pneumophila activates NF-κB signaling itself and critically depends on this signaling as demonstrated by CFU assay with the IKK inhibitor, we conclude that macrophages might be a better replication niche for L. pneumophila when NF-κB signaling has already been induced by a first stimulus.…”

Implication of the VirD4 Coupling Protein of the Lvh Type 4 Secretion System in Virulence Phenotypes of Legionella pneumophila

“…The Gateway cloning system uses lambda phage recombinase instead of restriction endonucleases and ligase to construct protein fusions between entry clones containing effector ORFs and a destination vector containing the CyaA domain. The entry clones of Legionella effector ORFs were described previously (44). The first step in construction of the destination vector was making a version of Gateway vector pDEST8 with kanamycin resistance (Km r ) instead of chloramphenicol resistance (Cm r ) as the drug marker.…”

“…To facilitate construction of cyclase fusions, we adapted the Invitrogen Gateway technology, which uses phage lambda recombinase to create fusions in high yield from donor and destination vectors containing the ORFs to be fused. A library of Gateway donor vectors containing full-length ORFs of Legionella effectors has been described previously (44). In the current work, a new destination vector was constructed for creating fusions to the cyclase CyaA.…”