Legionella translocates an E3 ubiquitin ligase that has multiple U‐boxes with distinct functions

Abstract

SummaryLegionella pneumophila has a Dot/Icm type IV secretion system used to translocate a number of 'effector proteins' which subvert host cell functions. In this study, we identified 19 novel Dot/Icm substrate proteins using a systematic screening technique. A BLAST analysis revealed that one of the substrates, which we named LubX (LegionellaU-box protein), contains two domains that have a remarkable similarity to the U-box, a domain found in eukaryotic E3 ubiquitin ligases. The expression of LubX is induced upon infection, and most of the LubX produced was translocated into the host cells. LubX has ubiquitin ligase activity in conjunction with UbcH5a or UbcH5c E2 enzymes and mediates polyubiquitination of host Clk1 (Cdc2-like kinase 1). We demonstrate that one of the U-boxes (U-box 1) is critical to the ubiquitin ligation, and the other U-box (U-box 2) mediates interaction with Clk1. Thus, the two U-boxes of LubX have distinct functions, and U-box 2 plays a non-canonical role in substrate binding. Although we demonstrate that inhibition of Clk kinase results in a marked reduction of Legionella growth within mouse macrophages, the consequence of Clk1 ubiquitination is still being elucidated. Together, these data suggest that Clk1 is the target host molecule which Legionella modulates during infection.

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

“…To determine whether LpSpl could mimic eukaryotic SPL, we first determined its subcellular localization. Efforts to directly determine the subcellular localization of LpSpl in the host cell were unsuccessful, consistent with previous studies showing that the levels of secretion of Dot/Icm effectors are very low (31)(32)(33). However, when transfected with an Xpress-tagged form of LpSpl or a FLAG-tagged form of mSPL, both clearly localized to the ER, as has been reported for hSPL.…”

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…99 For example, the effector LubX contains two U-box domains that ubiquitinate the host CDC2-like kinase 1. 124 In addition, AnkB, LegU1 and LegAU13 exhibit structural similarity to the eukaryotic F-box-containing E3 ligase. AnkB is crucial for L. pneumophila to acquire nutrition as it locates to the LCV membrane and recruits K48-linked polyubiquitinated proteins, the degradation of which yields amino acids for the bacteria.…”

Genome Dynamics in Legionella: The Basis of Versatility and Adaptation to Intracellular Replication

“…Oomycetes (4) Plants ( Moreover, there is evidence for the presence of a secretion signal in the carboxy terminal of the Dot/Icm substrates (Nagai et al 2005;Kubori et al 2008;Huang et al 2010); therefore, acquisition of this signal is also a challenge for these proteins to be secreted. Thus, although we start to discern how these eukaryotic proteins have appeared in the Legionella genomes, many questions with respect to the detailed mechanism of acquisition and integration remain unanswered.…”

“…LubX (for Legionella U-box) is a protein containing two U-box domains (U-box1 and Ubox2) similar to eukaryotic E3 ubiquitin ligases that function as a ubiquitin ligase in conjunction with host UbcH5a or UbcH5c E2 enzymes and mediates polyubiquitination of cellular Clk1 (Cdc2-like kinase) (Kubori et al 2008). U-box1 seems to be critical for ubiquitin ligation, whereas the U-box2 domain interacts with the substrate.…”