Saturday, December 21, 2024
12/21/2024
The pathogenic bacteria Legionella pneumophila (Lp), the causative agent of Legionnaires’ disease, injects nearly 330 effectors using its Dot/Icm protein secretion system into host cytosol to remodel cellular processes toward creating and maintaining a replicative organelle called the legionella-containing vacuole (LCV). Research in the last few years has shown some remarkable examples of ubiquitination chemistry catalyzed by some of these effectors that are unparalleled in the eukaryotic world. Two notable examples of ubiquitination of host targets have been identified where the mechanisms are independent of and radically different from the eukaryotic E1-E2-E3 system. This has added an exciting new layer of complexity to ubiquitin (Ub) manipulation by Lp, which was already known to possess several classical Ub E3 ligases and deubiquitinases among its effectors. This raises a question as to how far and deep Legionella’s manipulation of the host ubiquitin goes. Are there additional new Ub-manipulating proteins/enzymes in the large effector arsenal of the bacteria? We have performed an activity-based enrichment analysis to identify novel Ub-interacting proteins among Lp effectors to address this question. We have used Ub-derived activity-based probes (Ub-ABPs) to enrich proteins from the soluble proteome of Lp and have identified candidates using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This has led us to the Dot/Icm substrate MavL, with unknown biochemical and biological function, as a novel Ub-interacting effector. On further analysis, we find that MavL is a deADP-ribosyl glycohydrolase capable of removing ADP-ribose (ADPR) from an arginine side chain of ADP-ribosylated Ub (ADPR-Ub). We will use a proximity labeling biotinylation strategy implemented under infection conditions to capture MavL substrates. To complement this strategy, we will engineer an inactive mutant of the enzyme into an affinity tool for proteome-wide enrichment and identification of MavL interactors from host cells. Combining information from the proteomics datasets, we aim to arrive at a list of candidates which will be validated in cell-based assays under infection conditions. Further, we will try to provide the structural basis of Ub recognition by this novel pathogenic effector. Taken together, we will be able to characterize a unique Ub-recognizing deADP-ribosylating enzyme from a pathogenic bacterium and explore its host targets, thereby expanding the scope of reversible ADP-ribosylation in host-pathogen interaction.
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