Legionella pneumophila temporally regulates the activity of ADP/ATP translocases by reversible ADP‐ribosylation

Abstract

The mitochondrion is an important signaling hub that governs diverse cellular functions, including metabolism, energy production, and immunity. Among the hundreds of effectors translocated into host cells by the Dot/Icm system of Legionella pneumophila, several are targeted to mitochondria but the function of most of them remains elusive. Our recent study found that the effector Ceg3 inhibits the activity of ADP/ATP translocases (ANTs) by ADP‐ribosylation (ADPR). Here, we show that the effect of Ceg3 is antagonized by Larg1, an effector encoded by lpg0081, a gene that is situated next to ceg3. Larg1 functions to reverse Ceg3‐mediated ADPR of ANTs by cleaving the N‐glycosidic bond between the ADPR moiety and the modified arginine residues in ANTs, leading to restoration of their activity in ADP/ATP exchange. Structural analysis of Larg1 and its complex with ADPR reveals that this ADPR glycohydrolase harbors a unique macrodomain that catalyzes the removal of ADPR modification on ANTs. Our results also demonstrate that together with Ceg3, Larg1 imposes temporal regulation of the activity of ANTs by reversible ADPR during L. pneumophila infection.

Legionella pneumophilaexploits the endo-lysosomal network for phagosome biogenesis by co-opting SUMOylated Rab7

“…Digested peptides were analyzed by liquid chromatography (LC)-ESI-MS/MS using the Dionex UltiMate 3000 RSLC nano System coupled to the Q ExactiveTM HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) following the procedure described previously 80 . Briefly, peptides were separated using a trap column (300 μm ID × 5 mm) packed with 5 μm 100 Å PepMap C18 medium and a reverse-phase column (50 cm long × 75 μm ID) packed with 2 μm 100 Å PepMap C18 silica.…”

“…Total proteins of Hmg20a -/-BMDMs and the appropriate control cells were separated by SDS-PAGE gels for 10 min at 65 V. Protein bands visualized by Coomassie brilliant blue (CBB) staining were digested with trypsin as described previously 79 . Briefly, gel slices were cut into 1 mm cubes and incubated in de-staining buffer containing 50% Acetonitrile (ACN) and 50 mM NH4HCO3 at 37°C for 1 h. After reduction and alkylation, proteins were digested by trypsin (12 ng/μL trypsin in 50 mM NH4HCO3, 10% ACN) at 37°C for 1 h. Digested peptides were analyzed by liquid chromatography (LC)-ESI-MS/MS using the Dionex UltiMate 3000 RSLC nano System coupled to the Q ExactiveTM HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) following the procedure described previously 80 . Briefly, peptides were separated using a trap column (300 μm ID × 5 mm) packed with 5 μm 100 Å PepMap C18 medium and a reverse-phase column (50 cm long × 75 μm ID) packed with 2 μm 100 Å PepMap C18 silica.…”

The ever‐increasing necessity of mass spectrometry in dissecting protein post‐translational modifications catalyzed by bacterial effectors

“…Indeed, such a strategy has been applied in the study of the S. Typhimurium effector SopF (Xu et al, 2019) and the L. pneumophila effector Ceg3/Lpg0080 (Fu, Zhou, et al, 2022;Kubori et al, 2022), though in a more targeted manner as only those Af1521-enriched proteins with the size of interest were analyzed by LC-MS. Notably, Ceg3/Lpg0080 and Larg1/Lpg0081 were found to target ADP/ATP translocases by reversible ADP-ribosylation in host mitochondria to temporally regulate their ADP/ATP exchange activity during L. pneumophila infection (Fu, Li, et al, 2022;Fu, Zhou, et al, 2022;Kubori et al, 2022). Alternatively, the use of an NAD derivative, biotin-17-NAD, in in vitro ADP-ribosylation assays permits streptavidin pull-down of modified ART substrates prior to MS identification.…”

“…Salmonella kinase SteC remodels the host F-actin cytoskeleton by phosphorylating FMNL1 (Walch et al, 2021), whereas enteropathogenic Escherichia coli effector kinases NleH1 and NleH2 target Ser775 of host microvillus protein Eps8 for phosphorylation, thus inhibiting its bundling activity and driving its dispersal from the attaching and effacing lesion during infection (Pollock et al, 2022). In addition, Lpg0080/Ceg3 and Lpg0081/Larg1 from Legionella pneumophila coordinately modulate host mitochondrial ADP/ATP exchange via reversible ADP-ribosylation of ANTs (Fu, Li, et al, 2022;Fu, Zhou, et al, 2022;Kubori et al, 2022). Due to space limitation, only a few representative effectors and their associated pathways are shown in the figure . mammalian host cells, many bacterial pathogens have evolved effector proteins harboring kinase or phosphatase activities to directly engage with host phosphorylation signaling (Grishin et al, 2015).…”

The Legionella pneumophila Dot/Icm type IV secretion system and its effectors

“…LncP might collaborate with the effector Ceg3, which inhibits host ADP/ATP translocases by ADP ribosylation, to manage ATP transport across mitochondrial membranes and thus cellular energy levels [260]. The inhibition by Ceg3 can be relieved by the effector Larg1, which is an ADP-ribose glycohydrolase that removes the PTM [261]. Additional effectors such as Lpg1625 and Lpg0898, which localize to the mitochondria in transfected cells [262], could enable L. pneumophila to further tighten control over the cellular power plant.…”