A Legionella effector ADP-ribosyltransferase inactivates glutamate dehydrogenase

Abstract

ADP-ribosyltransferases (ARTs) are a widespread superfamily of enzymes frequently employed in pathogenic strategies of bacteria. Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaire's disease, has acquired over 330 translocated effectors that showcase remarkable biochemical and structural diversity. However, the ART effectors that influence L. pneumophila have not been well defined. Here, we took a bioinformatic approach to search the Legionella effector repertoire for additional divergent members of the ART superfamily and identified an ART domain in Legionella pneumophila gene0181, which we hereafter refer to as Legionella ADP-Ribosyltransferase 1 (Lart1) (Legionella ART 1). We show that L. pneumophila Lart1 targets a specific class of 120-kDa NAD+-dependent glutamate dehydrogenase (GDH) enzymes found in fungi and protists, including many natural hosts of Legionella. Lart1 targets a conserved arginine residue in the NAD+-binding pocket of GDH, thereby blocking oxidative deamination of glutamate. Therefore, Lart1 could be the first example of a Legionella effector which directly targets a host metabolic enzyme during infection.

Keywords: ADP-ribosylation; bacterial pathogenesis; bioinformatics; cell metabolism; enzyme kinetics; glutamate; glutamate dehydrogenase; infectious disease.

Figure 1

Lart1 is a mono-ADP-ribosyltransferase.A, domain schematic of Lart1. B, sequence logos (weblogos) illustrating the conservation of predicted catalytic residues (25) in 81 Lart1 homologs (left) and 453 members of the R-S-ExE clade of mARTs (pfam PF01375, enterotoxin a) (right). Ω: Aromatic amino acids, Φ: hydrophobic amino acids. C, growth curves depicting replication of WT (Lp02), ΔDotA (Lp03), and Lart1 KO (ΔLart1) Legionella strains in A. castellanii amoeba. Infected amoeba cells were lysed at the indicated time points and bacterial replication was quantified by plating serial dilutions of lysates. Results are obtained from triplicate conditions and are representative of three independent experiments. DotA, defect in organelle trafficking protein; mARTs, mono-ADP-Ribosyltransferases.

Figure 2

Lart1 ADP-ribosylates fungal and protozoan isoforms of glutamate dehydrogenase.A, incorporation of [³²P] from [³²P]-adenylate NAD+ into an unknown 120 kDa protein in a yeast lysate by Lart1, but not the E137A mutant. B, dendrogram depicting the four classes of glutamate dehydrogenase isoforms labeled by their subunit molecular weight, adapted from Miñambres et al. (26). Each class is annotated with its phylogenetic distribution (black), monomer composition (blue), and cofactor preference (magenta). C, incorporation of [³²P] from [³²P]-NAD+ by WT Lart1 or the inactive E137A mutant with a panel of glutamate dehydrogenases as substrates: the two Legionella glutamate dehydrogenases (Lpg1581 and Lpg0245), human Glud2 (HsGlud2), yeast (Gdh1p and Gdh2p), and Dictyostelium Glud2 (DdGlud2). Reaction products were separated by SDS-PAGE and incorporated radioactivity visualized by autoradiography. Lpg, Legionella pneumophila gene; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 3

Kinetic parameters and stoichiometry of the Lart1 transferase reaction.A, rate plot depicting the specific activity of WT Lart1 at saturating [DdGlud2] and varying [NAD+]. B, specific activity of Lart1 at saturating [NAD+] while varying [DdGlud2]. Km and Vmax (inset) are indicated along with a 95% confidence interval. The MW of Lart1 is 34997.6 g/mol. Reactions were performed in triplicate and are representative of three independent experiments. C, intact mass spectra of unmodified DdGlud2 (left) or after incubation with NAD+ and Lart1 (right). The theoretical MW of DdGlud2 is 117142.74 Da and the theoretical mass increase of ADP-ribosylation is 541 Da.

Figure 4

Lart1 targets a conserved arginine residue in the NAD+-binding pocket of glutamate dehydrogenase.A, fragmentation pattern of Gdh2p peptide containing phosphoribosyl-Arg800 identified by LC-MS/MS. The precursor [M+3H]³⁺ ion, m/z 657.30, is labeled with an asterisk (∗) and was subjected to HCD fragmentation to generate the spectrum shown. The modification site was localized to arginine 800 highlighted in red. The b10 fragment ion containing the modified residue shows neutral loss of the phosphoribosyl group (−212 Da). B, endpoint assays depicting incorporation of [³²P]-NAD+ by WT Lart1 into the indicated alanine mutants of Gdh2p. Mutation of the ADPR acceptor site R800 abolishes ADP-ribosylation. C, model of DdGlud2 built by Phyre (64) using Pyrococcus furiosus GDH (PDB 1HRD) as a template, indicating the position of the ADP-ribose acceptor residue Arg 763 (rendered in sticks). Bound NADH and glutamate (Glu), (modeled by alignment to liganded bovine GDH, PDB 6DHQ) are rendered in sticks. D, sequence logos depicting the conservation of R800 and the surrounding residues in GDH enzymes from fungi (top, based on MAFFT alignment of 125 sequences), amoeba (middle, based on MAFFT alignment of 19 sequences), and metazoans (bottom, based on MAFFT alignment of 145 sequences). Red arrows indicate the position of the arginine targeted by Lart1. LC-MS/MS, liquid chromatography/mass spectrometry.

Figure 5

ADP-ribosylation inactivates DdGlud2. Reaction progress curves showing (A) oxidative deamination of glutamate by unmodified (magenta) or ADP-ribosylated (teal) DdGlud2, generated by detecting NAD+ reduction to NADH (Abs 340 nm). Reactions contained excess NAD+ and glutamate. B, reaction progress curves showing reductive amination by unmodified (magenta) or ADP-ribosylated (teal) DdGlud2. Reactions contained excess ɑ-KG, NADH, and NH₄⁺. Plots display mean and SD of four replicates from two independent experiments.