Legionella pneumophila, a gram-negative intracellular pathogen, is the causative agent of a severe form of pneumonia called Legionnaires’ disease. Upon phagocytosis by alveolar macrophages, L. pneumophila secretes over 300 effector proteins into the host cell. These bacterial effector proteins hijack various host cellular processes to create an optimal niche, the Legionella-containing vacuole (LCV), to evade lysosomal-mediated degradation and promote intracellular bacterial growth. Recent studies revealed a family of L. pneumophila effectors that catalyze a novel mode of ubiquitination, termed phosphoribosyl-ubiquitination (PR-ubiquitination). Members of the Sde family of effectors modulate Legionella infection via the non-canonical PR-ubiquitination of host substrates, which has been shown to be critical for infection. As host ubiquitination is a preferential target of this intracellular pathogen, further characterization of effectors that target the ubiquitin system is crucial in understanding their roles during bacterial pathogenesis.

The activity of the Sde family has been shown to be antagonized by L. pneumophila effector SidJ. To elucidate how SidJ regulates PR-ubiquitination, we solved the crystal structure of SidJ in complex with human calmodulin 2, which revealed that SidJ adopts a protein kinase-like fold. Despite structural homology and conservation of protein kinase catalytic motifs, we were unable to demonstrate SidJ kinase activity. Through biochemical, mass spectrometric, and structural studies, we have shown that SidJ is a protein polyglutamylase that adds glutamate amino acids to a catalytic residue on SdeA. This posttranslational modification directly inhibits PR-ubiquitination and counteracts the function of the Sde family. This work has characterized a novel calmodulin-dependent pseudokinase that functions as a protein polyglutamylase, providing insights into the intricate regulation of the PR-ubiquitination pathway.

L. pneumophila employs a sophisticated strategy to co-opt the host ubiquitin system through canonical and non-canonical ubiquitination. During infection, the Sdc family of effectors, which function as canonical HECT-type E3 ligases, and the Sde family of PR-ubiquitin ligases work together to build mixed poly-ubiquitin chains, forming a “ubiquitin-coat” surrounding the pathogen. Despite the accumulation of ubiquitinated species, ubiquitin-binding autophagy adaptors are excluded from the LCV. Here, we show the Sde family is able to modify internal ubiquitin moieties in poly-ubiquitin chains with a phosphoribosyl group, prohibiting autophagy adaptor recognition of the ubiquitin-enriched vacuole. Furthermore, multiple host substrates can be covalently cross-linked to these modified poly-ubiquitin chains through PR-ubiquitination. Together, these results elucidate the nature of mixed poly-ubiquitin chains at the LCV and provide further mechanistic insights into how L. pneumophilaavoids clearance by autophagy.

Publications:

Sulpizio, A.*, Minelli, M.E.*, Wan, M.*, Burrowes, P.D., Wu, X., Sanford, E.J., Shin, J.H., Williams, B.C., Goldberg, M.L., Smolka, M.B., Mao, Y. Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ. Elife 8 (2019).

Sulpizio, A.G., Minelli, M.E. & Mao, Y. Glutamylation of Bacterial Ubiquitin Ligases by a Legionella Pseudokinase. Trends Microbiol 27, 967-969 (2019).

Sulpizio, A.G., Minelli, M.E. & Mao, Y. In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors. Bio Protoc 10, e3811 (2020).

Sulpizio, A.G., Shin, J.H., Minelli, M.E. & Mao, Y. Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ. Bio Protoc 10, e3770 (2020).

Wan, M.*, Minelli, M.E.*, Zhao, Q., Marshall, S., Yu, H., Smolka, M.B., Mao, Y. Phosphoribosyl modification of poly-ubiquitin chains at the Legionella-containing vacuole prohibiting autophagy adaptor recognition. In revision (2023).

Minelli, M.E. & Mao, Y. Characterization of a phosphodiesterase-domain-containing effector from Legionella pneumophila. In preparation.