Open Access

Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cells cytoplasm that hijack a plethora of cellular activities, including the hosts ubiquitination pathways. Effectors belonging to the SidE-family are involved in non-canonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires’ disease. This dynamic process is reversed by effectors called Dups that hydrolyse the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and x-ray crystallography approaches were used to identify the site of covalent crosslinking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.

With the emergence of immunotherapies, the understanding of functional HLA class I antigen presentation to T cells is more relevant than ever. Current knowledge on antigen presentation is based on decades of research in a wide variety of cell types with varying antigen presentation machinery (APM) expression patterns, proteomes and HLA haplotypes. This diversity complicates the establishment of individual APM contributions to antigen generation, selection and presentation. Therefore, we generated a novel Panel of APM Knockout Cell lines (PAKC) from the same genetic origin. After CRISPR/Cas9 genome-editing of ten individual APM components in a human cell line, we derived clonal cell lines and confirmed their knockout status and phenotype. We then show how PAKC will accelerate research on the functional interplay between APM components and their role in antigen generation and presentation. This will lead to improved understanding of peptide-specific T cell responses in infection, cancer and autoimmunity.