a, Raw264.7 macrophages were infected with wild-type, ΔSidJ or ΔSidE Legionella for 3 h. Lysates were used for immunoprecipitation with polyglutamylation antibody, followed by immunoblotting with SdeA. ni, samples that were not infected with bacteria. This experiment was repeated twice independently, with similar results. b, A549 cells were infected with different strains of L. pneumophila for 3 h. Cells were fixed and immunostained with antibodies against calnexin and polyglutamylation (GT335). DAPI staining marks the nucleus and cytosolic bacteria. Yellow arrows indicate bacteria in infected cells. The ROI is defined as calnexin-stained LCV. ROIs of 80 × 100 μm2 were chosen in the perinuclear region of cells, followed by quantification of Mander’s coefficient (m) using the Coloc2 plugin in FIJI. m represents the fraction of calnexin-positive LCVs that are also positive for polyglutamylation. Centre lines show the medians; box limits indicate the 25th and 75th percentiles (as determined by R software); whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles; and outliers are represented by dots. Number of ROIs (n) = 80 from 30 cells was used for quantification. ***P < 0.001 by two-tailed type-3 Student’s t-test. P value (wild type versus ΔsidJ) = 6.18 × 10−29; P value (wild type versus ΔsidE) = 1.09 × 10−5. This experiment was repeated twice independently, with similar results. c, Glutamylated proteins were isolated from wild-type, ΔsidE and ΔsidJ Legionella infection experiments using GT335 antibody and quantified using mass spectrometry. Correlation between wild-type versus ΔsidJ and ΔsidE versus ΔsidJ quantifications are plotted (inset), showing the most-correlated proteins in these two quantifications. Legionella infection and label-free liquid chromatograph–mass spectrometry analysis was performed with n = 3 biologically independent experiments. Significant differences between samples were detected by a corrected, two-sided Student's t-test with permutation-based false-discovery rate of 0.05. Proteins were labelled as significant if they were above the false-discovery-rate threshold of 0.05 in at least one comparison (ΔsidE and wild-type Legionella compared to ΔsidJ-infected cells). Proteins with a log 2 ratio above two (mean) in wild-type samples were labelled as highly enriched compared to ΔsidJ-infected cells in samples from wild-type and ΔsidE-infected cells. Source Data