Fig. 2: MavL is a metaeffector counteracting the SidE family.
A Deletion of mavL did not affect intracellular growth of L. pneumophila. Raw264.7 macrophages were infected with the indicated bacterial strains, and the colony-forming units (CFU) of each strain were monitored at 24-h intervals. n = 3 biologically independent samples. Error bars: standard deviation (SD) of the mean. B, C MavL reduces ADP-ribosylation on Ub under infection condition. HEK293T cells transfected with 3 × HA-Ub and antibody receptor FCγRII were infected with different L. pneumophila strains. At 2-h post infection, the HEK293T cells were lysed and ADP-ribosylation level on Ub was analyzed by immunoblotting against ADPR (shown in B). Expression of HA-Ub and translocation of Flag-tagged MavL were shown by immunoblotting against HA-tag and Flag-tag, respectively. Quantitation of three independent experiments was shown in (C). ADPR-Ub to Ub ratio was calculated by dividing the fluorescence intensity of anti-ADPR immunoblotting by that of anti-HA immunoblotting. Error bars: standard error of the mean (SEM). Statistical analysis was determined by two-tailed t-test, with the exact p-values marked in the graph. D Suppression of Legionella-infection-caused Rab33b PR-ubiquitination in HEK293T cells by MavL. HEK293T cells, transfected with Flag-Rab33b, antibody receptor FCγRII, and GFP-MavL (or empty vector), were infected with L. pneumophila. At 2-h post infection, the HEK293T cells were lysed and the modification of Rab33b was analyzed after anti-Flag IP. Expression of MavL and Rab33b is shown, with tubulin as a loading control. Experiments were performed three times independently with similar results. E Suppression of SdeA-mediated PR-ubiquitination by MavL. HEK293T cells were transfected with Flag-SdeA (or empty vector), GFP-MavL (or empty vector), and HA-UbAA. The PR-ubiquitination level in the cell lysate was probed by immunoblotting against HA-tag. Expression of SdeA and MavL is shown, with tubulin as a loading control. Experiments were performed three times independently with similar results. F MavL rescues yeast toxicity caused by wild-type SdeA and SdeA PDE mutant. SdeA and mutant were expressed in yeast under the galactose-inducible promotor with MavL or its mutant expressed constitutively. Expression of SdeA and MavL were shown by immunoblotting, with PGK as a loading control. Experiments were performed three times independently with similar results. G SidE-mediated noncanonical ubiquitination and its regulation.
