Figure 4. ADP-ribosylation of ADP/ATP translocases by Ceg3 occurs in cells infected with Legionella pneumophila.
(A) An intact mART motif in Ceg3 is required for its modification of ADP/ATP translocases during L. pneumophila infection. Bacteria of the indicated L. pneumophila strains were opsonized prior to infecting HEK293T cells transfected to express the FcγII receptor at an MOI of 100 for 2 hr. Samples of the mitochondrial fraction were probed for ADPR after SDS-PAGE (top). PDHA1 and tubulin were probed as controls to monitor the success of cell fractionation. One cytosolic fraction sample was included as an additional control (middle). The expression of Flag-Ceg3 in bacteria was analyzed with the Flag antibody, the metabolic enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control (bottom). (B) A functional Dot/Icm system is required for Ceg3-induced ADP-ribosylation of ADP/ATP translocases in infected cells. HEK293T cells expressing the FcγII receptor were infected with opsonized bacteria expressing Flag-Ceg3 at an MOI of 100 for 2 hr. Isolated mitochondrial proteins resolved by SDS-PAGE were probed for ADPR modification (top). The quality of cell fractionation was determined by probing for PDHA1 and tubulin (middle), respectively. The expression of Ceg3 in bacteria was detected with the Flag antibody, and ICDH was probed as a loading control (bottom). (C) ADP-ribosylation of ADP/ATP translocases is detectably induced by wild-type L. pneumophila at 6 hr post-infection. HEK293T cells expressing the FcγII receptor were infected with opsonized bacteria for the indicated periods of time at an MOI of 30. Mitochondrial proteins were analyzed by anti-ADPR and anti-PDHA1 Western blot. MOI, multiplicity of infection; NI, no infection.
Figure 4—figure supplement 1. Deletion of ceg3 did not detectably affect intracellular growth of Legionella pneumophila.
