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. 2021 Sep 7;12(5):e02316-21. doi: 10.1128/mBio.02316-21

FIG 1.

FIG 1

SdjA is constitutively expressed in L. pneumophila and impacts the function of members of the SidE effector family. (A) Gene organization of the loci harboring sidJ and sdjA on the chromosome of L. pneumophila. Each arrow represents the indicated gene; the enlargement shows the domain architecture of SdjA. The position of the IQ domain for calmodulin binding is indicated. Note that sidJ is situated next to dupA and sdjA is adjacent to dupB; both are involved in the regulation of the activity of the SidE family. (B) Expression of sdjA in broth-grown bacteria. A saturated culture was diluted 1:20 in fresh medium, and the growth of the culture was monitored by measuring absorbance at 600 nm. Samples with identical cell numbers were withdrawn at the indicated times and probed for SdjA by immunoblotting. The metabolic protein isocitrate dehydrogenase (ICDH) was detected as a loading control. Results are from one of two independent experiments with similar results. (C) Expression of CaM in L. pneumophila affects its virulence. The indicated bacterial strains were used to infect D. discoideum at a multiplicity of infection (MOI) of 0.1, and their intracellular replication was monitored at 24-h intervals for 72 h. Note that expression of CaM affects the virulence of the wild-type strain to a level comparable to that of the mutant lacking the sidE effector family. Similar results were obtained in at least three independent experiments, and the results shown are from one representative experiment done in triplicate. **, P < 0.01. (D and E) Evaluation of the impact of SdjA on the activity of SidEs by expressing CaM in L. pneumophila. Cells transfected to express Flag-Rab33b were infected with the indicated bacterial strains at an MOI of 10 for 2 h. Cell lysates were subjected to immunoprecipitation with agarose beads coated with Flag antibody, and the precipitates were detected by immunoblotting with the Flag antibody. Ubiquitinated Flag-Rab33b from three independent experiments was quantitated with ImageJ (D). Images from a representative experiment are shown (E). Note that expression of CaM in the ΔsidJ mutant led to detectable Rab33b ubiquitination, which suggests that the CaM-mediated activity of endogenous SdjA does not completely block the activity of SidEs. The expression and translocation of SdeA, SidJ, and SdjA were probed with the appropriate antibodies, with ICDH and metabolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being probed as loading controls. (F) SdjA differently impacts the activity of SdeA and SdeC. Ubiquitination of Flag-Rab33b was evaluated as described for panel E in cells infected with the indicated L. pneumophila strains. Modified Flag-Rab33b was judged by a shift in its molecular weight (top). The expression (lower three panels) and translocation (middle three panels) of SdeA, SdeC, and SdjA were probed by immunoblotting with the appropriate antibodies. ICDH and GAPDH were detected as loading controls. Bacterial strains: 1, ΔsidE ΔsidJ(vector, vector); 2, ΔsidE ΔsidJ(pSdeA, vector); 3, ΔsidE ΔsidJ(pSdeA, pCaM); 4, ΔsidE ΔsidJ(pSdeC, vector); 5, ΔsidE ΔsidJ(pSdeC; pCaM).