Second, the ratio between SidJ and SidEs becomes higher at later times after bacterial uptake (Figure 7), which allows the activity of SidJ to become dominant, converting substrates of SdeA to their unmodified form

Second, the ratio between SidJ and SidEs becomes higher at later times after bacterial uptake (Figure 7), which allows the activity of SidJ to become dominant, converting substrates of SdeA to their unmodified form. Temporal regulation of effector activity by effectors with antagonizing activity has been well documented in infection. not require the E1 and E2 enzymes of the host ubiquitination machinery. In this case, ubiquitin is first activated by ADP-ribosylation at Arg42 by a mono-ADP-ribosyltransferase activity; the intermediate is then cleaved by a phosphodiesterase activity also residing within SdeA, concomitant with the attachment of ubiquitin to serine residues of substrate proteins via a phosphoribosyl linker. Here we demonstrate that the effect of SidEs is antagonized by SidJ, an effector encoded by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ reverses ubiquitination of SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. SidJ also displays classical deubiquitinase activity but does not require catalytic cysteine residues. Further, these deubiquitinase activities of SidJ are essential for its role in infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a ubiquitin-deconjugating enzyme that functions to impose temporal regulation on the activity of SidE effectors. SidJ may be important in future studies of signaling cascades mediated by this unique ubiquitination, one that also potentially regulates cellular processes in eukaryotic cells. replication requires the Dot/Icm type IV secretion system, which delivers into the host cell hundreds of effectors that modulate various cellular processes such as vesicle trafficking, cell death, autophagy, phospholipid metabolism and ubiquitination, which benefit the bacterium6. Tcf4 Modulation of host ubiquitination pathways by Dot/Icm substrates has emerged as an important theme in the pathogenicity of treated with hydroxylamine25 was introduced into a yeast strain expressing SdeA from a galactose-inducible promoter20. Transformants unable to grow on inducing (galactose) medium harbor candidate SidJ mutants that have lost the suppressor activity. By screening 200 candidate mutants defective in such activity, we obtained five mutants which still encoded full-length proteins. Sequencing analysis revealed that these mutations (P290L, R536G, G544R, G569E, G719R) mapped onto three regions of SidJ, localized around the 290th, the 540th and the 719th residues, respectively (Figure 1A). Three of these mutations mapped to positions close to D542 and Eplivanserin mixture D545, which are critical for the ability of SidJ to rescue the yeast toxicity of SdeA24 (Figure 1B). None of these mutations affected the stability of SidJ, but all had lost the ability to suppress yeast toxicity by SdeA (Figure 1B). In addition, these mutants also failed to suppress SdeA-mediated inhibition of the secretion of the secreted embryonic alkaline phosphatase (SEAP) by mammalian cells (Figure 1C). These residues are either critical for the catalytic activity of SidJ or are important for SidJ to maintain its conformation. Open in a separate window Figure 1 Identification of SidJ substitution mutants unable to suppress SdeA yeast toxicity. (A) Distribution of substitution mutations that abolished the ability of SidJ to suppress the yeast toxicity of SdeA. Note the clustering of mutations around the 540th residue of SidJ. (B) Yeast strain expressing chromosomally integrated SdeA controlled by a galactose-inducible promoter was transformed with plasmids carrying Eplivanserin mixture WT or mutated SidJ controlled by Eplivanserin mixture the alcohol dehydrogenase (ADH) promoter. Serially diluted yeast cells were spotted onto glucose or galactose medium. Images were acquired 3 days after incubation at 30 C. Lower panel showed the expression of SdeA and SidJ, yeast cells grown in medium supplemented with glucose (1) were induced with galactose (2) for 8 h,.