Posttranslational modification of key host proteins by virulence factors is an important theme in bacterial pathogenesis. A remarkable example is the reversible modifications of the small GTPase Rab1 by multiple effectors of the bacterial pathogen Legionella pneumophila. Previous studies have shown that the effector SetA, dependent on a functional glucosyltransferase domain, interferes with host secretory pathways. However, the enzymatic substrate(s) of SetA in host cells remains unknown. Here, by using cross-linking mass spectrometry we uncovered Rab1 as the target of SetA during L. pneumophila infection. Biochemical studies establish that SetA covalently attaches a glucose moiety to Thr75 within the switch II region of Rab1, inhibiting its intrinsic GTPase activity. Moreover, we found that SetA preferentially modifies the GDP-bound form of Rab1 over its GTP-associated state and the modification of Rab1 inhibits its interaction with the GDP dissociation inhibitor GDI1, allowing for Rab1 activation. Our results thus add an extra layer of regulation on Rab1 activity and provide a mechanistic understanding of its inhibition of the host secretory pathways as well as cellular toxicity.

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The Fic (filamentation induced by cyclic AMP) domain is a widely distributed motif with a conserved sequence of HPFx[D/E]GN[G/K]R, some of which regulate cellular activity by catalyzing the transfer of the AMP moiety from ATP to protein substrates. Some Fic proteins, including Fic-1 from the soil bacterium Pseudomonas fluorescens strain 2P24, have been shown to inhibit bacterial DNA replication by AMPylating the subunit B of DNA gyrase (GyrB), but the biochemical activity and cellular target of most Fic proteins remain unknown. Here, we report that Fic-2, which is another Fic protein from strain 2P24 and Fic-1 AMPylate the topoisomerase IV ParE at Tyr 109. We also examined Fic proteins from several phylogenetically diverse bacteria and found that those from Yersinia pseudotuberculosis and Staphylococcus aureus AMPylate ParE and GrlB, the counterpart of ParE in Gram-positive bacteria, respectively. Modification by Fic-1 of P. fluorescens and FicY of Y. pseudotuberculosis inhibits the relaxation activity of topoisomerase IV. Consistent with the inhibition of ParE activity, ectopic expression of these Fic proteins causes cell filamentation akin to the canonical par phenotype in which nucleoids are assembled in the center of elongated cells, a process accompanied by the induction of the SOS response. Our results establish that Fic proteins from diverse bacterial species regulate chromosome division and cell separation in bacteria by targeting ParE.

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