Rho-family GTPases are essential eukaryotic signaling molecules that regulate cellular physiology. Virulence factors from various pathogens alter GTPase signaling by functioning as GTPase activating factors (GAPs), guanine exchange factors (GEFs) or direct covalent modifiers. Bacterial virulence factors that sense rather than alter Rho-family GTPase signaling states have not been previously described. Here, we report that the translocated Salmonellae virulence factor SseJ binds to the GTP bound form of RhoA with resultant stimulation of its enzymatic activity that results in host cell membrane cholesterol esterification. Therefore, GTPase mediated downstream activation is not exclusive to eukaryotic proteins, and a bacterial protein has evolved to recognize the GTPase signaling state of RhoA to regulate its enzymatic activity as part of the host-pathogen interaction.

Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15 N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly 233 -Asp 242 ). Residues Leu 186 -Thr 209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined ␣-helix and ␤-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions. Cytolethal distending toxin (CDT)4 is a DNA-damaging bacterial toxin produced by a number of important bacterial pathogens (1, 2). The cytopathic effects associated with CDT intoxication are a direct result of chromosomal DNA damage inflicted by the CdtB subunit, a homolog of eukaryotic type I DNase (3, 4). CdtB-mediated chromosome strand breakage signals the induction of an ATM (ataxia telangiectasia-mutated)-or ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent mitotic checkpoint resulting in the sequestration of Cdc25, a block in the cell cycle at the G 2 /M boundary, cellular distension, and ultimately cell death by endoreduplication or apoptosis (2, 5).The CDT holotoxin is a heterotrimer composed of CdtA, CdtB, and CdtC subunits. Although the catalyst for CDT-mediated apoptosis is ultimately attributed to the DNase activity of CdtB, all three subunits are required for cellular entry of CDT (6, 7). The CdtA and CdtC subunits are lectin-like domains that bind carbohydrate residues on the target cell surface, whereby CDT is subsequently internalized by receptor-mediated endocytosis (8, 9). The Haemophilus ducreyi holotoxin structure (HdCDT) has demonstrated that CDT is an AB type toxin, with the catalytically active A subunit of the AB toxin corresponding to the CdtB subunit (10). Inspection of this structure and of the structurally similar CDT holotoxin from Actinobacillus actinomycetemcomitans (AaCDT) (11) revealed that one face of the CdtA-CdtC dimer binds the CdtB subunit. Mutagenesis studies of HdCDT suggest that another face of the dimer with a grooved ricin B-chain fold mediates cell surface binding (10).Trafficking of CDT to the nucleus is thought to involve retrograde transport through the Golgi and the ultimate translocation of CdtB from the endoplasmic reticulum to the nuclear envelope by an apparent ERAD (endoplasmic reticulum-associated degradation)-independent step (9). CdtB traverse...

Cytolethal distending toxin (CDT) is a secreted protein toxin produced by several bacterial pathogens. The biologically active CDT subunit CdtB is an active homolog of mammalian type I DNase. Internalization of CdtB and subsequent translocation into the nucleus of target cells results in DNA-strand breaks, leading to cell-cycle arrest and apoptosis. CdtB crystals were grown using microbatch methods with polyethylene glycol 8000 as the precipitant. The CdtB crystals contain one molecule of MW 30.5 kDa per asymmetric unit, belong to space group P2(1)2(1)2(1) and diffract to 1.72 A.

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