Cytolethal distending toxins (CDTs) 6 are members of a group of bacterial toxins and effectors called "cyclomodulins" that interfere with the eukaryotic cell cycle rather than inducing overt cytotoxicity (1, 2). Inhibiting cell cycle disrupts many of the normal functions of rapidly dividing eukaryotic cells, including lymphocytes and epithelial cells, which provide immunity and physical barriers to microbial pathogens (3-5). Thus, it is not surprising that cdt genes are found in a diverse group of Gram-negative pathogens that colonize different niches within the host. Although a growing body of evidence supports the importance of CDTs in bacterial virulence and host-pathogen interactions (6), the manner in which individual CDTs interact with and intoxicate host cells remains poorly understood.CDTs are AB 2 toxins, consisting of a hetero-trimeric complex of three proteins (CdtA, CdtB, and CdtC) at a 1:1:1 molar ratio (5,7,8). The current model is that CdtA and CdtC are the binding "B" moieties that collaborate to facilitate binding and entry of the catalytic "A" subunit, CdtB, into mammalian cells. CdtB shares a common tertiary structure with DNase I and phosphatidylinositol 3,4,5-triphosphate phosphatase enzymes and displays both activities in cell-free systems (9 -13). It is not currently known which activity is of greater importance, and this may depend on the specific toxin and/or the host target cell type (12,14). CdtB enzymatic activity induces cell cycle arrest predominantly at the G 2 /M transition, resulting in cellular distension and ultimately cell death (5,15,16).Consistent with their proposed roles as binding subunits, CdtA and/or CdtC increase the ability of CdtB to associate with host cells and greatly enhance intoxication (7,(17)(18)(19)(20)(21)(22)(23)(24)(25). The identification of ricin-like lectin domains in CdtA and CdtC from structural and biochemical data first suggested that these subunits may interact with carbohydrates on the cell surface (13,26,27). Consistent with this hypothesis, CDT produced by Escherichia coli (Ec-CDT) was reported to require N-linked glycoproteins for binding and subsequent intoxication of HeLa cells (23). Moreover, Ec-CDT bound fucose in vitro, and fucose-specific lectins blocked Ec-CDT-mediated cell cycle arrest, presumably by preventing binding of toxin to its receptor. These findings suggested that fucose might serve as a binding determinant for Ec-CDT. Similarly, host glycans were reported to support Aggregatibacter actinomycetemcomitans (Aa-CDT) intoxication. Specifically, Aa-CDT bound three glycosphingolipids, GM1, GM2, and GM3, and intoxication of human monocytic U937 cells was blocked by preincubation of toxin with liposomes that contained G M3 (24). In addition, the CdtA subunit of Aa-CDT bound to the glycoprotein thyroglobulin (19). However, the functional significance of this binding is * This work was supported, in whole or in part, by National Institutes of Health Grants T32DE007296 (to A. E.), F31AI061837 (to F. J. M.-A.), and AI59095 (to S. R. B. ...

Summary The intracellular bacterial pathogen Francisella tularensis causes tularemia, a zoonosis that can be fatal. The type VI secretion system (T6SS) encoded by the Francisella pathogenicity island (FPI) is critical for the virulence of this organism. Existing studies suggest that the complete repertoire of T6SS effectors delivered to host cells is encoded by the FPI. Using a proteome-wide approach, we discovered that the FPI-encoded T6SS exports at least three effectors encoded outside of the island. These proteins share features with virulence determinants of other pathogens and we provide evidence that they can contribute to intramacrophage growth. The remaining proteins we identified are encoded within the FPI. Two of these FPI-encoded proteins constitute effectors, whereas the others form a unique complex required for core function of the T6SS apparatus. The discovery of secreted effectors mediating interactions between Francisella and its host significantly advances our understanding of the pathogenesis of this organism.

Many pathogenic intracellular bacteria manipulate the host phago-endosomal system to establish and maintain a permissive niche. The fate and identity of these intracellular compartments is controlled by phosphoinositide lipids. By mechanisms that have remained undefined, a Francisella pathogenicity island-encoded secretion system allows phagosomal escape and replication of bacteria within host cell cytoplasm. Here we report the discovery that a substrate of this system, outside pathogenicity island A (OpiA), represents a family of wortmannin-resistant bacterial phosphatidylinositol (PI) 3-kinase enzymes with members found in a wide range of intracellular pathogens, including Rickettsia and Legionella spp. We show that OpiA acts on the Francisella-containing phagosome and promotes bacterial escape into the cytoplasm. Furthermore, we demonstrate that the phenotypic consequences of OpiA inactivation are mitigated by endosomal maturation arrest. Our findings suggest that Francisella, and likely other intracellular bacteria, override the finely tuned dynamics of phagosomal PI(3)P in order to promote intracellular survival and pathogenesis.

Background: Cytolethal distending toxins (CDTs) produced by pathogenic bacteria are genotoxic. Results: CDTs exploit two different endocytic pathways to reach the nucleus. Conclusion: Individual members of the CDT superfamily interact with host cells by distinct mechanisms. Significance: Learning how CDTs interact with and modulate host cells and tissues is critical for understanding the strategies used by pathogenic bacteria during infection.

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.