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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. ...

Initiation of inhalation anthrax is believed to involve phagocytosis of Bacillus anthracis spores by alveolar macrophages, followed by spore germination within the phagolysosome. In order to establish a systemic infection, it is predicted that bacilli then escape from the macrophage and replicate extracellularly. Mechanisms utilized by B. anthracis to escape from the macrophage are not well characterized, but a role for anthrax toxin has been proposed. Here we report the isolation of an anthrax toxin-resistant cell line (R3D) following chemical mutagenesis of toxin-sensitive RAW 264.7 murine macrophage cells. Both R3D and RAW 264.7 cells phagocytize spores of a B. anthracis Sterne strain. However, RAW 264.7 cells are killed following spore challenge, whereas R3D cells survive. Resistance to toxin and spore challenge correlates with loss of expression of anthrax toxin receptor 2 (ANTXR2/CMG-2). When R3D cells are complemented with cDNA encoding either murine ANTXR2 or human anthrax toxin receptor 1 (ANTXR1/TEM-8), toxin and spore challenge susceptibility are restored, indicating that over-expression of either ANTXR can confer susceptibility to anthrax spore challenge. Taken together, these results indicate that anthrax toxin expression by the germinated spore enables B. anthracis killing of the macrophage from within.

Yersinia delivers Yops into numerous types of cultured cells, but predominantly into professional phagocytes and B cells during animal infection. The basis for this cellular tropism during animal infection is not understood. This work demonstrates that efficient and specific Yop translocation into phagocytes by Yersinia pseudotuberculosis (Yptb) is a multi-factorial process requiring several adhesins and host complement. When WT Yptb or a multiple adhesin mutant strain, ΔailΔinvΔyadA, colonized tissues to comparable levels, ΔailΔinvΔyadA translocated Yops into significantly fewer cells, demonstrating that these adhesins are critical for translocation into high numbers of cells. However, phagocytes were still selectively targeted for translocation, indicating that other bacterial and/or host factors contribute to this function. Complement depletion showed that complement-restricted infection by ΔailΔinvΔyadA but not WT, indicating that adhesins disarm complement in mice either by prevention of opsonophagocytosis or by suppressing production of pro-inflammatory cytokines. Furthermore, in the absence of the three adhesins and complement, the spectrum of cells targeted for translocation was significantly altered, indicating that Yersinia adhesins and complement direct Yop translocation into neutrophils during animal infection. In summary, these findings demonstrate that in infected tissues, Yersinia uses adhesins both to disarm complement-dependent killing and to efficiently translocate Yops into phagocytes.

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.

SummaryBacillus anthracis, the causative agent of anthrax, secretes two bipartite toxins that help the bacterium evade the immune system and contribute directly to pathogenesis. Both toxin catalytic moieties, lethal factor (LF) and oedema factor (OF), are internalized into the host-cell cytosol by a third factor, protective antigen (PA), which binds to cellular anthrax toxin receptors (ANTXRs). Oedema factor is an adenylate cyclase that impairs host defences by raising cellular cAMP levels. Here we demonstrate that oedema toxin (PA + OF) induces an increase in ANTXR expression levels in macrophages and dendritic cells resulting in an increased rate of toxin internalization. Furthermore, we show that increases in ANTXR mRNA levels depends on the ability of OF to increase cAMP levels, is mediated through protein kinase A-directed signalling and is monocyte-lineage-specific. To our knowledge, this is the first report of a bacterial toxin inducing host target cells to increase toxin receptor expression.

Summary A Yersinia pseudotuberculosis (Yptb) murine model of lung infection was previously developed using the serotype III IP2666NdeI strain, which robustly colonized lungs but only sporadically disseminated to the spleen and liver. We demonstrate here that a serotype Ib Yptb strain, IP32953, colonizes the lungs at higher levels and disseminates more efficiently to the spleen and liver compared to IP2666NdeI. The role of adhesins was investigated during IP32953 lung infection by constructing isogenic Δail, Δinv, ΔpsaE and ΔyadA mutants. An IP32953ΔailΔyadA mutant initially colonized but failed to persist in the lungs and failed to disseminate to the spleen and liver. Yptb expressing these adhesins selectively bound to and targeted neutrophils for translocation of Yops. This selective targeting was critical for virulence because persistence of the ΔailΔyadA mutant was restored following intranasal infection of neutropenic mice. Furthermore, Ail and YadA prevented killing by complement-mediated mechanisms during dissemination to and/or growth in the spleen and liver, but not in the lungs. Combined, these results demonstrate that Ail and YadA are critical, redundant virulence factors during lung infection, because they thwart neutrophils by directing Yop-translocation specifically into these cells.

Bacillus anthracis secretes two bipartite toxins, edema toxin (ET) and lethal toxin (LT), which impair immune responses and contribute directly to the pathology associated with the disease anthrax. Edema factor, the catalytic subunit of ET, is an adenylate cyclase that impairs host defenses by raising cellular cyclic AMP (cAMP) levels. Synthetic cAMP analogues and compounds that raise intracellular cAMP levels lead to phenotypic and functional changes in dendritic cells (DCs). Here, we demonstrate that ET induces a maturation state in human monocyte-derived DCs (MDDCs) similar to that induced by lipopolysaccharide (LPS). ET treatment results in downregulation of DC-SIGN, a marker of immature DCs, and upregulation of DC maturation markers CD83 and CD86. Maturation of DCs by ET is accompanied by an increased ability to migrate toward the lymph node-homing chemokine macrophage inflammatory protein 3␤, like LPS-matured DCs. Interestingly, cotreating with LT differentially affects the ET-induced maturation of MDDCs while not inhibiting ET-induced migration. These findings reveal a mechanism by which ET impairs normal innate immune function and may explain the reported adjuvant effect of ET.