Anthrax Lethal Toxin

The spore-forming, Gram-positive bacteria Bacillus anthracis contains two plasmids [for a review on the bacterium, please refer to the review by Mock and Fouet (Mock and Fouet, 2001)]. The three proteins that comprose the two toxins are encoded on one of these plasmids, pXO1. Together these proteins form a variation of the classical A-B toxins. Protective antigen (PA) and edema factor (EF) constitute the edema toxin and PA and lethal factor (LF) the lethal toxin (LeTx) (Collier and Young, 2003). The involvement of the lethal toxin and anthrax infection is evident as strains lacking the pXO1 plasmid are attenuated in virulence, and the lethal toxin alone manifests distinct symptoms of B. anthracis infection such as pleural effusions (Klein et al., 1962; Fish et al., 1968; Pezard et al., 1991; Moayeri et al., 2003; Cui et al., 2004). Therefore, much of anthrax research has focused on LeTx and its mechanism of action, and this toxin will also be the focus of this review.

LeTx entry into cells is now fairly well understood at the molecular level (Abrami et al., 2003, 2004) and is summarized in Figure 4.1. Only macrophages from a limited group of inbred mice have been shown to be sensitive to rapid lysis by LeTx and thus have until recently been suggested to be the target of LeTx action (Friedlander, 1986; Singh et al., 1989; Friedlander et al., 1993; Roberts et al., 1998). LeTx at sublytic concentrations induces macrophage apoptosis (Park et al., 2002; Popov et al., 2002b). Similar proapoptotic behavior is seen in human peripheral blood mononu-clear cells (PBMC) but these cells are not lysed by LeTx (Popov et al.,

Figure 4.1. Mechanism of action of anthrax lethal toxin. PA binds to two different receptors, tumor endothelial marker 8 (TEM-8) and capillary morphogenesis protein 2 (CMG-2) (Bradley et al., 2001; Scobie et al., 2003), which seem to be ubiquitously expressed. PA is then cleaved by the enzyme furin (Klimpel et al., 1992), heptamerizes, and then binds EF and/or LF (Singh et al., 1999).This complex is then internalized by clathrin-dependent raft-mediated endocytosis (Abrami et al., 2003), and the LF/EF are translocated across the endosomal membrane and into the cytosol via a pH- and voltage-dependent mechanism (Blaustein et al., 1989; Zhao et al., 1995; Wesche et al., 1998). Once inside the cell, the mechanism of action of LF is less well understood. It is known to cleave and inactivate members of the MAPKK family, and we now show that it can inactivate the glucocorticoid receptor probably by directly or indirectly destabilizing or interfering with GR binding to chromatin.

Figure 4.1. Mechanism of action of anthrax lethal toxin. PA binds to two different receptors, tumor endothelial marker 8 (TEM-8) and capillary morphogenesis protein 2 (CMG-2) (Bradley et al., 2001; Scobie et al., 2003), which seem to be ubiquitously expressed. PA is then cleaved by the enzyme furin (Klimpel et al., 1992), heptamerizes, and then binds EF and/or LF (Singh et al., 1999).This complex is then internalized by clathrin-dependent raft-mediated endocytosis (Abrami et al., 2003), and the LF/EF are translocated across the endosomal membrane and into the cytosol via a pH- and voltage-dependent mechanism (Blaustein et al., 1989; Zhao et al., 1995; Wesche et al., 1998). Once inside the cell, the mechanism of action of LF is less well understood. It is known to cleave and inactivate members of the MAPKK family, and we now show that it can inactivate the glucocorticoid receptor probably by directly or indirectly destabilizing or interfering with GR binding to chromatin.

2002a). Recent data has suggested that macrophage lysis is not essential for LeTx toxicity, however, it appears to potentially exacerbate the toxin's effects in mice (Moayeri et al., 2003, 2005). Most species harbor LeTx-resistant macrophages. However, a more complicated involvement of macrophage sensitivity in the pathogenesis of anthrax is suggested by discoveries showing that resistant C57BL/6J macrophages or human macrophages can be made sensitive to LeTx by treatment with poly-d-glutamic acid (the major component of the B. anthracis capsule), peptidoglycan (a component of Gram-positive bacterial cell walls), lipopolysaccharide (LPS; a component of Gram-negative bacterial cell walls), or TNF-a (Park et al., 2002; Popov et al., 2002a; Kim et al., 2003). Recent studies show that the differentiation state of human monocytic cells determines their LeTx sensitivity (Kassam et al., 2005). Therefore, it is possible to imagine sensitization of macrophages during the course of infection in hosts normally harboring LT-resistant macrophages may play a role in B. anthracis pathogenesis.

LF, a zinc metalloprotease, is known to cleave and inactivate some members of the mitogen activated protein (MAP) kinase kinase (MAPKK/MEK) family (Duesbery et al., 1998; Vitale et al., 1998;Pellizzari et al., 2000). The cleavage and inactivation of MAPKKs results in inhibition of downstream signaling pathways such as AP-1 and NFAT (Paccani et al., 2005). However, LF cleavage of MAPKKs alone cannot account for macrophage lysis as LF internalization (Menard et al., 1996; Roberts et al., 1998; Singh et al., 1989) and MAPKK cleavage in sensitive and resistant macrophages and cell lines (Pellizzari et al., 1999,2000; Watters et al., 2001) are the same. Inhibition of the proteolytic function of LF prevents LeTx toxicity in sensitive cells (Klimpel et al., 1994; Menard et al., 1996; Duesbery et al., 1998; Hammond and Hanna, 1998; Vitale et al., 1998) suggesting that cleavage of MAPKKs or other potentially unidentified substrates is necessary for LeTx macrophage lysis. It is possible that the response to MAPKK cleavage in different cells may lead to a different cascade of events that in sensitive cells leads to cell lysis but not in resistant cells, thereby defining differential sensitivity to LeTx. The resistance response, in turn, can be overcome by pretreatment with LPS or B. anthracis cell wall products. One such potential factor involved in this differential response may be the kinesin Kif1C, which was identified as the macrophage sensitivity locus for LeTx (Watters et al., 2001). The function of Kif1C is unknown, but it has been suggested to be involved in endoplasmic reticulum (ER) transport (Dorner et al., 1998). Although a single locus is linked to macrophage sensitivity to LeTx, another group has shown contribution of two additional loci, Ltxs2 and Ltxs3, to mouse susceptibility to LeTx (McAllister et al., 2003).The relative contribution of the different macrophage sensitivity loci to LeTx susceptibility has been shown to vary among different mouse strains (Moayeri et al., 2004).

Despite recent reports on endothelial cell sensitivity to LeTx (Kirby, 2004; Pandey and Warburton, 2004), most studies on the mechanism of LeTx-mediated cell death use the macrophage as a model. However, the mechanism by which LeTx induces apoptosis (Park et al., 2002) or necrosis (Kim et al., 2003) in macrophages or other cell types is currently unknown. Recent studies have focused on gene array and proteome changes in response to toxin using dying LeTx-sensitive macrophages at late time points, yielding a variety of changes primarily associated with stress responses or energy production in cells (Bergman et al., 2005; Chandra et al., 2005; Comer et al., 2005) and not providing too many clues to the early events involved in toxicity. Other studies on LeTx have reported on the toxin's ability to interfere with immune cell function through its inhibition of MAPKK function. For example, LeTx represses LPS-induced cytokine (TNF-a, IL-1a, IL-6, and IL-12) production in dendritic cells (Agrawal et al., 2003) and anthrax cell wall-induced cytokine release in peripheral blood mononuclear cells (PBMCs) (Popov et al., 2002a). Recently, the toxin has been shown to inhibit T-cell activation (Paccani et al., 2005).

Death from anthrax lethal toxin has been reported to occur from systemic shock (Smith et al., 1955), resembling cytokine-mediated LPS-induced shock (Hanna et al., 1993). However, the involvement of cytokines in LeTx toxicity has been a matter of some controversy. In sensitive macrophages, LeTx (1 pg/ml for 16 h) alone did not induce TNF-a, IL-6, IL-1a, or IL-1P (Erwin et al., 2001). However, others have shown that in a LeTx-sensitive macrophage cell line and in macrophages from ICR mice, sublytic LeTx concentrations (1 pg/ml for 6h) alone was able to induce TNF-a and IL-1P (Hanna et al., 1993; Shin et al., 2000). Analysis of more than 40 cytokines and inflammatory mediators in BALB/cJ and C57BL/6J mice after LeTx administration showed an early transitory increase of numerous factors in BALB/cJ but not C57BL/6J mice. However, no inflammatory cascade was induced and no TNF-a induction was seen (Moayeri et al., 2003) while animals from both strains were susceptible to LeTx. The transitory response seen in the Balb/c animals was directly linked to the macrophage lysis event (Moayeri et al., 2004). Inflammatory responses were also absent in a rat infusion model of LeTx killing (Cui et al., 2004). Knockout mice for the TNF or IL-1 receptors or iNOS did not differ in their susceptibility to anthrax infection compared with control mice (Kalns et al., 2002). In fact, in both sensitive and resistant macrophage cell lines, LeTx inhibits LPS/IFN-y stimulation of TNF-a and NO probably through inactivation of the MAPKK pathways (Pellizzari et al., 1999,2000; Erwin et al., 2001) much in the manner LPS-mediated cytokine responses are shut down by LT in dendritic cells (Agrawal et al., 2003). Although there is conflicting data regarding cytokine induction by LeTx alone, there seems to be consistency in that LeTx inhibits bacterial or LPS-induced cytokine release probably through inhibition of MAPKK. It is likely that an inflammatory cytokine release is not critical for LeTx lethality and induction of circulatory shock (Moayeri et al., 2003; Cui et al., 2004). However, cytokines are likely released in response to exposure to B. anthracis spores (Pickering and Merkel, 2004; Pickering et al., 2004; Popov et al., 2004) and may alter macrophage and other cell responses to LeTx in ways we currently cannot predict. In rats, LeTx-mediated killing can occur as rapidly as 45 min (Ezzell et al., 1984) and clearly does not require transcription-translation events, and thus cytokine expression. The method by which LeTx induces circulatory shock in different animal models remains to be elucidated.

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