Anthrax Lethal Factor Cleaves Mouse Nlrp1b in Both Toxin-Sensitive and Toxin-Resistant Macrophages

Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS ONE (Impact Factor: 3.23). 11/2012; 7(11):e49741. DOI: 10.1371/journal.pone.0049741
Source: PubMed


Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.

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    • "e ( MAPK ) kinases . These toxins alter host cell signaling , thereby paralyzing the immune response of the host and perturbing the endocrine and endothelial systems ( Tournier et al . , 2007 , 2009b ; Moayeri and Leppla , 2009 ) . Moreover , LF also cleaves and activates NLRP1 , another signaling module in certain inbred rodents , but not human ( Hellmich et al . , 2012 ; Levinsohn et al . , 2012 ; Chavarria - Smith and Vance , 2015 ) . It is now well established that both toxins play a critical role at two different stages of the infection : early in the infection to paralyze the immune system , and at a late stage to finally kill the host ( see reviews , by Guichard et al . , 2012 ; Liu et al . , 201"
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    ABSTRACT: Anthrax, caused by Bacillus anthracis, a Gram-positive spore-forming bacterium, is initiated by the entry of spores into the host body. There are three types of human infection: cutaneous, inhalational, and gastrointestinal. For each form, B. anthracis spores need to cross the cutaneous, respiratory or digestive epithelial barriers, respectively, as a first obligate step to establish infection. Anthrax is a toxi-infection: an association of toxemia and rapidly spreading infection progressing to septicemia. The pathogenicity of Bacillus anthracis mainly depends on two toxins and a capsule. The capsule protects bacilli from the immune system, thus promoting systemic dissemination. The toxins alter host cell signaling, thereby paralyzing the immune response of the host and perturbing the endocrine and endothelial systems. In this review, we will mainly focus on the events and mechanisms leading to crossing of the respiratory epithelial barrier, as the majority of studies have addressed inhalational infection. We will discuss the critical gaps of knowledge that need to be addressed to gain a comprehensive view of the initial steps of inhalational anthrax. We will then discuss the few data available on B. anthracis crossing the cutaneous and digestive epithelia.
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    • "LF belongs to the family of gluzincins and exerts its deleterious intracellular action by removing N-terminal segments from most members of the mitogen-activated protein kinase kinase (MAPKK) family of signaling proteins [9] [10], and from NOD-like receptor protein 1 (Nlrp1) resulting in the activation of the inflammasome and macrophage death [11] [12] [13]. The Zn 2 þ ion in the active site of LF is bound tetrahedrally to the side chains of His686, His690 and Glu735, and to the nucleophilic water molecule responsible for the cleavage of the scissile peptide bond in the substrates [14]. "
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    ABSTRACT: Anthrax lethal factor (LF) is a zinc-dependent endopeptidase which, through a process facilitated by protective antigen, translocates to the host cell cytosol in a partially unfolded state. In the current report, the influence of urea and guanidine hydrochloride (GdnHCl) on LF׳s catalytic function, fold and metal binding was assessed at neutral pH. Both urea and GdnHCl were found to inhibit LF prior to the onset of unfolding, with the inhibition by the latter denaturant being a consequence of its ionic strength. With the exception of demetallated LF (apoLF) in urea, unfolding, as monitored by tryptophan fluorescence spectroscopy, was found to follow a two-state (native to unfolded) mechanism. Analysis of the metal status of LF with 4-(2-pyridylazoresorcinol) (PAR) following urea or GdnHCl exposure suggests the enzyme to be capable of maintaining its metal ion passed the observed unfolding transition in a chelator-inaccessible form. Although an increase in the concentration of the denaturants eventually allowed the chelator access to the protein׳s zinc ion, such process is not correlated with the release of the metal ion. Indeed, significant dissociation of the zinc ion from LF was not observed even at 6 M urea, and only high concentrations of GdnHCl (>3 M) were capable of inducing the release of the metal ion from the protein. Hence, the current study demonstrates not only the propensity of LF to tightly bind its zinc ion beyond the spectroscopically determined unfolding transition, but also the utility of PAR as a structural probe.
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    • "EF is an adenylate cyclase; it converts adenosine triphosphate (ATP) to high concentrations of cyclic adenosine monophosphate (cAMP) that cause metabolic perturbations [32] [33]. LF is a metalloprotease that cleaves mitogen-activated protein kinase kinases (MAPKKs, or, as used herein, MEKs) [34] [35] and the NLRP1 inflammasome sensor [36] [37], the consequences of which are described in later sections of this chapter and shown in Figure 13.1. Anthrax toxins can be viewed as fitting the A/B model [38]. "

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