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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Available from: Inka Sastalla, Oct 07, 2015
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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.
    05/2015; 1:68-77. DOI:10.1016/j.bbrep.2015.03.004
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    • "Initially, Nlrp1b was assumed to just physically associate with cytosolic LF, but it was then shown that LF metalloprotease activity was needed for its recognition (Fink et al., 2008). Subsequent studies showed that LeTx cleaves Nlrp1b close to its N terminus (Chavarría- Smith and Vance, 2013; Hellmich et al., 2012). How removal of a short (4 kDa) peptide from the N terminus promotes inflammasome activation requires further analysis (Figure 3). "
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    ABSTRACT: Recent studies have offered a glimpse into the sophisticated mechanisms by which inflammasomes respond to danger and promote secretion of interleukin (IL)-1β and IL-18. Activation of caspases 1 and 11 in canonical and noncanonical inflammasomes, respectively, also protects against infection by triggering pyroptosis, a proinflammatory and lytic mode of cell death. The therapeutic potential of inhibiting these proinflammatory caspases in infectious and autoimmune diseases is raised by the successful deployment of anti-IL-1 therapies to control autoinflammatory diseases associated with aberrant inflammasome signaling. This Review summarizes recent insights into inflammasome biology and discusses the questions that remain in the field.
    Cell 05/2014; 157(5):1013-1022. DOI:10.1016/j.cell.2014.04.007 · 32.24 Impact Factor
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    • "It is unclear if NLRP1 mediated LT resistance exists in human cells, however it was mentioned in a recent review that the authors had never observed LT resistance in macrophages isolated from healthy human subjects (77). Studies in rats and mice, which also present cell death induced by LT depending on NLRP1, showed that LF-mediated cleavage of the N-terminal domain of NLRP1 leads to caspase-1 activation and IL-1β release (71, 78, 79). Interestingly, a recent study indicated that a direct cleavage of murine NLRP1b is sufficient to induce inflammasome activation in the absence of LF, and proposed that NLRP1 might function as a sensor of protease activity of multiple pathogens (79). "
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    ABSTRACT: The lung is a particularly vulnerable organ at the interface of the body and the exterior environment. It is constantly exposed to microbes and particles by inhalation. The innate immune system needs to react promptly and adequately to potential dangers posed by these microbes and particles, while at the same time avoiding extensive tissue damage. Nucleotide-binding oligomerization domain-like receptors (NLRs) represent a group of key sensors for microbes and damage in the lung. As such they are important players in various infectious as well as acute and chronic sterile inflammatory diseases, such as pneumonia, chronic obstructive pulmonary disease (COPD), acute lung injury/acute respiratory distress syndrome, pneumoconiosis, and asthma. Activation of most known NLRs leads to the production and release of pro-inflammatory cytokines, and/or to the induction of cell death. We will review NLR functions in the lung during infection and sterile inflammation.
    Frontiers in Immunology 11/2013; 4:393. DOI:10.3389/fimmu.2013.00393
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