Stephen H Leppla

National Institute of Allergy and Infectious Diseases, 베서스다, Maryland, United States

Are you Stephen H Leppla?

Claim your profile

Publications (256)1772.09 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Anthrax is caused by the spore-forming, gram-positive bacterium Bacillus anthracis. The bacterium's major virulence factors are (a) the anthrax toxins and (b) an antiphagocytic polyglutamic capsule. These are encoded by two large plasmids, the former by pXO1 and the latter by pXO2. The expression of both is controlled by the bicarbonate-responsive transcriptional regulator, AtxA. The anthrax toxins are three polypeptides-protective antigen (PA), lethal factor (LF), and edema factor (EF)-that come together in binary combinations to form lethal toxin and edema toxin. PA binds to cellular receptors to translocate LF (a protease) and EF (an adenylate cyclase) into cells. The toxins alter cell signaling pathways in the host to interfere with innate immune responses in early stages of infection and to induce vascular collapse at late stages. This review focuses on the role of anthrax toxins in pathogenesis. Other virulence determinants, as well as vaccines and therapeutics, are briefly discussed. Expected final online publication date for the Annual Review of Microbiology Volume 69 is September 08, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
    Annual review of microbiology 07/2015; DOI:10.1146/annurev-micro-091014-104523 · 13.02 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The immunogenicity of Bacillus anthracis capsule (poly-γ-d-glutamic acid, PGA) conjugated to recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two anthrax-naïve juvenile chimpanzees. In a previous study of these conjugates, highly protective monoclonal antibodies (mAbs) against PGA were generated. This study examines the polyclonal antibody response of the same animals. Preimmune antibodies to PGA with titers of >10(3) were detected in the chimpanzees. The maximal titer of anti-PGA was induced within 1-2 weeks following the 1(st) immunization, with no booster effects following 2(nd) and 3(rd) immunizations. Thus, the anti-PGA response in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 10(3). An anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA antibody titers and those following the 1(st) immunization were ≤300, with the antibodies peaking above 10(4) following the 2(nd) immunization. The polyclonal anti-PGA shared the mAb 11D epitope and, similar to the mAbs, exerted opsonophagocytic killing of B. anthracis. Most importantly, the PGA-TT-induced antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as second generation anthrax vaccines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Clinical and vaccine Immunology: CVI 06/2015; DOI:10.1128/CVI.00137-15 · 2.37 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background Lethal and edema toxin contribute to shock and lethality with Bacillus anthracis. We showed previously in a 96-h sedated canine model that raxibacumab, a monoclonal antibody against protective antigen, augmented hemodynamic support (HS) and improved survival with lethal toxin challenge. Here we study raxibacumab further. Using this model, we have now studied raxibacumab with 24 h edema toxin challenges (Study 1), and lethal and edema toxin challenges together (Study 2). Methods Using our canine model, we have now studied raxibacumab with 24h edema toxin challenges (Study-1), and lethal and edema toxin challenges together (Study-2). Results In Study 1, compared to no treatment, HS (titrated fluid and norepinephrine) increased mean arterial blood pressure (MAP, p ≤ 0.05) but not survival [0 of 10 (0/10) animals survived in each group] or median survival time [43.8 h (range 16.8 to 80.3) vs. 45.2 h (21.0 to 57.1)]. Compared to HS, HS with raxibacumab treatment at or 6 h after the beginning of edema toxin increased MAP and survival rate (6/7 and 7/8, respectively) and time [96.0 h (39.5 to 96.0) and 96.0 h (89.5 to 96.0), respectively]; (p ≤ 0.05). HS with raxibacumab at 12 h increased MAP (p ≤ 0.05) but not survival [1/5; 55.3 h (12.6 to 96.0)]. In Study-2, survival rate and time increased with HS and raxibacumab at 0 h (4/4) or 6 h after (3/3) beginning lethal and edema toxin compared to HS [0/5; 71.5 h (65 to 93)] (p = 0.01 averaged over raxibacumab groups). Conclusions Raxibacumab augments HS and improves survival during shock with lethal and edema toxin.
    02/2015; 3(1). DOI:10.1186/s40635-015-0043-4
  • Nolan K Maier, Stephen H Leppla, Mahtab Moayeri
    [Show abstract] [Hide abstract]
    ABSTRACT: Inflammasomes are cytosolic protein complexes that respond to diverse danger signals by activating caspase-1. The sensor components of the inflammasome, often proteins of the nucleotide-binding oligomerization domain-like receptor (NLR) family, detect stress, danger stimuli, and pathogen-associated molecular patterns. We report that the eicosanoid 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2) and related cyclopentenone PGs inhibit caspase-1 activation by the NLR family leucine-rich repeat protein (NLRP)1 and NLRP3 inflammasomes. This inhibition was independent of the well-characterized role of 15d-PGJ2 as a peroxisome proliferator receptor-γ agonist, its activation of NF erythroid 2-related factor 2, or its anti-inflammatory function as an inhibitor of NF-κB. Instead, 15d-PGJ2 prevents the autoproteolytic activation of caspase-1 and the maturation of IL-1β through induction of a cellular state inhibitory to caspase-1 proteolytic function. The eicosanoid does not directly modify or inactivate the caspase-1 enzyme. Rather, inhibition is dependent on de novo protein synthesis. In a mouse peritonitis model of gout, using monosodium urate crystals to activate NLRP3, 15d-PGJ2 caused a significant inhibition of cell recruitment and associated IL-1β release. Furthermore, in a murine anthrax infection model, 15d-PGJ2 reversed anthrax lethal toxin-mediated NLRP1-dependent resistance. The findings reported in this study suggest a novel mechanism for the anti-inflammatory properties of the cyclopentenone PGs through inhibition of caspase-1 and the inflammasome.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor (LF) and edema factor (EF). Antibodies (Abs) against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only Abs (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well-characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from 'pre-pore' to its SDS and heat-resistant 'pore' conformation while not preventing cleavage of full length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface generated PA63 subunit but not pre-formed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin, and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 01/2015; 290(10). DOI:10.1074/jbc.M114.627943 · 4.57 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Induction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1β (IL-1β), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1β in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. IMPORTANCE Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain “sensor” proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. We report here that T. gondii is a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Using mice deficient in IL-18 and IL-18R, we show that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine survival.
    mBio 12/2014; 5(1). DOI:10.1128/mBio.01117-13 · 6.88 Impact Factor
  • European Journal of Cancer 11/2014; 50:126. DOI:10.1016/S0959-8049(14)70520-9 · 4.82 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Detection of nucleic acids using antibodies is uncommon. This is in part because nucleic acids are poor immunogens and it is difficult to elicit antibodies having high affinity to each type of nucleic acid while lacking cross-reactivity to others. We describe the origins and applications of a variety of anti-nucleic acid antibodies, including ones reacting with modified nucleosides and nucleotides, single-stranded DNA, double-stranded DNA, RNA, DNA:RNA hybrids, locked-nucleic acids or peptide nucleic acid:nucleic acid hybrids. Carefully selected antibodies can be excellent reagents for detecting bacteria, viruses, small RNAs, microRNAs, R-loops, cancer cells, stem cells, apoptotic cells and so on. The detection may be sensitive, simple, rapid, specific, reproducible, quantitative and cost-effective. Current microarray and diagnostic methods that depend on cDNA or cRNA can be replaced by using antibody detection of nucleic acids. Therefore, development should be encouraged to explore new utilities and create a robust arsenal of new anti-nucleic acid antibodies.
    Expert Review of Molecular Diagnostics 07/2014; DOI:10.1586/14737159.2014.931810 · 4.27 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; Mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA- activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32%-87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5-3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers.
    Toxicology and Applied Pharmacology 06/2014; 279(2). DOI:10.1016/j.taap.2014.06.010 · 3.63 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knock-out strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and opens up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.
    Environmental Microbiology 06/2014; 17(3). DOI:10.1111/1462-2920.12548 · 6.24 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacillus anthracis pXO1 minireplicon (MR) plasmid consisting of ORFs GBAA_pXO1_0020 - GBAA_pXO1_0023 is not stably maintained in B. anthracis, whereas the full-size parent pXO1 plasmid (having 181,677 bp and 217 ORFs) is extremely stable under the same growth conditions. Two genetic tools developed for DNA manipulation in B. anthracis (Cre/loxP and Flp/FRT systems) were used to identify pXO1 regions important for plasmid stability. We localized a large segment of pXO1 that enables stable plasmid maintenance during vegetative growth. Further genetic analysis identified three genes that are necessary for pXO1 maintenance: amsP (GBAA_pXO1_0069), minP (GBAA_pXO1_0082), and sojP (GBAA_pXO1_0084). Analysis of conserved domains in the corresponding proteins indicated that only AmsP (Activator of Maintenance System of pXO1) is predicted to bind DNA, due to its strong helix-turn-helix domain. Two conserved domains were found in the MinP protein (Min protein from pXO1): an N-terminal domain having some similarity to the B. anthracis septum site-determining protein MinD, and a C-terminal domain that resembles a baculovirus single-strand DNA-binding protein. The SojP protein (Soj from pXO1) contains putative Walker-box motifs and belongs to the ParA family of ATPases. No sequences encoding other components of Type I plasmid partition systems, namely cis-acting centromere parS and its binding ParB protein, were identified within the pXO1 genome. A model describing the role of the MinP protein in pXO1 distribution between daughter cells is proposed.
    Journal of Bacteriology 06/2014; 196(16). DOI:10.1128/JB.01747-14 · 2.69 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We characterized an anti-cancer fusion protein consisting of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas exotoxin A by (i) mutating the N-terminal amino acids and by (ii) reductive methylation to dimethylate all lysines. Dimethylation of lysines was achieved quantitatively and specifically without affecting binding of the fusion protein to PA or decreasing the enzymatic activity of the catalytic moiety. Ubiquitination in vitro was drastically decreased for both the N-terminally mutated and dimethylated variants, and both appeared to be slightly more stable in the cytosol of treated cells. The dimethylated variant showed greatly reduced neutralization by antibodies to LF. The two described modifications offer unique advantages such as increased cytotoxic activity and diminished antibody recognition, and thus may be applicable to other therapeutic proteins that act in the cytosol of cells.
    Scientific Reports 04/2014; 4:4754. DOI:10.1038/srep04754 · 5.58 Impact Factor
  • Shihui Liu, Mahtab Moayeri, Stephen H Leppla
    [Show abstract] [Hide abstract]
    ABSTRACT: The pathophysiological effects resulting from many bacterial diseases are caused by exotoxins released by the bacteria. Bacillus anthracis, a spore-forming bacterium, is such a pathogen, causing anthrax through a combination of bacterial infection and toxemia. B. anthracis causes natural infection in humans and animals and has been a top bioterrorism concern since the 2001 anthrax attacks in the USA. The exotoxins secreted by B. anthracis use capillary morphogenesis protein 2 (CMG2) as the major toxin receptor and play essential roles in pathogenesis during the entire course of the disease. This review focuses on the activities of anthrax toxins and their roles in initial and late stages of anthrax infection.
    Trends in Microbiology 03/2014; DOI:10.1016/j.tim.2014.02.012 · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Upon infection of a mammalian host, Bacillus anthracis responds to host cues, and particularly to elevated temperature (37[degree sign]C) and bicarbonate/CO2 concentrations, with increased expression of virulence factors that include the anthrax toxins and extracellular capsular layer. This response requires the presence of the pXO1 virulence plasmid-encoded pleiotropic regulator AtxA. To better understand the genetic basis of this response, we utilized a controlled in vitro system and Next Generation sequencing to determine and compare RNA expression profiles of the parental strain and an isogenic AtxA-deficient strain in a 2 x 2 factorial design with growth environments containing or lacking carbon dioxide. We found 15 pXO1-encoded genes and 3 chromosomal genes that were strongly regulated by the separate or synergistic actions of AtxA and carbon dioxide. The majority of the regulated genes responded to both AtxA and carbon dioxide rather than to just one of these factors. Interestingly, we identified two previously unrecognized small RNAs that are highly expressed under physiological carbon dioxide concentrations in an AtxA-dependent manner. Expression levels of the two small RNAs were found to be higher than that of any other gene differentially expressed in response to these conditions. Secondary structure and small RNA-mRNA binding predictions for the two small RNAs suggest that they may perform important functions in regulating B. anthracis virulence. A majority of genes on the virulence plasmid pXO1 that are regulated by the presence of either CO2 or AtxA separately are also regulated synergistically in the presence of both. These results also elucidate novel pXO1-encoded small RNAs that are associated with virulence conditions.
    BMC Genomics 03/2014; 15(1):229. DOI:10.1186/1471-2164-15-229 · 4.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages.
    PLoS Pathogens 03/2014; 10(3):e1003927. DOI:10.1371/journal.ppat.1003927 · 8.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cytolethal distending toxin (Cdt) is produced by Gram-negative bacteria of several species. It is composed of three subunits, CdtA, CdtB, and CdtC, with CdtB being the catalytic subunit. We fused CdtB from Haemophilus ducreyi to the N-terminal 255 amino acids of Bacillus anthracis toxin lethal factor (LFn) to design a novel, potentially potent antitumor drug. As a result of this fusion, CdtB was transported into the cytosol of targeted cells via the efficient delivery mechanism of anthrax toxin. The fusion protein efficiently killed various human tumor cell lines by first inducing a complete cell cycle arrest in the G2/M phase, followed by induction of apoptosis. The fusion protein showed very low toxicity in mouse experiments and impressive antitumor effects in a Lewis Lung carcinoma model, with a 90% cure rate. This study demonstrates that efficient drug delivery by a modified anthrax toxin system combined with the enzymatic activity of CdtB has great potential as anticancer treatment and should be considered for the development of novel anticancer drugs.
    Cell Death & Disease 01/2014; 5(1):e1003. DOI:10.1038/cddis.2013.540 · 5.18 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6 J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; Mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA- activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32%-87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5-3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers.
    Toxicology and Applied Pharmacology 01/2014; · 3.63 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Inflammasomes are large cytoplasmic multiprotein complexes that activate caspase-1 in response to diverse intracellular danger signals. Inflammasome components termed nucleotide-binding oligomerization domain-like receptor (NLR) proteins act as sensors for pathogen-associated molecular patterns, stress, or danger stimuli. We discovered that arsenicals, including arsenic trioxide and sodium arsenite, inhibited activation of the NLRP1, NLRP3, and NAIP5/NLRC4 inflammasomes by their respective activating signals, anthrax lethal toxin, nigericin, and flagellin. These compounds prevented the autoproteolytic activation of caspase-1 and the processing and secretion of IL-1β from macrophages. Inhibition was independent of protein synthesis induction, proteasome-mediated protein breakdown, or kinase signaling pathways. Arsenic trioxide and sodium arsenite did not directly modify or inhibit the activity of preactivated recombinant caspase-1. Rather, they induced a cellular state inhibitory to both the autoproteolytic and substrate cleavage activities of caspase-1, which was reversed by the reactive oxygen species scavenger N-acetylcysteine but not by reducing agents or NO pathway inhibitors. Arsenicals provided protection against NLRP1-dependent anthrax lethal toxin-mediated cell death and prevented NLRP3-dependent neutrophil recruitment in a monosodium urate crystal inflammatory murine peritonitis model. These findings suggest a novel role in inhibition of the innate immune response for arsenical compounds that have been used as therapeutics for a few hundred years.
    The Journal of Immunology 12/2013; 192(2). DOI:10.4049/jimmunol.1301434 · 5.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Anthrax lethal toxin is a classical AB toxin comprised of two components: protective antigen (PA) and lethal factor (LF). Here, we show that following assembly and endocytosis, PA forms a channel that translocates LF, not only into the cytosol, but also into the lumen of endosomal intraluminal vesicles (ILVs). These ILVs can fuse and release LF into the cytosol, where LF can proteolyze and disable host targets. We find that LF can persist in ILVs for days, fully sheltered from proteolytic degradation, both in vitro and in vivo. During this time, ILV-localized LF can be transmitted to daughter cells upon cell division. In addition, LF-containing ILVs can be delivered to the extracellular medium as exosomes. These can deliver LF to the cytosol of naive cells in a manner that is independent of the typical anthrax toxin receptor-mediated trafficking pathway, while being sheltered from neutralizing extracellular factors of the immune system.
    Cell Reports 11/2013; 5. DOI:10.1016/j.celrep.2013.10.019 · 7.21 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacillus cereus is a spore-forming, Gram-positive bacterium commonly associated with outbreaks of food poisoning. It is also known as an opportunistic pathogen causing clinical infections such as bacteremia, meningitis, pneumonia, and gas gangrene-like cutaneous infections, mostly in immunocompromised patients. B. cereus secretes a plethora of toxins of which four are associated with the symptoms of food poisoning. Two of these, the non-hemolytic enterotoxin Nhe and the hemolysin BL (Hbl) toxin, are predicted to be structurally similar and are unique in that they require the combined action of three toxin proteins to induce cell lysis. Despite their dominant role in disease, the molecular mechanism of their toxic function is still poorly understood. We report here that B. cereus strain ATCC 10876 harbors not only genes encoding Nhe, but also two copies of the hbl genes. We identified Hbl as the major secreted toxin responsible for inducing rapid cell lysis both in cultured cells and in an intraperitoneal mouse toxicity model. Antibody neutralization and deletion of Hbl-encoding genes resulted in significant reductions of cytotoxic activity. Microscopy studies with Chinese Hamster Ovary cells furthermore showed that pore formation by both Hbl and Nhe occurs through a stepwise, sequential binding of toxin components to the cell surface and to each other. This begins with binding of Hbl-B or NheC to the eukaryotic membrane, and is followed by the recruitment of Hbl-L1 or NheB, respectively, followed by the corresponding third protein. Lastly, toxin component complementation studies indicate that although Hbl and Nhe can be expressed simultaneously and are predicted to be structurally similar, they are incompatible and cannot complement each other.
    PLoS ONE 10/2013; 8(10):e76955. DOI:10.1371/journal.pone.0076955 · 3.53 Impact Factor

Publication Stats

13k Citations
1,772.09 Total Impact Points

Institutions

  • 2003–2015
    • National Institute of Allergy and Infectious Diseases
      • Laboratory of Parasitic Diseases (LPD)
      베서스다, Maryland, United States
  • 1991–2014
    • National Institutes of Health
      • • Laboratory of Parasitic Diseases
      • • Branch of Oral and Pharyngeal Cancer
      • • Critical Care Medicine Department
      • • Branch of Oral Infection and Immunity
      • • Laboratory of Molecular Microbiology
      Maryland, United States
  • 2012
    • Medical College of Wisconsin
      Milwaukee, Wisconsin, United States
  • 2009–2012
    • The Catholic University of America
      • Department of Biology
      Washington, Washington, D.C., United States
    • Meso Scale Discovery
      Maryland, United States
  • 2010
    • University of California, San Francisco
      • Department of Pharmaceutical Chemistry
      San Francisco, California, United States
  • 2007
    • University of Chicago
      • Ben May Department for Cancer Research
      Chicago, Illinois, United States
  • 2006–2007
    • Scott & White
      Temple, Texas, United States
    • University of Texas at Austin
      • Department of Biomedical Engineering
      Austin, Texas, United States
  • 2005–2007
    • Van Andel Research Institute
      Grand Rapids, Michigan, United States
    • National Institute of Mental Health (NIMH)
      Maryland, United States
  • 2003–2007
    • National Institute of Allergy and Infectious Disease
      베서스다, Maryland, United States
  • 2002–2005
    • Wake Forest School of Medicine
      • Department of Internal Medicine
      Winston-Salem, NC, United States
  • 2004
    • Naval Medical Research Center
      Silver Spring, Maryland, United States
  • 2002–2004
    • Wake Forest University
      Winston-Salem, North Carolina, United States
  • 1996–2003
    • Northern Inyo Hospital
      BIH, California, United States
  • 1997–2001
    • University of Leicester
      • Department of Biochemistry
      Leicester, ENG, United Kingdom
    • University of Michigan
      Ann Arbor, Michigan, United States
  • 1999
    • Emory University
      Atlanta, Georgia, United States
  • 1998
    • NCI-Frederick
      Фредерик, Maryland, United States
  • 1984–1994
    • U.S. Army Medical Research Institute of Infectious Diseases
      Фредерик, Maryland, United States
    • University of Cincinnati
      • Department of Molecular Genetics, Biochemistry, and Microbiology
      Cincinnati, Ohio, United States
  • 1989
    • University of Virginia
      • Department of Pharmacology
      Charlottesville, Virginia, United States
  • 1986–1989
    • United States Army Medical Research Institute for Infectious Diseases
      Maryland, United States