Stephen H Leppla

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

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Publications (245)1651.78 Total impact

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    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; · 4.09 Impact Factor
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    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; · 3.98 Impact Factor
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    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; · 6.24 Impact Factor
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    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;
  • Shihui Liu, Mahtab Moayeri, Stephen H Leppla
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    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; · 8.43 Impact Factor
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    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. · 4.40 Impact Factor
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    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. · 8.14 Impact Factor
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    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:e1003. · 6.04 Impact Factor
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    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 01/2014; 5(1). · 6.88 Impact Factor
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    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 01/2014; 4:4754. · 5.08 Impact Factor
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    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; · 3.98 Impact Factor
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    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; · 5.52 Impact Factor
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    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; · 7.21 Impact Factor
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    ABSTRACT: Bacillus anthracis, the causative agent of anthrax disease, is lethal owing to the actions of two exotoxins: anthrax lethal toxin (LT) and oedema toxin (ET). The key tissue targets responsible for the lethal effects of these toxins are unknown. Here we generated cell-type-specific anthrax toxin receptor capillary morphogenesis protein-2 (CMG2)-null mice and cell-type-specific CMG2-expressing mice and challenged them with the toxins. Our results show that lethality induced by LT and ET occurs through damage to distinct cell types; whereas targeting cardiomyocytes and vascular smooth muscle cells is required for LT-induced mortality, ET-induced lethality occurs mainly through its action in hepatocytes. Notably, and in contradiction to what has been previously postulated, targeting of endothelial cells by either toxin does not seem to contribute significantly to lethality. Our findings demonstrate that B. anthracis has evolved to use LT and ET to induce host lethality by coordinately damaging two distinct vital systems.
    Nature 08/2013; · 38.60 Impact Factor
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    ABSTRACT: The monoclonal antibody S9.6 binds DNA-RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. A single-chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA-RNA and, surprisingly, 2.7 nM for RNA-RNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA-RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
    Journal of Molecular Recognition 08/2013; 26(8):376-81. · 3.01 Impact Factor
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    ABSTRACT: Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with sub-protective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection, and demonstrate the potential for small molecule therapeutics targeting these proteins.
    Antimicrobial Agents and Chemotherapy 06/2013; · 4.57 Impact Factor
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    ABSTRACT: BACKGROUND: Signals of danger and damage in the cytosol of cells are sensed by NOD-like receptors (NLRs), which are components of multiprotein complexes called inflammasomes. Inflammasomes activate caspase-1, resulting in IL-1-beta and IL-18 secretion and an inflammatory response. To date, the only known activator of rodent Nlrp1 is anthrax lethal toxin (LT), a protease secreted by the bacterial pathogen Bacillus anthracis. Although susceptibility of mouse macrophages to LT has been genetically linked to Nlrp1b, mice harbor two additional Nlrp1 paralogs in their genomes (Nlrp1a and Nlrp1c). However, little is known about their expression profile and sequence in different mouse strains. Furthermore, simultaneous expression of these paralogs may lead to competitional binding of Nlrp1b interaction partners needed for inflammasome activation, thus influencing macrophages susceptibility to LT. To more completely understand the role(s) of Nlrp1 paralogs in mice, we surveyed for their expression in a large set of LT-resistant and sensitive mouse macrophages. In addition, we provide sequence comparisons for Nlrp1a and report on previously unrecognized splice variants of Nlrp1b. RESULTS: Our results show that macrophages from some inbred mouse strains simultaneously express different splice variants of Nlrp1b. In contrast to the highly polymorphic Nlrp1b splice variants, sequencing of expressed Nlrp1a showed the protein to be highly conserved across all mouse strains. We found that Nlrp1a was expressed only in toxin-resistant macrophages, with the sole exception of expression in LT-sensitive Cast/EiJ macrophages. Consistently, when widening Nlrp1a expression analysis to different mouse tissues, we found expression in most tissues of C57BL/6J, but not in those of Balb/cJ mice. CONCLUSIONS: Our data present a complex picture of Nlrp1 protein variations and provide a basis for elucidating their roles in murine macrophage function. Furthermore, the high conservation of Nlrp1a implies that it might be an important inflammasome sensor in mice.
    BMC Genomics 03/2013; 14(1):188. · 4.40 Impact Factor
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    ABSTRACT: Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. By screening a highly directed library of PA mutants, we identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases, and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins, and potentially has wide application.
    Journal of Biological Chemistry 02/2013; · 4.65 Impact Factor
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    ABSTRACT: In this study, we attempt to target the mitogen-activated protein kinase (MAPK) pathway in acute myeloid leukemia (AML) cells using a recombinant anthrax lethal toxin (LeTx). LeTx consists of protective antigen (PrAg) and lethal factor (LF). PrAg binds cells, is cleaved by furin, oligomerizes, binds three to four molecules of LF, and undergoes endocytosis, releasing LF into the cytosol. LF cleaves MAPK kinases, inhibiting the MAPK pathway. We tested potency of LeTx on a panel of 11 human AML cell lines. Seven cell lines showed cytotoxic responses to LeTx. Cytotoxicity of LeTx was mimicked by the specific mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitor U0126, indicating that LeTx-induced cell death is mediated through the MEK1/2-extracellular signal-regulated kinase (ERK1/2) branch of the MAPK pathway. The four LeTx-resistant cell lines were sensitive to the phosphatidylinositol 3-kinase inhibitor LY294002. Co-treatment of AML cells with both LeTx and LY294002 did not lead to increased sensitivity, showing a lack of additive/synergistic effects when both pathways are inhibited. Flow cytometry analysis of MAPK pathway activation revealed the presence of phospho-ERK1/2 only in LeTx-sensitive cells. Staining for Annexin V/propidium iodide and active caspases showed an increase in double-positive cells and the absence of caspase activation following treatment, indicating that LeTx-induced cell death is caspase-independent and nonapoptotic. We have shown that a majority of AML cell lines are sensitive to the LF-mediated inhibition of the MAPK pathway. Furthermore, we have demonstrated that LeTx-induced cytotoxicity in AML cells is nonapoptotic and dependent on phospho-ERK1/2 levels.
    Translational oncology 02/2013; 6(1):25-32. · 3.40 Impact Factor
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    ABSTRACT: ABSTRACT Anthrax toxin proteins from Bacillus anthracis constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit their potency. We created fusion proteins containing modified ubiquitins with their C-terminal regions fused to the Pseudomonas exotoxin A catalytic domain (PEIII) in order to achieve delivery and release of PEIII to the cytosol. Fusion proteins in which all seven lysines of wild-type ubiquitin were retained while the site cleaved by cytosolic deubiquitinating enzymes (DUBs) was removed were nontoxic, apparently due to rapid ubiquitination and proteasomal degradation. Fusion proteins in which all lysines of wild-type ubiquitin were substituted by arginine had high potency, exceeding that of a simple fusion lacking ubiquitin. This variant was less toxic to nontumor tissues in mice than the fusion protein lacking ubiquitin and was very efficient for tumor treatment in mice. The potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequence cleaved by DUBs. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination is an effective strategy for enhancing the potency of tumor-targeting toxins. IMPORTANCE Bacterial toxins typically have highly efficient mechanisms for cellular delivery of their enzymatic components. Cytosolic delivery of therapeutic enzymes and drugs is an important topic in molecular medicine. We describe anthrax toxin fusion proteins containing ubiquitin as a cytosolic cleavable linker that improves the delivery of an enzyme to mammalian cells. The ubiquitin linker allowed modulation of potency in cells and in mice. This effective strategy for enhancing the intracellular potency of an enzyme may be useful for the cytosolic delivery and release of internalized drugs.
    mBio 01/2013; 4(3). · 6.88 Impact Factor

Publication Stats

10k Citations
1,651.78 Total Impact Points

Institutions

  • 2003–2014
    • National Institute of Allergy and Infectious Diseases
      • Laboratory of Parasitic Diseases (LPD)
      Maryland, United States
  • 1991–2014
    • National Institutes of Health
      • • Laboratory of Parasitic Diseases
      • • Critical Care Medicine Department
      • • Branch of Oral and Pharyngeal Cancer
      • • Program in Developmental and Molecular Immunity
      • • Branch of Oral Infection and Immunity
      • • Laboratory of Molecular Microbiology
      • • Laboratory of Molecular Biology
      Maryland, United States
  • 2013
    • École Polytechnique Fédérale de Lausanne
      • Global Health Institute
      Lausanne, Vaud, Switzerland
  • 2011
    • U.S. Food and Drug Administration
      • Laboratory of Molecular Virology
      Washington, Washington, D.C., United States
  • 2007–2010
    • Scott & White
      Temple, Texas, United States
    • University of Chicago
      • Ben May Department for Cancer Research
      Chicago, Illinois, United States
  • 2009
    • Meso Scale Discovery
      Maryland, United States
  • 2006–2009
    • The Catholic University of America
      • Department of Biology
      Washington, D. C., DC, United States
  • 2003–2006
    • University of Geneva
      • Department of Microbiology and Molecular Medicine (MIMOL)
      Genève, GE, Switzerland
  • 2005
    • National Institute of Mental Health (NIMH)
      Maryland, United States
  • 2002–2005
    • Wake Forest School of Medicine
      • Department of Biochemistry
      Winston-Salem, NC, United States
  • 2004
    • Naval Medical Research Center
      Silver Spring, Maryland, United States
  • 1997
    • University of Michigan
      Ann Arbor, Michigan, United States
  • 1985–1996
    • U.S. Army Medical Research Institute of Infectious Diseases
      Maryland, United States
  • 1989–1992
    • United States Army Medical Research Institute for Infectious Diseases
      Maryland, United States
    • Brigham Young University - Provo Main Campus
      • Department of Chemistry and Biochemistry
      Provo, UT, United States