PubMed Health⌕ Search

Biomedical subjects

Jeremy Mogridge

Publications and source records attributed to Jeremy Mogridge.

16 recordsLinked to original sources
PubMed2006

Polyvalent inhibitors of anthrax toxin that target host receptors.

Resistance of pathogens to antimicrobial therapeutics has become a widespread problem. Resistance can emerge naturally, but it can also be engineered intentionally, which is an important consideration in designing therapeutics for bioterrorism agents. Blocking host receptors used by pathogens represents a powerful strategy to overcome this problem, because extensive alterations to the pathogen may be required to enable it to switch to a new receptor that can still support pathogenesis. Here, we demonstrate a facile method for producing potent receptor-directed antitoxins. We used phage display to identify a peptide that binds both anthrax-toxin receptors and attached this peptide to a synthetic scaffold. Polyvalency increased the potency of these peptides by >50,000-fold in vitro and enabled the neutralization of anthrax toxin in vivo. This work demonstrates a receptor-directed anthrax-toxin inhibitor and represents a promising strategy to combat a variety of viral and bacterial diseases.

Acetylation↗
PubMed2006

Self-association of the transmembrane domain of an anthrax toxin receptor.

Protective antigen (PA), lethal factor (LF) and edema factor (EF) are secreted individually by Bacillus anthracis. These components of anthrax toxin must then assemble into complexes to intoxicate mammalian cells. Toxin assembly initiates when molecules of PA bind mammalian receptors ANTXR1/2 and are cleaved by surface proteases into 20 kDa and 63 kDa fragments. After PA20 dissociates, receptor-bound PA63 homo-oligomerizes into heptamers. Oligomeric PA63 binds EF and LF and these complexes are internalized into an acidic compartment where the two enzymatic components are translocated across the membrane by a channel formed by heptameric PA63. Since oligomerization of PA63 is required to bind and translocate the enzymatic components, we sought to determine whether interactions between toxin receptors could facilitate the assembly process. In the present work, we performed a co-immunoprecipitation experiment to demonstrate that ANTXR1 is oligomeric in mammalian cells. Computer modeling predicted the self-association of the ANTXR1 transmembrane domain and we detected oligomerization of ANTXR1 transmembrane domain peptides in the membrane-mimetic environment of SDS micelles using fluorescence resonance energy transfer. Furthermore, the ANTXR1 transmembrane domain mediated oligomerization of a reporter protein construct in a bacterial membrane. In both assays, mutations that disrupted the interaction were consistent with the interaction being mediated through an asymmetric binding interface. Mutations that impaired self-association of the transmembrane domain reduced the rate of PA63 heptamer formation on the mammalian cell surface. Our findings indicate that ANTXR1 transmembrane domains self-associate and that these interactions may stabilize intermediate oligomerization states of ANTXR1-PA63 complexes.

Amino Acid Sequence↗
PubMed2006

Statistical pattern matching facilitates the design of polyvalent inhibitors of anthrax and cholera toxins.

Numerous biological processes involve the recognition of a specific pattern of binding sites on a target protein or surface. Although ligands displayed by disordered scaffolds form stochastic rather than specific patterns, theoretical models predict that recognition will occur between patterns that are characterized by similar or "matched" statistics. Endowing synthetic biomimetic structures with statistical pattern matching capabilities may improve the specificity of sensors and resolution of separation processes. We demonstrate that statistical pattern matching enhances the potency of polyvalent therapeutics. We functionalized liposomes with an inhibitory peptide at different densities and observed a transition in potency at an interpeptide separation that matches the distance between ligand-binding sites on the heptameric component of anthrax toxin. Pattern-matched polyvalent liposomes inhibited anthrax toxin in vitro at concentrations four orders of magnitude lower than the corresponding monovalent peptide, and neutralized this toxin in vivo. Statistical pattern matching also enhanced the potency of polyvalent inhibitors of cholera toxin. This facile strategy should be broadly applicable to the detection and neutralization of toxins and pathogens.

Animals↗
PubMed2006

Synthesis of polyvalent inhibitors of controlled molecular weight: structure-activity relationship for inhibitors of anthrax toxin.

We describe a novel method to synthesize activated polymers of controlled molecular weight and apply this method to investigate the relationship between the structure and activity of polyvalent inhibitors of anthrax toxin. In particular, we observe an initial sharp increase in potency with increasing ligand density, followed by a plateau where potency is independent of ligand density. Our simple strategy for designing polyvalent inhibitors of controlled molecular weight and ligand density will be broadly applicable for designing inhibitors for a variety of pathogens and toxins, and for elucidating structure-activity relationships in these systems. Our results also demonstrate a role for kinetics in influencing inhibitory potency in polyvalent systems. Finally, our work presents a synthetic route to polyvalent inhibitors that are more structurally defined and effective in vivo. This control over inhibitor composition will be generally useful for the optimization of inhibitor potency and pharmacokinetics, and for the eventual application of these molecules in vivo.

Animals↗
PubMed2006

Inhibition of mitogen-activated protein kinase signalling by Bacillus anthracis lethal toxin causes destabilization of interleukin-8 mRNA.

Bacillus anthracis must overcome host innate immune defences to establish a systemic anthrax infection. This is facilitated in part by lethal toxin (LT), a secreted virulence factor that consists of a cell-binding moiety, protective antigen (PA), and an enzymatic moiety, lethal factor (LF). PA binds cells through protein receptors and mediates the delivery of LF to the cytosol. LF is a protease that cleaves amino-terminal fragments from mitogen-activated protein kinase kinases (MAPKKs), preventing phosphorylation of their downstream targets. Here we report that LT reduces the amount of interleukin (IL)-8 produced and secreted by human endothelial cells. The reduction of IL-8 levels by LT was not attributable to reduced expression from the IL-8 promoter, but resulted from destabilization of IL-8 mRNA. Destabilization by LT was mediated through the 3' untranslated region of the IL-8 transcript and could be mimicked by pharmacological inhibitors of MAPK pathways. LT diminished the induction of IL-8 mRNA and protein by lipopolysaccharide, indicating that the toxin can impair the ability of these cells to initiate an immune response. Destabilization of a cytokine transcript represents a new interference strategy used by either a bacterial or viral pathogen to reduce cytokine expression and may help B. anthracis to evade host immune defences.

3' Untranslated Regions↗
PubMed2006

Production and characterization of neutralizing monoclonal antibodies that recognize an epitope in domain 2 of Bacillus anthracis protective antigen.

Antibodies against the protective antigen (PA) of Bacillus anthracis play a key role in response to infection by this important pathogen. The aim of this study was to produce and characterize monoclonal antibodies (mAbs) specific for PA and to identify novel neutralizing epitopes. Three murine mAbs with high specificity and nanomolar affinity for B. anthracis recombinant protective antigen (rPA) were produced and characterized. Western immunoblot analysis, coupled with epitope mapping using overlapping synthetic peptides, revealed that these mAbs recognize a linear epitope within domain 2 of rPA. Neutralization assays demonstrate that these mAbs effectively neutralize lethal toxin in vitro.

Animals↗
PubMed2005

Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin.

Anthrax lethal toxin (LT) is comprised of protective antigen and lethal factor. Lethal factor enters mammalian cells in a protective antigen-dependent process and cleaves mitogen-activated protein kinase kinases. Although LT has no observable effect on many cell types, it causes necrosis in macrophages derived from certain mouse strains and apoptosis in activated mouse macrophages. In this study, we observed that LT treatment of three different human monocytic cell lines U-937, HL-60 and THP-1 did not induce cell death. Cells did become susceptible to the toxin, however, after differentiation into a macrophage-like state. Treatment with LT resulted in decreased phosphorylation of p38, ERK1/2 and JNK in both undifferentiated and differentiated HL-60 cells, suggesting that the change in susceptibility does not result from differences in toxin delivery or substrate cleavage. Death of differentiated HL-60 cells was accompanied by chromosome condensation and DNA fragmentation, but was not inhibited by the pan-caspase inhibitor Z-VAD-FMK. In addition, we observed that the macrophage differentiation process could be inhibited by LT. Our results indicate that LT-mediated death of mouse and human macrophages may occur through distinct processes and that the differentiation state of human cells can determine susceptibility or resistance to LT.

Amino Acid Chloromethyl Ketones↗
PubMed2004

Using light scattering to determine the stoichiometry of protein complexes.

The stoichiometry of a protein complex can be calculated from an accurate measurement of the complex's molecular weight. Multiangle laser light scattering in combination with size-exclusion chromatography and interferometric refractometry provides a powerful means for determining the molecular weights of proteins and protein complexes. In contrast to conventional size-exclusion chromatography and analytical centrifugation, measurements do not rely on the use of molecular weight standards and are not affected by the shape of the proteins. The technique is based on the direct relationship between the amount of light scattered by a protein in solution and the product of its concentration and molecular weight. A typical experimental configuration includes a size-exclusion column to fractionate the sample, a light-scattering detector to measure scattered light, and an interferometric refractometer to measure protein concentration. The determination of the molecular weight of an anthrax toxin complex will be examined to illustrate how multiangle laser light scattering can be used to determine the stoichiometry of protein complexes.

Bacillus anthracis↗
PubMed2003

Binding of anthrax toxin to its receptor is similar to alpha integrin-ligand interactions.

The secreted protein toxin produced by Bacillus anthracis contributes to virulence of this pathogen and can cause many of the symptoms seen during an anthrax infection, including shock and sudden death. The cell-binding component of anthrax toxin, protective antigen, mediates entry of the toxin into cells by first binding directly to the extracellular integrin-like inserted (I) domain of the cellular anthrax toxin receptor, ATR. Here we report that this interaction requires an intact metal ion-dependent adhesion site (MIDAS) in the receptor as well as the presence of specific divalent cations. Also, we demonstrate that the toxin-receptor interaction is critically dependent on the Asp-683 carboxylate group of protective antigen, which projects from the receptor binding surface. We propose that this carboxylate group completes the coordination of the MIDAS metal of ATR, mimicking integrin-ligand interactions.

Animals↗
PubMed2002

The lethal and edema factors of anthrax toxin bind only to oligomeric forms of the protective antigen.

The three proteins that comprise anthrax toxin, edema factor (EF), lethal factor (LF), and protective antigen (PA), assemble at the mammalian cell surface into toxic complexes. After binding to its receptor, PA is proteolytically activated, yielding a carboxyl-terminal 63-kDa fragment (PA(63)) that coordinates assembly of the complexes, promotes their endocytosis, and translocates EF and LF to the cytosol. PA(63) spontaneously oligomerizes to form symmetric ring-shaped heptamers that are capable of binding three molecules of EF and/or LF as competing ligands. To determine whether binding of these ligands depends on oligomerization of PA(63), we prepared two oligomerization-deficient forms of this protein, each mutated on a different PA(63)-PA(63) contact face. In solution or when bound to receptors on Chinese hamster ovary K1 cells, neither mutant alone bound ligand, but a mixture of them did. After the two mutants were proteolytically activated and mixed with ligand in solution, a ternary complex was isolated containing one molecule of each protein. Thus EF and LF bind stably only to PA(63) dimers or higher order oligomers. These findings are relevant to the kinetics and pathways of assembly of anthrax toxin complexes.

Adenylyl Cyclases↗
PubMed2002

Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen.

Assembly of anthrax toxin complexes at the mammalian cell surface involves competitive binding of the edema factor (EF) and lethal factor (LF) to heptameric oligomers and lower order intermediates of PA(63), the activated carboxyl-terminal 63-kDa fragment of protective antigen (PA). We used sequence differences between PA(63) and homologous PA-like proteins to delineate a region within domain 1' of PA that may represent the binding site for these ligands. Substitution of alanine for any of seven residues in or near this region (R178, K197, R200, P205, I207, I210, and K214) strongly inhibited ligand binding. Selected mutations from this set were introduced into two oligomerization-deficient PA mutants, and the mutants were used in various combinations to map the single ligand site within dimeric PA(63). The site was found to span the interface between two adjacent subunits, explaining the dependence of ligand binding on PA oligomerization. The locations of residues comprising the site suggest that a single ligand molecule sterically occludes two adjacent sites, consistent with the finding that the PA(63) heptamer binds a maximum of three ligand molecules. These results elucidate the process by which the components of anthrax toxin, and perhaps other binary bacterial toxins, assemble into toxic complexes.

Adenylyl Cyclases↗
PubMed2002

Stoichiometry of anthrax toxin complexes.

After being proteolytically activated, the protective antigen (PA) moiety of anthrax toxin self-associates to form symmetric, ring-shaped heptamers. Heptameric PA competitively binds the enzymatic moieties of the toxin, edema factor and lethal factor, and translocates them across the endosomal membrane by a pH-dependent process. We used two independent approaches to determine how many of the seven identical EF/LF binding sites of the PA heptamer can be occupied simultaneously. We measured isotope ratios in complexes assembled from differentially radiolabeled toxin subunits, and we determined the molecular masses of unlabeled complexes by multiangle laser light scattering. Both approaches yielded the same value: the PA heptamer in solution binds three molecules of protein ligand under saturating conditions. This suggests that each bound ligand sterically occludes the binding sites of two PA subunits. According to this model, a ligand-saturated heptamer is asymmetric, with the sites of six of the seven subunits occluded. These results contribute to the conceptual framework for understanding the mechanism of membrane translocation by anthrax toxin.

Antigens, Bacterial↗
PubMed2002

2001: a year of major advances in anthrax toxin research.

Anthrax is caused when spores of Bacillus anthracis enter a host and germinate. The bacteria multiply and secrete a tripartite toxin causing local edema and, in systemic infection, death. In nature, anthrax is primarily observed in cattle and other herbivores; humans are susceptible but rarely affected. In 2001, anthrax spores were used effectively for the first time in bioterrorist attacks, resulting in 11 confirmed cases of human disease and five deaths. These events have underscored the need for improved prophylaxis, therapeutics and a molecular understanding of the toxin. The good news about anthrax is that several decisive discoveries regarding the toxin have been reported recently. Most notably, the toxin receptor was identified, the 3-D structures of two of the toxin subunits were solved and potent in vivo inhibitors were designed. These findings have improved our understanding of the intoxication mechanism and are stimulating the design of strategies to fight disease in the future.

Animals↗
PubMedDate not supplied

Synthesis of potent inhibitors of anthrax toxin based on poly-L-glutamic acid.

We report the synthesis of biodegradable polyvalent inhibitors of anthrax toxin based on poly-L-glutamic acid (PLGA). These biocompatible polyvalent inhibitors are at least 4 orders of magnitude more potent than the corresponding monovalent peptides in vitro and are comparable in potency to polyacrylamide-based inhibitors of anthrax toxin assembly. We have elucidated the influence of peptide density on inhibitory potency and demonstrated that these inhibitory potencies are limited by kinetics, with even higher activities seen when the inhibitors are preincubated with the heptameric receptor-binding subunit of anthrax toxin prior to exposure to cells. These polyvalent inhibitors are also effective at neutralizing anthrax toxin in vivo and represent attractive leads for designing biocompatible anthrax therapeutics.

Absorbable Implants↗
PubMedDate not supplied

Functional characterization of peptide-based anthrax toxin inhibitors.

We have identified an optimized peptide inhibitor that can be used to develop potent anthrax toxin therapeutics. Anthrax toxin, an essential virulence factor of Bacillus anthracis, elicits many of the symptoms associated with the disease, and is responsible for death. The toxin is composed of a cell-binding component, protective antigen, and two enzymatic components, edema factor and lethal factor. The three proteins are secreted individually by the bacterium and then assemble into functional complexes on the surface of mammalian cells. These complexes are endocytosed, and the enzymatic components are translocated into the cytosol, where they exert their activities. We screened a phage display library for peptides that can bind the heptameric cell-binding subunit of anthrax toxin, and identified a novel peptide that can block toxin assembly. We made a series of mutant peptides and attached these peptides to polymer backbones to assess their inhibitory activities in vitro. This series of truncated peptide mutants was used to identify a minimal peptide sequence, TYWWLD, that can be used to develop potent polyvalent inhibitors of anthrax toxin.

Amino Acid Sequence↗