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      High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures

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          Abstract

          Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3–9.9 % coefficient of variation), and accurate (0.1–12.7 %error; n ≥ 25). Diagnostic sensitivity was 100 % ( n = 27 animal/clinical cases). Diagnostic specificity was 100 % ( n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics.

          Graphical Abstract

          Method schematic for analysis of anthrax lethal toxin activity by ID-MALDI-TOF MS

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          Most cited references33

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          Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.

          Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.
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            Anthrax toxin: receptor binding, internalization, pore formation, and translocation.

            Anthrax toxin consists of three nontoxic proteins that self-assemble at the surface of receptor-bearing mammalian cells or in solution, yielding a series of toxic complexes. Two of the proteins, called Lethal Factor (LF) and Edema Factor (EF), are enzymes that act on cytosolic substrates. The third, termed Protective Antigen (PA), is a multifunctional protein that binds to receptors, orchestrates the assembly and internalization of the complexes, and delivers them to the endosome. There, the PA moiety forms a pore in the endosomal membrane and promotes translocation of LF and EF to the cytosol. Recent advances in understanding the entry process include insights into how PA recognizes its two known receptors and its ligands, LF and EF; how the PA:receptor interaction influences the pH-dependence of pore formation; and how the pore functions in promoting translocation of LF and EF across the endosomal membrane.
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              Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.

              S H Leppla (1982)
              Anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins individually cause no known physiological effects in animals but in pairs produce two toxic actions. Injection of PA with LF causes death of rats in 60 min, whereas PA with EF causes edema in the skin of rabbits and guinea pigs. The mechanisms of action of these proteins have not been determined. It is shown here that EF is an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] produced by Bacillus anthracis in an inactive form. Activation occurs upon contact with a heat-stable eukaryotic cell material. The specific activity of the resulting adenylate cyclase nearly equals that of the most active known cyclase. In Chinese hamster ovary cells exposed to PA and EF, cAMP concentrations increase without a lag to values about 200-fold above normal, remain high in the continued presence of toxin, and decrease rapidly after its removal. The increase in cAMP is completely blocked by excess LF. It is suggested that PA interacts with cells to form a receptor system by which EF and perhaps LF gain access to the cytoplasm.
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                Author and article information

                Contributors
                jbarr@cdc.gov
                Journal
                Anal Bioanal Chem
                Anal Bioanal Chem
                Analytical and Bioanalytical Chemistry
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                1618-2642
                1618-2650
                12 February 2015
                12 February 2015
                2015
                : 407
                : 10
                : 2847-2858
                Affiliations
                [ ]Centers for Disease Control and Prevention, Atlanta, GA 30341 USA
                [ ]Battelle-Technical On-Site Professional Services, Atlanta, GA 30329 USA
                Article
                8509
                10.1007/s00216-015-8509-5
                4369318
                25673244
                36a093e0-5f9d-4bff-86ec-12836fb6c1ec
                © The Author(s) 2015

                Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

                Categories
                Research Paper
                Custom metadata
                © Springer-Verlag Berlin Heidelberg 2015

                Analytical chemistry
                anthrax,maldi-tof ms,quantification,lethal toxin,lethal factor,protective antigen,diagnostic,bacillus anthracis

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