863. A Serum Carboxypeptidase Contributes to the Processing of the Bacillus anthracis Protective Antigen
Session: Poster Abstract Session: Bacterial Infections: Pathogenesis and Immunity
Friday, October 9, 2015
Room: Poster Hall
Posters
  • poster-template-48x36-horizontal.pdf (1.0 MB)
  • Background:

    Bacillus anthracis secretes 2 bipartite toxins that require a protective antigen (PA) component to mediate cell entry.  The current paradigm holds that B. anthracis secretes PA83, which binds cell surface receptors and undergoes cleavage by a cell-associated furin into 2 fragments, PA20 and PA63. In contrast to this notion, we report that serum effectively cleaves PA83 via 2-step process that involves a carboxypeptidase. 

    Methods:

    Recombinant PA was obtained from Wadsworth laboratories.  Serum was obtained and stored at −80°C with approval from the Committee of Clinical Investigations at Albert Einstein College of Medicine.  Mass spectrometric (MS) measurements and liquid chromatographic separations were obtained on the LTQ linear ion trap mass spectrometer and the Rapid Separation LC 3000.   Inhibition of serum-mediated processing of PA was studied using a variety of reagents, including: competitive inhibitors of furin, a peptide library, PA-specific mAbs and the carboxypeptidase inhibitors, 2-guanidinoethylmercaptosuccinic acid and potato tuber carboxypeptidase inhibitor.  Digestion of recombinant PA using commercially obtained carboxpeptidase B was also studied.  

    Results:

    Serum digestion of PA produced 2 fragments similar in size to those seen following furin digestion.  Serum-treatment, however  consistently produced a smaller PA20-like fragment when compared with furin digestion.  MS analysis of this PA20-like fragment revealed a mass of 23,603 Da, which is within 0.06% of molecular weight of PA20, assuming all basic amino acids are removed from the carboxy-terminal.   Serum-mediated digestion of PA83 was prevented by furin-inhibitors and antibodies directed near the furin site.  Likewise, both carboxypeptidase inhibitors inhibited serum processing of PA20.   Finally, treatment of PA83with furin and carboxypeptidase B produced a digestion pattern similar to that seen with serum.  

    Conclusion:

    Our studies indicate that serum contains furin and carboxypeptidase-like activities that actively digest PA83.  Serum is known to contain 2 carboxypeptidases (N and B2) that have redundant activities.  The contribution of serum processing of PA to the pathogenesis of anthrax remains to be determined.  Therapeutic strategies targeting PA processing need to take into account serum proteolytic activity.

    Ahmed Elshazly, MBBCh1, Antonio Nakouzi, BA2, Johanna Rivera, PhD2, Arturo Casadevall, MD, PhD3 and David Goldman, MD4, (1)Pediatrics Infectious Diseases, Albert Einstein College of medicine, Bronx, NY, (2)Albert Einstein College of Medicine, Bronx, NY, (3)Chair, IDSA Annual Meeting Program Committee, Albert Einstein College of Medicine, New York, NY, (4)Children's Hospital at Montefiore, Bronx, NY

    Disclosures:

    A. Elshazly, None

    A. Nakouzi, None

    J. Rivera, None

    A. Casadevall, None

    D. Goldman, None

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