Analysis of Group B Streptococcal Fibrinogen Cleavage

Louisiana State Univ Hsc Shreveport, Shreveport LA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): Group B steptococcus is an important human pathogen, and is the leading cause of invasive neonatal disease including pneumonia, sepsis, and meningitis. GBS has also been recently recognized as a cause of invasive disease in the immunocompromised and eldedy. While some key virulence factors of GBS have been characterized, such as the polysaccharide capsule and the hemolysin, relatively few other virulence factors have been thoroughly characterized. In this proposal, we characterize a novel streptococcal virulence factor. We have discovered a streptococcal protease (CspA) that is necessary for full virulence in a neonatal rat sepsis model, is necessary for full resistance to opsonophagocytosis by human neutrophils, and mediates cleavage of human fibrinogen. In this proposal, we seek to prove that CspA is the protease responsible for the cleavage of human fibrinogen by purification of the CspA protease and characterization of how this important virulence factor cleaves fibrinogen. The first aim seeks to define the biochemical reaction mediated by CspA by determining the site of CspA-mediated cleavage of fibrinogen. It is our hypothesis that CspA cleaves fibrinogen in a manner similar to thrombin to generate a substance that coats the bacterium and protects it from opsonophagocytosis. We will investigate the characteristics of the fibrinogen cleavage reaction by two-dimensional electrophoresis, N-terminal sequencing of the fibrinogen reactants and products, and characterization by mass spectrometry of the fibrinogen peptide. The second aim seeks to prove that CspA is the protease responsible for the cleavage of fibrinogen by over expressing the protein in Bacillus subtilis, purifying the protease to homogeneity, and demonstrating that purified CspA can promote the fibrinogen cleavage reaction characterized in the first aim. The availability of the purified protein will facilitate future biochemical structure-function studies on this important virulence factor of GBS. [unreadable] [unreadable]