Biochemical Assays Of The Neisseria Meningitidis Serogroup W Capsule Polymerase
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The leading cause of bacterial meningitis is one of three bacteria: Streptococcus pneumonia, Haemophilus influenza or Neisseria meningitidis. Our goal in this work is to gain insight into the molecular mechanism of Neisseria meningitidis serogroup W capsule polymerase enzyme. This enzyme is crucial for building the carbohydrate-rich capsule that surrounds the outer surface of the bacteria. Capsular polysaccharides are an important structure that help the bacteria protect itself against the human immune system. The serogroup W capsule polymerase creates sialic acid galactose heteropolymers and uses CMP-sialic acid and UDP-galactose as nucleotide sugar donors. Better understanding of how this enzyme works can pave the way for drug and vaccine development against N. meningitidis. One goal of this research was to determine suitable fluorescent acceptors of this enzyme to follow product formation using HPLC with fluorescence detection. Many acceptors were investigated but the best acceptor was found to be fluorescently labelled serogroup W polysaccharide fragments. An-other goal of this research was to determine the kinetic parameters (Km and Vmax) for serogroup W capsule polymerase enzyme with its donor sugars. These kinetic parameters were determined for UDP-Galactose however further work is necessary for determining those for CMP-Sialic Acid. Finally, initial studies were performed to use molecular biology techniques to introduce mutations into the serogroup W capsule polymerase enzyme. Future work will build upon these studies to use more sensitive assays to determine kinetic parameters and create site-directed mutants of the enzyme.