Acetylation as a Regulatory Mechanism of Chitinase Activity in Francisella tularensis subsp. novicida
Abstract
Francisella tularensis is the causative agent of tularemia and is considered a Category A, Tier 1 biological threat agent. We and others have shown that Francisella tularensis forms biofilms in vitro , and we have previously demonstrated that biofilm formation is negatively regulated by chitinase expression. Given Francisella ’s classification as a Category A bioterrorism threat and the lack of understanding surrounding environmental persistence, a better understanding of biofilm formation and dispersal is crucial. Our hypothesis is that the activity of chitinase may be regulated by the post-translational modification acetylation, which has been shown to regulate other enzymes and occur in bacteria. A full analysis of the acetylation of the Francisella tularensis subsp. novicida proteome has been conducted by the van Hoek lab, resulting in the identification numerous lysine acetylation sites on chitin-binding protein A, Chitinase A, and Chitinase B. Given acetylation’s role on enzymatic regulation, we attempted to clone Francisella tularensis subsp. novicida U112 chitin-binding protein A and chitinases A, B, and D for enzymatic analysis upon chemical acetylation. Chitinase B and chitin-binding protein were successfully expressed for further experimentation. Concentrated Francisella secreted proteins containing chitinases A and B were chemically acetylated using lithium potassium acetyl phosphate, which resulted in a significant decrease in chitinase activity compared to unacetylated protein concentrate and Trichoderma viride chitinase (P=0.0067, P=0.000014). Additionally, by treating preformed biofilm with acetylated chitinase limited biofilm dispersal was visualized relative to chitinase treatment (P=0.000017). In conclusion, this study suggests that the inhibition of chitinase activity through acetylation may be one mechanism of regulation of chitinase activity, and thus biofilm formation levels in Francisella .
Description
This thesis has been embargoed for 5 years and will not be available until April 2024 at the earliest.
Keywords
Chitinase, Francisella tularensis, Biofilm, Acetylation, Post-translational modification, Bacterial cloning