ACETYLATION AS A REGULATORY MECHANISM OF CHITINASE ACTIVITY IN FRANCISELLA TULARENSIS SUBSP. NOVICIDA

Francisella tularensis is a serious bacterial pathogen known to form biofilms, which are structures that often contribute to environmental persistence and resistance. Previous work by the van Hoek lab established that chitinase enzymes are negative regulators of this biofilm—essentially, they help the bacteria disperse the biofilm. This study investigated whether post-translational N-epsilon-lysine acetylation, a common modification in bacteria, acts as a mechanism to regulate chitinase activity in F. tularensis subsp. novicida.

The researcher identified multiple lysine acetylation sites on chitinases (ChiA and ChiB) and a chitin-binding protein (CbpA) using mass spectrometry data. To test the hypothesis that acetylation inhibits these enzymes, the team expressed and purified F. novicida chitinases and treated concentrated secretomes with lithium potassium acetyl phosphate to induce chemical acetylation. They then performed chitinase activity assays and biofilm dispersal assays to compare the performance of acetylated versus non-acetylated proteins.

Chemical acetylation led to a statistically significant decrease in endochitinase activity within the concentrated protein secretome compared to untreated controls. Furthermore, when pre-formed bacterial biofilms were treated with these acetylated secreted proteins, the ability of the proteins to disperse the biofilm was significantly hindered. These results suggest that acetylation serves as an inhibitory "off-switch" for chitinase activity, which in turn modulates the stability of F. tularensis biofilms.

Understanding how F. tularensis regulates its biofilm dispersal is critical for addressing its environmental persistence and potential as a biological threat agent. Identifying acetylation as a regulatory mechanism provides a new target for understanding how the pathogen modulates its lifestyle in response to environmental cues. Future studies using site-directed mutagenesis of specific lysine residues will be necessary to confirm the biological relevance of these findings in vivo.