Citrullination mediated by PPAD constrains biofilm formation in P. gingivalis strain 381

Porphyromonas gingivalis is the only known human-associated prokaryote that produces a peptidylarginine deiminase (PPAD), a protein-modifying enzyme that is secreted along with a number of virulence factors via a type IX secretion system (T9SS). While the function of PPAD in P. gingivalis physiology is not clear, human peptidylarginine deiminases are known to convert positively charged arginine residues within proteins to neutral citrulline and, thereby, impact protein conformation and function. Here, we report that the lack of citrullination in a PPAD deletion mutant (Δ8820) enhances biofilm formation. More Δ8820 cells attached to the surface than the parent strain during the early stages of biofilm development and, ultimately, mature Δ8820 biofilms were comprised of significantly more cell–cell aggregates and extracellular matrix. Imaging by electron microscopy discovered that Δ8820 biofilm cells secrete copious amounts of protein aggregates. Furthermore, gingipain-derived adhesin proteins, which are also secreted by the T9SS were predicted by mass spectrometry to be citrullinated and citrullination of these targets by wild-type strain 381 in vitro was confirmed. Lastly, Δ8820 biofilms contained more gingipain-derived adhesin proteins and more gingipain activity than 381 biofilms. Overall, our findings support the model that citrullination of T9SS cargo proteins known to play a key role in colonization, such as gingipain-derived adhesin proteins, is an underlying mechanism that modulates P. gingivalis biofilm development. Biofilms: Porphyromonas gingivalis biofilms limited by citrullination The oral bacteria Porphyromonas gingivalis , implicated in periodontal disease, secretes the citrullination enzyme peptidylarginine deiminase (PPAD), whose surprising role in modulating biofilm formation has been uncovered. Mary Davey and colleagues at the University of Florida show that a strain of P. gingivalis with mutated PPAD (which is unable to promote citrullination, or the conversion of arginine residues to citrulline in target proteins) shows enhanced biofilm formation, with mature biofilms from the mutated strain secreting large amounts of proteins and having more cells and deposited extracellular matrix than biofilms from the wild-type strain. Importantly, secreted adhesion proteins known to be involved in P. gingivalis colonization were found to be citrullination targets and to be secreted in larger amounts by the PPAD mutant strain. These findings uncover