Investigating the Function of Fimbriae-forming Lipoprotein in Porphyromonas gingivalis - Porphyromonas gingivalis (Pg) is a highly proteolytic Gram-negative anaerobe implicated in periodontal disease, which is one of the most common chronic biofilm-based infections that result in destruction of hard and soft tissues of the periodontium and, ultimately, in loss of teeth. Although Pg has been historically classified as nonmotile, it was recently shown that fimbriated strains are capable of surface translocation when sandwiched between soft agar and a glass or plastic surface. Through genetic studies, it was determined that the type IX secretion system (T9SS) and T9SS cargo are integral to Pg’s migration behavior. Our working model is that during the early stages of surface translocation, T9SS cargo proteins which are known to be transported to the cell surface and released into the environment on outer membrane vesicles (OMVs) modify the surroundings and this promotes migration. However, the cooperation and function of the various OMV cargo proteins during the transition to surface translocation is still unknown. In a transcriptome analysis, PG1881, a predicted fimbriae- forming lipoprotein, was one of the most highly upregulated genes during the initial stages of surface translocation, yet its function is not clear. Importantly, PG1881 has been shown to be enriched on sphingolipid (SL) containing OMVs. Predicted post-translational modifications of PG1881 include palmitoylation, providing a link to SLs, as well as citrullination by PPAD (Porphyromonas peptidylarginine deiminase) which converts L- arginine residues to L-citrulline within peptides. It was previously shown that citrullination by PPAD promotes OMV biogenesis as well as surface translocation. Therefore, this study aims to investigate the function of PG1881 during the early stages of surface translocation. My preliminary data examining OMVs from surface translocating cells lacking PG1881 revealed distinct properties via fluorescence and transmission electron microscopy. Therefore, the central hypothesis is that PG1881 influences the biogenesis and properties of OMVs, in particular the protein cargo carried on OMVs, which impacts the initial stages of surface translocation. This hypothesis will be tested through two specific aims: 1) Characterize PG1881 and 2) Investigate the function of PG1881 in the context of surface translocation. In Aim 1, palmitoylation of PG1881 will be confirmed by using my PG1881 polyclonal antibody to perform immunoprecipitation followed by mass spectrometry. Citrullination of PG1881 will be confirmed using recombinant PG1881 as a substrate for a colorimetric assay that measures citrullination. In Aim 2, the spatial localization of PG1881 on surface translocating cells will be determined using my polyclonal PG1881 antibody. I will compare gingipains protein levels and enzymatic activity from surface translocating cells in the parent strain and PG1881 deletion mutant. The experiments outlined above will give insight on a new aspect of Pg physiology and characterize a predicted fimbriae-forming lipoprotein associated with known virulence determinants including OMVs and T9SS. Ultimately, this research proposal is the basis of a doctoral dissertation and will enhance the training of a student with an interest in oral health research.
