Porphyromonas gingivalis : keeping the pathos out of the biont

Bacteria alter the biophysical properties of their membrane lipids in response to environmental cues, such as shifts in pH or temperature. In essence, lipid composition determines membrane structure, which in turn influences many basic functions, such as transport, secretion, and signaling. Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis possesses the ability to synthesize a variety of novel membrane lipids, including species of dihydroceramides that are distinct, yet similar in structure to sphingolipids produced by the human host. The role of dihydroceramides in the physiology and pathogenic potential of the human microbiota is only beginning to be explored; yet there is increasing data indicating that these lipids play a role in human diseases, such as periodontitis and multiple sclerosis. Here, we report on the identification of a gene (PG1780) in the chromosome of P. gingivalis strain W83 encoding a putative serine palmitoyltransferse, the enzyme that catalyzes the first step in sphingolipid biosynthesis. While we were able to detect dihydroceramides in whole lipid extracts of P. gingivalis cells as well as crude preparations of outer membrane vesicles, sphingolipids were absent in the PG1780 mutant strain. Moreover, we show that the synthesis of sphingolipids plays an essential role in the long-term survival of the organism as well as its resistance to oxidative stress. Further, a PG1780 mutant displayed much lower activity of cell-associated arginine and lysine gingipains, yet slightly higher activity in the corresponding culture supernates, which we hypothesize is due to altered membrane properties and anchoring of these proteases to the cell surface. In addition, we determined that sphingolipid production is critical to the presentation of surface polysaccharides, with the mutant strain displaying less K-antigen capsule and more anionic polysaccharide (APS). Overall, we have discovered that, in addition to their role in pathogenicity, the synthesis of sphingolipids is critical to the cellular homeostasis and persistence of this important dental pathogen.

Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection.

Gram-negative bacteria produce nanosized OMVs that are actively released into their surroundings. The oral anaerobe P. gingivalis is prolific in OMV production, and many of the proteins packaged in these vesicles are proteolytic or protein-modifying enzymes.

In order to persist, bacteria need to adjust their physiological state in response to external and internal cues. External stimuli are often referred to as stressors. The stringent response, mediated by the alarmone (p)ppGpp, is central to the stress response in many bacteria; yet, there is limited knowledge regarding the role of (p)ppGpp signaling in bacteria belonging to the phylum Bacteroidetes. Like its counterparts in the gut (e.g., Bacteroides thetaiotaomicron and Bacteroides fragilis), Porphyromonas gingivalis persists in close association with its human host. Given the potential for numerous perturbations in the oral cavity, and the fact that P. gingivalis can enter and replicate within host cells, we hypothesized that (p)ppGpp is a key signaling molecule for stress adaptation and persistence. Here, we show that accumulation of ppGpp in P. gingivalis is governed by two homologous enzymes, designated Rel, and RshB, and that ppGpp signaling affects growth rate, survival, biofilm formation, production of outer membrane vesicles, and expression of genes encoding type IX secretion structural and cargo proteins. Overall, our findings provide a potential mechanism by which biofilm formation and virulence of P. gingivalis are integrated via ppGpp signaling, a regulatory mechanism central to bacterial survival in dynamic environments.

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.