Objectives: Porphyromonas gingivalis (Pg) is a periodontal pathogen that has been shown to invade and persist within HCAEC in vitro. Tetrapyrroles such as cobalamin are critical for survival and function as signaling and regulatory molecules. PG0242 is a putative tetrapyrrole methyltransferase that is up-regulated during invasion. Bioinformatic projections indicate that PG0242 is part of the cobalamin biosynthesis pathway; however this pathway is thought to be non-functional in Pg W83.
Methods: Microarray analysis followed by RT-PCR revealed that gene PG0242 was up-regulated during invasion of HCAEC. A mutant was constructed and assessed for growth characteristics, invasiveness, and trafficking. The PG0242 mutant was assessed for growth by measuring OD550 over a 36-hour time course. The invasion of W83 and the PG0242 mutant in HCAEC was evaluated using conventional antibiotic protection assays. Activation of autophagy and trafficking to the autophagic pathway in HCAEC was examined by electron microscopy and quantitative analysis. The presence of PG0242 and known components of the cobalamin biosynthesis pathway in four strains of Pg was determined by Megablast, BlastN, PsiBlast and BlastP sequence analysis.
Results: Growth was found to be inhibited. After 2.5 hours of invasion by the PG0242 mutant, there was a 4-fold reduction in recoverable CFU. Trafficking to the autophagic pathway was also impaired, with fewer Pg observed in autophagic vacuoles. PG0242 was detected by sequence homology in all four strains, however few traces of the known cobalamin pathway were detectable.
Conclusion: Expression of PG0242 is regulated during invasion, and the mutant has a distinct phenotype related to invasion and growth. However, the pathway PG0242 is strongly associated with is thought to be defunct in Pg, with few components detectable in the sequence data. This suggests that what remains of the pathway is still affecting Pg virulence, and may have been re-purposed to fill a different role.
Keywords: Bacterial, Cardiovascular disease, Cobalamin Biosynthesis, Enzymes and Microbiology