Effect of O2-stress on phosphoproteome and LPS biosynthesis in P.gingivalis

Objectives: The Gram-negative anaerobic bacterium Porphyromonas gingivalis is able to survive within periodontal pockets, where it is exposed to oxidative conditions. Previously, we described proteomics of the effect of O2 stress on protein expression in P. gingivalis W83 using two dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). However, the effect of O2 stress on virulence of P. gingivalis is still unclear. In the present study, we analyzed O2 stress-induced alterations of P. gingivalis phosphoproteome using 2-DE and MS. In addition, to clarify the effect of O2 stress on the virulence of P. gingivalis, we also examined LPS production by P. gingivalis subjected O2 stress. Methods: P. gingivalis W83 was grown anaerobically and incubated under aerobic conditions. Total protein of P. gingivalis was extracted with TCA/acetone method and subjected to 2-DE. Following 2-DE, the gels were stained using Pro-Q® Diamond and CBB. Protein spots showing different expression profiles with or without aeration were identified by using MALDI-TOF MS and Mascot. The roles of identified proteins were examined by KEGG pathway database. Moreover, aeration-induced changes in extracted LPS was analyzed using SDS-PAGE. Results: Using 2-DE and MALDI-TOF MS, we identified two enzymes, phosphomannomutase (PG2010) and fructose bisphosphate aldolase class I (PG1755), which contribute to the biosynthesis of LPS. Moreover, phosphorylation of these proteins was significantly enhanced under aerobic conditions. This increase in protein phosphorylation of PG2010 and PG1755 was followed by an increase in the LPS production of P. gingivalis. Conclusion: Subjecting P. gingivalis to O2 stress led to increased phosphorylation of PG2010 and PG1755 that preceded an increase in LPS biosynthesis. This suggests that O2 stress-induced protein phosphorylation is key determinant of the virulence in P. gingivalis.