Crp/Fnr-type regulator PG0396 aggravates periapical bone destruction via suppression of antioxidant defenses and gingipain production in Porphyromonas gingivalis

Porphyromonas gingivalis(P. gingivalis) is a key oral pathogen. While Crp/Fnr family transcription factors are widespread in bacteria and P. gingivalis encodes three members (PG1053, PG1573, and PG0396)—with PG1053 involved in nitrosative stress and PG1573 in iron acquisition and biofilm regulation—PG0396 harbors a helix-turn-helix domain distinct from the other two, suggesting non-redundant regulatory functions. However, whether PG0396 acts as a transcriptional regulator and how its biological pathways compare to known members remain unclear. This study aimed to characterize the role of PG0396 in virulence regulation and pathogenesis to elucidate the functional specialization within this family.

A PG0396 deletion mutant (ΔPG0396:: ermF) was constructed in P. gingivalis W83 via homologous recombination, using a PG2125 deletion mutant (ΔPG2125::ermF) as a control. Growth kinetics were monitored by optical density at 600 nm[OD(600)] every 2 h for 24 h. Oxidative stress tolerance was assessed in BHI-HV medium supplemented with 0.25 mmol/L H₂O₂. Arginine- and lysine-specific gingipain activities (Rgp and Kgp) were measured using chromogenic substrates BAPNA and ALNA, respectively. Hemagglutination titers against sheep erythrocytes were determined by serial dilution. Eighteen 6- to 8-week-old male SPF C57BL/6 mice were randomized into three groups (n=6 each): control (pulp exposure + sterile saline), W83 group (pulp exposure + W83 suspension), and ΔPG0396 group (pulp exposure+ ΔPG0396 suspension). Following pulp exposure of the right mandibular first molar, OD(600)=1.0 bacterial suspensions or saline were applied via cotton pellet compression for 3 min, followed by drying, application of a self-etching adhesive, light-curing for 20 s, and resin restoration. Alveolar bone loss was quantified by micro-computed tomography. Serum interleukin-6 and P. gingivalis-specific IgG levels were measured by ELISA on days 0, 7, 14, and 21 post-inoculation. Transcriptomic profiling of ΔPG0396 and W83 was performed with subsequent GO and KEGG enrichment analyses. Recombinant His-PG0396 protein was purified and its binding to target gene promoters was verified by electrophoretic mobility shift assay.

Compared with the W83 strain, ΔPG0396 showed no significant difference in growth curves in BHI-HV medium, consistent with the phenotype of the ΔPG2125 control strain. Under 0.25 mmol/L H₂O₂ stress, ΔPG0396 entered the stationary phase roughly 4 h eralier than the W83 strain. Gingipain activities were significantly elevated in ΔPG0396: OD(405) values for Rgp increased by around 60% (P<0.05), and OD(405) values for Kgp rose by about 34% after 120 min of incubation (P<0.05); no significant alterations were detected in the ΔPG2125 mutant. Hemagglutination titer decreased from 1:8 to 1:4. In the murine apical periodontitis model, alveolar bone loss volume in the ΔPG0396 group was 85% greater than that in the W83 group (0.280±0.062 vs 0.151±0.019 mm³, P<0.05). Serum IL-6 levels in the ΔPG0396 group were significantly higher than those in the W83 group on days 14 and 21 post-modeling (P<0.05), and serum P. gingivalis-specific IgG levels were also significantly elevated on day 21 (P<0.05). Transcriptome sequencing revealed 30 differentially expressed genes in ΔPG0396 (10 upregulated, 20 downregulated), which were mainly enriched in pathways related to oxidative stress response and lipopolysaccharide biosynthesis. Electrophoretic mobility shift assay (EMSA) confirmed that His-PG0396 specifically bound to the promoter regions of the differentially expressed genes PG1777 and PG0901.

PG0396 functions as a Crp/Fnr family transcription factor in P. gingivalis that negatively regulates oxidative stress responses, gingipain activity, and pathogenicity in experimental apical periodontitis.