High Molecular Weight Hyaluronic Acid Reduces the Expression of Virulence Genes fimA, mfa1, hagA, rgpA, and kgp in the Oral Pathogen Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis (P. gingivalis) is a cornerstone pathogen in the development and progression of periodontal and peri-implant tissue destruction. It is capable of causing dysbiosis of the microbial biofilm and modulation of the host immune system. Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan found in all living organisms. It is well known and has been used for improving tissue healing. In addition, some studies have suggested that there may be an antimicrobial potential to HA. The aim of this study was to evaluate the effect of hyaluronic acid, azithromycin (AZM), and chlorhexidine (CHX) on the expression of genes (i.e., fimA, mfa1, hagA, rgpA, rgpB, and kgp) related to the virulence and adhesion of P. gingivalis. The study groups were divided into four: (1) HA treated group; (2) AZM treated group; (3) CHX treated group; and (4) untreated group to serve as a negative control. P. gingivalis ATCC 33277 was cultured and then exposed to four different concentrations (100% MIC, 50% MIC, 25% MIC, and 12.5% MIC) of HA, AZM, and CHX for 24 h. The expression levels of the aforementioned genes were measured using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Relative fold-change values were calculated and compared between groups. The fold-change values of all genes combined were 0.46 ± 0.33, 0.31 ± 0.24, and 0.84 ± 0.77 for HA, AZM, and CHX, respectively. HA has downregulated all the genes by mostly a half-fold: 0.35 ± 0.20, 0.47 ± 0.35, 0.44 ± 0.25, 0.67 ± 0.46, 0.48 ± 0.33 and 0.35 ± 0.22 with fimA, mfa1, hagA, rgpA, rgpB and kgp, respectively. The effect of HA was significant on all genes except rgpB compared to the untreated control. Lower concentrations of HA tended to exhibit greater downregulation with 1 mg/mL being the most effective. High molecular weight (1.5 MDa) hyaluronic acid has a potent effect on P. gingivalis by downregulating fimA, mfa1, hagA, rgpA, and kgp. The effect of HA was generally less than that of AZM but greater than that of CHX.

Keywords: azithromycin; chlorhexidine; gingipains; hyaluronic acid; peri-implantitis; peri-mucositis; periodontal disease; periodontitis; porphyromonas gingivalis; sodium hyaluronate.

Figure 1

(A) A diagram depicting the distribution of 5 µL drops from 12 wells that were diluted by a two-fold factor. ‘C’ refers to untreated control; (B) Hyaluronic acid from 4 mg/mL to 2 µg/mL; (C) Azithromycin from 25 µg/mL to 0.012 µg/mL; (D) Chlorhexidine from 1 mg/mL to 0.49 µg/mL.

Figure 2

Overall fold-change values (mean ± SD) for all genes combined. >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****).

Figure 3

Overall fold-change values (mean ± SD) for independent genes. >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****).

Figure 4

Fold-change values (mean ± SD) of different concentrations on expression of fimA. >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****). Statistically significant compared to control p < 0.05 (†).

Figure 5

Fold-change values (mean ± SD) of different concentrations on expression of mfa1. >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****). Statistically significant compared to control p < 0.05 (†).

Figure 6

Fold-change values (mean ± SD) of different concentrations on expression of hagA. >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****). Statistically significant compared to control p < 0.05 (†).

Figure 7

Fold-change values (mean ± SD) of different concentrations on expression of rgpA. >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****). Statistically significant compared to control p < 0.05 (†).

Figure 8

Fold-change values (mean ± SD) of different concentrations on expression of rgpB. >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****). Statistically significant compared to control p < 0.05 (†).

Figure 9

Fold-change values (mean ± SD) of different concentrations on expression of kgp. >0.05 (ns), ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), ≤0.0001 (****). Statistically significant compared to control p < 0.05 (†).