The Study About Sialidase Inhibitor N-acetyl-2,3-dehydro-2-Deoxyneuraminic Acid Affecting On The Pathogenicity Of Porphyromonas Gingivalis

Objective: Chronic periodontitis is a multifactorial disease involving periodontal microorganisms as initiating factors and the immune host defense.Porphyromonas gingivalis(P.gingivalis)is implicated as a major pathogens periodontitis.The sialidase gene in P.gingivalis is the only gene encoding P.gingivalis encoding sialidase,which plays an important role in the initiation and progression of chronic periodontitis.Knocking out this gene can seriously affect its capsule synthesis,biofilm formation and gingipains activity,and reduce the ability to resist host immune defense.Therefore,inhibition of the activity of sialidase gene transcription,expression or its product(sialidase)is expected to be a new direction for preventing or treating chronic periodontitis.2-deoxy-2,3-didehydro-N-acetylneuraminic acid(DANA)is a broad-spectrum sialidase inhibitor with high inhibitory potency against some bacteria.The aim of this study is to clarify the effect of DANA on the sialidase activity,growth,bacterial biofilm and virulence factors of P.gingivalis in order to explore the mechanism about DANA affecting on the pathogenicity of P.gingivalis.It will provide a new idea for prevention and treatment of chronic periodontitis.Methods: P.gingivalis W83 was chosen as the experimental bacterial.The toxic effect of different concentration DANA or Oseltamivir on U937 cells were detected by MTS assay.And the inhibitory effect of different concentration DANA or Oseltamivir on P.gingivalis sialidase was verified by using fluorometric assay.1 m M DANA was added in P.gingivalis cultural medium(experiment groups).The control group was the untreated P.gingivalis.The growth curves were measured by recording the absorbance at 600 nm,and the cell morphology was viewed by transmission electron microscopy(TEM).Biofilms were formed on polystyrene plates,stained with a Live/Dead Bac Light bacterial viability kit and imaged with a confocal laser scanning microscope.The expressions of fim A,fim R,fim S genes were detected by real-time PCR.The expressions changes of virulence genes expression were detected by real-time PCR.Arg-gingipains(Rgps)and Lys-gingipain(Kgp)activity were determined using the synthetic substrates Nα-benzoyl-DL-arginine p-nitroanilide hydrochloride(BAPNA) and N-(p-tosyl)-Gly-Pro-Lys 4-nitroanilide acetate salt.LPS activity was measured by Chromogenic End-point Tachypleus Amebocyte Lysate Kit.Results: 1.The results of MTS assay showed that the relative proliferation rates of U937 cells treated with 0.1,1,10 m M DANA were 97.61 ± 2.68%,93.75 ± 7.87%,60.98 ±5.80%,respectively.The relative proliferation rates of U937 treated with 0.1,1,10 m M oseltamivir were 96.59 ± 2.93%,93.35 ± 3.86%,76.93 ± 10.05%,respectively.DANA has a potent inhibitory effect on P.gingivalis sialidase activity and its inhibition rates are higher than those of Oseltamivir.When the concentrations of DANA were 0.2,0.5,1.0,1.5,2.0,2.5 m M,the inhibition rate of sialidase activity were 47.13%,59.15%,72.01%,72.69%,72.79% and 71.77% respectively.Therefore,1 m M DANA was used for the following experiments because 1 m M DANA showed no toxic effects on U937 cells and significantly inhibited sialidase activity in P.gingivalis.2.The growth of P.gingivalis inhibited by a concentration of 1 m M DANA significantly.Compared to the biofilms of P.gingivalis W83,those formed by P.gingivalis W83 treated with 1 m M DANA were decreased and discontinuous.And the biofilms formed by P.gingivalis W83 treated with 1 m M DANA were thinner than those of P.gingivalis W83.3.Image of TEM showed that P.gingivalis cells were intact with clear cell membrances and cell walls,even after treated with 1 m M DANA,and the cell structure was similar in these two groups.4.Compared with P.gingivalis W83,the expressions of fimA,fimR and fimS genes in P.gingivalis W83 treated with 1 m M DANA were decreased and the differences were statistically significant(P<0.05).The Kgp activities of P.gingivalis W83 treated with 1m M DANA and the control group were 0.53 ±0.02 and 0.62 ± 0.03,respectively.The Rgps activities of them were 0.72 ± 0.07 and 0.96 ± 0.08,respectively.The differences of Kgp and Rgps activities were statistically significant between these two groups.But there were no significant differences in LPS activity.Conclusion: DANA exhibited potent sialidase inhibitory activities in P.gingivalis,which affected the growth and biofilm formation of P.gingivalis,decreased the expression of fimbria genes,and the activity of gingipains.It is expected to become a new resource for preventment and treatment of periodontitis.