Acyl homoserine lactone-mediated quorum sensing in the oral cavity: a paradigm revisited

Abstract

Acyl homoserine lactones (AHLs), the quorum sensing (QS) signals produced by Gram-negative bacteria, are currently considered to play a minor role in the development of oral biofilm since their production by oral pathogens has not been ascertained thus far. However, we report the presence of AHLs in different oral samples and their production by the oral pathogen Porphyromonas gingivalis. The importance of AHLs is further supported by a very high prevalence of AHL-degradation capability, up to 60%, among bacteria isolated from dental plaque and saliva samples. Furthermore, the wide-spectrum AHL-lactonase Aii20J significantly inhibited oral biofilm formation in different in vitro biofilm models and caused important changes in bacterial composition. Besides, the inhibitory effect of Aii20J on a mixed biofilm of 6 oral pathogens was verified using confocal microscopy. Much more research is needed in order to be able to associate specific AHLs with oral pathologies and to individuate the key actors in AHL-mediated QS processes in dental plaque formation. However, these results indicate a higher relevance of the AHLs in the oral cavity than generally accepted thus far and suggest the potential use of inhibitory strategies against these signals for the prevention and treatment of oral diseases.

Figure 1

Detection of acyl homoserine lactones (AHLs) in saliva samples (a) and extracted teeth (b) obtained from the same patients using HPLC-MS. P = periodontal disease; C = caries.

Figure 2

Effect of the AHL-lactonase Aii20J (20 µg/mL) on in vitro oral biofilm obtained from the saliva of a healthy donor as measured using the xCELLigence system. The culture was done in BHI (a) and BHI supplemented with 0.1% sucrose (BHIs) (b). Biofilm formation was expressed in Cell Index units. Data are representative of 3 independent experiments.

Figure 3

Effect of the AHL-lactonase Aii20J (20 µg/mL) on in vitro oral biofilm formed using saliva samples obtained from healthy (S0 and S1), and periodontal patients (S2-S6) in BHI and BHI supplemented with 0.1% sucrose (BHIs) and quantified by the xCELLigence on line measurement system. Measures are shown after 8 h of incubation and expressed as percentage of the control cultures (n = 2).

Figure 4

Formation of in vitro oral biofilm obtained from saliva samples from a healthy patient with and without the AHL-lactonase Aii20J (20 µg/mL) using the Active Attachment model and measured with the crystal violet staining assay. (a) Biofilms incubated in aerobic conditions at 37 °C using BHI and BHI supplemented with 0.1% sucrose (BHIs). Aerobic biofilms were sampled at 24 h, 48 h and 72 h; (b) Biofilms incubated in anaerobic conditions at 37 °C using BHI and BHI-2 (BHI supplemented with 2.5 g/L mucin, 1 g/L yeast extract, 0.1 g/L cysteine, 2 g/L sodium bicarbonate, 5 mg/L hemin, 1 mg/L menadione and 0.25% (v/v) glutamic acid). Anaerobic biofilms were sampled at 24 h, 48 h, 72 h and 96 h at 37 °C for BHI. BHI-2 biofilms could only be stained after 24h. Significantly different values are indicated with an asterisk (Student’s t-test, p < 0.05) (n = 3).

Figure 5

Formation of in vitro oral biofilms obtained from saliva samples from a healthy (a) and a periodontal (b) donor with and without the AHL-lactonase Aii20J (20 µg/mL) using the Active Attachment model and measured with the crystal violet staining assay (O.D. 590 nm). The cultures were done in aerobic conditions using BHI and BHI supplemented with 0.1% sucrose (BHIs) for 24 h (n = 3) and in anaerobic conditions for 24 and 48 h using BHI (n = 1). Significantly different values are indicated with an asterisk (Student’s t-test, p < 0,05).

Figure 6

Bacterial diversity of the in vitro oral biofilms obtained from saliva samples from a healthy (a) and a periodontal (b) donor with and without the AHL-lactonase Aii20J (20 µg/mL). The cultures were incubated in aerobic conditions using BHI and BHI supplemented with 0.1% sucrose (BHIs) for 24 h and in aerobic conditions for 24 and 48 h using BHI.

Figure 7

Principal components analysis of the species composition of in vitro oral biofilms obtained from healthy (a) and periodontal (b) saliva samples with and without the QQ enzyme Aii20J. Biofilms were cultured in BHI or BHI supplemented with 0.1% sucrose under aerobic and in BHI during 24 h and 48 h under anaerobic conditions. Closed symbols represent the control biofilms while open symbols represent the biofilms treated with the QQ enzyme Aii20J. Squares = BHI aerobic conditions, circles = BHI anaerobic conditions, triangles = BHI supplemented with 0.1% sucrose in aerobic conditions and diamonds = 48 h BHI in anaerobic conditions in both panels.

Figure 8

Confocal microscopy visualization (63×) of the effect of the QQ enzyme Aii20J (20 µg/mL) on multi-species biofilms formed by the oral pathogens A. naeslundii, A. actinomycetemcomitans, F. nucleatum, P. gingivalis S. oralis and V. parvula using fluorescence dyes Syto9 and propidium iodide. The biofilms were cultured in BHI-2 during 3 and 4 days in anaerobic conditions at 37 °C.