Differential Modulation of Saliva-Derived Microcosm Biofilms by Antimicrobial Peptide LL-31 and D-LL-31

Abstract

Microbiome modulation, aiming to restore a health-compatible microbiota, is a novel strategy to treat periodontitis. This study evaluated the modulation effects of antimicrobial peptide LL-31 and its D-enantiomer (D-LL-31) on saliva-derived microcosm biofilms, spiked with or without Porphyromonas gingivalis. To this end, one-day-old biofilms were incubated for 24 h with biofilm medium alone, or medium containing 40 µM LL-31 or D-LL-31, after which biofilms were grown for 5 days. Biofilms were assessed at 1 day and 5 days after intervention for the total viable cell counts, dipeptidyl peptidase IV (DPP4) activity, P. gingivalis amount (by qPCR) and microbial composition (by sequencing). The results showed that D-LL-31, not LL-31, significantly reduced the total viable cell counts, the P. gingivalis amount, and the DPP4 activity of the biofilms spiked with P. gingivalis, but only at 1 day after intervention. In the biofilms spiked with P. gingivalis, D-LL-31 tended to reduce the α-diversity and the compositional shift of the biofilms in time as compared to the control and LL-31 groups. In conclusion, D-LL-31 showed a better performance than LL-31 in biofilm modulation. The biofilm modulation function of the peptides could be impaired when the biofilms were in a severely dysbiotic state.

Keywords: 16S rRNA gene amplicon sequencing; Porphyromonas gingivalis; oral microbiome; periodontitis; saliva-derived microcosm biofilms.

Figure 1

Experimental scheme. Pg, P. gingivalis; S biofilms, saliva-derived microcosms alone; SPg biofilms, saliva-derived microcosms spiked with P. gingivalis. Biofilms were treated with peptide LL-31 (40 µM), peptide D-LL-31 (40 µM), or MQ water (negative control, NC).

Figure 2

The influences of LL-31 and D-LL-31 on the growth of P. gingivalis, F. nucleatum and S. mitis in planktonic cultures. Peptides were tested at concentrations of 20, 40 and 80 µM. Data are presented as mean ± standard deviation of percentage bacterial growth (%), relative to the negative control group (MQ water). Statistically significant differences as compared to the negative control group are indicated by asterisks: * p < 0.05; ** p < 0.005; *** p < 0.0005 (one-way ANOVA followed by Bonferroni post hoc test).

Figure 3

(A) The amount of P. gingivalis in the SPg biofilms at 1 day and 5 days after peptide intervention as determined by qPCR; data are presented as the calculated DNA concentration (ng/µL) of P. gingivalis. (B) Relative abundance of OTU_2 in SPg biofilms at 1 day and 5 days after peptide interventions. The representative sequence of OTU_2 was confirmed to be identical to the spiked strain P. gingivalis ATCC 33277 by blasting against the expanded HOMD database (http://www.homd.org (accessed on 23 December 2022); HOMD 16S rRNA RefSeq version 15.22) using default parameters. (C) Total viable-cell counts of S biofilms (left) and SPg biofilms (right) at 1 day and 5 days after peptide interventions. Data are represented as log₁₀ CFU counts. S biofilms, saliva-derived microcosms alone; SPg biofilms, saliva-derived microcosms spiked with P. gingivalis; L, intervention by peptide LL-31; D, intervention by peptide D-LL-31. All data represent mean ± standard deviation of three independent experiments. Statistically significant differences between different groups at each time point are indicated by asterisks: * p < 0.05 (one-way ANOVA followed by Bonferroni post hoc test).

Figure 4

(A) Total protease activity of S biofilms (left) and SPg biofilms (right) at 1 day and 5 days after peptide interventions; data are presented as relative fluorescence (RF) values per min; (B) DPP4 activity of S biofilms (left) and SPg biofilms (right) at 1 day and 5 days after peptide interventions; data are presented as fluorescence intensity (FI) values. S biofilms, saliva-derived microcosms alone; SPg biofilms, saliva-derived microcosms spiked with P. gingivalis; L, intervention by peptide LL-31; D, intervention by peptide D-LL-31. All data represent mean ± standard deviation of three independent experiments. Statistically significant differences between different groups at each time point are indicated by asterisks: ** p < 0.005; *** p < 0.0005 (one-way ANOVA followed by Bonferroni post hoc test).

Figure 5

Relative abundance of top 15 most abundant bacterial genera or higher taxa (remaining genera are grouped as “Others”) in: (A) S biofilms; (B) SPg biofilms, at 1 day and 5 days after peptide intervention. Data represent an average of replicate samples of three independent experiments. S biofilms, saliva-derived microcosms alone; SPg biofilms, saliva-derived microcosms spiked with P. gingivalis; L, intervention by peptide LL-31; D, intervention by peptide D-LL-31.

Figure 6

The α-diversity analyses of biofilms: (A) Species richness of S biofilms (left) and SPg biofilms (right) at 1 day and 5 days after peptide interventions; (B) Shannon diversity index of S biofilms (left) and SPg biofilms (right) at 1 day and 5 days after peptide interventions. S biofilms, saliva-derived microcosms alone; SPg biofilms, saliva-derived microcosms spiked with P. gingivalis; L, intervention by peptide LL-31; D, intervention by peptide D-LL-31. Statistically significant differences are indicated by asterisks: * p < 0.05; ** p < 0.005; *** p < 0.0005; one-way ANOVA followed by Bonferroni post hoc test was used for comparison between different groups within each time point; two-way ANOVA followed by Bonferroni post hoc test was used for comparison between time points.

Figure 7

Principal component analysis (PCA) plot of S biofilms (symbols in circles) and SPg biofilms (symbols in triangles) at 1 day and 5 days after peptide interventions. S biofilms, saliva-derived microcosms alone; SPg biofilms, saliva-derived microcosms spiked with P. gingivalis; L, intervention by peptide LL-31; D, intervention by peptide D-LL-31.

Figure 8

Linear discriminant analysis effect size (LEfSe) of OTUs that were differentially abundant in S biofilms (A) and SPg biofilms (B) as the biofilm aged. The default settings were used when performing LEfSe, except that the LDA threshold was set to 4. S biofilms, saliva-derived microcosms alone; SPg biofilms, saliva-derived microcosms spiked with P. gingivalis; L, intervention by peptide LL-31; D, intervention by peptide D-LL-31. Statistically significant differences are indicated by asterisks: * p < 0.05; ** p < 0.005; *** p < 0.0005 (two-way ANOVA followed by Bonferroni post hoc test).

Figure 9

Linear discriminant analysis effect size (LEfSe) of OTUs that were differentially abundant between different treatment groups at 5 days after peptide treatments in S biofilms (A) and SPg biofilms (B). The default settings were used when performing LEfSe, except that the LDA threshold was set to 4 and one-against-all was used as the strategy for multi-class analysis. S biofilms, saliva-derived microcosms alone; SPg biofilms, saliva-derived microcosms spiked with P. gingivalis; L, intervention by peptide LL-31; D, intervention by peptide D-LL-31. Statistically significant differences are indicated by asterisks: * p < 0.05; ** p < 0.005 (one-way ANOVA followed by Bonferroni post hoc test).