Identification of the antibacterial action mechanism of diterpenoids through transcriptome profiling

Abstract

Effective antibacterial substances of Aralia continentalis have anti-biofilm and bactericidal activity to the oral pathogen Streptococcus mutans. In this study, three compounds extracted from A. continentalis were identified as acanthoic acid, continentalic acid, and kaurenoic acid by NMR and were further investigated how these diterpenoids affect the physiology of the S. mutans. When S. mutans was exposed to individual or mixed fraction of diterpenoids, severe growth defects and unique morphology were observed. The proportion of unsaturated fatty acids in the cell membrane was increased compared to that of saturated fatty acids in the presence of diterpenoids. Genome-wide gene expression profiles with RNA-seq were compared to reveal the mode of action of diterpenoids. Streptococcus mutans commonly enhanced the expression of 176 genes in the presence of the individual diterpenoids, whereas the expression of 232 genes was considerably reduced. The diterpenoid treatment modulated the expression of genes or operon(s) involved in cell membrane synthesis, cell division, and carbohydrate metabolism of S. mutans. Collectively, these findings provide novel insights into the antibacterial effect of diterpenoids to control S. mutans infection, which causes human dental caries.

Keywords: Aralia continentalis; Streptococcus mutans; antibacterial activity; diterpenoids; transcriptome analysis.

Figure 1

Antibacterial compounds derived from Aralia continentalis.

Figure 2

The effect of diterpenoids on the growth of S. mutans. Diterpenoids were added to the bacteria in (A) the lag phase and (B) the mid exponential phase.

Figure 3

Scanning electron microscopy (SEM) micrographs of S. mutans treated with DM. (A–C), 1 h treatment; (D–F), 6 h treatment; (G–I), 24 h treatment. (A,D,G), untreated; (B,E,H), treated with 4 μg/ml; (C,F,I), treated with 40 μg/ml. Red and orange arrows indicate aberrant morphology and abnormal extracellular coating by DM treatment, respectively.

Figure 4

DEGs detected in comparison between diterpenoid-untreated and diterpenoid-treated samples. Streptococcus mutans was treated with DM, AA, CA, and KA. (A) Up- and downregulated number of DEGs, (B) Venn diagram showing unique and shared DEGs. DEGs with >1.0-fold change and p < 0.05 were clustered across all samples.

Figure 5

Cluster of Orthologous Groups (COG) classification of the identified DEGs. The alphabetical codes represent the following functional categories: C, energy production and conversion; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme metabolism; I, lipid metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis, transport, and catabolism; D, cell division and chromosome partitioning; M, cell envelope biogenesis and outer membrane; N, cell motility and secretion; O, post-translational modification, protein turnover, and chaperones; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; and V, defense mechanisms; J, translation, ribosomal structure, and biogenesis; K, transcription; L, DNA replication, recombination, and repair; S, function unknown (|log₂| ≥ 1, p_adj < 0.05).