RNA-seq analysis of antibacterial mechanism of Cinnamomum camphora essential oil against Escherichia coli

Materials

The 98% C. camphora essential oil used in this study was purchased from Chengdu Aikeda Chemical Reagent Co. Ltd and the acetone was from Xilong Scientific Co. Ltd. E. coli ATCC8739, provided by the Strain Preservation Center, was cultured in Luria-Bertani (1% sodium chloride, 1% peptone, and 0.5% yeast extract).

Evaluation of antibacterial activity

Determination of MIC and MBC

Minimum inhibitory concentration (MIC) determination is to determine the lowest concentration of the essential oil that inhibits the growth of bacteria. MIC was defined as the concentration for the transition from a plate full of colonies to a dense single colony. Minimum bactericidal concentration (MBC) determination is to determine the minimum concentration of the essential oil that kills bacteria, which was defined the minimum concentration for no bacterial colony growth as the MBC. After treating E. coli with acetone (100% concentration) and water, the wet and dry weights of them were measured to determine the inhibitory effect of the essential oil on the growth of them. After different concentrations of acetone and the essential oil were applied to E. coli, the inhibitory effects of the different concentrations of acetone on the growth of E. coli were determined according to the growth of the plate colony and turbidity of the culture medium during culture. All the experiments were repeated in triplicate.

Effects of the essential oil on the cell membrane and cell wall of E. coli

With water as a control, E. coli ATCC8739 was treated with acetone and 1/2 MIC of the essential oil to determine the cell integrity and effect of the essential oil on the cell permeability. In order to detect β-galactosidase (β-Gal) and alkaline phosphatase (AKP) leakage in the supernatant, the β-galactosidase enzyme activity and alkaline phosphatase assay kits (Solarbio) were used respectively.

RNA-seq analysis

E. coli was first treated with lysozyme for 5 min at room temperature. Then, total RNA was extracted using RNAiso (TakaRa, D9108A) according to the manufacturer’s instructions. Then the biomass was stored at −80 ^∘C until further treatment. RNA purity was assessed using a NanoDrop ND-1000 Spectrophotometer (Isogen Life Science) and 1% agarose gel electrophoresis. Then the high-quality RNA was sent to the Beijing Novogene Bioinformatics Technology Co., Ltd (Beijing, China).

In order to obtain the high-quality clean data, the raw reads from the Beijing Novogene Bioinformatics Technology Co., Ltd were initially processed for removing the adapter sequences and low-quality reads. Clean Reads were quickly and accurately compared with the reference genome of E. coli by using Bowtie2 to obtain the localization information of Reads on the reference genome. After the quantification of gene expression was completed, DESeq2 was used to conduct statistical analysis on the expression data, and the genes with significantly different expression levels in different states were screened. GOseq was used in Gene Ontology (GO) enrichment analysis which was based on Wallenius non-central hyper-geometric distribution. KOBAS (2.0) was used for Pathway significant enrichment analysis, which took pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database as units, and applied hypergeometric test to find the pathways that showed significant enrichment in differentially expressed genes compared with the whole genome background.

Real-time polymerase chain reaction

Total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Japan). Real-time polymerase chain reaction (RT-PCR) was performed using the SYBR Green Premix Ex Taq II (Takara, Japan) and Applied Biosystems StepOnePlus real-time PCR System (Applied Biosystems, Carlsbad, CA, USA).

Antibacterial activity

To investigate the bacteriostatic activity, the Oxford cup method was used to detect the bacteriostatic circle. Compared with water and solvent acetone, which have no bacteriostatic phenomenon, the essential oil and ampicillin solution show stronger bacteriostatic activity (Fig. 1A). The antibacterial activity of ampicillin solution was slightly higher than that of the essential oil, but there was no significant difference, and the inhibitory zone size was between 11-11.5 mm (Table 1). This result suggested that the essential oil (100% concentration) had antibacterial effect on E. coli, and had similar antibacterial activity as 0.1 mg/ml ampicillin solution.

Comparing the control and acetone, the dry and wet weights of E. coli reduced significantly after treatment with the essential oil (Figs. 1B, 1C). The dry and wet weights of E. coli treated with acetone, the solvent of the essential oil, were also less than those treated with water. This indicated that the essential oil exerted a strong antibacterial effect on E. coli, and acetone also had certain inhibitory effect on the growth of E. coli. Therefore, different concentrations of acetone were applied to E. coli, and the inhibition effect of the different concentrations of acetone on the growth of E. coli was determined according to the growth of the plate colony and turbidity of the culture medium during culture. (Table 2). The results showed that acetone did not exert a strong bacteriostatic effect. To ensure the minimum effect of the solvent on the bacteriostatic phenomenon and at the same time to dissolve the essential oil, 5% acetone concentration was selected as the concentration of dissolved the essential oil.

The MIC and MBC of the essential oil are shown in Table 3. This indicated that the essential oil exerted a strong bacteriostatic effect on E. coli, and the MIC value was 0.625%. The MBC of the oil against E. coli was 2.5%. Therefore, to explore the antibacterial mechanism of the essential oil on E. coli, the 1/2 MIC of the essential oil concentration was selected to treat E. coli. In this case, the E. coli plate colony showed growth, but the growth rate was much lower than that of the control.

Effects of the subinhibitory concentration of the essential oil on the cell membrane and cell wall of E. coli

Beta-galactosidase is an intracellular enzyme which could not be detected in supernatant. Figure 2A shows that the content of β-galactosidase enzyme in the supernatant was very low during the 24 h analysis. The consistency occurs in three treatments. This indicated that 1/2 MIC of the essential oil and acetone did not inhibit the growth of E. coli by destroying the cell membranes.

Alkaline phosphatase (AKP) is a protease that exists between cell membranes and cell walls. It is almost impossible for AKP to pass through the cell wall of a healthy bacterium. Moreover, AKP activity was higher in the control group than in the acetone and the essential oil (Fig. 2B). Therefore, acetone and 1/2 MIC of the essential oil did not destroy the cell wall of E. coli and even prevented the cell wall from being destroyed.

Identification of differentially expressed genes

The difference in expression is determined by understanding the difference genes between different treatment to clarify the gene regulation. Then the DEseq package and setting the parameter padj at < 0.05 were adopted to identify the differentially expressed genes.

To investigate the effect of the essential oil on the gene expression of E. coli, upregulated and downregulated genes were assessed (Figs. 3A–3D). The results showed that there were only 81 different genes in the acetone-treated compared with the control, consistent with previous results. Nevertheless, there were 1745, 2311, and 2359 differential genes in the 1/8 MIC, 1/4 MIC, and 1/2 MIC of the essential oil-treated compared with the control, respectively. For the concentration of essential oil, the number of the different genes were increased. Among them, the downregulated genes increased with an increase in concentration, but the upregulated genes increased first and then stabilized. There was little difference in the upregulated genes, compared with the 1/4 MIC and 1/2 MIC of the essential oil. This result indicated that the concentration of 1/4 MIC of the essential oil was an equilibrium point. Perhaps at this concentration, the regulation of growth in E. coli reached its maximum value.

To analyze gene expression patterns of differentially expressed genes in different processing states, cluster analysis was adopted. Genes with similar expression patterns may have similar functions, or participate in the same metabolic process, or participate in the same cell pathway. Therefore, these genes with similar expression patterns were grouped into classes to examine the changes in gene expression under different treatment conditions and the gene expression changes were examined after gene normalization. The results showed that the gene expression patterns were different among the different treatments, indicating that the metabolic pathways were different among the different treatments (Fig. 3E).

To understand how the genes were classified, a Venn diagram of the different genes was constructed. The results showed 1,414 identical differentially expressed genes in E. coli treated with three concentrations of the essential oil compared with the control (Fig. 3F). The function of these common differentially expressed genes may be related to E. coli resistance.

GO analysis of the differential genes

To explore the antibacterial mechanism of the essential oil, GO term enrichment analysis was performed on the differential genes of treatment with 1/8, 1/4, and 1/2 MIC of the essential oil (Fig. 4). The differential genes under different treatments were introduced into the R Programming; the GOseq package was employed to enrichment analysis and the hmmscan package was used for annotation. The 30 GO terms with the most significant enrichment were selected and plotted in the graph. In addition, to understand the GO terms which were significantly enriched in the GO analysis, a directed acyclic graph (DAG) analysis was performed to draw the DAG diagrams of the biological process (BP), molecular function (MF), and cellular component (CC). Data files were given as Figs. S1–S3. The non-analytic and chaotic nodes were screened out and analytic and significantly enriched nodes were sorted out for viewing by using Visio. In Figs. S1–S3, each ellipse node represents a GO term, and the box represents the GO with an enrichment degree of TOP 10. For the 1/8 MIC, the downregulated pathways were enriched in the process of bacterial chemotaxis, anabolic metabolism, signal transduction, etc., while the upregulated pathways were mostly enriched in productive metabolic activities and resistance activities. For the 1/4 MIC, the downregulated pathways were enriched in the regulation of gene expression, the process of macromolecule anabolic metabolism, and signal transduction, while the upregulated pathways were enriched in the production capacity reaction and the synthesis of some resistant substances. For the 1/2 MIC, the downregulated pathways were enriched in the synthesis, signal transduction, and metabolism processes of macromolecules, while the upregulated pathways were enriched in the synthesis and translation processes of some resistant substances.

KEGG pathway analysis of differentially expressed genes

To explore the antibacterial mechanism of the essential oil, KEGG pathway enrichment analysis (Fig. 5). According to KEGG pathway analysis, the growth of E. coli was inhibited through inhibiting the anabolic, chemotaxis, signal transduction, and other life activities of E. coli, while the activity was maintained by strengthening its production capacity and repair in the 1/8 MIC of the essential oil. In the 1/4 MIC of the essential oil, the growth of E. coli was inhibited by inhibiting metabolism, movement, signal transduction, and other life activities as well as some resistance activities, while life activities were maintained by increasing production capacity and enhancing the synthesis of resistant substances. In the 1/2 MIC of the essential oil, the growth of E. coli was inhibited by inhibiting metabolism, chemotaxis, and certain resistance reactions, while life activities were sustained by enhancing productive reaction, the translation process, and the synthesis of resistant substances. These results indicated that the essential oil exerted a strong antibacterial effect against E. coli, and this inhibitory effect was realized by inhibiting the life activity, signal transduction and inhibitory activity and enhanced with an increase in concentration.

Analysis of the bacteriostatic mechanism

To explore the bacteriostatic mechanism, several genes which encode metabolism and resistance in E. coli were selected and measured by RT-PCR. Eighteen functionally relevant genes (dadA, raiA, dadX, csgD, tar, cph2, rstA, purD, purF, purL, hdeD, evgs, zraS, zraP, asr, cysK, ompC, and lamB) were randomly selected to evaluate the transcription levels. For quantitative real-time PCR, the results showed that the dadA, dadX, asr, cysK, ompC, and lamB expression levels were significantly higher in treated with essential oil than the control (Fig. 6). Nevertheless, the expression of raiA, csgD, cph2, tar, rstA, purD, purF, purL, hdeD, evgs, zraS, and zraP were significantly inhibited (Fig. 6).