Comprehensive gene expression analysis of erythritol-treated S. pseudintermedius
We recently revealed that erythritol at concentrations above 5% (w/w) inhibited the growth of a standard strain of S. pseudintermedius, JCM 17571 (SP) [8]. In this study, comprehensive gene expression analysis using RNA sequencing (RNA-seq) was performed for SP cultured in the presence or absence of erythritol (n = 3). To avoid complete suppression of cellular metabolism, a weak inhibiting concentration (5% [w/w]) was selected. After filtering the raw reads, we found that there were 19,142,672, 20,940,942, and 22,024,908 clean reads of the transcriptome in the control samples, whereas 19,068,865, 19,060,494, and 19,393,064 clean reads were obtained from the erythritol-treated samples. A complete list of all reads is shown in Additional file 1. Among these genes, a total of 625 differentially expressed genes were identified using the calculated gene expression levels (|Log2 FC (fold change)| ≥ 1, p-value ≤ 0.05), with 244 genes markedly up-regulated and 381 genes markedly down-regulated in SP following erythritol treatment. A PCA plot showed a clear split between the control and erythritol-treated samples, with 95% of the variance explained by PC1 (Fig. 1a). A volcano plot showed that the differential expressions of the top up-regulated and down-regulated genes were statistically significant (Fig. 1b).
The top 15 up-regulated and down-regulated genes in response to erythritol
The predicted functional descriptions and GO biological process classes for the top 15 up-regulated and top 15 down-regulated genes in response to erythritol are shown in Table 2. Among the top 15 up-regulated genes with a predicted GO biological process class, genes related to two biological functional groups were present. Three genes (Up-Regulated Genes group 1, URGs1), BJK46_009280 (ptsG), BJK46_000300 (ppdK), and BJK46_000295 (ppdkR), were predicted to encode proteins related to PTS. The ptsG gene encodes the glucose-specific EIICBA component of PTS (PtsG), and the ppdK and ppdkR genes encode pyruvate phosphate dikinase (PPDK) and PPDK regulatory protein (PPDKR), respectively, which are involved in the regeneration of PEP that is required to drive PTS. Moreover, the other four genes (URGs2), BJK46_008080 (vraT), BJK46_008075 (vraS), BJK46_008070 (vraR), and BJK46_008015 (sgtB) were predicted to belong to the same group. The vraTSR genes belonging to a single gene cluster are predicted to comprise a three-component regulatory system necessary to promote resistance to cell wall agents, and sgtB is predicted to encode a glycosyltransferase associated with peptidoglycan biosynthesis. The GO classes of the biological process could not be predicted in the remaining 8 up-regulated genes.
Of the top 15 down-regulated genes, 11 genes (Down-Regulated Genes, DRGs), BJK46_008815 (purD), BJK46_008820 (purH), BJK46_008825 (purN), BJK46_008830 (purM), BJK46_008835 (purF), BJK46_008840 (purL), BJK46_008845 (purQ), BJK46_008850 (purS), BJK46_008855 (purC), BJK46_008860 (purK), and BJK46_008865 (purE), were located on a single pur-operon and assigned GO biological process classes predicted to be associated with 'de novo' IMP biosynthetic process. The GO biological process classes could not be predicted in the remaining 4 down-regulated genes.
The expression of the genes BJK46_008775 (encoding a PEP-protein phosphotransferase; PTS system enzyme I, E1), BJK46_002265 (encoding a histidine-containing phosphocarrier protein (HPr)), and BJK46_011055 (encoding a pur-operon repressor (PurR)) were not strongly induced or repressed by erythritol, exhibiting log2FC values of 0.550, 1.22, and 0.206, respectively, as shown in Additional file 1.
Glucose supplementation restored erythritol-induced up-regulation of PTS- and cell wall-related gene transcription and growth inhibition of SP
A real-time quantitative PCR (qPCR) analysis further confirmed the up-regulation of genes belonging to URGs1 and URGs2 in the presence of erythritol (Fig. 2). Because PTS contributes to glucose uptake, we thought that the up-regulation of PTS-related gene transcription was caused by glucose starvation in SP. Therefore, we compared the expression of PTS-related genes in SP between glucose-free and glucose-supplemented (1% [w/w]) conditions. We found that up-regulation of transcription of genes belonging to URGs1 in response to erythritol was restored under glucose-supplemented conditions, suggesting that transcription of PTS-related genes was up-regulated in response to glucose starvation. We also found that up-regulation of transcription of genes belonging to URGs2 in response to erythritol was restored under glucose-supplemented conditions. Moreover, supplementation of 0.1% glucose partially restored growth inhibition by erythritol (Fig. 3). Even when 1% glucose was added, the suppression of growth inhibition remained at the same level as when 0.1% glucose was added.
Erythritol inhibited biofilm formation in SP
Down-regulation of genes belonging to DRGs was also confirmed by qPCR analysis (Fig. 2). Previous reports suggested that down-regulation of pur-operon genes is associated with inhibition of biofilm formation in staphylococci [10]. Therefore, we hypothesized that erythritol inhibits biofilm formation in SP. Erythritol significantly inhibited the growth of SP (Fig. 4a) and the amount of biofilm formation (Fig. 4b) in a dose-dependent manner. In the condition with 5% erythritol, the biofilm inhibition effect (41.8% of OD570 vs. control) is notably greater than the growth inhibition effect (74.9% of OD660 vs. control).