Determination of key time point of interaction between B. subtilis YPS-32 and S. scabies
The inhibitory effect of B. subtilis YPS-32 on S. scabies growth increased with co-culture time, reaching a maximum of 92% ± 6.1% inhibition at 24 h (Fig. 1). There was no significant change in inhibition rate after co-culturing for 24 h; thus, 24 h was selected as the key interaction time point between the two strains. Samples for transcriptomic and proteomic sequencing were then selected from a co-culture of B. subtilis YPS-32 and S. scabies at 0 h (control) and 24 h (treatment group).
Overview of transcriptome sequencing of B. subtilis YPS-32 co-cultured with S. scabies
In this study, the DNBSEQ platform was used to sequence six samples, with an average of 13.84 M reads generated per sample. The average comparison rate of the sample comparison genome was 90.30%, and the average comparison rate of the comparison gene set was 52.81%. A total of 3969 genes were detected. Compared with the control group, 1772 DEGs were found in the treatment group under pathogen stress. Among them, 809 were up-regulated and 963 were down regulated.
GO functional annotation and enrichment analysis of B. subtilis YPS-32 DEGs
GO functional annotation is divided into three categories: cellular component, molecular function and biological process. The GO functional annotation analysis of the 1772 DEGs showed that in the biological process, the number of genes involved in cellular process and metabolic process was the largest; Among the molecular functions, the number of genes involved in catalytic activity, binding, and transporter activity was the largest. Among cellular components, the number of genes involved in cellular analytical entity and intracellular and protein containing complex was the largest.
GO enrichment analysis of 809 up-regulated DEGs revelaed the top 20 GO terms with the most significant enrichment (Fig. 2A). The up-regulated DEGs were mainly enriched in cell differentiation, sporulation resulting in formation of a cellular spore, developmental process, sporulation, anatomical structure formation involved in morphogenesis, cellular developmental process, anatomical structure morphogenesis, anatomical structure development, cellular amino acid metabolic process, alpha-amino acid metabolic process, alpha-amino acid biosynthetic process, and cell wall synthesis.
GO enrichment analysis of 963 down-regulated DEGs revealed the top 20 GO terms with the most significant enrichment (Fig. 2B). The results showed that the down regulated DEGs were mainly enriched in cellular process, RNA binding, cellular metabolic process, heterocyclic compound binding, organic cyclic compound binding, cellular nitrogen compound metabolic process, macromolecule metabolic process, and nucleobase-containing compound metabolic process.
KEGG pathway function annotation and enrichment analysis of B. subtilis YPS-32 DEGs
Using the KEGG metabolic pathway analysis, DEGs were mainly categorized into five branches: cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems. Among them, pathways related to metabolism included the most DEGs, which were associated with carbohydrate metabolism, amino acid metabolism, energy metabolism, metabolism of cofactors and vitamins, lipid metabolism, nucleotide metabolism and biosynthesis of other secondary metabolites.
A total of 809 up-regulated DEGs were subjected to KEGG pathway analysis. The up-regulated DEGs were located in 105 KEGG pathways, of which the top 20 KEGG pathways with the most significant enrichment are shown in Fig. 3A. These included histidine metabolism, biosynthesis of amino acids, cysteine and methionine metabolism, ascorbate and aldarate metabolism, polyketide sugar unit biosynthesis, sulfur metabolism, phosphotransferase system (PTS), fructose and mannose metabolism, 2-oxocarboxylic acid metabolism, pentose and glucuronate interconversions, starch and sucrose metabolism, and streptomycin biosynthesis. Among them, the category “polyketide sugar unit biosynthesis” comprised the upregulated genes that was related to bacillaene sugar unit biosynthesis, such as spsL (fold change = 3.46), rfbD (fold change = 3.63), rfbB (fold change = 3.46), and spsI (fold change = 3.41).
KEGG pathway enrichment analysis were also performed on the down-regulated DEGs. The down-regulated DEGs were located in 104 KEGG pathways, of which the top 20 KEGG pathways with the most significant enrichment are shown in Fig. 3B. These included flagellar assembly, oxidative phosphorylation, photosynthesis, RNA degradation, bacterial chemotaxis, bacterial secretion system, ribosome and two-component system.
qRT-PCR verification of transcriptome sequencing results
To verify the accuracy of the transcriptome sequencing results, 15 highly expressed DEGs were selected for qRT-PCR analysis. A comparison of the log2Fold Change values showed that the results of qRT-PCR results were consistent in terms of up- versus down-regulation with those of transcriptome sequencing for each of the 15 DEGs analyzed (Fig. 4). These results support the reliability of the transcriptome sequencing results.
Proteomic analysis of B. subtilis YPS-32 co-cultured with S. scabies
A total of 301181 secondary spectrograms were generated by proteomic analysis, and 16547 peptide segments corresponding to 2394 proteins were identified under the 1% false discovery rate (FDR) filtration standard at the protein level. Compared with the control group, there were 1188 DEPs in the treatment group; 579 were up-regulated and 609 were down-regulated.
GO function annotationl of B. subtilis YPS-32 DEPs
In the GO functional annotation of the 1188 DEPs, the biological process and the number of proteins involved in cellular process and metabolic process were maximum. Among the molecular functions, the number of proteins involved in catalytic activity, binding and transporter activity weres maximum. Among the cell components, the number of proteins related to cell, cell part, and membrane were maximum (Fig. 5).
KEGG pathway function annotation and enrichment analysis of B. subtilis YPS-32 DEPs
The KEGG pathway annotation analysis results of 1188 DEPs are shown in Fig. 6A. DEPs were mainly enriched in metabolic pathways, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, biosynthesis of amino acids, starch and sucrose metabolism, oxidative phosphorylation, glycine, serine and threonine metabolism, pentose phosphate pathway, arginine and proline metabolism and phosphotransferase system.
The KEGG pathway enrichment analysis of the 1188 DEPs was also performed, and the top 13 KEGG pathways with the most significant enrichment are shown in Fig. 6B. These included glycine, serine and threonine metabolism, biosynthesis of secondary metabolites, synthesis and degradation of ketone bodies, ubiquinone and other terpenoid − quinone biosynthesis, metabolic pathways, arginine and proline metabolism, oxidative phosphorylation, biosynthesis of amino acids, nonribosomal peptide structures, 2 − oxocarboxylic acid metabolism, flagellar assembly, starch and sucrose metabolism and histidine metabolism. Among the category “biosynthesis of secondary metabolites”, five proteins (WP_160214990.1 (fold change = 1.98), WP_080015512.1 (fold change = 1.65), WP_160214989.1 (fold change = 1.85), WP_032723105.1 (fold change = 1.77), and WP_089172562.1 (fold change = 1.75)) involved in the biosynthetic pathway of fengycin were significantly up-regulated in B. subtilis YPS-32 after 24 h co-culturing with S. scabies. Furthermore, two bacilysin biosynthesis-related proteins, WP_003244300.1 (fold change = 1.62) and WP_003227505.1 (fold change = 1.40) were also significantly up-regulated when B. subtilis YPS-32 was co-cultured with S. scabies. In addition, the protein, WP_003245811.1 (fold change = 5.50) involved in bacillibactin export was also significantly upregulated.