Analysis of Akkermansia muciniphila in Mulberry Galacto-Oligosaccharide Medium via Comparative Transcriptomics

Akkermansia muciniphila is a common member of the human gut microbiota and belongs to the phylum Verrucomicrobia. Decreased levels of A. muciniphila are associated with many diseases, so it is thought to be a beneficial resident of the intestinal mucosal layer. In this study, we found that different prebiotics promoted the proliferation of A. muciniphila, and mulberry galacto-oligosaccharide (MGO) had the greatest effect. We cultured A. muciniphila in a brian heart infusion (BHI) medium containing 5% galactooligosaccharides (GOS), mulberry polysaccharide solution (MPS), and MGO, and transcriptomic analyses were performed. The results revealed that, after 6 days of cultivation, the numbers of upregulated functional genes (based on Gene Ontology) were approximately 0.7 and 19% higher with MPS and MGO, respectively, than with GOS. Analysis using the Kyoto Encyclopedia of Genes and Genomes showed that, when A. muciniphila was cultured with MGO, genes that were upregulated were enriched in the carbohydrate metabolism, the metabolism of cofactors and vitamins, the energy metabolism, the amino acid metabolism, and the lipid metabolism. Upregulated genes included galM and pfkA in the galactose metabolism, and pgi, pfk, fbaA, tpiA, gapA, pgk, gpml, eno, pyk, and lpd in the glycolysis/gluconeogenesis pathway. Real-time quantitative PCR results were consistent with the RNA-Seq data. This work provides valuable knowledge which can be available for the functional application of A. muciniphila and MGO.


Introduction
In recent years, research on the biological activities of plant functional polysaccharides has mainly focused on the regulation of sugar and lipid homeostasis, enhancing immunity, and improving the intestinal microbiota. Because plant functional polysaccharides have extremely low toxicity and few side effects, they are particularly widely used in the prevention and treatment of chronic diseases such as diabetes, obesity, and metabolic syndrome in humans [1,2]. Mulberry is widely cultivated in Asia, and it has various health benefits such as regulating sugar homeostasis, immune regulation, and acting as a laxative for humans [3,4]. Mulberry polysaccharides have been confirmed to play an important role in many biological activities of mulberry [5]. Chen et al. reported that mulberry polysaccharide has good antioxidant activity and can inhibit the absorption of glucose by inhibiting the activities of α-amylase and α-glucosidase in vitro [6]. Mulberry polysaccharide can upregulate the expression of the insulin receptor InsR, insulin substrate receptor IRS-2, and glucose transporter in type II diabetic rats and lower insulin resistance [7]. with different individual enzymes (5%; pectinase, glucoamylase, β-mannanase, xylanase, β-glucanase, and α-amylase) at 50 • C for 4 h. Mulberry oligosaccharides (MOSs) produced from MPS via digestion with β-mannanase had the greatest effect on the proliferation of A. muciniphila in experiments. Mulberry oligosaccharides (MOSs) were prepared by incubating MPS with 5% (w/v) β-mannanase at 50 • C for 4 h, and then lyophilized.
MOSs were dissolved in water (10% w/v) and then loaded onto a DEAE-52 cellulose column (2.5 × 25 cm), previously equilibrated with water. The column was eluted with water and a step gradient of 0.1 and 0.3 mol/L NaCl at a flow-rate of 1 mL/min. The eluates were collected with an automatic collector (10 mL in one tube). The elution profile was detected using the phenol-sulfuric acid assay. It could be divided into three main elution peaks, namely MOS-I, MOS-II, and MOS-III, which were then lyophilized. MOS-I was selected for further fractionation because of its higher proliferation rate on A. muciniphila proliferation. Size-exclusion chromatography on a Sephadex G-100 column (2.5 × 25 cm) with water at a flow-rate of 1 mL/min yielded a fraction which was named MOS-Ia and then lyophilized. On the basis of previous results, the purified oligosaccharide was composed of galactose. The average molecular weight was 987 Da [19]. Because it solely contains galactose, it was named mulberry galacto-oligosaccharide (MGO).
The number of colony-forming units of colonies of A. muciniphila in BHI agar medium with no added oligosaccharide was defined as having a proliferation level of 100%, where the proliferation level was calculated as: The number of colonies in BHI agar medium with oligosaccharide The number of colonies in BHI agar medium with no oligosaccharide × 100%

Effects of Different Prebiotics on the Growth of A. muciniphila
The positive control group was the GOS group. The negative control group was medium without any added oligosaccharide. GOS, MPS, or MGO were added to BHI medium to achieve the concentration at 5% (w/v). Then, the medium was anaerobically cultured and inoculated with 5% (v/v) of culture of A. muciniphila for 5 days. Samples were plated every day to calculate the number of colonies. The maximum biomass stage of A. muciniphila growth (after 6 days of anaerobic culture) was selected as the sampling time point for comparative transcriptomics. There were three replicates per group. A total RNA extractor extracted the total RNA following the method described by Li et al. [9]. RNA quality was determined with a 2100 Bioanalyser (Agilent Technologies Co., Ltd., Colorado Springs, CO, USA) and quantified using an ND-2000 (NanoDrop Technologies/Thermo Scientific, Wilmington, DE, USA). An RNA-Seq transcriptome library was prepared with the TruSeq RNA Sample Preparation Kit from Illumina (San Diego, CA, USA) using 2 µg of total RNA. Shortly after preparation, ribosomal (r)RNA depletion was performed using the Ribo-Zero Magnetic Kit (Epicenter); then, all mRNAs were broken into short fragments by adding fragmentation buffer. The data generated by the Illumina platform would be used to analyze bioinformatics. The data were analyzed using the online platform Majorbio Cloud Platform (www.Majorbo. com (accessed on 3 September 2021)) at Shanghai Majorbio Bio-pharm Technology Co., Ltd., Shanghai China. For each data set, and for each alignment and quantification protocol, we identified differentially expressed genes (DEGs) by using the edgeR, DESeq2, and DESeq packages.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis
DEGs between the control group (BHI medium group) and treatment groups (5% (w/v) GOS added to the BHI medium group, 5% (w/v) MPS added to the BHI medium group, and 5% (w/v) MGO added to BHI medium group) were assigned using GO and KEGG.

Validation of RNA-Seq Results by RT-qPCR
Thirteen DEGs associated with the galactose metabolism (galM, pfkA, and pgm) or glycolysis/gluconeogenesis (pgi, pfk, fbaA, tpiA, gapA, pgk, gpml, eno, pyk, and lpd) were employed to validate the RNA-seq results. Gene names and primer sequences used for RT-qPCR are shown in Table 1. The PCR programs included 40 cycles of 95 • C for 5 s, annealing at 55 • C for 30 s, and extension at 72 • C for 40 s. The 16S rRNA gene was an internal standard to normalize gene expression. Three independent repetitions were performed for each sample, and the 2 −∆∆Ct method was used to calculate the relative expression levels of genes.

Statistical Analysis
All experiments were represented as the mean ± standard deviation of three replicated measurements, and the results were analyzed using SPSS-19 software (Chicago, IL, USA). Statistical significance (p < 0.05) between treatments was analyzed using one-way analysis of variance, followed by Duncan's multiple-range test.

Proliferation of A. muciniphila Cultured with Different Carbohydrates
In this study, MOS was produced from MPS via digestion with different kinds of enzymes (pectinase, glucoamylase, β-mannanase, xylanase, β-glucanase, and α-amylase). Using commercial prebiotics (GOS and IMO) as controls, the effects of mulberry oligosaccharides with different concentrations on the proliferation of A. muciniphila were investigated (Table 2). It was found that the effect of mulberry oligosaccharide prepared by adding 5% β-mannanase was the greatest on the proliferation of A. muciniphila. The proliferation of the bacterium following treatment with mulberry oligosaccharide produced using 5% β-mannanase (652% ± 9.9%) was superior to that following treatment with GOS (582% ± 10.2%). We speculate that the composition of the sugar chain after enzymatic hydrolysis is an important factor affecting the proliferation of A. muciniphila. Table 2. Proliferation of A. muciniphila cultured in medium containing different prebiotic carbohydrates after 5 days of incubation at 37 • C.

Carbohydrate Concentration (%)
A. muciniphila Proliferation The data are presented as the mean ± SD of three replicates. Means marked with the same letter are not significantly different (p < 0.05) according to Duncan's multiple range test. Abbreviations: MPS, crude mulberry polysaccharide solution.
The mulberry oligosaccharides prepared using β-mannanase were separated into three fractions-MOS-1, MOS-2, and MOS-3 via DEAE-cellulose column chromatography ( Figure 1A). When adding 5% (w/v) MOS-1, the proliferation level of A. muciniphila was 781% ± 10.5 (Table 3). MOS-I was further separated via Sephadex G-100 chromatography in distilled water ( Figure 1B). A fraction termed MOS-Ia was obtained, which showed a single peak in the chromatogram. On the basis of our previous research, MOS-Ia is composed of galactose. The average molecular weight was 987 Da. We named it mulberry galactooligosaccharide (MGO), because the oligosaccharide only contains galactose units [19]. Figure 1C shows the growth curves of A. muciniphila in different BHI media when adding 5% (w/v) GOS, MPS, or MGO. When cultured for 6 days, the total number of colonies reached a maximum value of about 7.8×10 8 colony-forming units (CFU)/mL with the addition of 5% (w/v) MGO. The number of CFU increased about by fivefold in MGO medium compared with the control (BHI with no added prebiotics, 1.5 × 10 8 CFU/mL). Since the total number of A. muciniphila colonies reached the maximum value on the sixth day, the following transcriptome sequencing experiments were performed after 6 days of culture. 245% ± 6.8c MOS-III 1 132% ± 2.3a 3 201% ± 4.8b 5 253% ± 6.1c The data are presented as the mean ± SD of three replicates. Means marked with the same letter not significantly different (p < 0.05) according to Duncan's multiple range test. The degree of polymerization affects the proliferation of probiotics caused prebiotics. Prebiotics with a low degree of polymerization are more readily available probiotics. Compared with MPS, IMO, or GOS, it was found that MGO had the low degree of polymerization, which had a greater proliferative effect on A. muciniphila addition to the degree of polymerization, the chemical structure, monosacchar composition, degree of branching, and water solubility have an impact on the prebi effects. Therefore, a simple chemical structure, high water solubility, and more branc ending in carbohydrate chains in oligosaccharides are considered to have be proliferative effects on probiotics. Curve of Akkermansia muciniphila growth in BHI medium supplemented with different prebiotics (C). Table 3. Proliferation of A. muciniphila cultured in medium containing different elution fractions of mulberry oligosaccharides prepared using β-mannanase after incubation for 5 days at 37 • C.

Carbohydrate Concentration (%)
A. muciniphila Proliferation The data are presented as the mean ± SD of three replicates. Means marked with the same letter are not significantly different (p < 0.05) according to Duncan's multiple range test.
The degree of polymerization affects the proliferation of probiotics caused by prebiotics. Prebiotics with a low degree of polymerization are more readily available to probiotics. Compared with MPS, IMO, or GOS, it was found that MGO had the lowest degree of polymerization, which had a greater proliferative effect on A. muciniphila. In addition to the degree of polymerization, the chemical structure, monosaccharide composition, degree of branching, and water solubility have an impact on the prebiotic effects. Therefore, a simple chemical structure, high water solubility, and more branches ending in carbohydrate chains in oligosaccharides are considered to have better proliferative effects on probiotics.

Cluster Analysis of Differential Gene Expression
The Pearson correlation coefficient is shown in Figure 2. The higher the intensity of the red color, the higher the correlation between two samples and a smaller difference between The Pearson correlation coefficient is shown in Figure 2. The higher the intensity of the red color, the higher the correlation between two samples and a smaller difference between them. The biological duplication from each group had good repeatability, and the group composed of BHI and GOS was significantly different from the groups composed of MPS and MGO.

Identification of DEGs
In order to clarify the gene expression responses of A. muciniphila in different prebiotic treatments, transcriptomic analysis was carried out to identify the DEGs after 6 days of culture. A total of 1277, 1216, and 1240 genes were upregulated when GOS, MPS, and MGO were added to the BHI medium, respectively. Furthermore, 1150, 1205, and 1185 genes were shown to be downregulated. In the respective media, the expression of 159, 165, and 161 genes did not change. Statistical analyses are shown in volcano plots in Figure 3.

Identification of DEGs
In order to clarify the gene expression responses of A. muciniphila in different prebiotic treatments, transcriptomic analysis was carried out to identify the DEGs after 6 days of culture. A total of 1277, 1216, and 1240 genes were upregulated when GOS, MPS, and MGO were added to the BHI medium, respectively. Furthermore, 1150, 1205, and 1185 genes were shown to be downregulated. In the respective media, the expression of 159, 165, and 161 genes did not change. Statistical analyses are shown in volcano plots in Figure 3.  As shown in supplementary excel 1, 119, 195, and 187 genes were upregulated when GOS, MPS, or MGO was added to the BHI medium, with a more than 2 log2fold change. Genes AMUC_RS05445, AMUC_RS06065, AMUC_RS07280, AMUC_RS07405, AMUC_RS08975, and AMUC_RS09930, which, respectively, encode a hypothetical protein, M15 family metallopeptidase, DUF1778 domain-containing protein, acyltransferase, nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase, and DUF2075 domain-containing protein, were highly expressed. The M15 family metallopeptidase of the zinc-binding metallopeptidase family, which contains mostly carboxypeptidases and dipeptidases, is involved in bacterial cell wall biosynthesis and metabolism [20]. Acyltransferase A was found to only have affinity for short-chain aliphatic amides with maximum activity towards acetamide [21]. Nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase catalyzes the synthesis of alpha-ribazole-5 -phosphate from nicotinate mononucleotide and 5,6-dimethylbenzimidazole [22].

GO Annotation Analysis of DEGs
To clarify the changes that occur in biological processes of A. muciniphila on treatment with different prebiotics, GO term enrichment analysis was performed on the up-and downregulated genes identified via RNA-Seq analysis. As shown in Figure 4, upregulated GO functional gene enrichment analysis resulted in a list of affected biological processes, cellular components, and molecular functions. The molecular function category contained the greater part of the GO annotations, followed by the biological process and cellular component categories. In molecular function, the main affected categories were ATP binding, DNA binding, and metal ion binding. In the cellular component ontology, most DEGs were associated with the integral components of membrane, cytoplasm, and ribosome. Among the biological process, most enriched DEGs were in translation, cell redox homeostasis, and glycolytic processes. The upregulated GO functional gene component categories for the GOS, MPS, and MGO treatments contained 556, 560, and 659 genes, respectively; that is, the value was highest when MGO was added to A. muciniphila cultures.

KEGG Analysis of DEGs
To further identify changes in biochemical pathways following different prebiotic treatments, we used the KEGG database to map DEGs [23]. KEGG pathways containing

KEGG Analysis of DEGs
To further identify changes in biochemical pathways following different prebiotic treatments, we used the KEGG database to map DEGs [23]. KEGG pathways containing DEGs were identified for analysis ( Figure 5). Among them, the maximum number of DEGs of five pathways when the BHI medium was cultured with MGO were the carbohydrate metabolism, the metabolism of cofactors and vitamins, the energy metabolism, the amino acid metabolism, and the lipid metabolism. MGO is an oligosaccharide which only contains galactose units. Combined with the KEGG pathology analysis results, we supposed that the MGO-dependent proliferation mechanism of A. muciniphila stimulates genetic changes which correlate with the carbohydrate metabolism, especially the galactose metabolism and glycolysis/gluconeogenesis. Thus, in order to understand the molecular mechanisms underlying the effects of MGO on the galactose metabolism and glycolysis/gluconeogenesis, we screened DEGs related to these two pathways in the transcriptomic sequencing results (Table 4).  MGO is an oligosaccharide which only contains galactose units. Combined with the KEGG pathology analysis results, we supposed that the MGO-dependent proliferation mechanism of A. muciniphila stimulates genetic changes which correlate with the carbohydrate metabolism, especially the galactose metabolism and glycolysis/gluconeogenesis. Thus, in order to understand the molecular mechanisms underlying the effects of MGO on the galactose metabolism and glycolysis/gluconeogenesis, we screened DEGs related to these two pathways in the transcriptomic sequencing results (Table 4).
Two DEGs (galM and pfkA) were upregulated and one (pgm) was downregulated in relation to the galactose metabolism. RT-qPCR data ( Figure 6A) were almost the same as the gene expression obtained from RNA-Seq, indicating that the data from the RNA-seq were reliable. In RT-qPCR, the mRNA levels of galM and pfkA increased by 396% and 357%, respectively, and that of pgm was decreased to 63% when the addition of MGO into the BHI medium reached 4% (w/v). In the KEGG, galM is assigned as galactose mutarotase. Galactose mutarotase is an important metabolism-related enzyme in bacteria which catalyzes the change in the optical rotation of α-D-galactose to generate β-D-galactose, the only substrate of galactose kinase. Galactose mutarotase is thus a key enzyme in the process of galactose metabolism and an important member of the galactose operon [24][25][26][27]. The KEGG pathway assignment of pfkA is 6-phosphofructokinase. The 6-phosphofructokinase enzyme is the rate-limiting enzyme of the glycolytic pathway; its activity strictly controls the rate of glycolysis and greatly affects the use of hexoses by bacteria. The 6-phosphofructokinase enzyme is a key node in the glucose metabolism, which is accompanied by a large amount of energy consumption. The loss of 6-phosphofructokinase leads to an imbalance of reducing power, which affects downstream metabolic pathways, including pathways related to nitrogen fixation. Therefore, in bacteria, it directly affects the use of carbon sources [28][29][30][31]. In the KEGG, galM is assigned as galactose mutarotase. Galactose mutarotase is an important metabolism-related enzyme in bacteria which catalyzes the change in the optical rotation of α-D-galactose to generate β-D-galactose, the only substrate of galactose kinase. Galactose mutarotase is thus a key enzyme in the process of galactose metabolism and an important member of the galactose operon [24][25][26][27]. The KEGG pathway assignment of pfkA is 6-phosphofructokinase. The 6-phosphofructokinase enzyme is the rate-limiting enzyme of the glycolytic pathway; its activity strictly controls the rate of glycolysis and greatly affects the use of hexoses by bacteria. The 6-phosphofructokinase enzyme is a key node in the glucose metabolism, which is accompanied by a large amount of energy consumption. The loss of 6-phosphofructokinase leads to an imbalance of reducing power, which affects downstream metabolic pathways, including pathways related to nitrogen fixation. Therefore, in bacteria, it directly affects the use of carbon sources [28][29][30][31].
The pgi in KEGG pathway is glucose-6-phosphate isomerase. Glucose-6-phosphate isomerase exists in eukaryotes and prokaryotes, and it is a multifunctional enzyme whose main function is to catalyze the mutual conversion between glucose-6-phosphate and fructose-6-phosphate in the process of glycolysis [32][33][34][35][36]. tpiA encodes triose-phosphate isomerase, an enzyme in the glycolytic pathway. In this pathway, 1,6-diphosphate fructose reacts with aldolase to generate dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Dihydroxyacetone phosphate and glyceraldehyde 3-phosphate can be interconverted under the catalysis of triose-phosphate isomerase; glyceraldehyde 3-phosphate will continue to be catalyzed to pyruvic acid [37][38][39]. The KEGG pathway assignment of eno is phosphopyruvate hydratase. Phosphopyruvate hydratase is a key enzyme in the glucose metabolism pathway and is expressed abundantly in the cytoplasm of many organs. Its role is to convert 2-phospho-glycerate into phosphoenolpyruvate [40,41]. The KEGG pathway assignment of pyk is pyruvate kinase. Pyruvate kinase is involved in the last reaction of glycolysis and is one of the main rate-limiting enzymes in the process [42]. It catalyzes the synthesis of one molecule of adenosine triphosphate and pyruvate from adenosine diphosphate and phosphoenolpyruvic acid, which requires the participation of Mg 2+ [43]. Pyruvate kinase is produced in all cells and tissues capable of glycolysis, and its metabolites, adenosine triphosphate and pyruvate, are used in numerous biosynthetic pathways [44,45]. Pyruvate kinase is a master regulator that controls adenosine triphosphate production in glycolysis and is considered a potential drug target [46,47]. However, because of the conserved structure of the active site of pyruvate kinase and its central role in glycolysis in all organisms, the development of selective inhibitors against the active site of this enzyme has been difficult.

Conclusions
In the present study, MGO was found to be a good stimulus to proliferate A. muciniphila. We discovered that after adding MGO to the BHI medium, GO functional analysis revealed that 659 genes were upregulated in A. muciniphila. These genes were mainly enriched in ATP binding, an integral component of membranes, and translation. When DEGs were mapped to the KEGG database, the upregulated genes were enriched in the carbohydrate metabolism, the metabolism of cofactors and vitamins, the energy metabolism, the amino acid metabolism, and the lipid metabolism. In the significantly up-regulated genes, galM and pfkA are relevant to the galactose metabolism, and pgi, pfk, fbaA, tpiA, gapA, pgk, gpml, eno, pyk, and lpd are relevant to the glycolysis/gluconeogenesis pathway. Our research provides a more in-depth theoretical basis for the application of A. muciniphila and MGO.

Data Availability Statement:
The data are available from the corresponding author.

Conflicts of Interest:
The authors declare that they have no conflict of interest.