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Sign in to access free PDF. Although adenine is the most important methylation target, e. These are found in cytosine and depend on the modification site: m4C N 4 -methyl-cytosine and m5C C 5 -methyl-cytosine Murphy et al.

In general, m6A has been used as the main signal for epigenetic regulation in bacteria, m5C has been mainly described in mammals and plant studies Kim et al. DNA methylation plays an important role in the epigenetic regulation of eukaryotes via gene expression modulation Moore et al.

Indeed, methylation of promoter and gene body can either silence or promote expression, respectively Yang et al. In bacteria, DNA methylation may also play regulatory roles in the control of DNA replication or transcription Reisenauer et al. Single-molecule real-time SMRT sequencing Pacific Biosciences is a recently developed DNA sequencing method where kinetic data can also be used to distinguish base modifications, e.

Several bacterial methylomes have been determined using SMRT sequencing technology Fang et al. Specifically, a methylome analysis of M.

tuberculosis complex MTBC was reported by Zhu et al. Vitamins are organic compounds that cannot be produced by the host organism. Even when they are produced, concentrations are low and external supplementation from the diet or from commensal bacteria is required.

Vitamins have been found to regulate immunity, and a number of studies have reported the effect of vitamins as adjunct to treat tuberculosis Parida et al. For example, vitamins A, C and D have been used as adjunct to complement anti-tuberculosis drugs Dini and Bianchi, ; Vilcheze et al.

The active form of thiamin Vitamin B1, V B 1 , thiamin diphosphate ThDP Begley et al. In addition, V B 1 plays an important role in the activation of the immune system, nerve tissue repair, neuronal communication and cell-membrane dynamics Gibson and Blass, ; Manzetti et al.

Recent studies have shown the effects of V B 1 both in vitro and in vivo ; V B 1 inhibits in vitro growth of BCG with an MIC of 8 mM, whereas it limits Mtb in vivo growth by regulating innate immunity in a peroxisome proliferator-activated receptor γ-dependent manner Hu et al.

V C also can hamper the growth of Mtb by a mechanism that involves the reduction of ferric to ferrous ion and subsequent production of reactive oxygen species i. BCG is also sensitive to Vc, with an MIC is 0. Microbial metabolomics constitutes an integrated component of systems biology.

By studying the complete set of metabolites within a microorganism and monitoring the global outcome of interactions between its development processes and the environment, metabolomics can potentially provide a more accurate snap shot of the actual physiological state of the cell.

Furthermore, when these data are interpreted in combination with genomics, proteomics and transcriptomics data and so on, using what is termed a systems biology approach, a more holistic understanding of these systems can be achieved.

Up to now, the metabolomics has contributed to characterize Mtb in terms of metabolism, growth and replication, pathogenicity, and drug resistance, from the perspective of systems biology Swanepoel and Loots, For example, Meissner-Roloff compared the metabolomes of a hypo- and hyper-virulent Beijing Mtb strain and subsequently identified a reduction in various metabolite markers in the relatively hyper-virulent strain Meissner-Roloff et al.

Loots used metabolomics approach to identify potentially new metabolic pathways and metabolite markers to explain many of the phenotypical characteristics associated with a katG mutation and the resulting isoniazid-resistance in Mtb Loots du, Additionally, metabolomics was to identify individual metabolites or metabolite profiles that could be used as biomarkers of early MAP Mycobacterium avium subsp.

Paratuberculosis infection in ruminants De Buck et al. However, despite transcriptomics and proteomics efforts have been studied Shiloh et al. DNA methylation and transcriptional regulation are tightly related and play an important role in the epigenetics of living organisms, whereas vitamins have an anti-tuberculosis effect.

These prompted us to study the methylome, transcriptome and changes in metabolism of Mycobacterium bovis BCG after V B 1 and V C stimulation. Mycobacterium bovis BCG str. Tokyo strain was grown in Middlebrook 7H9 medium Becton Dickinson, supplemented with 0. When OD nm reached 0. This was followed by incubation at 37°C for 48 h.

At least three replicates were prepared for each condition. Different concentrations 0, 1, 2, and 4 mM of cysteine were used to test the effect for BCG growth. These samples were then sonicated for 5 min and incubated at 37°C for 1 h.

RNase was added to the sonicated samples and incubated for 5 min at room temperature, followed by protease K addition, according to manufacturer instructions TIANamp Bacteria DNA Kit protocol, Tiangen Biotech, Beijing, China.

The concentration of DNA samples was determined using a NanoDrop ND spectrophotometer. The extracted genomic DNA from BCG was sequenced using the Pacific Biosciences RSII DNA sequencing system Pacific Biosciences, Menlo Park, CA, United States.

One SMRT cell was used for each sequencing. De novo assembly of the insert reads was performed with the Hierarchical Genome Assembly Process HGAP.

Circularization was achieved by manual comparison and removal of an overlap region. The final genome was confirmed by remapping of sequence data. Promoter regions were analyzed using the Neural Network Promoter Prediction tool 1 and PePPER 2.

The SMRT Portal searches close to maximize through of all possible motifs, progressively testing longer motifs using a branch-and-bound search. The 60 mg pellets were collected by centrifugation and the supernatant were removed, the metabolites are measured by LC-MS as described Song et al.

After membrane filtration 0. Separation was achieved in a Shimadzu LCA system equipped with an ACQUITY UPLC HSS T3 × 2. The temperature of the autosampler was 4°C. Gradient elution of analytes was carried out with 0. After equilibration, 5 μL of each sample were injected.

Gas1 and gas2 were both set at 50 psi and curtain gas was 35 psi. The source temperature was °C. The amino acids of samples treated with V B 1 and V C were detected by HR-UPLC-MS high resolution ultra-performance liquid chromatography-mass spectrometry as described Song et al.

Briefly, an equimolar standard mixture of 20 amino acids i. Pellets of bacteria mg were ground in liquid nitrogen, and amino acids were extracted with 1 mL HCl overnight. The supernatant was collected by centrifugation at 12, rpm for 5 min. Chromatographic separation was achieved with an ACQUITY UPLC system equipped with a 1.

Gradient elution of the analytes was implemented with solvent A 0. Five μL of sample were injected following equilibration.

The ESI source was applied in positive mode by multiple reaction monitoring MRM. The ion source capillary voltage and cone voltage were set as 3, and 20 V, respectively, and the desolvation temperature was set at °C.

Since methylation and transcriptomics are both ways of studying epigenetics and they have the close relationship, to further investigate how V B 1 and V C treatment affect BCG growth, RNA-seq was used to compare the transcriptome of the three replicates of each BCG sample V B 1 -treated, V C -treated and control.

RNA-seq was performed as described Song et al. Cell pellets were lysed and homogenized by high-speed agitation in a bead mill in presence of glass beads and lysis buffer. Total RNA was extracted using RNeasy Mini Kit Qiagen according to the instructions with one on-column DNase I treatment Qiagen at 37°C for 30 min, in order to remove any contaminating genomic DNA.

DNase I was removed with RNeasy mini kit, according to the clean-up procedure Song et al. The RNA integrity number RIN was used to inspect RNA integrity by an Agilent Bioanalyzer Agilent Technologies, Santa Clara, CA, United States. The concentration of RNA samples was determined with a NanoDrop ND spectrophotometer.

The strand-specific library was constructed using TruSeq ® Stranded Total RNA Sample Preparation kit Illumina, United States. The ribosomal RNA was removed and the RNA fragments were cleaved, whereas the first and second strand cDNA were synthesized.

The purified libraries were quantified using Qubit ® 2. The cluster was generated by cBot, and RNA sequencing was performed using a bp pair-end strategy with the Illumina Hiseq X10 platform Illumina, United States to generate three billion bases per sample.

Thereafter, raw data was acquired, followed by pretreatment using Septk1. The clean reads were mapped to the Mycobacterium bovis BCG str. Tokyo genome using Bowtie2 Langmead and Salzberg, Differential expression analysis was performed using edgeR package Robinson et al.

Differentially expressed genes were defined as experiencing more than a 2-fold change and a FDR false discovery rate of less than 0. Enrichment of KEGG pathways for a given gene list was calculated using a classical hypergeometric distribution statistical comparison of a query gene list against a reference gene list.

KEGG pathways fulfilling this condition were defined as significantly enriched in regulated genes. The raw data was deposited in the GEO repository, under accession numbers GSE V B 1 -treated and GSE V C -treated. The experiments were performed at least in biological triplicate and the results are presented as the mean ± standard deviation SD.

Since the CFU counting of BCG is similar after V B 1 8 mM and V C 5 mM treatment for 48 h and the differentiation continues comparing with the control group Figure 1 , all the omics studies were performed with final concentration of 8 mM for V B 1 and 5 mM for Vc.

SMRT sequencing was used to determine the genome-wide distribution of methylated bases in V C -treated, V B 1 -treated and control BCG.

The average sequencing coverage was X Supplementary Table 1. Analysis of the SMRT-sequenced data detected N 6 -methyl-adenine m6A in the three samples, whereas m4C sites, 5,, were only detected in the Vc-treated BCG sample Supplementary Table 2.

Figure 1. CFU assays of BCG treated with V B 1 8 mM and Vc 5 mM. In total, 5, m6A sites were detected in the three BCG samples and 4, of them Five m6A sequence motifs were detected Supplementary Table 3.

A total of 3, adenines in this motif A total of 1, protein coding sequences CDS were affected by m6A, accounting for COG class enrichment analysis also revealed that m6A was present in genes participating in cell envelope biogenesis and outer membrane, also suggesting that the m6A modification has a protective role.

In contrast, un-methylated genes i. COG class enrichment analysis also revealed abundant un-methylated genes with unknown biological function.

Surprisingly, m4C was observed only in samples treated with V C , with 5, m4C sites in three motifs Supplementary Table 3. Among these, 1, CDSs were also methylated at adenine sites i.

This suggests that, since V C treatment hampers BCG growth, genes involved in cell envelope biogenesis become more protected. In eukaryotes, promoter methylation has been suggested to inhibit gene expression. Our analysis discovered 4, m6A sites located in CDSs, and 7.

Since the promoter regions in bacteria are usually located within bp upstream of the start codon Heyden et al. Only m6A modifications located within bp upstream of the start codon were assumed to affect genes.

In this way, only out of the m6A sites in IGRs probably affected the promoters of genes Supplementary Table In BCG treated with V C , m4C were located in IGRs, where affected promoters of genes Supplementary Table Figure 2. Distances relative to the start codon in intergenic region of m6A sites A and m4C sites B basing on the methylation sites in V B 1 and V C treated BCG samples.

To investigate how V B 1 and V C treatment affects BCG growth, RNA-seq was used to compare the transcriptome of the three replicates of each BCG sample V B 1 -treated, V C -treated and control. The mean FPKM Fragments Per Kilo bases per Million reads was A total of genes were regulated in V B 1 -treated BCG, and in Vc-treated BCG Figure 3.

Of these, more down-regulated genes were found for V B 1 -treated BCG, which were enriched in pathways like Nitrogen metabolism and Two-component system Supplementary Table 7. In contrast, V C -treated BCG showed more up-regulated genes involved in replication and repair.

In the Vc-treated BCG samples, lower expression was found in genes involved in energy metabolism. In addition, we note that expression of genes involved in biosynthesis of siderophore group non-ribosomal peptides was inhibited by V B 1 , but stimulated by V C Supplementary Table 7.

The same pattern was observed for another six genes, with two of them mycobactin polyketide synthetase gene MbtB and MbtC participating in this pathway. Figure 3. Significantly regulated BCG genes after vitamin treatment.

The Y -axis indicates the gene number of significantly changed genes. Partial omics data of V B 1 can be found in this published paper Song et al.

To investigate the relationship between gene methylation and gene expression, we analyzed the methylation changes in significantly regulated genes. There were 19 unique m6A sites in V B 1 -treated BCG, but only one of them was located within the coding region of significantly down-regulated genes, away from the promoter region.

For the 20 unique m6A sites in V C -treated BCG, two were located in the promoter region of significantly up-regulated genes Supplementary Table In that sample, m4C was found in the coding region of 31 down-regulated genes These results suggest that gene expression is not affected when methylation is in the coding region, but when m4C is located in the promoter region it contributes to up-regulate BCG gene expression.

To validate this hypothesis, we compared the gene expression levels of methylated and non-methylated genes, based on their FPKM.

Liquid chromatography-mass spectrometry LC-MS was used to analyze six bacterial sediments of each of the three BCG samples V B 1 -treated, V C -treated and control.

After V B 1 or V C treatment, the number of differential metabolites was and 3,, respectively Figure 4. Figure 4. The diagram for differential metabolites found in the three BCG samples examined.

We identified 41 metabolites with significant variation, 10 in V B 1 -treated BCG and 21 in Vc-treated BCG Figure 5. Enrichment analysis revealed that variant metabolites were mainly clustered in amino acid metabolism pathways Supplementary Table 8.

After V B 1 treatment, variant metabolites were significantly enriched in cysteine and methionine metabolism Supplementary Table 7. Figure 5. Correlation of significantly variant metabolites of BCG treated with V B 1 A and BCG treated with Vc B relative to control.

Since exposure to vitamins led to variant metabolites enrichment in amino acid metabolism pathways, we performed additional amino acids content analysis using six bacterial sediments of each of the three BCG samples V B 1 -treated, V C -treated and control. Exposure to vitamins significantly reduced V B 1 or increased V C amino acid content Supplementary Table 9.

The expression changes associated with amino acid metabolism were also analyzed, as described below. Another significantly down-regulated gene was chorismate mutase [5.

We then examined the pyruvate metabolism and glycolysis pathways. In the pyruvate metabolism pathway, no significant up-regulation was observed, indicating that the conversion of pyruvate to other metabolites did not increase. However, in the glycolysis pathway, a fold reduced expression was observed for 6-phosphofructokinase 2 [2.

For histidine synthesis, although no significant gene expression change was observed, genes involved in five out of nine steps were down-regulated. Genes involved in the synthesis of other amino acids also showed the same pattern, where at least one gene encoding a key enzyme was down-regulated Supplementary Table V C addition caused ornithine to increase 1.

Also, three out of four genes encoding enzymes that convert ornithine to arginine doubled their expression levels. V C addition also increased the content of aspartate 2. L -asparaginase [3.

Thus, the observed increase of Thr might be due to an increase in Asp content Supplementary Figure 6 and to a down-regulation of genes degrading Thr Supplementary Table The tryptophan synthase [4. Strikingly, the cysteine synthase A [2.

Finally, we found no direct association between base modification in coding or promoter regions and expression of genes involved in amino acid metabolism Supplementary Table Taken together, we found significant regulation of several genes directly involved in amino acid biosynthesis and metabolism, consistent with amino acid content changes.

However, most gene expression changes were not significant. We speculate that treatment with V B 1 and V C changes amino acid levels by altering precursor concentrations. Following the observation that vitamins treatment increase the transcription levels of cysteine synthase A, we determined the effect of cysteine on BCG replication.

Different concentrations of cysteine 0, 1, 2, and 4 mM were used for CFU calculation and the results are summarized in Figure 6. The growth curve demonstrates that cysteine inhibits BCG growth in a concentration-dependent manner.

Figure 6. BCG treatment with cysteine at concentrations 0, 1, 2, and 4 mM. Aliquots taken at indicated times and plated to determine CFUs. Vitamins such as biotin and thiamine are essential for M. tuberculosis growth and infection Tyagi et al.

At the same time, supplementation of some vitamins together with anti-TB drugs has been shown to improve chemotherapy outcomes Vilcheze et al. Clinical trials have shown that Vc supplementation improves the healing process in tuberculosis patients Tjandrawinata et al.

Therefore, the mechanism by which some vitamins can ammeliorate TB requires further investigation. Mtb growth is inhibited by vitamins A V A and V D Greenstein et al. Thus, due to the potential supplementary role of certain vitamins in TB treatment, analysis of the BCG methylome can help elucidate the relationship between base methylation, gene expression and metabolic products.

Many DNA methylation enzymes are part of RM systems, and are involved in the defense against phages and viruses.

We found Regulation of gene expression by DNA methylation occurs via more than one mechanism. DNA methylation hinders the interaction between DNA and regulatory proteins by direct atomic steric effects Sanchez-Romero et al. Additionally, some DNA-binding proteins inhibit methylation of specific DNA sequences by binding to unmethylated DNA with high affinity.

In the present study, we found that genes Additionally, 65 genes that harbored m4C sites in the promoter region showed expression levels significantly higher than the control no m4C sites , based on FPKM values.

This suggests that m4C can promote gene transcription to some extent, but using mechanisms that are still unknown. The m4C modification was observed in type I R-M systems, suggesting that, in addition to m6A modifications, they can also use this modification for host protection Morgan et al.

Recent studies have shown that N 4 -cytosine DNA methylation can regulate transcription and pathogenesis in Helicobacter pylori Kumar et al.

Indeed, in human gastric adenocarcinoma cells, H. pylori mutant with increasing m4C methylation increased adhesion, while the bacteria without m4C methyltransferase halved the adhesion rate and significantly reduced H.

pylori -induced apoptosis. Additionally, in Cyanobacterium Synechocystis sp. PCC m4C, methylation is involved in the regulation of gene expression, fine-tuning of DNA replication and DNA repair mechanisms Gartner et al.

Until now, however, the role of m4c modification in Mtb has been uncertain. In our study, we found 11 up-regulated genes harboring an m4c modification in the promoter region Supplementary Table 12 of BCG genome are homologous with these genes which had been studied to show important roles in Mtb.

This is proposed as an energy source during dormancy, and its disruption prevents triacylglycerol accumulation under inducing conditions Sirakova et al. In metabolomics, we found that the concentrations of several metabolites including tryptophan, inosine, NAD and ADP decreased after V B 1 and Vc treatment.

Tryptophan is classified as an essential amino acid in humans and must be acquired through the diet. This requirement alleviates the concern of common targets within humans. Recent studies found that tryptophan synthase which catalyzes the final step in tryptophan biosynthesis is one potentially novel anti-tubercular drug target Abrahams et al.

In addition, treatment with 6-FABA 2-aminofluoro-benzoic acid 6-FABA chemically induces Mtb tryptophan auxotrophy.

Together with immune- mediated trypothan starvation, this results in mycobacterial death. The results provide genetic and chemical validation of the tryptophan biosynthesis pathway as a target for highly active antibiotics Zhang et al.

Inosine is made up of deaminizing adenosine and can be released extracellularly under inflammatory conditions Jabs et al.

Inosine has been reported to exert nutritional and neuroprotective functions in nerve cells, and stimulates mast cell degranulation by activating adenosine A3 receptor Jin et al. Moreover, inosine exhibits anti-inflammatory activity by inhibiting the release of inflammatory cytokine from activated T cells and promoting the formation of IL Clinical trials have proposed that the use of inosine to raise serum urate levels may have benefits for at least some multiple sclerosis patients.

The effect of this treatment is likely to be a consequence of inactivation of peroxynitrite-dependent free radicals Markowitz et al. NAD P is an indispensable cofactor for all organisms and its biosynthetic pathways are proposed as promising novel antibiotics targets against pathogens such as Mtb.

Since the synthesis pathways of these significantly reduced metabolites are mostly drug targets for the treatment of tuberculosis, we hypothesized that the addition of vitamins may directly or indirectly affect the synthesis or metabolic processes of these products.

This could potentially inhibit bacterial growth. A striking result in this study is that V B 1 treatment of BCG resulted in content reduction of almost all types of amino acids, whereas Vc treatment caused the opposite effect. Through RNA-seq analysis, we found that the gene encoding cysteine synthase A was significantly up-regulated after vitamin treatment, suggesting that Cys synthesis is promoted.

High levels of intracellular cysteine can induce ROS production, causing DNA damage and oxidation of cysteine to cystine in E. coli Park and Imlay, Indeed, transition metals, such as copper or iron, catalyze the oxidation of cysteine into cystine, leading to the production of hydrogen peroxide H 2 O 2 , superoxide, and hydroxyl radicals Kachur et al.

This high concentration of cysteine could therefore initiate cationic stress, resulting in the formation of ROS and DNA damage Imlay et al. In our study, the gene encoding cysteine synthase A was significantly up-regulated after V B 1 and Vc treatment and this hinting that the synthesis of cysteine is promoted.

In addition, in vitro growth test show that cysteine inhibit the growth of BCG in a concentration-dependent manner. Therefore, we hypothesize that V B 1 and Vc inhibit BCG growth by increasing the concentration of cysteine, which can eventually damage DNA via Fenton reaction.

This hypothesis is consistent with the transcriptomics results, where Vc induced the expression of genes related to DNA repair, suggesting that Vc may indirectly affect DNA integrity. Notably, the scientists found that Vc sterilized Mtb cultures via Fenton reaction and increased iron concentration, which correlates with the bactericidal activity of V C Vilcheze et al.

The results shown in the present study suggest that BCG growth inhibition may not be due just to high levels of iron, but also to cysteine. In addition, decreased concentrations of tryptophan, NAD P and other metabolites are also potential reasons for vitamin inhibition of BCG growth Figure 7.

Our study also identifies another possible mechanism by which vitamins inhibit BCG growth. Therefore, V B 1 and Vc supplementation may enhance the action of anti-tuberculosis drugs.

Figure 7. Schematic representation of the mechanism of action of vitamins against BCG growth. The datasets generated for this study can be found in the Gene Expression Omnibus, accession numbers GSE and GSE NS designed this study and wrote the manuscript. YZ analyzed the data and prepared the figures.